WO2001023431A1 - Derivatives of antibody against ganglioside gm2 - Google Patents

Derivatives of antibody against ganglioside gm2 Download PDF

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Publication number
WO2001023431A1
WO2001023431A1 PCT/JP2000/006775 JP0006775W WO0123431A1 WO 2001023431 A1 WO2001023431 A1 WO 2001023431A1 JP 0006775 W JP0006775 W JP 0006775W WO 0123431 A1 WO0123431 A1 WO 0123431A1
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antibody
region
chain
amino acid
seq
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PCT/JP2000/006775
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French (fr)
Japanese (ja)
Inventor
Nobuo Hanai
Kazuyasu Nakamura
Rinpei Niwa
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Kyowa Hakko Kogyo Co., Ltd.
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Priority to AU74492/00A priority Critical patent/AU7449200A/en
Publication of WO2001023431A1 publication Critical patent/WO2001023431A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to an antibody against ganglioside G2 (hereinafter, referred to as G2) and an antibody fragment thereof.
  • the invention further relates to DNA sequences encoding the above derivatives and antibody fragments.
  • the present invention relates to an expression vector comprising the DNA sequence, and a cell transformed by the expression vector.
  • the present invention further relates to a method for producing the above derivative and antibody fragment using the transformed cell, and a diagnostic and therapeutic agent for cancer using the above derivative and antibody fragment.
  • HAMA Human Anti Mouse Antibody
  • a non-human animal antibody can be used as a human-type chimeric antibody or a human-type complementarity determining region (hereinafter referred to as CDR) transplanted antibody, etc.
  • CDR human-type complementarity determining region
  • a human chimeric antibody is an antibody in which the antibody variable region (hereinafter, referred to as V region) is an antibody of a non-human animal and the constant region (hereinafter, referred to as C region) is a human antibody. Idings 'Ob The National' Academy 'Ob' Science (Proc. Natl. Acad. Sci.
  • Human CDR-grafted antibodies are antibodies from non-human animals This is an antibody obtained by grafting the amino acid sequence of the CDR in the V region of the above to an appropriate position of a human antibody [Nature, 321, 522 (1986)].
  • These humanized antibodies have various advantages in human clinical application compared to non-human animal antibodies such as mouse antibodies. For example, in terms of immunogenicity and stability in blood, it has been reported that the human chimeric antibody, when administered to humans, has an approximately six-fold increase in blood half-life compared to the mouse antibody [Procy. One Ding's' Op 'The National' Academy 'Ob' Science (Proc. Natl. Acad. Sci.
  • ADCC activity complement-dependent cytotoxicity
  • the high cytotoxic activity such as antibody-dependent cytotoxic activity (hereinafter referred to as ADCC activity) is important for its therapeutic effect. Since the Fc region of a human antibody can more efficiently activate human complement components, Fc receptors of monocytes, macrophages, and NK cells on the cell surface, than the Fc region of It is reported to be superior.
  • a mouse antibody against ganglioside GD2 (hereinafter referred to as GD2) (hereinafter referred to as anti-GD2 mouse antibody) is a human chimeric antibody having an Fc region of a human antibody (hereinafter referred to as anti-GD2 chimeric antibody).
  • anti-GD2 mouse antibody is a human chimeric antibody having an Fc region of a human antibody (hereinafter referred to as anti-GD2 chimeric antibody).
  • Ganglioside which is a kind of glycolipid containing sialic acid, constitutes the cell membrane of an animal, and is a molecule composed of a sugar chain, which is a sex side chain, and sphingosine and a fatty acid, which are sex side chains. It is known that the type and expression level of gangliosides vary depending on cell types, organ types, animal types, and the like. It is also known that the expression of gangliosides changes quantitatively and qualitatively in the process of canceration of cells [Cancer's Research (Cancer Res.), 45, 2405 (1985)].
  • GM2 which has been noted in the present invention, is present in only a very small amount in normal cells, but is present in large amounts in cancer cells such as lung cancer, melanomas, gliomas, and neuroblastomas [Cancer-Research (Cancer- Res.), 45, 2405 (1985); Proceedings of the 'The' National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 80, 5392 (1983); Cancer Research (J. Natl.
  • an antibody against GM2 (hereinafter referred to as anti- GM2 antibody) is considered to be useful for the diagnosis and treatment of these cancers [Lancet, I, 786 (1989)]. Furthermore, in recent years, the usefulness of GM2 as a vaccine in the treatment of melanoma has been shown, and a high anti-GM2 antibody titer has been observed in the serum of administered patients [Procedures of the National ' Academy of Sciences (Proc. Natl. Acad. Sci. USA), 92, 2805 (1995)].
  • anti-GM2 humanized antibody a humanized antibody against GM2 having the same activity as mouse antibody (hereinafter referred to as anti-GM2 humanized antibody) was produced.
  • anti-GM2 human chimeric antibody hereinafter referred to as anti-GM2 quinula antibody
  • anti-GM2 quinula antibody an anti-GM2 human chimeric antibody
  • anti-GM2 quinula antibody an anti-GM2 human chimeric antibody
  • Anti-GM2 chimeric antibody KM966 and anti-GM2 CDR ⁇ -implanted antibody KM8969 have lower immunogenicity in humans than anti-GM2 mouse antibodies, have a longer half-life in blood, and have a longer-lasting therapeutic effect, and even stronger cells A high antitumor effect based on the damaging activity is expected.
  • humanized antibodies against tumors are isolated by binding of complement components via the Fc region of human antibodies, CDC activity based on activation and ADCC activity based on binding to Tc receptors, etc.
  • Therapeutic effect is also expected from the use of, but it is being studied to further enhance the effect by using it in combination with other molecules.
  • cytokines are used as one of these molecules. Cytokine is a general term for various humoral factors that control cell-cell interactions in the immune response.
  • ADCC activity is carried by mononuclear cells, macrophages, and effector cells having the Fc receptor of NK cells on the cell surface [J.
  • cytokine interleukin 2 (hereinafter referred to as hIL-2) or human granulocyte-macrophage colony stimulating factor (hereinafter referred to as hGM-CSF)
  • hIL-2 human cytokine interleukin 2
  • hGM-CSF human granulocyte-macrophage colony stimulating factor
  • a protein was prepared by fusing an antibody molecule and cytokine in order to efficiently reach the cytokinin only to the target tissue.
  • hIL-2 a fusion protein with anti-GD2 chimeric antibody chl.18.18 was genetically engineered, and its tumor effect was demonstrated in mice using an anti-GD2 chimeric antibody chl4.18 and hIL-2. It has been reported that it is superior to co-administration [Procedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 89, 1428 (1992); Proceedings' ob 'the national' aka Natl. Acad. Sci.
  • Radiopharmaceuticals 3, 60 (1990); Surgery, 106, 533 (1989)].
  • These derivatives provide more effective diagnosis with less side effects by accumulating proteins (cytokines, toxin enzymes, etc.), radioisotopes, low-molecular-weight drugs, etc. around the target tissue in accordance with the isomerism of the antibody molecule. It is expected that treatment will be possible.
  • cytokines, toxin enzymes, etc. proteins
  • radioisotopes low-molecular-weight drugs, etc.
  • a higher diagnostic and therapeutic effect than the antibody alone can be expected.
  • antibody fragments single-chain antibodies derived from anti-GD2 mouse antibodies have been produced, Hybridoma, 16, 335 (1997)], and there were no reports of the production of other antibody fragments.
  • antibody fragments derived from anti-G2 antibodies antibody fragments derived from mouse antibodies and humanized antibodies There is no report of production. Therefore, if a fragment derived from an anti-GM2 humanized antibody can be prepared, it is expected that humans will have excellent targetability and immunogenicity will be reduced. Furthermore, the therapeutic effect of fragments derived from the anti-GM2 antibody can be further improved if various derivatives can be produced by fusing the above-mentioned proteins (cytokines, toxins, enzymes, etc.), radioisotopes, low molecular drugs, etc. It is expected to increase. Disclosure of the invention
  • an antibody fragment of the known anti-GM2 antibody was prepared for the purpose of enhancing the therapeutic effect of the antibody. Furthermore, an antibody derivative in which the anti-G2 antibody or the antibody fragment was bound to a radioisotope, protein or low-molecular drug was prepared. Specifically, a cDNA was constructed in which the cDNA encoding hIL-2 was linked to the 3 'end of the cDNA encoding the H chain of the anti-GM2CDR ⁇ planted antibody KM8969, and the cDNA and the L chain of 1M8969 were constructed.
  • the encoded cDNA is cloned into an expression vector for animal cells, and an expression vector for a fusion protein of the anti-GM CDR ⁇ plant antibody KM8969 and hIL-2 (hereinafter referred to as KM8969—hIL-2) is obtained. It was constructed. By introducing the KM8969-hIL-2 expression vector into animal cells, a transformed clone KM8969ML2 (FEM BP-6792) producing KM8969_hIL-2 in the culture supernatant was prepared.
  • FEM BP-6792 transformed clone KM8969ML2
  • K8969-ML-2 was purified from the culture supernatant of the transformed clone KM8969hIL2, and KM8969-hIL-2 had the same antigen-binding activity, anti-GM2CDR ⁇ antibody as KM8969, 3 ⁇ 43 ⁇ 4 binding specificity, and hIL-2-dependent growth was found to exhibit the same growth supporting activity as that of hIL-2 against fine JK. Furthermore, it was confirmed that the activity of M8969-hIL-2 was enhanced in the cytotoxic activity using the human peripheral blood mononuclear cell fraction as compared to the anti-G2CDR ⁇ plant antibody KM8969, and the present invention was completed. .
  • the present invention relates to the following (1) to (75).
  • a monoclonal antibody that specifically reacts with ganglioside GM The derivative of the antibody according to (1), which is an antibody selected from an antibody produced by a lidoma, a humanized antibody, and a human antibody.
  • CDR1, CDR2, and CDR3 of the heavy chain (H chain) variable region (V region) of the monoclonal antibody are SEQ ID NOs: 9, 10, and 11, respectively, and CDM, CDR2, and CDR3 of the light chain (L chain) V region
  • the human chimeric antibody comprises the amino acid sequences of the H chain V region and L chain V region of a monoclonal antibody produced by a hybridoma against ganglioside GM2,
  • the human chimeric antibody is composed of the H chain V region and 1 chain V region of a monoclonal antibody produced by a hybridoma against ganglioside G, and the H chain constant region (C region) and 1 chain C region of a human antibody.
  • the human CDR ⁇ -grafted antibody binds to the CDRs of the H chain V region and 1 chain V region of the monoclonal antibody against ganglioside GM2 and the framework region (FR) of the H chain V region and 1 chain V region of the human antibody
  • Human CDR ⁇ -grafted antibodies were converted to the CDRs of the H chain V region and single chain V region of the monoclonal antibody against ganglioside GM2, the FRs of the H chain V region and single chain V region of the human antibody, and A derivative of the human CDR ⁇ -grafted antibody according to the above (7), comprising the amino acid sequence of the H chain C region and the single chain C region of a human antibody.
  • CDR1, CDR2, and CDR3 of the H chain V region of the antibody include the amino acid sequences represented by SEQ ID NOs: 9, 10, and 11, respectively, and CDR1, CDR2, and CDR3 of the L chain V region correspond to the SEQ ID NO:
  • the antibody fragment is an antibody fragment selected from Fab-Fari, F (ab,) 2 , single-chain antibody (scFv), disulfide-stabilized V region fragment (dsFv) and a peptide containing CDR. Certain derivatives of the antibody fragment according to (1) above.
  • the antibody fragment comprises the amino acid sequences of the H chain V region and L chain V region of a monoclonal antibody produced by a hybridoma to ganglioside GM2, and the H chain C region and 1 chain C region of a human antibody.
  • the antibody fragment, wherein the CDR1, CDR2, and CDR3 of the H chain V region of the antibody include the amino acid sequences represented by SEQ ID NOs: 9, 10, and 11, respectively, and the CDR1, CDR2, and CDR3 of the L chain V region each have the SEQ ID NO:
  • the transformant according to (48) or (49) above is cultured in a medium, and the derivative of the monoclonal antibody according to any one of (1) to (45) or the antibody thereof is contained in the culture.
  • the antibody fragment is an antibody fragment selected from Fab, Fab ⁇ F (ab,) 2 , a single-chain antibody (scFv), a disulfide-stabilized V region fragment (dsFv) and a peptide containing CDR; (51) The antibody fragment according to (1).
  • the antibody fragment is derived from the H chain V region and single chain V region of a monoclonal antibody produced by a hybridoma against ganglioside GM2, and the H chain C region and single chain C region of a human antibody.
  • the antibody fragment according to (53) or (54), wherein the antibody H chain V region comprises the amino acid sequence represented by SEQ ID NO: 7.
  • the antibody fragment comprises the HilV region and the 1-chain V region of the human CDR ⁇ -grafted antibody against ganglioside GM2, and the amino acid sequence of the H-chain C region and the 1-chain C region of the human antibody;
  • the antibody fragment wherein the CDR1, CDR2, and CDR3 of the H chain V region of the antibody include the amino acid sequences represented by SEQ ID NOS: 9, 10, and 11, respectively, and the CDR1, CDR2, and CDR3 of the L chain V region each have the SEQ ID NO:
  • a method for producing an antibody fragment comprising collecting the antibody fragment.
  • a medicament comprising the derivative of the monoclonal antibody and the derivative of the antibody fragment thereof according to (1) to (45), and at least one selected from the antibody fragments according to (51) to (68).
  • the present invention relates to a derivative of an antibody in which a monoclonal antibody specifically reacting with GM2 or an antibody fragment thereof is bound to a radioisotope, a protein or a low-molecular-weight drug.
  • a monoclonal antibody specifically reacting with GM2 or an antibody fragment thereof is bound to a radioisotope, a protein or a low-molecular-weight drug.
  • the monoclonal antibody include an antibody produced by a hybridoma, a humanized anti-human antibody, and the like.
  • a hybridoma is a monoclonal antibody having a desired antigen-specificity, which is obtained by cell fusion of B cells obtained by immunizing a mammal other than human with myeloma cells derived from a mouse or the like. Means a producing cell.
  • Humanized antibodies include human chimeric antibodies and human CDR ⁇ -planted antibodies.
  • the human chimeric antibody is composed of a non-human animal H chain V region (hereinafter referred to as HV or VH) and an antibody L chain V region (hereinafter referred to as LV or VL) and a human antibody H chain C Region (hereinafter referred to as CH) and an L chain C region (hereinafter referred to as CL) of a human antibody.
  • HV or VH non-human animal H chain V region
  • LV or VL antibody L chain V region
  • CH human antibody H chain C Region
  • CL L chain C region
  • the human chimeric antibody of the present invention obtains cDNAs encoding VH and "VL" from a hybridoma producing a monoclonal antibody that specifically reacts with GM2, and encodes human antibody CH and human antibody CL.
  • a human-type chimeric antibody expression vector can be constructed by inserting each into an expression vector for animal cells having the gene to be expressed, and the expression can be produced by introducing the expression vector into animal cells.
  • any CH may be used as long as it belongs to human immunoglobulin (hereinafter, referred to as hlg), but those of the hlgG class are preferable, and hIgGl, hIgG2, hIgG3 belonging to the hlgG class are preferable.
  • HIgG4 can be used.
  • the CL of the human chimeric antibody may be any CL as long as it belongs to hlg, and a class or in-class CL can be used.
  • Specific examples of the anti-GM2 chimeric antibody include KM966 described in JP-A-6-205694.
  • the human CDR-grafted antibody means an antibody obtained by grafting the amino acid sequences of VH and 7L CDR of an antibody of a non-human animal to appropriate positions of VH and VL of a human antibody.
  • the anti-G2CD ⁇ -planted antibody of the present invention encodes a V region obtained by grafting the VH and VL CDR sequences of a non-human animal antibody specifically reacting with GM2 into the VH and VL CDR sequences of any human antibody. And constructing a human CDR ⁇ plant antibody expression vector by inserting the cDNA into an expression vector for animal cells having genes encoding human antibody CH and human antibody CL, respectively. A human CDR-grafted antibody can be expressed and produced by introducing the expression vector into animal cells.
  • any CH may be used as long as it belongs to hlg, but the hlgG class is preferable, and further, subclasses such as hIgGl, hIgG2, hIgG3, and hIgG4 belonging to the hlgG class. Either can be used.
  • the CL of the human CDR ⁇ -planted antibody may be any CL as long as it belongs to hlg, and a class or human class CL can be used.
  • anti-GM2CDR ⁇ plant antibodies include KM8969 described in JP-A-10-257893. It is.
  • Human antibodies originally mean antibodies naturally occurring in the human body, but human antibody phage libraries and human antibodies created by recent advances in genetic engineering, cell engineering, and developmental engineering technology Antibodies obtained from the producing transgenic animal are also included.
  • Antibodies present in the human body can be obtained, for example, by isolating human peripheral blood lymphocytes, infecting EB virus and the like, immortalizing them, and cloning the cells to produce the antibody-producing lymphocytes. Antibodies can be purified.
  • the human antibody phage library is a library in which antibody fragments such as Fab and scFv are expressed on the phage surface by inserting an antibody gene prepared from human B cells into the phage gene.
  • a phage expressing an antibody fragment having a desired fc "source binding activity can be recovered from the library by using the binding activity to the substrate on which ⁇ 3 ⁇ 4 ⁇ is immobilized as an indicator. Furthermore, it can be 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 refers to an animal in which the human antibody gene is inserted into cells.
  • a human antibody-producing transgenic animal can be produced by introducing a human antibody gene into mouse ES cells, transplanting the ES cells into an early embryo of another mouse, and then developing the embryo.
  • Human antibodies can be produced from human antibody-producing transgenic animals by preparing human antibody-producing hybridomas by the usual method for producing hybridomas used in mammals other than humans, and culturing them in culture. Can produce and accumulate human antibodies.
  • antibody fragment examples include Fab, Fab ⁇ F (ab,) 2 , scFv, dsFv, a peptide containing CDR, and the like.
  • Fab is a fragment obtained by treating IgG with proteolytic enzyme papain (which is cleaved at the 224th amino acid residue of H ⁇ ). About half of the N-terminal side of the H chain and the entire L chain are dimerized. It is an antibody fragment having a molecular weight of about 50,000 and a source binding activity linked by a sulfide bond.
  • the Fab of the present invention can be obtained by treating an antibody specifically reacting with GM2 with proteolytic enzyme papain.
  • DNA encoding the Fab of the antibody is inserted into a prokaryotic expression vector or a 3 ⁇ 4 biological expression vector, and the vector is a prokaryote. The protein can be expressed by introducing it into eukaryotes to produce Fab.
  • F (ab ') 2 is a fragment obtained by treating IgG with the protease pepsin (which is cleaved at the 234th amino acid residue in the H chain), and Fab is a disulfide bond in the hinge region.
  • This is an antibody fragment having a molecular weight of about 100,000 and having an antigen-binding activity, which is slightly larger than that bound through the DNA.
  • the F (ab ') 2 of the present invention can be obtained by treating an antibody that specifically reacts with GM2 with the protease pepsin. Alternatively, it can be prepared by making the following Fab 'a thioether bond or a disulfide bond.
  • Fab ' is an antibody fragment having a molecular weight of about 50,000 and a source binding activity in which a disulfide bond in the hinge region of HF (ab') 2 is cleaved.
  • the Fab of the present invention can be obtained by treating F (ab,) 2 that specifically reacts with GM2 with a reducing agent dithiothreitol.
  • DNA encoding the Fab ′ fragment of the antibody is inserted into a prokaryotic expression vector or a eukaryotic expression vector, and the vector is introduced into a prokaryotic or difficult organism to produce a Fab. 'Can be expressed and produced.
  • scFv refers to a VH-P-VL or VL-P-VH polypeptide in which one VH and one VL are linked using an appropriate peptide linker (hereinafter, referred to as P).
  • P an appropriate peptide linker
  • VH and 7L contained in the scFv used in the present invention any of antibodies produced by hybridomas, humanized antibodies, and human antibodies can be used.
  • the scFv of the present invention obtains cDNA encoding VH and VL of an antibody that specifically reacts with GM2, constructs a DNA encoding the scFv, and uses the DNA for prokaryotic expression vector or
  • the scFv can be expressed and produced by inserting it into a prokaryotic expression vector by inserting it into a eukaryotic expression vector and introducing the expression vector into a eukaryote.
  • dsFv refers to a polypeptide in which one amino acid residue in each of VH and VL has been substituted with a cysteine residue, which is linked via a disulfide bond between the cysteine residues.
  • the amino acid residue to be substituted for the cysteine residue was determined by the method described by Reiter et al.
  • the antibody can be selected based on the prediction of the three-dimensional structure of the antibody.
  • VH and VL contained in the dsFv of the present invention any of an anti-humanized antibody and a human antibody produced by a hybridoma can be used.
  • the dsFv of the present invention is obtained by obtaining cDNAs encoding VH and VL of an antibody specifically reacting with GM2, constructing a DNA encoding the dsFv, and using the DNA for prokaryotic expression vector or eukaryote.
  • DsFv can be produced by inserting the expression vector into a biological expression vector and introducing the expression vector into a prokaryote or a difficult organism to express.
  • the peptide containing the CDR comprises at least one region of the H chain or L chain CDR.
  • a plurality of CDRs can be linked directly or via an appropriate peptide linker.
  • the peptide containing the CDR of the present invention can be obtained by obtaining a cDNA encoding VH and VL of an antibody specifically reacting with GM2, and then using the cDNA for expression in a prokaryotic expression vector or eukaryotic expression vector.
  • the expression vector can be inserted into a prokaryote or eukaryote for expression to produce a peptide containing a CDR.
  • the peptide containing CDR can also be produced by a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method), the tBoc method (t-butyloxycarbonyl method), and the like.
  • the derivative of the antibody of the present invention may be a derivative of the antibody or antibody fragment which specifically reacts with GM2, N-terminal or C-terminal of the H chain or L chain, an appropriate substituent or side chain in the antibody or antibody fragment,
  • thigh isotopes, proteins, and low-molecular-weight drugs are linked to the sugar chains in the antibody or antibody fragment by a chemical method [Introduction to Antibody Engineering (Osamu Kanemitsu, 1994; Jinjinshokan Co., Ltd.)] By doing so, it can be produced.
  • the derivative of the antibody of the present invention is obtained by ligating a DNA encoding an antibody or an antibody fragment specifically reacting with GM2 with a DNA encoding a protein to be bound and inserting the DNA into an expression vector. It can also be produced by a genetic engineering technique for introducing a vector into a host cell.
  • the 3 ⁇ 4 ⁇ isotope, 131 1, 125 1, and the like, for example, more chloramine ⁇ method, can be attached to the antibody.
  • low molecular drugs examples include alkylating agents such as Nitrogen 'mustard and cyclophosphamide, antagonists such as 5-fluorouracil and methotrexate, and antibiotics such as daunomycin, bleomycin, mitomycin C, daunorubicin, and doxorubicin.
  • alkylating agents such as Nitrogen 'mustard and cyclophosphamide
  • antagonists such as 5-fluorouracil and methotrexate
  • antibiotics such as daunomycin, bleomycin, mitomycin C, daunorubicin, and doxorubicin.
  • plant alkaloids such as vincristine, vinblastine, vindesine, hormonal agents such as evening moxifen, dexamethasone [Clinical Oncology (Japanese Society for Clinical Oncology, 1996, Cancer and Chemotherapy)], or steroids such as hydrocotisone and prednisone, non-steroids such as aspirin and indomethacin;
  • Immunomodulators such as gold thiomalate and penicillamine, immunosuppressants such as cyclophosphamide and azathioprine, and anti-inflammatory agents such as antihistamines such as chlorpheniramine maleate and clemacitin [Inflammation and anti-inflammatory Therapy 1977, Itoyaku Shuppan Publishing Co., Ltd.].
  • a method for binding daunomycin to an antibody a method for binding between daunomycin and the amino group of the antibody via gludealdehyde, and a method for binding the amino group of daunomycin to the carboxyl group of the antibody via water-soluble carbodiimide And the like.
  • Suitable proteins include cytokines that activate immunocompetent cells, such as hIL-2, hGM-CSF, human macrophage colony stimulating factor (hereinafter abbreviated as hM-CSF), Leukin 12 (hereinafter referred to as hIL-12) and the like.
  • toxins such as ricin and diphtheria toxin can be used to directly damage cancer cells.
  • a fusion antibody with a protein a cDNA encoding the protein is linked to a cDNA encoding the antibody or antibody fragment, a DNA encoding the fusion antibody is constructed, and the DNA is converted to a prokaryotic or eukaryotic organism.
  • the expression vector is inserted into an expression vector, and the expression vector is introduced into a prokaryotic cell by introducing it into a eukaryote, whereby a fusion antibody can be prepared.
  • the derivative of the humanized anti-GM2 antibody include a fusion protein of the H chain and hIL-2 of the antibody having the amino acid sequence of SEQ ID NO: 1, and the V region of the L chain of the antibody having the amino acid sequence of SEQ ID NO: 2.
  • KM8969-ML-2 which is a fusion protein of the anti-GM2CDR ⁇ -planted antibody KM8969 and hIL-2 having the sequence.
  • a humanized antibody expression vector is an expression vector for animal cells into which genes encoding human antibody CH and CL have been incorporated. It can be constructed by cloning genes coding for body CH and CL, respectively.
  • the C region of a human antibody can be CH and CL of any human antibody.
  • the C region of the IgGl subclass of the H chain of a human antibody hereinafter referred to as hC ⁇ 1
  • the L chain of a human antibody Class C region hereinafter referred to as hC / c.
  • genes encoding CH and CL of the human antibody chromosomal DNA consisting of exons and introns can be used, and cDNA can also be used.
  • any expression vector can be used as long as it can express the gene encoding the C region of the human antibody.
  • pAGE107 [Cytotechnology, 3, 133 (1990)]
  • pAGE103 [Journal of Biochemistry (J. Biochem.), 101, 1307 (1987)]
  • pHSG274 [Gene , 27, 223 (1984)]
  • pKCR Procedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 78, 1527 (1981)]
  • pSGl? d2-4 [Cytotechnology, 4, 173 (1990)].
  • Promoters and enhancers used for expression vectors for animal cells include the early promoters and enhancers of SV40 [Journal of Ob 'Biochem. (J. Biochem.), 101, 1307 (1987) Moroni monoleukemia virus LTR promoter Ichiyuichi and Enhansa I [Biochemical 'and Biophysical' Research Communications (Biochem. Biophys. Res. Co. thigh un.), 149, 960 (1987)], Immunization Examples include the globulin H chain promoter [Cell, 41, 479 (1985)] and Enhansa I [Cell, 717 (1983)].
  • the vector for expression of humanized antibody may be of the type in which the antibody H chain and 1 chain are present on separate vectors or of the type present on the same vector (hereinafter referred to as tandem type). Tandem type because of the ease of construction of a humanized antibody expression vector, ease of introduction into animal cells, and balanced expression of antibody H chain and single chain in animal cells. For expression of a humanized antibody, one is more preferred [Journal of Immunological Methods], J. Immunol. Methods, 167, 271 (1994).
  • the constructed humanized antibody expression vector can be used for expression of a human chimeric antibody and a human CDR ⁇ -planted antibody in animal cells.
  • MRNA is extracted from hybridoma cells producing mouse antibodies, and cDNA is synthesized.
  • the synthesized cDNA is cloned into a vector such as a phage or a plasmid to prepare a cDNA library.
  • Each of the replacement plasmids is isolated.
  • the entire nucleotide sequence of VH and L of the target mouse antibody on the recombinant phage or recombinant plasmid is determined, and the entire amino acid sequence of VH and 7L is estimated from the nucleotide sequence.
  • any animal such as mouse, rat, hamster, and rabbit can be used as long as hybridoma cells can be produced.
  • Methods for preparing total RNA from hybridoma cells include guanidine thiocyanate-cesium trifluoroacetate method [Methods in Enzymol., 154, 3 (1987)], and mE from total RNA. The method is as follows: Oligo (dT) immobilized cellulose column method [Molecular Cloning: A Laboratory Manual], Cold Spring Harbor Lab. Press New York, 1989, below. , Molecular cloning: a, laboratory, manual, etc.]. Examples of kits for preparing mRNA from hybridoma cells include Fast Track mRNA Isolation Kit (manufactured by Invitrogen), Quick Prep mB Purification Kit (manufactured by Pharmacia), and the like.
  • Methods for cDNA synthesis and cDNA library preparation are routine methods [Molecular 'Clothing: A Laboratory' Manual; Current 'Protocols' in' Molecular Protocols in Molecular Biology, Supplement 1-34, hereinafter referred to as current 'protocols' in 'molecular' biologic) or a commercially available kit such as Super Script TM Plasmid System for cDNA Synthesis and Plasmid Cloning (GIBCO BRL) or ZAP -A method using a cDNA Synthesis Kit (Stratagene) can be used.
  • mRNA extracted from hybridoma cells was Any vector can be used as a vector into which the cDNA synthesized in this manner can be incorporated, so long as the vector can incorporate the cDNA.
  • ZAP Express [Strategies, 5, 58 (1992)], pBluescript II SK (+) [Nucleic Acids Research, 17, 9494 (1989)], Azap II (Stratagene GtlO, Agtll [DNA Cloning: A Practical Approach], I, 49 (1985)], Lambda BlueMid (Clontech), ExCell, pT7T3 18U (Pharmacia) ), PcD2 [Molecular 'and' Cellular 'Biology (Mol. Cell. Biol.), 3, 280 (1983)], pUC18 [Gene, 33, 103 (1985)] and the like are used. .
  • any Escherichia coli can be used as long as the cDNA library can be introduced, expressed and maintained.
  • XLl-Blue MRF '[Strategies, 5, 81 (1992)], C600 [Genetics, 39, 440 (1954)], Y1088, Y1090 [Science, 222, 778] (1983)], NM522 [Journal 'ob' Molecular 'Biology (J. Mol. Biol.), 166, 1 (1983)], concealed [journal' ob 'Molecular' Biology (J. Mol. Biol.), 16, 118 (1966)] and JM105 [Gene, 38, 275 (1985)].
  • Methods for selecting cDNA clones encoding VH and "VL" of non-human animal antibodies from a cDNA library include colony hybridization or plaque using an isotope or fluorescently labeled probe.
  • ⁇ Hybridization method molecular ⁇ cloning: laboratory ⁇ manual ⁇ manual
  • primers and use the cDNA or cDNA library synthesized from mRNA as type III to obtain the Polymerase Chain Reaction ( Hereinafter, it is referred to as PCR method; molecular-cloning: a laboratory-manual; current-protocols-in-molecular-biology) can be used to prepare cDNAs encoding VH and VL. .
  • the cDNA selected by the above method is digested with an appropriate restriction enzyme or the like, and then cloned into a plasmid such as pBluescript SK (-) (manufactured by Stratagene), and a commonly used nucleotide sequence analysis method, for example, Sangaichi (Sanger , F.) et al. [Procedings of the 'National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 74, 5463 (1977)], and analyzed using an automatic nucleotide sequence analyzer, for example, ALF DNA sequencer (Pharmacia) to obtain the nucleotide sequence of the cDNA. Can be determined.
  • a plasmid such as pBluescript SK (-) (manufactured by Stratagene)
  • a commonly used nucleotide sequence analysis method for example, Sangaichi (Sanger , F.) et al. [Procedings of the
  • amino acid sequence 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 “ob” "proteins” "ob” immunological ""
  • amino acid sequences “ob” proteins
  • amino acid sequence at the ⁇ -terminal can be estimated, and the subgroup to which they belong can be known.
  • amino acid sequences of VH and each CDR of known amino acids with VH and 7L amino acid sequences of known antibodies (sequences of proteins, proteins of immunological and in rest). Can be found by:
  • the VH and "VL" of a non-human animal antibody are coded upstream of the gene encoding CH and CL of the human antibody of the humanized antibody expression vector described in 1 (1) of this section.
  • a human chimeric antibody expression vector can be constructed by cloning the cDNA encoding the VH and 7L of a non-human animal antibody with the VH and VL of a non-human animal antibody. 3, consisting of the base sequence on the terminal side and the base sequence on the terminal side of CH and CL of the human antibody and having a recognition sequence for an appropriate restriction enzyme at both ends.
  • the humanized chimeric antibody is cloned so that it can be expressed in an appropriate form upstream of the genes encoding CH and CL of the human antibody of the humanized antibody expression vector according to 1 (1) of this section.
  • An expression vector can be constructed.
  • CDNA encoding VH and 7L of human CD side plant antibody is constructed as follows be able to. First, the amino acid sequences of the framework regions (hereinafter referred to as FR) of the VH and VL of the human antibody to which the amino acid sequences of the VH and 7L CDRs of the antibody of the target non-human animal are transplanted are selected. As the amino acid sequence of human antibody VH and 7L FR, any amino acid sequence can be used as long as it is derived from a human antibody.
  • FR amino acid sequences of VH and VL of human antibodies registered in the Database of Protein Data Bank, etc., and common amino acid sequences of FR subgroups of VH and VL of human antibodies (Sequences of 'Proteins', of 'Immological', and 'Inrest Rest').
  • sequences of 'Proteins', of 'Immological', and 'Inrest Rest' common amino acid sequences of FR subgroups of VH and VL of human antibodies.
  • the amino acid sequences of the target non-human animal antibody VH and "VL CDR" were grafted to the VH and VL FR amino acid sequences of the selected human antibody, and the human CDR-grafted antibody VH and 7L Designing the amino acid sequence
  • the designed amino acid sequence is converted into a DNA sequence in consideration of the codon usage (sequences of proteins, proteins, proteins, immunologicals, etc.) found in the nucleotide sequence of the antibody gene.
  • Convert and design a DNA sequence encoding the amino acid sequence of VH and 1 VL of the human CDR ⁇ plant antibody Based on the designed DNA sequence, synthesize several synthetic DNAs of about 100 bases in length. In this case, it is preferable to design a total of six DNAs for both the H chain and the L chain in view of the reaction efficiency in the PCR and the length of DNA that can be synthesized.
  • ⁇ easily cloned into the humanized antibody expression vector constructed in (1) on page 1 be able to.
  • the amplified product is cloned into a plasmid such as pBluescript SK (-) (Stratagene), and the nucleotide sequence is determined by the method described in (2) of this section 1; A plasmid having a DNA sequence encoding the amino acid sequences of VH and VL is obtained.
  • the human CDR ⁇ -grafted antibody has a binding activity of only the non-human animal antibody VH and VL CDRs transplanted into the human antibody VH and VL FR, and the binding activity of the original non-human animal It is known to be lower than antibodies [Biotechnology-(BI0 / TECHN0L0GY), 9, 266 (1991)]. This can be caused by the original non-human animal In the VH and "VL" antibodies, not only the CDRs but also some amino acid residues of the FR are directly or indirectly involved in the antigen-binding activity. It is thought that the transplantation changes these amino acid residues into amino acid residues having different FRs in the human antibody, resulting in a decrease in the binding activity.
  • Amino acid residues that are directly involved in binding to f3 ⁇ 4l amino acid residues that interact with the amino acid residues of CDR in the amino acid sequence of human antibody VH and 7L FR, and antibody construction
  • amino acid residues that are indirectly involved in binding to ⁇ 3 ⁇ 41 modify them to amino acid residues of the original non-human animal antibody, and increase the reduced ⁇ 3 ⁇ 4 binding activity
  • Biotechnology BIO / TECHNOLOGY), 9, 266 (1991)
  • the most important point is how to efficiently identify the amino acid residues of FR involved in the binding activity. Crystallographic analysis [J. Mol. Biol. Biol. (J. Mol.
  • the modification of the amino acid residues of FRs of VH and VL of a human antibody can be achieved by performing the PCR method described in (5) of this section 1 using the modification compound ⁇ 3 ⁇ 4) ⁇ .
  • the nucleotide sequence of the amplified product after PCR is determined by the method described in (2) of this section 1 to confirm that the desired modification has been performed.
  • the cDNA encoding VH and 7L of the antibody can be cloned to construct a human CDR-transplanted antibody expression vector.
  • the 5 'end of the synthetic DNA located at both ends is used.
  • Suitable restriction yeast By introducing the elemental recognition sequence, the appropriate form can be placed upstream of the genes encoding the CH and CL of the human antibody in the humanized antibody expression vector described in (1) on page 1 * 1. It is possible to construct a human-type 011-planted antibody expression vector by cloning to express in step (1).
  • the humanized antibody expression vector described in 1 (4) and (7) or an expression vector obtained by modifying them was used. Can be used to effect transient expression of a humanized antibody.
  • the host cell into which the expression vector is introduced any cell can be used as long as it can express a humanized antibody. However, due to its high expression level, COS-7 cells (ATCC CRL1651) can be used. Commonly used [Methods in Nucleic Acids Res., CRC press, 283 (1991)].
  • Methods for introducing an expression vector into COS-7 cells include the DEAE-dextran method [Methods in Nucleic Acids Res., CRC press, 283 (1991)], Lipofexion method Proc. 'Ob' The 'National' Academia 'Ob' Science (Proc. Natl. Acad. Sci. USA), 84, 7413 (1987)].
  • Humanized antibody expressed in YB2 / 0 cells TTADCC activity is preferable because it increases.
  • the expression level of humanized antibody and antigen-binding activity in the culture supernatant are measured by enzyme-linked immunosorbent assay [hereinafter referred to as ELISA method].
  • Antibodies A 'Laboratory-Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Chapter 14, 1988, Monochrome' Antibodies: Principle Norez and Practice : Principles and Practice), Academic Press Limited, 1996].
  • Examples of a method for introducing an expression vector into a host cell include an electrolysis method [Japanese Patent Laid-Open No. 257891/1990, Cytotechnology, 3, 133 (1990)] and the like. It is.
  • any cell can be used as long as it can express the humanized antibody.
  • mouse SP2 / 0-Agl4 cells ATCC CRL1581
  • mouse P3X63-Ag8.653 cells ATCC CRL1580
  • CH0 cells deficient in the dihydrofolate reductase gene hereinafter referred to as dhfr
  • dhfr dihydrofolate reductase gene
  • YB2 / 3HL.P2.G11.16Ag.20 cells ATCC CRL1662, hereinafter referred to as YB2 / 0 cells
  • the transformant which stably expresses the humanized antibody can be transformed with a drug such as G418 sulfate (hereinafter referred to as G418: manufactured by SIGMA) according to the method disclosed in JP-A-2-2577891.
  • G418 manufactured by SIGMA
  • GIBC0 BRL Hybridoma-SFM A medium
  • FBS fetal calf serum
  • the humanized antibody can be purified from the culture supernatant of the transformant using a protein A column.
  • a purification method usually used for protein purification can be used.
  • purification can be performed by a combination of gel filtration, ion exchange chromatography and ultrafiltration.
  • the molecular weight of the purified humanized antibody H-chain, L-chain or the whole antibody molecule can be determined by polyacrylamide gel electrophoresis [hereinafter referred to as SDS-PAGE: Nature, 227, 680 (1970)] or Western plotty.
  • SDS-PAGE polyacrylamide gel electrophoresis
  • the binding activity of the purified humanized antibody to ⁇ 3 ⁇ 41 and the binding activity to culture media were determined by ELISA and photoantibodies [Cancer 'Immunology I' Immunotherapy (Cancer Immunol. I Thigh unother.), 36, 373 (1993)]. Cytotoxic activity against ⁇ 3 ⁇ 4positive cells can be evaluated by measuring CDC activity, ADCC activity and the like [Cancer Immunology 1 (Cancer Immunol.
  • Antibody fragments can be produced by genetic engineering or protein chemical techniques based on the humanized antibodies described on page J * 1 or antibodies produced by hybridomas and human antibodies.
  • Examples of the antibody fragment include Fab, F (ab,) 2 , Fab ′, scFv, dsFv, peptides including CDR, and the like. These antibody fragments can be prepared using protein chemistry or genetics techniques.
  • Fab can be prepared by treating IgG with the protease, papain. After papain treatment, if the original antibody is an IgG subclass that has protein A binding properties, it can be separated from IgG molecules and Fc fragments by passing through a protein A column and recovered as a uniform Fab. [Monoclonal Antibodies: Principles and Practice, third edition, 1995, hereinafter referred to as "Monoclonal Antibodies: Third Edition"]. In the case of an IgG subclass antibody that does not have protein A binding, Fab can be recovered in a fraction eluted at a low salt concentration by ion exchange chromatography (monoclonal antipodes 3rd edition) ).
  • Fab is prepared by cloning a DNA encoding the antibody V region described in (1) (2) and (5) of this section into a Fab expression vector, producing a Fab expression vector, and introducing the expression vector into a host. By doing so, it can be expressed and produced.
  • Any Fab expression vector can be used as long as it can incorporate and express Fab DNA.
  • An example is PIT106 [Science, 240, 1041 (1988)]. You.
  • Any host cell such as eukaryotes and prokaryotes can be used, but Escherichia coli is preferred.
  • the Fab expression vector can be introduced into an appropriate host cell to produce and accumulate Fab in the inclusion body or Veriplasma layer. From the inclusion body, an active Fab can be converted to the active Fab by the refolding method usually used for proteins, and when expressed in the periplasm layer, the active Fab leaks into the culture supernatant. Get out. After refolding or from the culture supernatant, it is possible to purify a uniform Fab by using a column to which is bound [Antipod Engineering 'A' Practical Kasole 'Guide (Antibody Engineering, A Practical Guide) , WH Freeman and Company, 1992].
  • F (ab ') 2 can be produced by treating IgG with the protease pepsin. After treatment with pepsin, it can be recovered as uniform F (ab ') 2 by the same purification procedure as Fab (monoclonal' Antibodies 3rd edition). Further, a method of treating Fab ′ described in (3) of * ⁇ shell 2 described later with o-PDM or a maleimide such as bismaleimide hexane and the like to form a thioether bond, treatment with DTNB, and disulfide It can also be prepared by a method of bonding [Antibody Engineering, A Practical Approach], IRL PRESS, 1996, hereinafter referred to as Antibody 'Engineering'.
  • Fab ′ can be obtained by treating F (ab,) 2 described in (2) of this section 2 with a reducing agent such as dithiothreitol.
  • Fab ′ was cloned into a vector for Fab ′ expression using a DNA encoding the antibody V region described in (2) and (5) of ffll to prepare Fab and an expression vector. It can be expressed and produced by introducing the expression vector into a host cell.
  • any vector can be used as long as it can incorporate and express Fab' DNA.
  • pAK19 Biotechnology I (BI0 / TECHN0L0GY), 10, 163 (1992)] and the like can be mentioned.
  • the host cell any cells such as eukaryotes and prokaryotes can be used, and Escherichia coli is preferred.
  • the Fab 'expression vector can be introduced into an appropriate host cell to produce and accumulate Fab in the inclusion body or periplasm layer.
  • active Fab 'can be obtained by the refolding method usually used for proteins, and when expressed in the periplasma layer, partial digestion with lysozyme, osmotic shock, sonication Bacteria can be crushed by treatment with one shot or the like and collected outside the cells.
  • uniform Fab 'can be purified from the lysate of the fungi by using a protein G column or the like (Antibody' Engineering ').
  • DNA encoding the antibody V region described in (2) and (5) on page 1 is cloned into a scFv expression vector to prepare an scFv expression vector, and the expression vector is It can be expressed and produced by introducing it into host cells.
  • scFv expression vector can be used as long as it can incorporate and express scFv DNA.
  • pCANTAB5E Pulacia
  • pHFA Humidity Antibody Hybridoma, 5, 48 (1994)
  • pHFA Humidity Antibody Hybridoma, 5, 48 (1994)
  • any cells such as eukaryotes and prokaryotes can be used, and Escherichia coli is preferred.
  • scFv By introducing the scFv expression vector into an appropriate host cell and infecting it with a helper phage, a phage that expresses the scFv on the phage surface in a form fused with the phage surface protein can be obtained.
  • scFv can be produced and accumulated in the inclusion body of Escherichia coli or the periplasm layer into which the scFv expression vector has been introduced.
  • the inclusion body can be converted into an active scFv by the refolding method usually used for proteins, and when expressed in the periplasmic layer, it can be used for partial digestion with lysozyme, osmotic shock, sonication, etc.
  • the bacteria can be crushed by the treatment and collected outside the cells. Uniform scFv can be purified after refolding or from the lysate of bacteria by using cation exchange chromatography, etc.
  • dsFv dsFv can be prepared as follows. First, a mutation is introduced into an appropriate position of the DNA encoding the antibodies VH and VL described in (2) and (5) of this section 1 to prepare a DNA in which the encoding amino acid residue is substituted with cysteine. Each of the prepared DNAs is cloned into a dsFv expression vector, VH and L expression vectors are prepared, and the expression vector is introduced into a host cell to be expressed and prepared.
  • the vector for dsFv expression any vector can be used as long as it can incorporate and express DNA for dsFv. An example is pULI9 [Protein Engineering, 7, 697 (1994)].
  • any cells such as eukaryotes and prokaryotes can be used, and Escherichia coli is preferred.
  • VH and expression vector can be introduced into a suitable host cell to produce and accumulate dsFv in inclusion bodies or periplasmic layers.
  • VH and 7L can be obtained from the inclusion body or periplasma layer, mixed, and converted into active dsFv by the refolding method usually used for proteins. After refolding, it can be further purified by ion exchange chromatography, gel filtration, etc. [Protein Engineering, 7, 697 (1994)].
  • the peptide containing CDR can be prepared by a chemical synthesis method such as the Fmoc method or the tBoc method.
  • a DNA encoding a peptide containing CDR can be prepared, and the prepared DNA can be cloned into an appropriate expression vector to prepare a CDR peptide expression vector.
  • Any expression vector can be used as long as it can incorporate and express the DNA encoding the CDR peptide. For example, pLEX (Invitrogen), pAX4a + (Invitrogen) and the like can be mentioned.
  • any cells such as eukaryotes and prokaryotes can be used, and Escherichia coli is preferred.
  • the expression vector can be introduced into a suitable host cell to produce and accumulate the CDR peptide in the inclusion body or Veriplasma layer.
  • CDR peptides can be obtained from inclusion bodies or Veriplasma layers and purified by ion-exchange chromatography and gel filtration, etc. [Protein Engineering, 7, 697 (intestine)]. (7) Evaluation of antibody fragment activity
  • the binding activity of the obtained antibody fragment to ⁇ and the binding activity to culture media were determined by ELISA and immunofluorescence [Cancer 'Immunology-1' immunotherapy (Cancer).
  • the antibody fragment of the present invention specifically binds to GM2 expressed in human-derived cultured JK, it can be used for diagnosis of human cancers such as lung cancer, malignant black ⁇ 1, flil tumor, and neuroblastoma. It may be useful in treatment. Since antibody fragments have a smaller molecular weight than IgG molecules, they are expected to show rapid target transfer in the human body.
  • the antibody fragment of the present invention can be administered alone, it is usually mixed with one or more pharmacologically acceptable carriers and is well known in the technical field of pharmaceuticals. It is desirable to provide it as a pharmaceutical preparation produced by any method.
  • intravenous administration can be preferably mentioned.
  • Dosage forms include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like.
  • Formulations suitable for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like.
  • Liquid preparations such as L-agents and syrup U include water, sugars such as sucrose, sorbitol, fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil, and soybean oil. And preservatives such as p-hydroxybenzoic acid esters, and flavors such as strawberry flavor and peppermint as additives.
  • Capsules, tablets, powders, granules, etc. are excipients such as lactose, glucose, sucrose, mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate, talc, polyvinyl Binders such as alcohol, hydroxypropyl cellulose and gelatin, surfactants such as fatty acid esters, and glycerin It can be produced using a plasticizer or the like as an additive.
  • Formulations suitable for parenteral administration include injections, suppositories, sprays and the like.
  • An injection is prepared using a carrier comprising a salt solution, a glucose solution, or a mixture of both.
  • Suppositories are prepared using carriers such as cocoa butter, hydrogenated fats or carboxylic acids. Sprays are prepared using the antibody fragment itself or a carrier that does not irritate the oral and respiratory mucosa of the recipient and disperses the antibody fragment as fine particles to facilitate absorption.
  • the carrier include lactose and glycerin.
  • Formulations such as aerosols and dry powders can be made depending on the properties of the antibody fragment and the carrier used. In these parenteral preparations, the components exemplified as additives in the oral preparation can be added.
  • the dosage or frequency of administration varies depending on the desired therapeutic effect, administration method, treatment period, age, body weight, etc., but is usually 10 g / kg to 8 mg / kg per adult per day.
  • a gene encoding a fusion protein of a humanized antibody and a cytokine can be constructed by linking to the 3 'end.
  • a recognition sequence for an appropriate restriction enzyme is introduced into the 5 'end of the amplification primer, and the gene encoding the H chain or L chain of the humanized antibody is obtained.
  • a gene encoding a fusion protein of a humanized antibody and a cytokine can be constructed.
  • the gene encoding a cytokine either chromosomal DNA or cDNA can be used.
  • the nucleotide sequence of the constructed gene encoding the fusion protein of the humanized antibody and cytokine is determined by the method described in (1) on page 1 to confirm that it is the desired sequence.
  • a gene encoding a fusion protein of CH and cytokine of the humanized antibody is constructed by connecting the gene to the humanized antibody expression vector described in (4) and (7) of W1.
  • An expression vector can be constructed by substituting the gene encoding CH of the humanized antibody in (1).
  • the activity of the humanized antibody portion that is, the binding activity to and the binding activity to culture medium can be measured by an ELISA method, a fluorescent antibody method, or the like.
  • cytotoxic activity against antigen-positive cultured cancer cell lines can be evaluated by measuring CDC activity, ADCC activity and the like.
  • the activity of the cytokine portion can be evaluated, for example, using the growth supporting activity of a culture cell exhibiting a concentration-dependent growth for the cytokine as an index. 'National Academy' of Science (Proc. Natl. Acad. Sci. USA), 91, 9626 (1994)].
  • a fusion protein of a humanized antibody and a cytokine can be evaluated for its tumor effect by, for example, administering it to a wild-type mouse transplanted with a cultured mouse cancer cell expressing GM2.
  • cytodynamic antibody alone or co-administration of humanized antibody and cytokine stronger anti-tumor effect in vivo can be evaluated [Cancer-Immunology-Immunotherapy (Cancer) Immunol., Innnunother.), 42, 88 (1996)] 0
  • the fusion protein of the humanized antibody of the present invention and cytokine specifically binds to GM2 expressed in a human-derived cultured cancer cell line and exhibits cytotoxic activities such as CDC activity and ADCC activity. It is considered to be useful in the diagnosis and treatment of human cancers such as lung cancer, malignant melanoma, brain tumors, and neuroblasts.
  • Humanized antibodies have a strong antitumor effect in the human body and do not show immunogenicity because most of the portion derived from the amino acid sequence of the human antibody compared to antibodies from non-human animals. It is expected to last for a long time, and furthermore, the activity of the fused cytokine can activate immunocompetent cells in the vicinity of the cancer.
  • the anti-tumor effect is expected to be stronger than that of the simultaneous administration of site force, and the side effects are expected to be reduced compared to the systemic administration of site force.
  • the fusion protein of the humanized antibody of the present invention and cytokines can be administered alone, it is usually mixed with one or more pharmacologically acceptable carriers to prepare a preparation. It is desirable to provide it as a pharmaceutical preparation manufactured by any method well known in the technical field of science.
  • intravenous administration can be preferably used.
  • Dosage forms include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, agents, ointments, tablets and the like.
  • Formulations suitable for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like.
  • Emulsions and liquid preparations such as syrup can be obtained from sugars such as water, sucrose, sorbitol, fructose, glycols such as polyethylene glycol and propylene glycol, sesame oil, olive oil, soybean oil, etc. It can be produced using preservatives such as oils and p-hydroxybenzoic acid esters, and flavors such as strawberry flavor and peppermint as additives.
  • Capsules, tablets, powders, granules, etc. are excipients such as lactose, glucose, sucrose, mannitol, disintegrants such as starch, sodium alginate, mag stearate It can be manufactured using lubricants such as nesidum and talc, binders such as polyvinyl alcohol, hydroxypropyl cellulose and gelatin, surfactants such as fatty acid esters, and plasticizers such as glycerin as additives.
  • Formulations suitable for parenteral administration include injections, suppositories, sprays and the like.
  • the wing preparation is prepared using a carrier comprising a salt solution, a glucose solution, or a mixture of both.
  • Suppositories are prepared using carriers such as cocoa butter, hydrogenated fats or carboxylic acids.
  • a spray is prepared using the fusion protein itself or a carrier which does not irritate the oral and respiratory tract mucosa of the recipient and disperses the fusion protein as fine particles to facilitate absorption.
  • the carrier include lactose and glycerin.
  • preparations such as aerosols and dry powders can be made.
  • the components exemplified as additives in the oral preparation can be added.
  • the dose or frequency of administration varies depending on the desired therapeutic effect, administration method, treatment period, age, body weight, etc., but is usually lO zg / k Smg / kg per adult per day.
  • FIG. 1 is a diagram showing a construction process of plasmid pBSA-B.
  • FIG. 2 is a view showing a construction process of plasmid pBS ihCat IL-2.
  • FIG. 3 is a diagram showing a construction process of plasmid pBShCIL-2.
  • FIG. 4 is a view showing a construction process of plasmid pKANTEX8969-hIL2.
  • FIG. 5 is a diagram showing an SDS-PAGE (using a 4 to 15% gradient gel) electrophoresis pattern of the purified anti-GM2CD antibody KM8969 and the purified fusion protein M8969-hIL-2.
  • the left side shows the results of electrophoresis under non-reducing conditions, and the right side shows electrophoresis under reducing conditions.
  • Lane 1 shows the migration patterns of low molecular weight proteins, 2 or 1 M8969, 3 shows IM8969-hIL-2, 4 shows low molecular weight markers 1 and 5 «8969, 6 or 1M8969-hIL-2, respectively.
  • FIG. 6 is a graph showing the binding activity of the purified anti-GM2CD artificial antibody KM8969 and the purified fusion protein KM8969-hi 2 to GM2 measured by varying the antibody concentration.
  • the vertical axis shows the binding activity to GM2, and the horizontal axis shows the antibody concentration.
  • ⁇ is KM8969, reference is KM8969—hIL-2 activity Are respectively shown.
  • FIG. 7 shows the results of measuring the growth supporting activity of hIL-2 and the purified fusion protein KM8969-hIL-2 on hIL-2-dependent cell CTLL-2 by varying the concentration of each protein.
  • the vertical axis shows the growth supporting activity, and the horizontal axis shows the protein concentration.
  • ⁇ indicates hIL-2 and reference indicates KM8969—activity of hIL-2.
  • FIG. 8 is a graph showing the activation of human EFX-1 cells by the purified anti-GM2CDR ⁇ -implanted antibody KM8969 and the purified fusion protein KM8969-hIL-2 and the resulting cytotoxicity.
  • the vertical axis indicates the cytotoxic activity, and the horizontal axis indicates the protein used.
  • the mouth shows the activity of KM8969, and the garden shows the activity of KM8969-hIL-2.
  • a fusion protein of a humanized anti-GM2 antibody and a cytokine KM8969-hIL-2
  • a fusion protein of the anti-GM2 CDR ⁇ plant antibody KM8969 and hIL-2 was prepared as follows and its activity was evaluated.
  • reaction solution was fractionated by agarose gel electrophoresis, and about 2 g of an Apal-BamHI fragment of about 2.95 kb was recovered using a QIAquick Gel Extraction Kit (manufactured by QIAGEN) according to the attached instruction.
  • Shiro NA having the nucleotide sequence of SEQ ID NO: 3 or 4 was synthesized using an automatic DNA synthesizer (380A, manufactured by Applied Biosystems). 0.3 of the obtained fi3 ⁇ 4DNA ⁇ G each was added to 15 zl of sterilized water, and heated at 65 ° C for 5 minutes. The reaction solution was left at room temperature for 30 minutes, and 2 ⁇ 1 10-fold buffer [500 IDM Tris-hydrochloric acid (pH7.6), 100 mM Shiridani Magnesium, 50 mM DTT] and 2 zl lOmM ATP were added.
  • IDM Tris-hydrochloric acid pH7.6
  • 100 mM Shiridani Magnesium, 50 mM DTT 100 mM Shiridani Magnesium, 50 mM DTT
  • 2 zl lOmM ATP were added.
  • T4 Polynucleotide Kinase manufactured by Takara Shuzo Co., Ltd.
  • 10 units of T4 Polynucleotide Kinase (manufactured by Takara Shuzo Co., Ltd.) was added and reacted at 37 ° C. for 30 minutes to phosphorylate the 5 ′ end.
  • 0.1 g of the Apal-BajnHI fragment derived from the plasmid pBluescript SK (-) obtained above and 0.05 g of phosphoric acid conjugated fi3 ⁇ 4A were added to a total of 101 sterile water, and the DNA ligation Kit was used.
  • Ver.2 (Takara Shuzo) was connected according to the instruction manual.
  • Escherichia coli DH5 strain (manufactured by Stratagene) was transformed using the recombinant plasmid DNA solution obtained in this manner to obtain a plasmid pBSA-B shown in FIG.
  • a plasmid pBSA-B shown in FIG.
  • the AutoRead Sequencing Kit Pharmacia
  • ALF electrophoresis
  • plasmid pBSA-B obtained above was added to a buffer consisting of 101 50 mM Tris-HCl (PH7.5), 100 mM sodium chloride, 100 mM magnesium chloride and ImM DTT, and an additional 10 units of the buffer were added.
  • the restriction enzyme EcoRI (Takara Shuzo) was added and reacted at 37 ° C for 1 hour.
  • the reaction solution was precipitated with ethanol, and added to 10 ⁇ 1 of a buffer solution consisting of 33 mM Tris-acetic acid (pH 7.9), 66 mM potassium acetate, magnesium lOniMS, 0.5 mM DTT and 100 / g / ml BSA.
  • the restriction enzyme Smal (Takara Shuzo) was added thereto and reacted at 30 ° C for 1 hour.
  • the reaction solution was fractionated by agarose gel electrophoresis, and about 2 ⁇ g of an EcoRI-Smal fragment of about 3.00 kb was recovered using a QIAquick Gel Extraction Kit (manufactured by QIAGEN) according to the specifications.
  • plasmid pILL4 [Agricultural 'and' biological 'Chemistry (Agri Biol. Chem.), 51, 1135 (1987)] containing the mature full-length cDNA of hIL-2 PCR was performed as shown in the following.
  • Lng of plasmid pILL4 was added to a 100 zl reaction solution (1x concentration of Ex Taq buffer (Takara Shuzo), 200 / M dNTPs ⁇ 1.0 / M revl primer (SEQ ID NO: 5), 1.0 ⁇ M fw2 primer (SEQ ID NO: 5) 6) and 2.5 units of TaXaRa Ex Taq DNA polymerase (Takara Shuzo Co., Ltd.)), cover with 100 ⁇ 1 mineral oil, set in a DNA thermal cycler (PJ480, PERKIN ELMER), and set at 94 ° C. After reacting for 3 minutes, 30 cycles of 96 ° C. for 30 seconds, 55 ° C. for 1 minute, 72 ° C.
  • PJ480, PERKIN ELMER DNA thermal cycler
  • reaction solution was precipitated with ethanol, and 30 1 of 50 mM Tris- In addition to a buffer solution consisting of hydrochloric acid (pH 7.5), 100 mM sodium chloride, 100 mM magnesium chloride and ImMDTT, 10 units of a restriction enzyme EcoRI (Takara Shuzo Co., Ltd.) was further added and reacted at 37 ° C for 1 hour.
  • the reaction was precipitated with ethanol and added to 10 1 of a buffer consisting of 33 mM Tris-acetic acid (pH 7.9), 66 mM potassium acetate, 10 mM magnesium acetate, 0.5 mM DTT and 100 / g / ml BSA.
  • the enzyme Smal (Takara Shuzo) was added and reacted at 30 ° C for 1 hour.
  • the reaction solution was fractionated by agarose gel electrophoresis, and about 1 zg of an EcoRI-Smal fragment of about 0.41 kb was recovered using a QIAquick Gel Extraction Kit (manufactured by QIAGEN) according to the attached instruction.
  • Each plasmid DNA was prepared from 10 clones of the transformation » followed by reaction with the AutoRead Sequencing Kit (Pharmacia) according to the attached instructions, followed by electrophoresis using ALF DNA Sequencer (Pharmacia), and the inserted cDNA.
  • plasmid pBSAhCat IL-2 shown in FIG. 2 having the target nucleotide sequence was obtained.
  • the reaction solution was burned with ethanol, added to 10 ⁇ 1 of a buffer solution consisting of 50 mM Tris-HCl (pH 7.5), 10 mM magnesium chloride, lmM DTT and 100 mM sodium chloride, and further added 10 units of restriction enzyme EcoT22I ( (Takara Shuzo) and reacted at 37 C for 1 hour.
  • the reaction solution was fractionated by agarose gel electrophoresis, and about lg of an ApaI-EcoT22I fragment of about 0.92 kb was recovered using a QIAquick Gel Extraction Kit (manufactured by QIAGEN) according to the attached instruction.
  • 3 g of the plasmid pBSAhCat IL-2 obtained in paragraph (1) of Example 1 above was composed of 10 1 lOmM Tris-HCl (pH 7.5), 10 mM magnesium chloride, and lmM DTT.
  • the reaction solution was precipitated with ethanol, added to a buffer solution consisting of 10 ⁇ 1 of 50 mM Tris-HCl (pH 7.5), 10 mM magnesium chloride, ImM DTT and 100 mM sodium chloride, and further added 10 units of restriction enzyme EcoT22I (Takara Shuzo Co., Ltd.). ) was added and reacted at 37 ° C for 1 hour.
  • the reaction solution was fractionated by agarose gel electrophoresis, and QIAquick Gel
  • 0.1 g of the ApaI-EcoT22I fragment of the plasmid pBShC ⁇ 1 obtained above and 0.1 zg of the ApaI-EcoT22I fragment of the plasmid pBS muhCat IL-2 were added to 10 1 of sterile water, and the DNA was added.
  • Ligation Kit Ver.2 (Takara Shuzo Co., Ltd.) was used in accordance with the instruction manual.
  • Escherichia coli DH5o strain was transformed using the recombinant plasmid DNA solution obtained in this manner to obtain plasmid pBShCatIL-2 shown in FIG.
  • 3 ⁇ g of plasmid pANTEX796HM2Lm-28No.l described in 10-257893 is added to a buffer consisting of 101 lOmM Tris-HCl (pH 7.5), 10 mM magnesium chloride and lmMDTT, and an additional 10 units
  • the restriction enzyme Apal (Takara Shuzo) was added and reacted at 37 ° C for 1 hour.
  • the reaction solution is added to ethanol, added to a buffer containing 10/1 2 tris-hydrochloric acid (pH 8.5), 10 mM magnesium salt, ImM DTT and potassium salt, and further 10 units of restriction enzyme.
  • BamHI (manufactured by Takara Shuzo) was added and reacted at 30 ° C.
  • the reaction solution was fractionated by agarose gel electrophoresis, and about 2 g of an Apal-BamHI fragment of about 12.57 kb was recovered using a QIAquick Gel Extraction Kit (manufactured by QIAGEN) according to the attached instruction.
  • 3 zg of the plasmid pBShCat IL-2 obtained in section 1 (2) of Example 1 was buffered with 10 1 of 10 mM Tris-hydrochloride ( ⁇ 7.5), ⁇ magnesium chloride and ImM DTT.
  • 10 units of the restriction enzyme Apal (Takara Shuzo) was further added and reacted at 37 ° C for 1 hour.
  • the reaction mixture was diluted with ethanol and added to a buffer consisting of 10 ⁇ 1 of 20m Tris-hydrochloric acid (pH 8.5), lOmM magnesium chloride, ImM DTT and lOOmM potassium chloride, and further 10 units of restriction enzyme Bail (Takara Shuzo) and reacted at 30 ° C. for 1 hour.
  • the reaction solution was fractionated by agarose gel electrophoresis, and about 2 / g of an Apal-BamHI fragment of about 1.45 kb was recovered using a QIAquick Gel Extraction Kit (manufactured by QIAGEN) according to the attached instruction.
  • KM8969-hIL-2 in animal cells was carried out as follows using pANTEX8969-hIL2, which is a stable expression vector of KM8969-hIL-2 obtained in paragraph (2) of Example 1 above. Was.
  • G418 was added to 0.5 g of G418 in order to increase the amount of antibody expression using the dhfr amplification system.
  • MTX methotrexate
  • DHFR dihydrofolate reductase
  • the GM2 binding activity derived from the anti-GM2CDR ⁇ planted antibody # 8969 in the culture supernatant was measured by the ELISA method described in Example 1, section 3.
  • the MTX concentration was increased to 100 nM and 200 nM in the same manner as above, and finally A transformant capable of growing on an RPMI1640-FBS (IO) medium containing G418 at a concentration of 0.5 mg / ml and MTX at a concentration of 200 nM and highly expressing KM8969-hIL-2 was obtained.
  • KM8969WL2 was deposited as FERM BP-6792 on July 22, 1999 with the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology (1-3-1 Tsukuba East, Ibaraki, Japan).
  • KM8969-hIL-2 About 30 mg was purified from about 4 L of the culture supernatant using a Prosep-A (manufactured by Bioprocessin) column according to the attached instructions. About 4 jiig of the obtained KM8969_hIL-2 and anti-6121201 plant antibody KM8969 were subjected to electrophoresis according to a known method [Nature, 227, 680 (1970)], and the molecular weight and production accuracy were examined. The results are shown in FIG. As shown in FIG. 5, purified KM8969-hIL-2 had a molecular weight of about 180 Kd under non-reducing conditions, and two bands of about 65 Kd and about 25 Kd were observed under reducing conditions.
  • the binding activity of the antibody to various gangliosides was measured as follows. Dipalmitoyl phosphatidylcholine with 2 nmol of various gangliosides
  • the 1% BSA-PBS was discarded, and the culture supernatant of the transformant or various dilutions of the purified antibody were added at 50 ⁇ l and reacted at room temperature for 1 hour. After the reaction, wash each well with PBS containing 0.05% Tween20 (hereinafter referred to as Tween-PBS), and then dilute 3000 times with 1% BSA-PBS to peroxidase-labeled goat anti-human IgG (H & L). An antibody solution (manufactured by American Qualex) was added as a secondary antibody solution at 50/1 / well, and reacted at room temperature for 1 hour.
  • Tween-PBS PBS containing 0.05% Tween20
  • KM8969-hIL-2 The reactivity of purified KM8969-hIL-2 to GM2 (DIA-IATR0N) was measured according to the method described in Example 1, section 3.
  • Figure 6 shows that the amount of GM2 adsorbed to each well of the ELISA plate was fixed at 20 pmol / well, and the reactivity was examined by changing the concentration of the added anti-GM2CDR ⁇ plant antibody KM8969 and KM8969-hIL-2. This is the result.
  • KM8969-hIL-2 was shown to have a binding activity to GM2 equal to or higher than that of the anti-GM2CDR ⁇ plant antibody KM8969.
  • the activity of purified KM8969-hIL-2 as hIL-2 was measured according to the method described below.
  • a mouse T cell line CTLL-2 (ATCC TIB214) showing concentration-dependent growth for hIL-2 was suspended in RPMI1640-FBS (10) medium at a concentration of 2 ⁇ 10 5 cells / ml, Evening Dispensed 50 ⁇ 1 / ⁇ l into an iterative plate (manufactured by Limestone Bright). The 50 ⁇ 1 solution diluted to various concentrations added to each ⁇ E Le hIL- 2 (R & D Ltd.
  • RPMI1640- FBS and lx lO 6 cells of human small Hosokoboshi City cancer cell cultured in (IO) Medium strain SBC-3 (JCRB 0818) was prepared, the N 51 Cr0 4 is a TO substance added 1.85MBq eq
  • the cells were allowed to react at 37 ° C for 1 hour to release the cells.
  • the suspension was washed with RPMI1640-FBS (IO) medium three times by suspending and centrifuging, resuspended in the medium, and left on ice at 4 ° C for 30 minutes to allow natural dissociation of radioactive substances.
  • 5 ml of RPMI1640-FBS (IO) medium was added to adjust to 2 ⁇ 10 5 cells / ml, and used as a target cell solution.
  • the ratio of effector cells to target cells is 5: 1.
  • the plate was centrifuged, and the amount of 51 Cr in the supernatant was measured at a county.
  • the amount of spontaneously dissociated 51 Cr was determined by performing the same operation as above using only the medium instead of the effector cell solution and the antibody solution, and measuring the amount of 51 Cr in the supernatant.
  • ADCC activity (%) X 100
  • a derivative of an antibody that specifically reacts with GM2 is provided.
  • SEQ ID No. 3 Description of Artificial Sequence: Synthetic DNA
  • SEQ ID No. 4 Description of Artificial Sequence: Synthetic S3 ⁇ 4DNA
  • SEQ ID No. 5 Description of Artificial Sequence: Synthetic SgDNA SEQ ID NO.

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Abstract

Derivatives of an antibody against ganglioside GM2 (hereinafter referred to as GM2); fragments of the antibody against GM2; and diagnostics and remedies for cancer with the use of the above derivatives and antibody fragments.

Description

明 細 書  Specification
ガングリオシド GM 2に対する抗体の誘導体 技術分野  Derivatives of antibodies to ganglioside GM 2
本発明は、 ガングリオシド G 2 (以下、 G 2と表記する） に対する抗体およびそ の抗体断片に関する。 本発明は更に、 上記の誘導体および抗体断片をコードする DNA配列に関する。 本発明は、 該 DNA配列を含んでなる発現ベクター、 および該発 現べク夕一により形質転換された細胞に関する。 本発明は更に、 該形質転換細胞 を用いた上記の誘導体および抗体断片の製造方法、 ならびに上記の誘導体および 抗体断片を用レ、る癌の診断薬および治療薬に関する。 背景技術  The present invention relates to an antibody against ganglioside G2 (hereinafter, referred to as G2) and an antibody fragment thereof. The invention further relates to DNA sequences encoding the above derivatives and antibody fragments. The present invention relates to an expression vector comprising the DNA sequence, and a cell transformed by the expression vector. The present invention further relates to a method for producing the above derivative and antibody fragment using the transformed cell, and a diagnostic and therapeutic agent for cancer using the above derivative and antibody fragment. Background art
ハイプリドーマ法による抗体作製技術の確立により、 ヒ卜以外の動物を免疫し て、所望の 結^異性を有する抗体を作製することが可能となった [ネィチヤ -(Nature), 256, 495 ( 1975)]。 そして、 抗体はその高い抗原結合活性、 抗原結 異性および物理的安定性から、 ヒトの各種疾患の予防、 診断および治療への 応用が検討された。 しかし、 ヒト以外の動物の抗体、 例えばマウス抗体をヒトに 投与すると、 異物として認識されることにより、 ヒト体内にマウス抗体に対する ヒト抗体 (Human Anti Mouse Antibody：以下、 HAMAと表記する） が誘導されてし まう。 誘導された HAMAは投与されたマウス抗体と反応し、 副作用を引き起こした り [ジャーナル'ォブ 'クリニカル'オンコロジ一(J. Clin. Oncol. ), 2, 881 ( 1984) ；ブラッド（Blood), 65, 1349 (1985)；ジャーナル'ォブ'ザ'ナショナル'キャン サ一'インスティテユート（J. Natl. Cancer Inst. ) , 80, 932 (1988)；プロシ一 ディングス 'ォブ 'ザ'ナショナル 'アカデミー'ォブ 'サイエンス（Proc. Natl. Acad. Sci. U. S.A. ), 82, 1242 ( 1985)]、 投与されたマウス抗体の血中からの消失を速 め [ジャーナル'ォブ'ニュークレア一'メデイシン（J. Nucl. Med. ), 26, 1011 (1985)；ブラヅド（Blood), 65, 1349 ( 1985)；ジャーナル'ォブ'ザ 'ナショナル' キャンサ一.インスティテユート（J. Natl. Cancer Inst. ), 80, 937 ( 1988)]、 マ ウス抗体の治療効果を減じてしまうことが知られている [ザ'ジャーナル 'ォプ 'ィ ムノロジ一 (J. Immunol. ) , 135, 1530 ( 1985) ;キャンサー.リサーチ (Cancer Res. ), 46, 6489 ( 1986)]。 The establishment of antibody production technology using the hybridoma method has made it possible to immunize animals other than humans and produce antibodies having the desired isomerism [Nichia-(Nature), 256, 495 (1975) )]. Antibodies have been studied for their application in the prevention, diagnosis and treatment of various human diseases because of their high antigen-binding activity, antigen isomerism and physical stability. However, when an antibody from a non-human animal, such as a mouse antibody, is administered to a human, a human antibody against the mouse antibody (Human Anti Mouse Antibody: hereinafter, referred to as HAMA) is induced in the human body by being recognized as a foreign substance. I will. Induced HAMA reacts with the administered mouse antibody, causing side effects and [J. Clin. Oncol., 2, 881 (1984); Blood, 65, 1349 (1985); Journal of the National Institute of Cancer, J. Natl. Cancer Inst., 80, 932 (1988); Proceedings of the National of Cancer Inst. 'Academy' of Science '(Proc. Natl. Acad. Sci. USA), 82, 1242 (1985)], speeding up the disappearance of administered mouse antibodies from the blood [Journal of Obcle's Nuclear 'Medicine (J. Nucl. Med.), 26, 1011 (1985); Blood, 65, 1349 (1985); Journal' Ob 'The' National 'Cancer Institute. J. Natl. Cancer Inst.), 80, 937 (1988)], which is known to reduce the therapeutic effect of mouse antibodies [The Journal of Opin. J. Immunol.), 135, 1530 (1985); Cancer Res. 46, 6489 (1986)].
これらの問題点を解決するため、 遺伝子組換え技術を利用してヒト以外の動物 の抗体をヒト型キメラ抗体或いはヒト型相補性決定領域 （Complementarity Determining Region：以下、 CDRと表記する）移植抗体などのヒト化抗体にするこ とが試みられている。 ヒト型キメラ抗体とは、 抗体可変領域（以下、 V領域と表記 する） がヒト以外の動物の抗体で、 定常領域（以下、 C領域と表記する） がヒト抗 体である抗体であり [プロシ一ディングス 'ォブ ·ザ'ナショナル 'アカデミー'ォブ 'サイエンス（Proc. Natl. Acad. Sci. U. S.A. ), 81, 6851 ( 1984)]、 ヒト型 CDR 移植抗体とは、 ヒト以外の動物の抗体の V領域中の CDRのアミノ酸配列をヒト抗体 の適切な位置に移植した抗体である [ネィチヤ一 (Nature) , 321, 522 ( 1986) ]。 こ れらのヒト化抗体は、 マウス抗体等のヒト以外の動物の抗体に比較してヒトへの 臨床応用上、 様々な利点を有している。 例えば、 免疫原性および血中での安定性 に関しては、 ヒト型キメラ抗体では、 ヒトに投与した場合、 マウス抗体に比べて 血中半減期が約 6倍伸びたことが報告されている [プロシ一ディングス 'ォプ 'ザ' ナショナル 'アカデミー'ォブ'サイエンス（Proc. Natl . Acad. Sci. U. S.A. ), 86, 4220 ( 1989)] 0 ヒト型 CDR^植抗体では、 サルを用いた実験でマウス抗体に比べ免 疫原性が低下し、 血中半 期が 4〜5倍伸びたことが報告されている [ザ'ジャーナ ル'ォブ 'ィムノロジー (J. Iimniinol. ), 147, 1352 ( 1991 )]。 即ち、 ヒト化抗体は、 ヒト以外の動物の抗体に比べ、 副作用が少なく、 その治療効果が長期間持続する ことが期待される。 また、 特に抗腫瘍抗体としての応用を考えた場合、 抗体の Fc 領域 （抗体重鎖のヒンジ領域以降の領域） を介した補体依存性細胞障害活性 （以 下、 CDC活性と表記する）や抗体依存性細胞障害活性（以下、 ADCC活性と表記する ) 等の細胞障害活性の高さがその治療効果に重要であるが、 こうした細胞障害活 性に関しても、 ヒトにおいてはヒト以外の動物の抗体の Fc領域よりも、 ヒト抗体 の Fc領域の方がヒト補体成分や、 単核球、 マクロファージ、 NK細胞の Fc受容体を 細胞表面に有するヒトエフェクター細胞をより効率的に活性化できる為、 より優 れていることが報告されている。 例えば、 ガングリオシド GD2 (以下、 GD2と表記 する） に対するマウス抗体（以下、 抗 GD2マウス抗体と表記する）は、 ヒト抗体の Fc領域を有するヒト型キメラ抗体（以下、抗 GD2キメラ抗体と表記する）に変換す ることにより、 ヒトエフヱクタ一細胞による腫瘍細胞障害活性が上昇することが 報告されており [ザ'ジャーナル'ォブ 'ィムノロジ一( Immunol.), 144, 1382 (1990)]、 また、 CAMPATH-li¾lに対するヒト型 CDR^植抗体についても同様の結果 が報告されている [ネイチヤー (Nature), 332, 323 (1988)]。 In order to solve these problems, using a gene recombination technique, a non-human animal antibody can be used as a human-type chimeric antibody or a human-type complementarity determining region (hereinafter referred to as CDR) transplanted antibody, etc. An attempt has been made to make this a humanized antibody. A human chimeric antibody is an antibody in which the antibody variable region (hereinafter, referred to as V region) is an antibody of a non-human animal and the constant region (hereinafter, referred to as C region) is a human antibody. Idings 'Ob The National' Academy 'Ob' Science (Proc. Natl. Acad. Sci. USA), 81, 6851 (1984)], Human CDR-grafted antibodies are antibodies from non-human animals This is an antibody obtained by grafting the amino acid sequence of the CDR in the V region of the above to an appropriate position of a human antibody [Nature, 321, 522 (1986)]. These humanized antibodies have various advantages in human clinical application compared to non-human animal antibodies such as mouse antibodies. For example, in terms of immunogenicity and stability in blood, it has been reported that the human chimeric antibody, when administered to humans, has an approximately six-fold increase in blood half-life compared to the mouse antibody [Procy. One Ding's' Op 'The National' Academy 'Ob' Science (Proc. Natl. Acad. Sci. USA), 86, 4220 (1989)] 0 It has been reported that immunogenicity is lower than that of mouse antibodies and that blood half-life is increased by 4 to 5 times [The 'Journal of the Immuninol' (J. Iimniinol.), 147, 1352 ( 1991)]. That is, humanized antibodies are expected to have fewer side effects and to have a long-lasting therapeutic effect compared to antibodies of non-human animals. In particular, when considering its application as an antitumor antibody, complement-dependent cytotoxicity (hereinafter referred to as CDC activity) via the Fc region of the antibody (the region following the hinge region of the antibody heavy chain) and The high cytotoxic activity such as antibody-dependent cytotoxic activity (hereinafter referred to as ADCC activity) is important for its therapeutic effect. Since the Fc region of a human antibody can more efficiently activate human complement components, Fc receptors of monocytes, macrophages, and NK cells on the cell surface, than the Fc region of It is reported to be superior. For example, a mouse antibody against ganglioside GD2 (hereinafter referred to as GD2) (hereinafter referred to as anti-GD2 mouse antibody) is a human chimeric antibody having an Fc region of a human antibody (hereinafter referred to as anti-GD2 chimeric antibody). To increase the tumor cell-damaging activity of a single human effector cell. [The 'Journal' of Immunology (Immunol.), 144, 1382 (1990)], and similar results have been reported for human CDR ^ -planted antibodies against CAMPATH-li¾l [ Nature, 332, 323 (1988)].
以上の結果は、 ヒトへの臨床応用に用いる抗体としては、 ヒト化抗体の方がマ ウス抗体等のヒト以外の動物の抗体より望ましいことを明確に示している。  The above results clearly show that humanized antibodies are more preferable than non-human animal antibodies such as mouse antibodies for use in human clinical applications.
シアル酸を含有する糖脂質の一種であるガングリオシドは動物の細胞膜を構成 しており、 性側鎖である糖鎖と、 性側鎖であるスフインゴシンおよび脂 肪酸とから構成される分子である。 ガングリオシドの種類と発現量は、 細胞種、 臓器種、 動物種等によって異なることが知られている。 更に細胞が癌化する過程 においては、 ガングリオシドの発現が量的および質的に変化を起こすことも知ら れている [キャンサー 'リサーチ (Cancer Res. )， 45, 2405 (1985)]。 特に、 本発明 で注目した GM2は、 正常細胞にはごく微量にしか存在しないが、肺癌、 メラノ一マ 、 グリオ一マ、 ニューロブラストーマ等の癌細胞では多量に存在し [キャンサー- リサーチ (Cancer Res.), 45, 2405 (1985)；プロシーディングス ·ォブ 'ザ 'ナ ショナル ·アカデミー ·ォブ ·サイエンス（Proc. Natl. Acad. Sci. U.S.A.), 80, 5392 (1983)；ジャーナル ·ォブ ·ナショナル ·キャンサー ·ィンスティテュート (J. Natl. Cancer Inst.), 78, 45 (1987)；キャンサー 'リサーチ（Cancer Res. )， 50, 7444 (1990)]、 GM2に対する抗体 （以下、 抗 GM2抗体と表記する） は、 これら の癌の診断、 治療に有用であると考えられている [ランセット（Lancet), I, 786 (1989)]。 更に近年、 GM2のメラノ一マ治療におけるワクチンとしての有用性が示 され、 投与された患者血清中に高い抗 GM2抗体価の上昇が観察されている [プロシ 一ディングス ·ォブ'ザ 'ナショナル ·アカデミー'ォブ'サイエンス（Proc. Natl. Acad. Sci. U.S.A.), 92, 2805 (1995)]。 また、 サイ トメガロウィルスの感染に 伴って抗 GM2抗体価の上昇が認められることも報告され [ジャーナル'ォブ'ザ '二 ユーロロジカル.サイェンシィズ (J. Neurological Sciences), 154， 14 (1998) ；アナルズ'ォブ'ザ 'ニューヨーク 'アカデミー'ォブ'サイェンシィズ (Annals New York Academy Sciences), 845, 423 (1998)]、 各種の細菌、 ウィルス感染に おける GM2の役割が注目され、抗 GM2抗体の各種感染症への適応も期待されている。 以上の観点から、本発明者らは抗 GM2抗体のより効果的なヒトへの臨床応用を目 的として、 本発明者らが作製した抗 GM2マウス抗体 KM796 (特開平 6-205694) を基 に、 マウス抗体と同等の活性を有する GM2に対するヒト化抗体 （以下、 抗 GM2ヒト 化抗体と表記する）の作製を試み、 その結果、 抗 GM2ヒト型キメラ抗体（以下、 抗 GM2キヌラ抗体と表記する） KM966および抗 GM2ヒト型 CDR^植抗体（以下、抗 GM2CDR 移植抗体と表記する） KM8969を作製している （特開平 10-257893) 。 抗 GM2キメラ 抗体 KM966および抗 GM2CDR^植抗体 KM8969は、 抗 GM2マウス抗体に比べヒトにおい て免疫原性が低下し、 血中半減期が延長して長期間に渡る治療効果、 更にはより 強い細胞障害活性に基づく高い抗腫瘍効果が期待されている。 Ganglioside, which is a kind of glycolipid containing sialic acid, constitutes the cell membrane of an animal, and is a molecule composed of a sugar chain, which is a sex side chain, and sphingosine and a fatty acid, which are sex side chains. It is known that the type and expression level of gangliosides vary depending on cell types, organ types, animal types, and the like. It is also known that the expression of gangliosides changes quantitatively and qualitatively in the process of canceration of cells [Cancer's Research (Cancer Res.), 45, 2405 (1985)]. In particular, GM2, which has been noted in the present invention, is present in only a very small amount in normal cells, but is present in large amounts in cancer cells such as lung cancer, melanomas, gliomas, and neuroblastomas [Cancer-Research (Cancer- Res.), 45, 2405 (1985); Proceedings of the 'The' National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 80, 5392 (1983); Cancer Research (J. Natl. Cancer Inst.), 78, 45 (1987); Cancer 'Research (Cancer Res.), 50, 7444 (1990)], an antibody against GM2 (hereinafter referred to as anti- GM2 antibody) is considered to be useful for the diagnosis and treatment of these cancers [Lancet, I, 786 (1989)]. Furthermore, in recent years, the usefulness of GM2 as a vaccine in the treatment of melanoma has been shown, and a high anti-GM2 antibody titer has been observed in the serum of administered patients [Procedures of the National ' Academy of Sciences (Proc. Natl. Acad. Sci. USA), 92, 2805 (1995)]. It has also been reported that an increase in anti-GM2 antibody titer was observed following cytomegalovirus infection [Journal 'ob' the '2 Eurological Sciences (J. Neurological Sciences), 154, 14 (1998) Anals 'ob' the 'New York' Academy ''ob'Sciences (Annals New York Academy Sciences), 845, 423 (1998)], the role of GM2 in various bacterial and viral infections has been noted, and anti-GM2 antibodies It is also expected to be applied to various infectious diseases. In view of the above, the present inventors have developed an anti-GM2 mouse antibody KM796 (JP-A-6-205694) prepared by the present inventors for the purpose of more effective clinical application of human anti-GM2 antibodies to humans. First, a humanized antibody against GM2 having the same activity as mouse antibody (hereinafter referred to as anti-GM2 humanized antibody) was produced. As a result, an anti-GM2 human chimeric antibody (hereinafter referred to as anti-GM2 quinula antibody) was obtained. KM966 and anti-GM2 human CDR-grafted antibody (hereinafter referred to as anti-GM2 CDR-grafted antibody) KM8969 have been produced (Japanese Patent Laid-Open No. 10-257893). Anti-GM2 chimeric antibody KM966 and anti-GM2 CDR ^ -implanted antibody KM8969 have lower immunogenicity in humans than anti-GM2 mouse antibodies, have a longer half-life in blood, and have a longer-lasting therapeutic effect, and even stronger cells A high antitumor effect based on the damaging activity is expected.
腫瘍に対するヒト化抗体は、 上記の様にヒト抗体の Fc領域を介した補体成分の 結合、活性ィ匕に基づく CDC活性および Tc受容体との結合に基づく ADCC活性等によつ て抗体単独の使用によっても治療効果が期待されているが、 更に他の分子との併 用により、 その効果をより高めることが検討されている。 例えば、 それら分子の 一つとしてサイ トカインが用いられている。 サイ トカインは免疫反応における細 胞間相互作用を司る種々の液性因子の総称である。 ADCC活性は、 単核球、 マクロ ファージ、 NK細胞の Fc受容体を細胞表面に有するエフェク夕一細胞によって担わ れており [ザ'ジャーナル'ォブ 'ィムノロジ一 (J. Iimnunol . ) , 138, 1992 ( 1987)]、 種々のサイトカインはこれらのエフェクター細胞を活性化することから、 ADCC活 性を高める目的で、 抗体と組み合わせて投与することが行われている。 例えば、 抗 GD2キメラ抗体 chl4. 18に関しては、サイ トカインであるヒトインターロイキン 2 (以下、 hIL-2と表記する）或いはヒト顆粒球一マクロファージコロニー刺激因子 (以下、 hGM-CSFと表記する） と組み合わせてヒトに投与することが行われた [キ ヤンサ一(Cancer), 80, 317 ( 1997)；キャンサー'ジャーナル 'フロム'サイェンテ ィフィック ·アメリカン（Cancer J. From Scientific American) , 3, S121 ( 1997) ] 。 しかし、 これらの併用療法は、 サイト力インの全身性の副作用から期待された 程の効果は認められていない。 そこで、 サイト力インを標的組織のみに効率的に 到達させるため、 抗体分子とサイ トカインを融合させた蛋白質が作製された。 hIL- 2に関しては、抗 GD2キメラ抗体 chl4. 18との融合蛋白質が遺伝子工学的に作 製され、 マウスを用いた実験でその ί¾®瘍効果が抗 GD2キメラ抗体 chl4.18および hIL- 2の同時投与よりも優れていることが報告されている [プロシ一ディングス · ォブ ·ザ ·ナショナル ·アカデミー ·ォブ ·サイエンス（Proc. Natl. Acad. Sci . U. S.A. ) , 89， 1428 ( 1992) ;プロシ一ディングス 'ォブ 'ザ ·ナショナル 'ァカ デミ一 'ォブ 'サイエンス（Proc. Natl. Acad. Sci. U.S.A.), 91, 9626 (1994) ；キヤンサ一'ィムノロジ一'ィムノセラピー (Cancer Immunol. , Immunother. ), 42, 88 (1996)；ブラッド（Blood), 91, 1706 (1998)]。 現在では、 サイトカイン に限らず他の蛋白質 （トキシン、 酵素等） 、 更には 性同位元素、 低分子の薬 剤等を抗体に結合させた各種誘導体の作製も行われ、 それらの臨床応用が検討さ れている [ニューィングランド ·ジャーナル'ォブ 'メディシン（New Eng. J. Med.), 329, 459 (1993)；アンチキャンサー · リサーチ (Anticancer Res.), 17, 1735 (1997)；ブラッ K (Blood), 78, 1173 (1991)；ジャーナル ·ォブ'クリニカル · オンコロジ一 ( Clin. Oncol. ), 15, 723 (1997)；バイオコンジュゲート 'ケミ ストリー (Biocon jugate Chem.), 7, 606 (1997)；キャンサー (Cancer), 61, 881 (1988)；ジャパニーズ ·ジャーナル 'ォブ'キヤンサ一 ·リサーチ (Jpn. J. Cancer Res.), 85, 167 (1994)；アンチボディ ·ィムノコンジユゲーヅ 'アンド 'ラディ オファーマシューティカノレズ (Antibody Immunocon ugates and As described above, humanized antibodies against tumors are isolated by binding of complement components via the Fc region of human antibodies, CDC activity based on activation and ADCC activity based on binding to Tc receptors, etc. Therapeutic effect is also expected from the use of, but it is being studied to further enhance the effect by using it in combination with other molecules. For example, cytokines are used as one of these molecules. Cytokine is a general term for various humoral factors that control cell-cell interactions in the immune response. ADCC activity is carried by mononuclear cells, macrophages, and effector cells having the Fc receptor of NK cells on the cell surface [J. Iimnunol., 138, 1992 (1987)], since various cytokines activate these effector cells, they have been administered in combination with antibodies for the purpose of enhancing ADCC activity. For example, for the anti-GD2 chimeric antibody chl.18, the human cytokine interleukin 2 (hereinafter referred to as hIL-2) or human granulocyte-macrophage colony stimulating factor (hereinafter referred to as hGM-CSF) Administration to humans in combination has been performed [Cancer, 80, 317 (1997); Cancer J. From Scientific American, 3, S121 (1997). )]. However, these combinations have not been as effective as expected due to the systemic side effects of cytoforcein. Thus, a protein was prepared by fusing an antibody molecule and cytokine in order to efficiently reach the cytokinin only to the target tissue. Regarding hIL-2, a fusion protein with anti-GD2 chimeric antibody chl.18.18 was genetically engineered, and its tumor effect was demonstrated in mice using an anti-GD2 chimeric antibody chl4.18 and hIL-2. It has been reported that it is superior to co-administration [Procedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 89, 1428 (1992); Proceedings' ob 'the national' aka Natl. Acad. Sci. USA, 91, 9626 (1994); Cyansa's Immunology, Immunother., 42, 88 (1996); Blood. (Blood), 91, 1706 (1998)]. At present, not only cytokines but also other proteins (toxins, enzymes, etc.), as well as various derivatives in which sex isotopes, low-molecular-weight drugs, etc. are bound to antibodies are being produced, and their clinical application is being studied. [New Eng. J. Med., New Eng. J. Med., 329, 459 (1993); Anticancer Research, 17, 1735 (1997); Black K (Blood), 78, 1173 (1991); Journal of Clinical Oncol., 15, 723 (1997); Bioconjugate Chem., 7, 606. (1997); Cancer, 61, 881 (1988); Japanese Journal 'Ob' Kyansa Research (Jpn. J. Cancer Res.), 85, 167 (1994); Antibody Simnocon Antibody Immunocon ugates and
Radiopharmaceuticals), 3, 60 (1990);サ一ジェリー（Surgery), 106, 533 (1989)] 。 これらの誘導体は、 抗体分子の結^異性に従って蛋白質 （サイトカイン、 ト キシス 酵素等） 、 放射性同位元素、 低分子の薬剤等を標的組織周辺に集積させ ることで、 より効果的で副作用の少ない診断、 治療を可能にすることが期待され ている。抗 GM2抗体に関しても、 蛋白質 （サイ トカイン、 トキシン、 酵素等）、放 射性同位元素、 低分子の薬剤等を融合させた誘導体が作製できれば、 抗体単独よ りも更に高い診断、 治療効果が期待されるが、 現在までにこれら誘導体の作製の 報告はない。 Radiopharmaceuticals), 3, 60 (1990); Surgery, 106, 533 (1989)]. These derivatives provide more effective diagnosis with less side effects by accumulating proteins (cytokines, toxin enzymes, etc.), radioisotopes, low-molecular-weight drugs, etc. around the target tissue in accordance with the isomerism of the antibody molecule. It is expected that treatment will be possible. For the anti-GM2 antibody, if a derivative can be produced by fusing proteins (cytokines, toxins, enzymes, etc.), radioisotopes, low-molecular-weight drugs, etc., a higher diagnostic and therapeutic effect than the antibody alone can be expected. However, there is no report on the production of these derivatives to date.
一方、 最近の蛋白質工学、 遺伝子工学の進歩により、 Fab、 Fab'、 F(ab5 )2、 一 本鎖抗体 (scFv) [サイエンス（Science), 242, 423 (1988)]、 ジスルフィ ド安定 化 V領域（dsFv)断片 [モレキュラー'ィムノロジ一 (Molecular Immunol.), 32, 249 (1995)]等の、 より分子量の小さい抗体断片の作製が可能となっている。これら抗 体断片は、 完全な抗体分子に比べ分子量が小さいため、 標的組織への移行性に優 れている [キャンサー'リサーチ（Cancer Res.), 52, 3402 (1992)]。 これらの抗体 断片についても、 ヒトへの臨床応用の場合には、 マウス抗体等のヒト以外の動物 の抗体よりもヒト化抗体に由来する方がより望ましいと考えられる。 On the other hand, recent protein engineering, advances in genetic engineering, Fab, Fab ', F ( ab 5) 2, single chain antibodies (scFv) [Science (Science), 242, 423 ( 1988)], disulfide stabilized V region (dsFv) fragments [Molecular Immunol. (Molecular Immunol.), 32, 249 (1995)] and other antibody fragments with smaller molecular weights can be produced. These antibody fragments have a smaller molecular weight than the whole antibody molecule, and are therefore excellent in transferability to target tissues [Cancer's Research (Cancer Res.), 52, 3402 (1992)]. Also in the case of clinical application to humans, it is considered that these antibody fragments are more preferably derived from humanized antibodies than non-human animal antibodies such as mouse antibodies.
抗体断片に関しては、 抗 GD2マウス抗体由来の一本鎖抗体は作製されているが [ ハイプリ ドーマ (Hybridoma), 16, 335 (1997)]、 それ以外の抗体断片の作製の報 告はなく、抗 G 2抗体由来の抗体断片に関してはマウス抗体およびヒト化抗体に由 来する抗体断片の作製の報告はなレ、。従って、抗 GM2ヒト化抗体に由来する断片が 作製できれば、 ヒトにおいて優れた標的 行性を有し、 かつ、 免疫原性が低 下することが期待される。更に抗 GM2抗体由来の断片についても、上記で述べた蛋 白質 （サイトカイン、 トキシン、 酵素等） 、 放射性同位元素、 低分子の薬剤等を 融合させた各種の誘導体が作製できれば、 その治療効果がより高まることが期待 される。 発明の開示 Regarding antibody fragments, single-chain antibodies derived from anti-GD2 mouse antibodies have been produced, Hybridoma, 16, 335 (1997)], and there were no reports of the production of other antibody fragments. For antibody fragments derived from anti-G2 antibodies, antibody fragments derived from mouse antibodies and humanized antibodies There is no report of production. Therefore, if a fragment derived from an anti-GM2 humanized antibody can be prepared, it is expected that humans will have excellent targetability and immunogenicity will be reduced. Furthermore, the therapeutic effect of fragments derived from the anti-GM2 antibody can be further improved if various derivatives can be produced by fusing the above-mentioned proteins (cytokines, toxins, enzymes, etc.), radioisotopes, low molecular drugs, etc. It is expected to increase. Disclosure of the invention
本発明ではこれまでに知られている抗 GM2抗体の治療効果の増強を目的に、該抗 体の抗体断片を作製した。 更に、 抗 G 2抗体または該抗体断片と放射性同位元素、 蛋白質または低分子の薬剤とを結合させた抗体の誘導体を作製した。具体的には、 抗 GM2CDR^植抗体 KM8969の H鎖をコードする cDNAの 3'末端に hIL- 2をコ一ドする cDNAを繋げた cDNAを構築し、該 cDNAおよひ 1M8969の L鎖をコードする cDNAを動物細 胞用発現べク夕一にクロ一ニングして抗 GM CDR^植抗体 KM8969と hIL- 2との融合 蛋白質（以下、 KM8969— hIL-2と表記する）の発現ベクターを構築した。該 KM8969 — hIL- 2発現べク夕一を動物細胞へ導入することにより KM8969 _hIL-2を培養上清 中に生産する形質転換クローン KM8969ML2 (FEM BP-6792) を作製した。 形質転 換クローン KM8969hIL2の培養上清より K 8969—ML- 2を精製し、 KM8969— hIL- 2が 抗 GM2CDR^植抗体 KM8969と同等の抗原結合活性、 ¾¾ 結合特異性および hIL- 2依存 性増殖を示す細 JKに対して hIL- 2と同等の増殖支持活性を示すことを見出した。 更にはヒト末梢血単核球画分を用いた細胞障害活性において抗 G 2CDR^植抗体 KM8969に比べ、 M8969— hIL-2の活性が増強されることを確認し、本発明を完成さ せた。  In the present invention, an antibody fragment of the known anti-GM2 antibody was prepared for the purpose of enhancing the therapeutic effect of the antibody. Furthermore, an antibody derivative in which the anti-G2 antibody or the antibody fragment was bound to a radioisotope, protein or low-molecular drug was prepared. Specifically, a cDNA was constructed in which the cDNA encoding hIL-2 was linked to the 3 'end of the cDNA encoding the H chain of the anti-GM2CDR ^ planted antibody KM8969, and the cDNA and the L chain of 1M8969 were constructed. The encoded cDNA is cloned into an expression vector for animal cells, and an expression vector for a fusion protein of the anti-GM CDR ^ plant antibody KM8969 and hIL-2 (hereinafter referred to as KM8969—hIL-2) is obtained. It was constructed. By introducing the KM8969-hIL-2 expression vector into animal cells, a transformed clone KM8969ML2 (FEM BP-6792) producing KM8969_hIL-2 in the culture supernatant was prepared. K8969-ML-2 was purified from the culture supernatant of the transformed clone KM8969hIL2, and KM8969-hIL-2 had the same antigen-binding activity, anti-GM2CDR ^ antibody as KM8969, ¾¾ binding specificity, and hIL-2-dependent growth Was found to exhibit the same growth supporting activity as that of hIL-2 against fine JK. Furthermore, it was confirmed that the activity of M8969-hIL-2 was enhanced in the cytotoxic activity using the human peripheral blood mononuclear cell fraction as compared to the anti-G2CDR ^ plant antibody KM8969, and the present invention was completed. .
本発明は、 以下の （1 ) 〜 （7 5 ) に関する。  The present invention relates to the following (1) to (75).
( 1 ) ガングリオシド GM2に特異的に反応するモノクローナル抗体またはその 抗体断片と、 放射性同位元素、 蛋白質または低分子の薬剤とを結合させた抗体の  (1) An antibody in which a monoclonal antibody or an antibody fragment thereof that specifically reacts with ganglioside GM2 and a radioisotope, protein, or low-molecular-weight drug is bound
( 2 ) ガングリオシド GMに特異的に反応するモノクローナル抗体が、ハイブ リドーマが産生する抗体、 ヒト化抗体およびヒト抗体から選ばれる抗体である、 上記 （ 1 ) 記載の抗体の誘導体。 (2) A monoclonal antibody that specifically reacts with ganglioside GM The derivative of the antibody according to (1), which is an antibody selected from an antibody produced by a lidoma, a humanized antibody, and a human antibody.
(3) モノクローナル抗体の H鎖 V領域の CDR1、 CDR2および CDR3がそれぞれ配 列番号 9、 10および 1 1で示されるアミノ酸配列を含む上記 （ 1) または （2 ) 記載の抗体の誘導体。  (3) The antibody derivative according to the above (1) or (2), wherein CDR1, CDR2, and CDR3 of the V region of the H chain of the monoclonal antibody comprise the amino acid sequences represented by SEQ ID NOS: 9, 10, and 11, respectively.
(4) モノクロ一ナル抗体の L鎖 V領域の CDR1、 CDR2および CDR3がそれぞれ配 列番号 12、 13および 14で示されるアミノ酸配列を含む上記 （1) または （ 2 ) 記載の抗体の誘導体。  (4) The antibody derivative according to the above (1) or (2), wherein CDR1, CDR2 and CDR3 of the L chain V region of the monoclonal antibody contain the amino acid sequences represented by SEQ ID NOs: 12, 13 and 14, respectively.
(5) モノクローナル抗体の重鎖 （H鎖） 可変領域 （V領域） の CDR1、 CDR2お よび CDR3がそれぞれ配列番号 9、 10および 1 1、軽鎖（L鎖） V領域の CDM、 CDR2 および CDR3がそれぞれ配列番号 12、 13および 14で示されるァミノ酸配列を 含む上記 （1) または （2) 記載の抗体の誘導体。  (5) CDR1, CDR2, and CDR3 of the heavy chain (H chain) variable region (V region) of the monoclonal antibody are SEQ ID NOs: 9, 10, and 11, respectively, and CDM, CDR2, and CDR3 of the light chain (L chain) V region The antibody derivative according to the above (1) or (2), which comprises an amino acid sequence represented by SEQ ID NO: 12, 13, or 14, respectively.
(6) ハイプリドーマが産生する抗体が "KM796 (FEEMBP-3340) である、 上記 ( 2 ) 記載の抗体の誘導体。  (6) The derivative of the antibody according to (2), wherein the antibody produced by the hybridoma is "KM796 (FEEMBP-3340).
(7) ヒト化抗体が、 ヒト型キメラ抗体またはヒト型 CDR^植抗体である、 上記 (2) 記載の抗体の誘導体。  (7) The derivative of the antibody according to (2), wherein the humanized antibody is a human chimeric antibody or a human CDR ^ -planted antibody.
(8) ヒト型キメラ抗体が、ガングリオシド GM2に対するハイプリ ドーマが産生 するモノクローナル抗体の H鎖 V領域および L鎖 V領域のァミノ酸配列を含む、 上記 (8) The human chimeric antibody comprises the amino acid sequences of the H chain V region and L chain V region of a monoclonal antibody produced by a hybridoma against ganglioside GM2,
( 7 ) 記載のヒト型キメラ抗体の誘導体。 (7) A derivative of the human chimeric antibody according to (7).
(9) ヒ卜型キメラ抗体が、ガングリオシド G に対するハイプリ ドーマが産生 するモノクロ一ナル抗体の H鎖 V領域および 1鎖 V領域、ならびにヒト抗体の H鎖定常 領域（C領域）および 1鎖 C領域のアミノ酸配列を含む、 上記（7)記載のヒト型キ メラ抗体の誘導体。  (9) The human chimeric antibody is composed of the H chain V region and 1 chain V region of a monoclonal antibody produced by a hybridoma against ganglioside G, and the H chain constant region (C region) and 1 chain C region of a human antibody. The derivative of the human chimeric antibody according to the above (7), comprising the amino acid sequence of:
(10) H鎖 V領域が配列番号 7で示されるアミノ酸配列を含む、 上記（ 8 ) また は （9) 記載のヒト型キメラ抗体の誘導体。  (10) The derivative of the human chimeric antibody according to (8) or (9), wherein the V region of the H chain comprises the amino acid sequence represented by SEQ ID NO: 7.
(1 1) L鎖 V領域が配列番号 8で示されるアミノ酸配列を含む、 上記（ 8 ) また は （9) 記載のヒト型キメラ抗体の誘導体。  (11) The derivative of the human chimeric antibody according to the above (8) or (9), wherein the L chain V region comprises the amino acid sequence represented by SEQ ID NO: 8.
(12) H鎖 V領域が配列番号 7で示されるアミノ酸配列を含み、 L鎖 V領域が配列 番号 8で示されるアミノ酸配列を含む上記（8) または（9)記載のヒト型キメラ 抗体の誘導体。 (13) H鎖 V領域が配列番号 7で示されるアミノ酸配列を含み、 L鎖 V領域が配列 番号 8で示されるアミノ酸配列を含む上記（8) または（9)記載のヒト型キメラ 抗体 KM966の誘導体。 (12) The derivative of the human chimeric antibody according to (8) or (9), wherein the H chain V region comprises the amino acid sequence represented by SEQ ID NO: 7, and the L chain V region comprises the amino acid sequence represented by SEQ ID NO: 8. . (13) The human chimeric antibody KM966 according to the above (8) or (9), wherein the H chain V region comprises the amino acid sequence represented by SEQ ID NO: 7, and the L chain V region comprises the amino acid sequence represented by SEQ ID NO: 8. Derivatives.
(14) ヒト型 CDR^植抗体が、 ガングリオシド GM2に対するモノクローナル抗 体の H鎖 V領域および 1鎖 V領域の CDRのアミノ酸配列を含む、 上記（7)記載のヒト 型 CDR^植抗体の誘導体。  (14) The derivative of the human CDR ^ -grafted antibody according to (7), wherein the human CDR ^ -grafted antibody comprises the amino acid sequences of the CDRs of the H chain V region and the single chain V region of the monoclonal antibody against ganglioside GM2.
( 15) ヒト型 CDR^植抗体が、 ガングリオシド GM2に対するモノクローナル抗 体の H鎖 V領域および 1鎖 V領域の CDRとヒト抗体の H鎖 V領域および 1鎖 V領域のフレ —ムワーク領域 (FR)のアミノ酸配列を含む、 上記（7)記載のヒト型 CDR^植抗 体の誘導体。  (15) The human CDR ^ -grafted antibody binds to the CDRs of the H chain V region and 1 chain V region of the monoclonal antibody against ganglioside GM2 and the framework region (FR) of the H chain V region and 1 chain V region of the human antibody The derivative of the human CDR ^ -planted antibody according to the above (7), comprising the amino acid sequence of
( 16) ヒト型 CDR^植抗体が、 ガングリオシド GM2に対するモノクロ一ナル抗 体の H鎖 V領域および 1鎖 V領域の CDR、 ヒト抗体の H鎖 V領域および 1鎖 V領域の FR、な らびにヒト抗体の H鎖 C領域および 1鎖 C領域のアミノ酸配列を含む、 上記 （7) 記 載のヒト型 CDR^植抗体の誘導体。  (16) Human CDR ^ -grafted antibodies were converted to the CDRs of the H chain V region and single chain V region of the monoclonal antibody against ganglioside GM2, the FRs of the H chain V region and single chain V region of the human antibody, and A derivative of the human CDR ^ -grafted antibody according to the above (7), comprising the amino acid sequence of the H chain C region and the single chain C region of a human antibody.
(17) 抗体の H鎖 V領域の CDR 1、 CDR2および CDR 3がそれぞれ配列番号 9、 10 および 11で示されるアミノ酸配列を含む、 上記 （14) 〜 （16) のいずれか 1 項に記載のヒト型 CDR^植抗体の誘導体。  (17) The method according to any one of the above (14) to (16), wherein CDR1, CDR2, and CDR3 of the V region of the H chain of the antibody include the amino acid sequences represented by SEQ ID NOS: 9, 10, and 11, respectively. Derivative of human CDR ^ plant antibody.
( 18) 抗体の L鎖 V領域の CDR1、 CDR2および CDR3がそれぞれ配列番号 12、 13お よび 14で示されるアミノ酸配列を含む、 上言己 （14) 〜 （16) のいずれか 1項 に記載のヒト型 CDR^植抗体の誘導体。  (18) The method according to any one of (14) to (16), wherein CDR1, CDR2, and CDR3 of the V region of the L chain of the antibody include the amino acid sequences represented by SEQ ID NOS: 12, 13, and 14, respectively. Derivative of human CDR ^ plant antibody.
( 19) 抗体の H鎖 V領域の CDR1、 CDR2および CDR3がそれぞれ配列番号 9、 10およ び 11で示されるアミノ酸配列を含み、 L鎖 V領域の CDR1、 CDR2および CDR3がそれぞ れ配列番号 12、 13および 14で示されるアミノ酸配列を含む、 上記 （14) 〜 （1 6) のいずれか 1項に記載のヒト型 CDR^植抗体の誘導体。  (19) CDR1, CDR2, and CDR3 of the H chain V region of the antibody include the amino acid sequences represented by SEQ ID NOs: 9, 10, and 11, respectively, and CDR1, CDR2, and CDR3 of the L chain V region correspond to the SEQ ID NO: The derivative of the human CDR ^ -planted antibody according to any one of the above (14) to (16), comprising the amino acid sequence represented by 12, 13, or 14.
(20) 抗体の H鎖 V領域が配列番号 15で示されるアミノ酸配列を含む上記 （ 1 4) 〜 （16) のいずれか 1項に記載のヒト型 CDR^植抗体の誘導体。  (20) The human CDR ^ -grafted antibody derivative according to any one of the above (14) to (16), wherein the H chain V region of the antibody comprises the amino acid sequence represented by SEQ ID NO: 15.
(21) 抗体の L鎖 V領域が配列番号 16で示されるァミノ酸配列を含む上記 （ 1 4) ~ (16) のいずれか 1項に記載のヒト型 CDR^植抗体の誘導体。  (21) The human CDR ^ -implanted antibody derivative according to any one of the above (14) to (16), wherein the L chain V region of the antibody comprises the amino acid sequence represented by SEQ ID NO: 16.
(22) 抗体の H鎖 V領域が配列番号 15で示されるアミノ酸配列を含み、 L鎖 V領 域が配列番号 16で示されるアミノ酸配列を含む上記 （14) 〜 （16) のいずれ か 1項に記載のヒト型 CDR^植抗体の誘導体。 (22) Any of the above (14) to (16), wherein the H chain V region of the antibody comprises the amino acid sequence represented by SEQ ID NO: 15, and the L chain V region comprises the amino acid sequence represented by SEQ ID NO: 16. Or a derivative of the human CDR ^ -planted antibody according to item 1.
(23) 抗体の H鎖 V領域が配列番号 15で示されるァミノ酸配列を含み、 L鎖 V領 域が配列番号 16で示されるアミノ酸酉己列を含む上記 （14) 〜 （ 16) のいずれ か 1項に記載のヒト型 CDR^植抗体 KM8969の誘導体。  (23) Any of the above (14) to (16), wherein the H chain V region of the antibody contains the amino acid sequence represented by SEQ ID NO: 15, and the L chain V region contains the amino acid sequence represented by SEQ ID NO: 16. Or a derivative of the human CDR ^ plant antibody KM8969 according to item 1.
(24) 抗体断片が、 Fabヽ Fa 'り，、 F(ab，）2、 一本鎖抗体 (scFv) 、 ジスルフィ ド安定化 V領域断片 （dsFv) および CDRを含むペプチドから選ばれる抗体断片であ る上記 （1) 記載の抗体断片の誘導体。 (24) The antibody fragment is an antibody fragment selected from Fab-Fari, F (ab,) 2 , single-chain antibody (scFv), disulfide-stabilized V region fragment (dsFv) and a peptide containing CDR. Certain derivatives of the antibody fragment according to (1) above.
(25) 抗体断片が、ガングリオシド GMに対するハイプリドーマが産生するモ ノクローナル抗体の H鎖 V領域および 1鎖 V領域のアミノ酸配列を含む、 上記 （24 ) 記載の抗体断片の誘導体。  (25) The derivative of the antibody fragment according to (24), wherein the antibody fragment comprises the amino acid sequences of the H chain V region and the 1 chain V region of a monoclonal antibody produced by a hybridoma against ganglioside GM.
(26) 抗体断片が、ガングリオシド GM2に対するハイプリドーマが産生するモ ノクローナル抗体の H鎖 V領域および L鎖 V領域、 ならびにヒト抗体の H鎖 C領域およ ひ 1鎖 C領域のアミノ酸配列を含む、 上記 （24) 記載の抗体断片の誘導体。  (26) The antibody fragment comprises the amino acid sequences of the H chain V region and L chain V region of a monoclonal antibody produced by a hybridoma to ganglioside GM2, and the H chain C region and 1 chain C region of a human antibody. A derivative of the antibody fragment according to (24).
(27) 抗体断片が、抗体の H鎖 V領域が配列番号 7で示されるアミノ酸配列を含 む、 上記 （25) または （26) 記載の抗体断片の誘導体。  (27) The derivative of the antibody fragment according to the above (25) or (26), wherein the antibody fragment comprises the amino acid sequence represented by SEQ ID NO: 7 in the V region of the H chain of the antibody.
(28) 抗体断片が、抗体の L鎖 V領域が配列番号 8で示されるアミノ酸配列を含 む、 上記 （25) または （26) 記載の抗体断片の誘導体。  (28) The derivative of the antibody fragment according to (25) or (26), wherein the antibody fragment comprises an amino acid sequence represented by SEQ ID NO: 8 in the L chain V region of the antibody.
(29) 抗体断片が、抗体の H鎖 V領域が配列番号 7で示されるアミノ酸配列を含 み、 抗体の L鎖 V領域が配列番号 8で示されるアミノ酸配列を含む、 上記（25) ま たは （26) 記載の抗体断片の誘導体。  (29) The method according to (25), wherein the antibody fragment comprises an H chain V region of the antibody comprising the amino acid sequence represented by SEQ ID NO: 7, and an L chain V region of the antibody comprising the amino acid sequence represented by SEQ ID NO: 8. Is a derivative of the antibody fragment according to (26).
(30) 抗体断片が、抗体の H鎖 V領域が配列番号 7で示されるアミノ酸配列を含 み、 抗体の L鎖 V領域が配列番号 8で示されるアミノ酸配列を含む、 上記（25)記 載のハイプリド一マが産生する抗体 KM796の抗体断片の誘導体。  (30) The antibody described in (25) above, wherein the antibody fragment has the H chain V region of the antibody containing the amino acid sequence represented by SEQ ID NO: 7, and the L chain V region of the antibody containing the amino acid sequence represented by SEQ ID NO: 8. A derivative of the antibody fragment of the antibody KM796 produced by the hybridoma.
(31) 抗体断片が、抗体の H鎖 V領域が配列番号 7で示されるアミノ酸配列を含 み、 抗体の L鎖 V領域が配列番号 8で示されるアミノ酸配列を含む、 上記（26)記 載のヒト型キメラ抗体 KM966の抗体断片の誘導体。  (31) The antibody described in (26) above, wherein the antibody fragment has the H chain V region of the antibody containing the amino acid sequence represented by SEQ ID NO: 7, and the L chain V region of the antibody containing the amino acid sequence represented by SEQ ID NO: 8. Derivative of antibody fragment of human chimeric antibody KM966.
(32) 抗体断片が、 ガングリオシド GM2に対するヒト型 CDR^植抗体の H鎖 V領 域および 1鎖 V領域のアミノ酸配列を含む、上記（ 24)記載の抗体断片の誘導体。 (32) The derivative of the antibody fragment according to (24), wherein the antibody fragment comprises the amino acid sequence of the H chain V region and the 1 chain V region of a human CDR ^ -planted antibody against ganglioside GM2.
(33) 抗体断片が、 ガングリオシド GM2に対するヒト型 CDR^植抗体の H鎖 V領 域および 1鎖 V領域、 ならびにヒト抗体の H鎖 C領域および 1鎖 C領域のアミノ酸配列 を含む、 上記 （ 24 ) 記載の抗体断片の誘導体。 (33) The amino acid sequence of the H chain V region and 1 chain V region of the human CDR ^ -planted antibody against ganglioside GM2, and the H chain C region and 1 chain C region of the human antibody A derivative of the antibody fragment according to the above (24), comprising:
(34) 抗体断片が、 抗体の H鎖 V領域の CDR1、 CDR2および CDR3がそれぞれ配列 番号 9、 10および 11で示されるアミノ酸配列を含む、 上記 （32) または （33) 記載の抗体断片の誘導体。  (34) The derivative of the antibody fragment according to (32) or (33), wherein the antibody fragment has CDR1, CDR2, and CDR3 of the V region of the H chain of the antibody including the amino acid sequences represented by SEQ ID NOS: 9, 10, and 11, respectively. .
(35) 抗体断片が、 抗体の L鎖 V領域の CDR1、 CDR2および CDR3がそれぞれ配列 番号 12、 13および 14で示されるアミノ酸配列を含む、 上記 （32) または （33 ) 記載の抗体断片の誘導体。  (35) The derivative of the antibody fragment according to the above (32) or (33), wherein the antibody fragment comprises CDR1, CDR2 and CDR3 of the V region of the L chain of the antibody comprising the amino acid sequences represented by SEQ ID NOS: 12, 13 and 14, respectively. .
(36) 抗体断片が、 抗体の H鎖 V領域の CDR1、 CDR2および CDR3がそれぞれ配列 番号 9、 10および 11で示されるアミノ酸配列を含み、 L鎖 V領域の CDR1、 CDR2および CDR3がそれぞれ配列番号 12、 13および 14で示されるアミノ酸配列を含む、 上記 （ 32) または （33) 記載の抗体断片の誘導体。  (36) The antibody fragment, wherein the CDR1, CDR2, and CDR3 of the H chain V region of the antibody include the amino acid sequences represented by SEQ ID NOs: 9, 10, and 11, respectively, and the CDR1, CDR2, and CDR3 of the L chain V region each have the SEQ ID NO: The derivative of the antibody fragment according to the above (32) or (33), comprising the amino acid sequence represented by 12, 13, or 14.
(37) 抗体断片が、 抗体の H鎖 V領域が配列番号 15で示されるアミノ酸配列を 含む、 上記 （32) または （33) 記載の抗体断片の誘導体。  (37) The derivative of the antibody fragment according to the above (32) or (33), wherein the antibody fragment has the V region of the H chain of the antibody comprising the amino acid sequence represented by SEQ ID NO: 15.
(38) 抗体断片が、 抗体の L鎖 V領域が配列番号 16で示されるアミノ酸配列を 含む、 上記 （32) または （33) 記載の抗体断片の誘導体。  (38) The derivative of the antibody fragment according to (32) or (33), wherein the antibody fragment has the V region of the L chain of the antibody comprising the amino acid sequence represented by SEQ ID NO: 16.
(39) 抗体断片が、 抗体の H鎖 V領域が配列番号 15、 抗体の L鎖 V領域が配列番 号 16で示されるアミノ酸配列を含む、 上記 （32) または （33) 記載の抗体断 片の誘導体。  (39) The antibody fragment according to (32) or (33), wherein the antibody fragment comprises an amino acid sequence represented by SEQ ID NO: 15 in the H chain V region of the antibody and SEQ ID NO: 16 in the L chain V region of the antibody Derivatives.
(40) 抗体断片が、 抗体の H鎖 V領域が配列番号 15で示されるアミノ酸配列を 含み、 抗体の L鎖 V領域が配列番号 16で示されるアミノ酸配列を含む、 上記 （32 ) または （33) 記載のヒト型 CDR^植抗体 KM8969の抗体断片の誘導体。  (40) The antibody fragment according to (32) or (33), wherein the antibody H chain V region comprises the amino acid sequence represented by SEQ ID NO: 15, and the antibody L chain V region comprises the amino acid sequence represented by SEQ ID NO: 16. ) A derivative of the antibody fragment of the human CDR ^ -planted antibody KM8969 described above.
(41) 蛋白質がサイト力インである、 上記 （1) 記載のモノクローナル抗体 の誘導体またはその抗体断片の誘導体。  (41) The derivative of the monoclonal antibody or the derivative of the antibody fragment thereof according to the above (1), wherein the protein is cytodynamic.
(42) サイトカインがヒトインターロイキン 2 (hIL-2) である上記 （41) 記載のモノク口一ナル抗体の誘導体またはその抗体断片の誘導体。  (42) The derivative of the monoclonal antibody or the antibody fragment derivative thereof according to the above (41), wherein the cytokine is human interleukin 2 (hIL-2).
(43) 抗体の H鎖 V領域と ML- 2との融合蛋白質が配列番号 1記載のアミノ酸配 列を有し、 抗体の L鎖 V領域が配列番号 2記載のアミノ酸配列を有する上記 （ 1) 記載の抗体の誘導体。  (43) The above (1), wherein the fusion protein of the H chain V region of the antibody and ML-2 has the amino acid sequence of SEQ ID NO: 1, and the L chain V region of the antibody has the amino acid sequence of SEQ ID NO: 2. A derivative of the described antibody.
(44) 抗体の誘導体が、 ヒト型 CDR^植抗体 KM8969と hIL- 2とからなる上記 （ (44) The above antibody derivative comprising the human CDR ^ -grafted antibody KM8969 and hIL-2
41) 〜 （43) のいずれか 1項に記載の抗体の誘導体。 (45) 抗体の誘導体が、 ヒト型 CD職植抗体 KM8969と hIL-2とからなる上記 （ 41) 〜 （43) のいずれか 1項に記載の抗体の誘導体 KM8969- hIL-2。 41) The derivative of the antibody according to any one of the items (43) to (43). (45) The derivative KM8969-hIL-2 of the antibody according to any one of the above (41) to (43), wherein the derivative of the antibody comprises a humanized CD-specific antibody KM8969 and hIL-2.
(46) 上記（ 1)〜（45)のいずれか 1項に記載のガングリオシド GM2に特 異的に反応するモノクローナル抗体の誘導体またはその抗体断片の誘導体をコ一 ドする DNA。  (46) A DNA encoding a derivative of the monoclonal antibody or a derivative of the antibody fragment thereof that specifically reacts with ganglioside GM2 according to any one of (1) to (45).
(47) 上記 （ 46 ) 記載の DNAを含有する組換えべクタ一。  (47) A recombinant vector containing the DNA according to (46).
(48) 上記 （47) 記載の組換えべクタ一を宿主細胞に導入して得られる形 質転換株。  (48) A transformant obtained by introducing the recombinant vector according to (47) into a host cell.
(49) 上記 （ 45 ) 記載の誘導体を生産する形質転換 OT8969hIL2 (FERM BP- 6792) 。  (49) A transformant OT8969hIL2 (FERM BP-6792) that produces the derivative according to (45) above.
(50) 上記 （ 48 ) または （ 49 ) 記載の形質転騰を培地に培養し、 培養 物中に上記 （1) 〜 (45) のいずれか 1項に記載のモノクローナル抗体の誘導 体またはその抗体断片の誘導体を生成蓄積させ、 該培養物から該抗体の誘導体ま たはその抗体断片の誘導体を採取することを特徴とする製造方法。  (50) The transformant according to (48) or (49) above is cultured in a medium, and the derivative of the monoclonal antibody according to any one of (1) to (45) or the antibody thereof is contained in the culture. A process for producing and accumulating a fragment derivative, and collecting the antibody derivative or the antibody fragment derivative from the culture.
(51) ガングリオシド GM2に特異的に反応する抗体断片。  (51) An antibody fragment that specifically reacts with ganglioside GM2.
(52) 抗体断片が、 Fab、 Fab\ F(ab，）2、 一本鎖抗体 (scFv) 、 ジスルフィ ド安定化 V領域断片 （dsFv) および CDRを含むペプチドから選ばれる抗体断片であ る上記 （51) 記載の抗体断片。 (52) the antibody fragment is an antibody fragment selected from Fab, Fab \ F (ab,) 2 , a single-chain antibody (scFv), a disulfide-stabilized V region fragment (dsFv) and a peptide containing CDR; (51) The antibody fragment according to (1).
(53) 抗体断片が、ガングリオシド GM2に対するハイプリ ドーマが産生するモ ノクロ一ナル抗体の H鎖 V領域および L鎖 V領域のアミノ酸配列を含む、 上記 （51 ) 記載の抗体断片。  (53) The antibody fragment according to (51), wherein the antibody fragment comprises the amino acid sequence of the H chain V region and L chain V region of a monoclonal antibody produced by a hybridoma against ganglioside GM2.
(54) 抗体断片が、ガングリオシド GM2に対するハイプリドーマが産生するモ ノクロ一ナル抗体の H鎖 V領域およひ 1鎖 V領域、 ならびにヒ卜抗体の H鎖 C領域およ ひ 1鎖 C領域のアミノ酸配列を含む、 上記 （51) 記載の抗体断片。  (54) The antibody fragment is derived from the H chain V region and single chain V region of a monoclonal antibody produced by a hybridoma against ganglioside GM2, and the H chain C region and single chain C region of a human antibody. The antibody fragment according to the above (51), comprising an amino acid sequence.
(55) 抗体断片が、抗体の H鎖 V領域が配列番号 7で示されるアミノ酸配列を含 む、 上記 （53) または （54) 記載の抗体断片。  (55) The antibody fragment according to (53) or (54), wherein the antibody H chain V region comprises the amino acid sequence represented by SEQ ID NO: 7.
(56) 抗体断片が、抗体の L鎖 V領域が配列番号 8で示されるァミノ酸配列を含 む、 上記 （53) または （54) 記載の抗体断片。  (56) The antibody fragment according to (53) or (54) above, wherein the L chain V region of the antibody comprises an amino acid sequence represented by SEQ ID NO: 8.
(57) 抗体断片が、抗体の H鎖 V領域が配列番号 7で示されるァミノ酸配列を含 み、 抗体の L鎖 V領域が配列番号 8で示されるアミノ酸配列を含む、 上記（53) ま たは （54) 記載の抗体断片。 (57) The antibody fragment described in (53) or (53) above, wherein the antibody H chain V region contains the amino acid sequence shown in SEQ ID NO: 7, and the antibody L chain V region contains the amino acid sequence shown in SEQ ID NO: 8. Or the antibody fragment of (54).
(58) 抗体断片が、抗体の H鎖 V領域が配列番号 7で示されるアミノ酸配列を含 み、 抗体の L鎖 V領域が配列番号 8で示されるアミノ酸配列を含む、 上記（53)記 載のハイプリドーマが産生する抗体 KM796の抗体断片。  (58) The antibody described in (53) above, wherein the antibody fragment has the H chain V region of the antibody containing the amino acid sequence represented by SEQ ID NO: 7, and the L chain V region of the antibody containing the amino acid sequence represented by SEQ ID NO: 8. An antibody fragment of the antibody KM796 produced by the hybridoma.
(59) 抗体断片が、抗体の H鎖 V領域が配列番号 7で示されるアミノ酸配列を含 み、 抗体の L鎖 V領域が配列番号 8で示されるアミノ酸配列を含む、 上記（53) ま たは （54) 記載のヒト型キメラ抗体 KM966の抗体断片。  (59) The antibody according to (53) or (53), wherein the antibody H-chain V region comprises the amino acid sequence represented by SEQ ID NO: 7, and the antibody L-chain V region comprises the amino acid sequence represented by SEQ ID NO: 8. Is an antibody fragment of the human chimeric antibody KM966 according to (54).
(60) 抗体断片が、 ガングリオシド GM2に対するヒト型 CDR^植抗体の H鎖 V領 域および "L鎖 V領域のアミノ酸配列を含む、 上記 （52) 記載の抗体断片。  (60) The antibody fragment according to the above (52), wherein the antibody fragment comprises the amino acid sequence of the H chain V region and the "L chain V region" of a human CDR ^ -planted antibody against ganglioside GM2.
(61) 抗体断片が、 ガングリオシド GM2に対するヒト型 CDR^植抗体の HilV領 域およひ 1鎖 V領域、 ならびにヒト抗体の H鎖 C領域およひ 1鎖 C領域のァミノ酸配列 を含む、 上記 （ 52 ) 記載の抗体断片。  (61) the antibody fragment comprises the HilV region and the 1-chain V region of the human CDR ^ -grafted antibody against ganglioside GM2, and the amino acid sequence of the H-chain C region and the 1-chain C region of the human antibody; The antibody fragment according to the above (52).
(62) 抗体断片が、 抗体の H鎖 V領域の CDR1、 CDR2および CDR3がそれぞれ配列 番号 9、 10および 11で示されるアミノ酸配列を含む、 上記 （60) または （61) 記載の抗体断片。  (62) The antibody fragment according to the above (60) or (61), wherein CDR1, CDR2, and CDR3 of the V region of the H chain of the antibody include the amino acid sequences represented by SEQ ID NOS: 9, 10, and 11, respectively.
(63) 抗体断片が、 抗体の L鎖 V領域の CDR1、 CDR2および CDR3がそれぞれ配列 番号 12、 13および 14で示されるアミノ酸配列を含む、 上記 （60) または （61 ) 記載の抗体断片。  (63) The antibody fragment according to (60) or (61), wherein CDR1, CDR2, and CDR3 of the V region of the L chain of the antibody include the amino acid sequences represented by SEQ ID NOs: 12, 13, and 14, respectively.
(64) 抗体断片が、 抗体の H鎖 V領域の CDR1、 CDR2および CDR3がそれぞれ配列 番号 9、 10および 11で示されるアミノ酸配列を含み、 L鎖 V領域の CDR1、 CDR2および CDR3がそれぞれ配列番号 12、 13および 14で示されるアミノ酸配列を含む、 上記 （ 60) または （61) 記載の抗体断片。  (64) the antibody fragment, wherein the CDR1, CDR2, and CDR3 of the H chain V region of the antibody include the amino acid sequences represented by SEQ ID NOS: 9, 10, and 11, respectively, and the CDR1, CDR2, and CDR3 of the L chain V region each have the SEQ ID NO: The antibody fragment according to the above (60) or (61), comprising the amino acid sequence represented by 12, 13, or 14.
(65) 抗体断片が、 抗体の H鎖 V領域が配列番号 15で示されるアミノ酸配列を 含む、 上記 （60) または （61) 記載の抗体断片。 .  (65) The antibody fragment according to the above (60) or (61), wherein the antibody H chain V region comprises the amino acid sequence represented by SEQ ID NO: 15. .
(66) 抗体断片が、 抗体の L鎖 V領域が配列番号 16で示されるアミノ酸配列を 含む、 上記 （60) または （61) 記載の抗体断片。  (66) The antibody fragment according to (60) or (61), wherein the L chain V region of the antibody comprises the amino acid sequence represented by SEQ ID NO: 16.
(67) 抗体断片が、 抗体の H鎖 V領域が配列番号 15で示されるアミノ酸配列を 含み、 抗体の L鎖 V領域が配列番号 16で示されるアミノ酸配列を含む、 上記 （60 ) または （61) 記載の抗体断片。  (67) The antibody according to (60) or (61), wherein the antibody H chain V region comprises the amino acid sequence represented by SEQ ID NO: 15, and the antibody L chain V region comprises the amino acid sequence represented by SEQ ID NO: 16. ).
(68) 抗体断片が、 抗体の H鎖 V領域が配列番号 15で示されるアミノ酸配列を 含み、 抗体の L鎖 V領域が配列番号 16で示されるアミノ酸配列を含む、 上記 （60(68) the antibody fragment, wherein the H chain V region of the antibody has the amino acid sequence represented by SEQ ID NO: 15; The above (60) wherein the L chain V region of the antibody comprises the amino acid sequence represented by SEQ ID NO: 16.
) または （61)記載のヒト型 CDR^植抗体 KM8969の抗体断片。 Or the antibody fragment of the human CDR ^ -planted antibody KM8969 according to (61).
(69) 上記 （51)〜 （68)のいずれか 1項に言己載のガングリオシド GM2 に特異的に反応する抗体断片をコードする DNA。  (69) A DNA encoding an antibody fragment specifically reacting with ganglioside GM2 described in any one of the above (51) to (68).
(70) 上記 （ 69 )記載の DNAを含有する組換えべクタ一。  (70) A recombinant vector containing the DNA according to (69).
(71) 上記 （70)記載の組換えべクタ一を宿主細胞に導入して得られる形 質転換株。  (71) A transformed strain obtained by introducing the recombinant vector according to (70) into a host cell.
(72) 上記 （ 71 ) 記載の形質転換株を培地に培養し、 培養物中に上記 （ 5 2)〜 （68)のいずれか 1項に記載の抗体断片を生成蓄積させ、 該培養物から 抗体断片を採取することを特徴とする抗体断片の製造方法。  (72) culturing the transformant according to the above (71) in a medium, producing and accumulating the antibody fragment according to any one of the above (52) to (68) in the culture; A method for producing an antibody fragment, comprising collecting the antibody fragment.
(73)上言己 （1) ~ (45)記載のモノクローナル抗体の誘導体およびその抗 体断片の誘導体、 ならびに上記 （51) 〜 （68)記載の抗体断片から選ばれる 少なくとも 1種からなる医薬。  (73) A medicament comprising the derivative of the monoclonal antibody and the derivative of the antibody fragment thereof according to (1) to (45), and at least one selected from the antibody fragments according to (51) to (68).
(74) 上記 （ 1 ) ~ ( 45 ) 記載のモノクロ一ナル抗体の誘導体およびその 抗体断片の誘導体、 ならびに上記 （51)〜 （68)記載の抗体断片から選ばれ る少なくとも 1種を有効成分として含有する癌の治療薬。  (74) As the active ingredient, at least one selected from the monoclonal antibody derivatives and the antibody fragment derivatives described in the above (1) to (45) and the antibody fragments described in the above (51) to (68). A therapeutic agent for cancer containing.
(75) 上記 （ 1 ) 〜 （ 45 ) 記載のモノク口一ナル抗体の誘導体およびその 抗体断片の誘導 ならびに上記 （51)〜 （68)記載の抗体断片から選ばれ る少なくとも 1種を有効成分として含有する癌の診断薬。  (75) Inducing the derivative of the monoclonal antibody and the antibody fragment thereof described in (1) to (45) above, and at least one selected from the antibody fragments described in (51) to (68) as an active ingredient A diagnostic agent for cancer containing.
本発明は GM2に特異的に反応するモノクローナル抗体またはその抗体断片と、放 射性同位元素、蛋白質または低分子の薬剤とを結合させた抗体の誘導体に関する。 モノクローナル抗体としては、 ハイブリ ド一マが産生する抗体、 ヒト化抗 ヒト抗体などがあげられる。  The present invention relates to a derivative of an antibody in which a monoclonal antibody specifically reacting with GM2 or an antibody fragment thereof is bound to a radioisotope, a protein or a low-molecular-weight drug. Examples of the monoclonal antibody include an antibody produced by a hybridoma, a humanized anti-human antibody, and the like.
ハイプリドーマとは、ヒト以外の哺乳動物に を免疫して取得された B細胞と 、 マウスなどに由来するミエローマ細胞とを細胞融合させて得られる、 所望の抗 原特異性を有したモノクローナル抗体を産生する細胞を意味する。  A hybridoma is a monoclonal antibody having a desired antigen-specificity, which is obtained by cell fusion of B cells obtained by immunizing a mammal other than human with myeloma cells derived from a mouse or the like. Means a producing cell.
ヒト化抗体としては、ヒト型キメラ抗体およびヒト型 CDR^植抗体などがあげら れる。  Humanized antibodies include human chimeric antibodies and human CDR ^ -planted antibodies.
ヒト型キメラ抗体は、 ヒト以外の動物の抗体 H鎖 V領域 （以下、 HVまたは VHと表 記する）および抗体 L鎖 V領域（以下、 LVまたは VLと表記する） とヒト抗体の H鎖 C 領域 （以下、 CHと表記する） およびヒト抗体の L鎖 C領域 （以下、 CLと表記する） とからなる抗体を意味する。 ヒト以外の動物としては、 マウス、 ラット、 ハムス 夕一、 ラビット等、 ハイプリドーマを作製することが可能であれば、 いかなるも のも用いることができる。 The human chimeric antibody is composed of a non-human animal H chain V region (hereinafter referred to as HV or VH) and an antibody L chain V region (hereinafter referred to as LV or VL) and a human antibody H chain C Region (hereinafter referred to as CH) and an L chain C region (hereinafter referred to as CL) of a human antibody. As animals other than humans, any mouse, rat, hamus, and rabbit can be used as long as they can produce hybridomas.
本発明のヒト型キメラ抗体は、 GM2に特異的に反応するモノクローナル抗体を生 産するハイブリド一マより、 VHおよび "VLをコードする cDNAを取得し、 ヒト抗体 CH およびヒト抗体 CLをコ一ドする遺伝子を有する動物細胞用発現ベクターにそれぞ れ挿入してヒト型キメラ抗体発現ベクターを構築し、 該発現ベクターを動物細胞 へ導入することにより発現させ、 作製することができる。  The human chimeric antibody of the present invention obtains cDNAs encoding VH and "VL" from a hybridoma producing a monoclonal antibody that specifically reacts with GM2, and encodes human antibody CH and human antibody CL. Thus, a human-type chimeric antibody expression vector can be constructed by inserting each into an expression vector for animal cells having the gene to be expressed, and the expression can be produced by introducing the expression vector into animal cells.
ヒト型キメラ抗体の CHとしては、 ヒトイムノグロブリン （以下、 hlgと表記す る） に属すればいかなるものでもよいが、 hlgGクラスのものが好適であり、 更に hlgGクラスに属する hIgGl、 hIgG2、 hIgG3、 hIgG4といったサブクラスのいずれも 用いることができる。 また、 ヒト型キメラ抗体の CLとしては、 hlgに属すればいか なるものでもよく、 クラスあるいは入クラスのものを用いることができる。 抗 GM2キメラ抗体の具体例としては、 特開平 6- 205694に記載の KM966があげられ る。  As the CH of the human chimeric antibody, any CH may be used as long as it belongs to human immunoglobulin (hereinafter, referred to as hlg), but those of the hlgG class are preferable, and hIgGl, hIgG2, hIgG3 belonging to the hlgG class are preferable. , HIgG4 can be used. The CL of the human chimeric antibody may be any CL as long as it belongs to hlg, and a class or in-class CL can be used. Specific examples of the anti-GM2 chimeric antibody include KM966 described in JP-A-6-205694.
ヒ卜型 CDR 植抗体は、 ヒト以外の動物の抗体の VHおよひ 7Lの CDRのアミノ酸配 列をヒト抗体の VHおよび VLの適切な位置に移植した抗体を意味する。  The human CDR-grafted antibody means an antibody obtained by grafting the amino acid sequences of VH and 7L CDR of an antibody of a non-human animal to appropriate positions of VH and VL of a human antibody.
本発明の抗 G 2CD ^植抗体は、 GM2に特異的に反応するヒト以外の動物の抗体の VHおよび VLの CDR配列を任意のヒト抗体の VHおよび VLの CDR配列に移植した V領域 をコードする cDNAを構築し、 ヒト抗体の CHおよびヒト抗体の CLをコ一ドする遺伝 子を有する動物細胞用発現べクタ一にそれぞれ挿入してヒト型 CDR^植抗体発現 ベクタ一を構築し、 該発現べクタ一を動物細胞へ導入することによりヒト型 CDR 移植抗体を発現させ、 作製することができる。  The anti-G2CD ^ -planted antibody of the present invention encodes a V region obtained by grafting the VH and VL CDR sequences of a non-human animal antibody specifically reacting with GM2 into the VH and VL CDR sequences of any human antibody. And constructing a human CDR ^ plant antibody expression vector by inserting the cDNA into an expression vector for animal cells having genes encoding human antibody CH and human antibody CL, respectively. A human CDR-grafted antibody can be expressed and produced by introducing the expression vector into animal cells.
本発明のヒト型 CDR^植抗体の CHとしては、 hlgに属すればいかなるものでもよ いが、 hlgGクラスのものが好適であり、 更に hlgGクラスに属する hIgGl、 hIgG2、 hIgG3、 hIgG4といったサブクラスのいずれも用いることができる。 また、 該ヒト 型 CDR^植抗体の CLとしては、 hlgに属すればいかなるものでもよく、 クラスあ るいは人クラスのものを用いることができる。  As the CH of the human CDR ^ -grafted antibody of the present invention, any CH may be used as long as it belongs to hlg, but the hlgG class is preferable, and further, subclasses such as hIgGl, hIgG2, hIgG3, and hIgG4 belonging to the hlgG class. Either can be used. In addition, the CL of the human CDR ^ -planted antibody may be any CL as long as it belongs to hlg, and a class or human class CL can be used.
抗 GM2CDR^植抗体の具体例としては、特開平 10-257893に記載の KM8969があげら れる。 Specific examples of anti-GM2CDR ^ plant antibodies include KM8969 described in JP-A-10-257893. It is.
ヒト抗体は、 元来、 ヒト体内に天然に存在する抗体を意味するが、 最近の遺伝 子工学的、 細胞工学的、 発生工学的な技術の進歩により作製されたヒト抗体ファ ージライブラリ一およびヒト抗体産生トランスジエニック動物から得られる抗体 等も含まれる。  Human antibodies originally mean antibodies naturally occurring in the human body, but human antibody phage libraries and human antibodies created by recent advances in genetic engineering, cell engineering, and developmental engineering technology Antibodies obtained from the producing transgenic animal are also included.
ヒト体内に存在する抗体は、 例えば、 ヒト末梢血リンパ球を単離し、 EBウィル ス等を感染させ不死化、 クローニングすることにより、 該抗体を産生するリンパ 球を培養でき、 培養物中より該抗体を精製することができる。  Antibodies present in the human body can be obtained, for example, by isolating human peripheral blood lymphocytes, infecting EB virus and the like, immortalizing them, and cloning the cells to produce the antibody-producing lymphocytes. Antibodies can be purified.
ヒト抗体ファージライブラリーは、ヒト B細胞から調製した抗体遺伝子をファー ジ遺伝子に挿入することにより Fab、 scFv等の抗体断片をファ一ジ表面に発現させ たライブラリーである。 該ライブラリ一より、 ί¾ ^を固定ィ匕した基質に対する結 合活性を指標として所望の fc"源結合活性を有する抗体断片を発現しているファー ジを回収することができる。該抗体断片は、 更に遺伝子工学的手法により、 2本の 完全な H鎖および 2本の完全な L鎖からなるヒト抗体分子へも変換することができ る。  The human antibody phage library is a library in which antibody fragments such as Fab and scFv are expressed on the phage surface by inserting an antibody gene prepared from human B cells into the phage gene. A phage expressing an antibody fragment having a desired fc "source binding activity can be recovered from the library by using the binding activity to the substrate on which ί¾ ^ is immobilized as an indicator. Furthermore, it can be converted into a human antibody molecule consisting of two complete H chains and two complete L chains by genetic engineering techniques.
ヒト抗体産生トランスジエニック動物は、 ヒト抗体遺伝子が細胞内に糸1&まれ た動物を意味する。 具体的には、 マウス ES細胞ヘヒト抗体遺伝子を導入し、 該 ES 細胞を他のマウスの初期胚へ移植後、 発生させることによりヒト抗体産生トラン スジヱニック動物を作製することができる。 ヒト抗体産生トランスジエニック動 物からのヒト抗体の作製方法は、 通常のヒト以外の哺乳動物で行われているハイ プリドーマ作製方法によりヒト抗体産生ハイプリドーマを得、 培養することで培 養物中にヒト抗体を産生蓄積させることができる。  A human antibody-producing transgenic animal refers to an animal in which the human antibody gene is inserted into cells. Specifically, a human antibody-producing transgenic animal can be produced by introducing a human antibody gene into mouse ES cells, transplanting the ES cells into an early embryo of another mouse, and then developing the embryo. Human antibodies can be produced from human antibody-producing transgenic animals by preparing human antibody-producing hybridomas by the usual method for producing hybridomas used in mammals other than humans, and culturing them in culture. Can produce and accumulate human antibodies.
抗体断片としては、 Fab、 Fab\ F(ab，）2、 scFv、 dsFv、 CDRを含むペプチドなど があげられる。 Examples of the antibody fragment include Fab, Fab \ F (ab,) 2 , scFv, dsFv, a peptide containing CDR, and the like.
Fabは、 I g Gを蛋白質分解酵素パパインで処理して得られる断片のうち （H^ の 224番目のアミノ酸残基で切断される） 、 H鎖の N末端側約半分と L鎖全体がジ スルフィ ド結合で結合した分子量約 5万のお源結合活性を有する抗体断片である。 本発明の Fabは、 GM2に特異的に反応する抗体を蛋白質分解酵素パパィンで処理 して得ることができる。 または、 該抗体の Fabをコードする DNAを原核生物用発現 ベクタ一あるいは ¾ 生物用発現べク夕一に挿入し、 該べク夕一を原核生物ある レヽは真核生物へ導入することにより発現させ、 Fabを作製することができる。Fab is a fragment obtained by treating IgG with proteolytic enzyme papain (which is cleaved at the 224th amino acid residue of H ^). About half of the N-terminal side of the H chain and the entire L chain are dimerized. It is an antibody fragment having a molecular weight of about 50,000 and a source binding activity linked by a sulfide bond. The Fab of the present invention can be obtained by treating an antibody specifically reacting with GM2 with proteolytic enzyme papain. Alternatively, DNA encoding the Fab of the antibody is inserted into a prokaryotic expression vector or a ¾ biological expression vector, and the vector is a prokaryote. The protein can be expressed by introducing it into eukaryotes to produce Fab.
F(ab' )2は、 I g Gを蛋白質分解酵素ペプシンで処理して得られる断片のうち （ H鎖の 234番目のアミノ酸残基で切断される）、 Fabがヒンジ領域のジスルフイ ド結 合を介して結合されたものよりやや大きい、 分子量約 10万の抗原結合活性を有す る抗体断片である。 F (ab ') 2 is a fragment obtained by treating IgG with the protease pepsin (which is cleaved at the 234th amino acid residue in the H chain), and Fab is a disulfide bond in the hinge region. This is an antibody fragment having a molecular weight of about 100,000 and having an antigen-binding activity, which is slightly larger than that bound through the DNA.
本発明の F(ab' )2は、 GM2に特異的に反応する抗体を蛋白質分解酵素ペプシンで 処理して得ることができる。 または、 下記の Fab'をチォエーテル結合あるいはジ スルフィ ド結合させ、 作製することができる。 The F (ab ') 2 of the present invention can be obtained by treating an antibody that specifically reacts with GM2 with the protease pepsin. Alternatively, it can be prepared by making the following Fab 'a thioether bond or a disulfide bond.
Fab'は、 上言 HF(ab' )2のヒンジ領域のジスルフィ ド結合を切断した分子量約 5万 の 源結合活性を有する抗体断片である。 Fab 'is an antibody fragment having a molecular weight of about 50,000 and a source binding activity in which a disulfide bond in the hinge region of HF (ab') 2 is cleaved.
本発明の Fab，は、 GM2に特異的に反応する F(ab，）2を還元剤ジチオスレィ トール 処理して得ることができる。または、該抗体の Fab'断片をコードする DNAを原核生 物用発現べクタ一あるいは真核生物用発現べクタ一に挿入し、 該ベクターを原核 生物ある ヽは難生物へ導入することにより Fab'を発現させ、 作製することがで ぎる。 The Fab of the present invention can be obtained by treating F (ab,) 2 that specifically reacts with GM2 with a reducing agent dithiothreitol. Alternatively, DNA encoding the Fab ′ fragment of the antibody is inserted into a prokaryotic expression vector or a eukaryotic expression vector, and the vector is introduced into a prokaryotic or difficult organism to produce a Fab. 'Can be expressed and produced.
scFvは、 一本の VHと一本の VLとを適当なぺプチドリンカー（以下、 Pと表記する ) を用いて連結した、 VH— P— VLないしは VL— P— VHポリペプチドを示す。 本発明 で使用される scFvに含まれる VHおよび 7Lは、 ハイプリドーマが産生する抗体、 ヒ ト化抗体、 ヒト抗体のいずれをも用いることができる。  scFv refers to a VH-P-VL or VL-P-VH polypeptide in which one VH and one VL are linked using an appropriate peptide linker (hereinafter, referred to as P). As VH and 7L contained in the scFv used in the present invention, any of antibodies produced by hybridomas, humanized antibodies, and human antibodies can be used.
本発明の scFvは、 GM2に特異的に反応する抗体の VHおよび VLをコ一ドする cDNA を取得し、 scFvをコードする DNAを構築し、 該 DNAを原核生物用発現べクタ一ある いは真核生物用発現べク夕一に挿入し、 該発現べク夕一を原核生物ある ヽは真核 生物へ導入することにより scFvを発現させ、 作製することができる。  The scFv of the present invention obtains cDNA encoding VH and VL of an antibody that specifically reacts with GM2, constructs a DNA encoding the scFv, and uses the DNA for prokaryotic expression vector or The scFv can be expressed and produced by inserting it into a prokaryotic expression vector by inserting it into a eukaryotic expression vector and introducing the expression vector into a eukaryote.
dsFvは、 VHおよび VL中のそれぞれ 1アミノ酸残基をシスティン残基に置換した ポリべプチドを該システィン残基間のジスルフィ ド結合を介して結合させたもの をいう。 システィン残基に置換するアミノ酸残基は Reiterらにより示された方法 dsFv refers to a polypeptide in which one amino acid residue in each of VH and VL has been substituted with a cysteine residue, which is linked via a disulfide bond between the cysteine residues. The amino acid residue to be substituted for the cysteine residue was determined by the method described by Reiter et al.
[プロテイン.エンジニアリング (Protein Engineering), 7, 697 ( 1994)] に従つ て、 抗体の立体構造予測に基づいて選択することができる。 本発明の dsFvに含ま れる VHおよび VLはハイブリド一マが産生する抗 ヒト化抗体、 ヒト抗体のいず れをも用いることができる。 本発明の dsFvは、 GM2に特異的に反応する抗体の VHおよび VLをコードする cDNA を取得し、 dsFvをコードする DNAを構築し、 該 DNAを原核生物用発現べクタ一ある いは真核生物用発現ベクターに挿入し、 該発現べクタ一を原核生物あるいは難 生物へ導入することにより発現させ、 dsFvを作製することができる。 According to [Protein Engineering, 7, 697 (1994)], the antibody can be selected based on the prediction of the three-dimensional structure of the antibody. As the VH and VL contained in the dsFv of the present invention, any of an anti-humanized antibody and a human antibody produced by a hybridoma can be used. The dsFv of the present invention is obtained by obtaining cDNAs encoding VH and VL of an antibody specifically reacting with GM2, constructing a DNA encoding the dsFv, and using the DNA for prokaryotic expression vector or eukaryote. DsFv can be produced by inserting the expression vector into a biological expression vector and introducing the expression vector into a prokaryote or a difficult organism to express.
CDRを含むぺプチドは、 H鎖または L鎖 CDRの少なくとも 1領域以上を含んで構成 される。複数の CDRは、直接または適当なベプチドリンカ一を介して結合させるこ とができる。  The peptide containing the CDR comprises at least one region of the H chain or L chain CDR. A plurality of CDRs can be linked directly or via an appropriate peptide linker.
本発明の CDRを含むぺプチドは、 GM2に特異的に反応する抗体の VHおよび VLをコ 一ドする cDNAを取得した後、 該 cDNAを原核生物用発現べクタ一あるいは真核生物 用発現べク夕一に挿入し、 該発現べクターを原核生物あるいは真核生物へ導入す ることにより発現させ、 CDRを含むぺプチドを作製することができる。  The peptide containing the CDR of the present invention can be obtained by obtaining a cDNA encoding VH and VL of an antibody specifically reacting with GM2, and then using the cDNA for expression in a prokaryotic expression vector or eukaryotic expression vector. The expression vector can be inserted into a prokaryote or eukaryote for expression to produce a peptide containing a CDR.
また、 CDRを含むペプチドは、 Fmoc法（フルォレニルメチルォキシカルボニル法 ) 、 tBoc法 （t-ブチルォキシカルボニル法） 等の化学合成法によって製造するこ ともできる。  The peptide containing CDR can also be produced by a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method), the tBoc method (t-butyloxycarbonyl method), and the like.
本発明の抗体の誘導体は、上述した GM2に特異的に反応する抗体または抗体断片 の H鎖或いは L鎖の N末端側或いは C末端側、 抗体または抗体断片中の適当な置換基 あるいは側鎖、さらには抗体または抗体断片中の糖鎖に腿性同位元素、蛋白質、 低分子の薬剤などを化学的手法 [抗体工学入門 （金光修著 1 9 9 4年 （株） 地人書館） ] により結合させることにより作製することができる。  The derivative of the antibody of the present invention may be a derivative of the antibody or antibody fragment which specifically reacts with GM2, N-terminal or C-terminal of the H chain or L chain, an appropriate substituent or side chain in the antibody or antibody fragment, In addition, thigh isotopes, proteins, and low-molecular-weight drugs are linked to the sugar chains in the antibody or antibody fragment by a chemical method [Introduction to Antibody Engineering (Osamu Kanemitsu, 1994; Jinjinshokan Co., Ltd.)] By doing so, it can be produced.
また本発明の抗体の誘導体は、 GM2に特異的に反応する抗体または抗体断片をコ ードする DNAと、 結合させたい蛋白質をコードする DNAを連結させて発現べクタ一 に挿入し、 該発現べクターを宿主細胞へ導入する遺伝子工学的手法によっても作 製することができる。  In addition, the derivative of the antibody of the present invention is obtained by ligating a DNA encoding an antibody or an antibody fragment specifically reacting with GM2 with a DNA encoding a protein to be bound and inserting the DNA into an expression vector. It can also be produced by a genetic engineering technique for introducing a vector into a host cell.
¾Ιί性同位元素としては、 1311、 1251等があげられ、 例えば、 クロラミン Τ法等に より、 抗体に結合させることができる。 The ¾Ιί isotope, 131 1, 125 1, and the like, for example, more chloramine Τ method, can be attached to the antibody.
低分子の薬剤としては、 ナイ トロジェン 'マスタード、 サイクロフォスフアミ ドなどのアルキル化剤、 5—フルォロウラシル、 メソトレキセ一トなどの代 抗剤、 ダウノマイシン、 ブレオマイシン、 マイ トマイシン C， ダウノルビシン、 ドキソルビシンなどの抗生物質、 ビンクリスチン、 ビンブラスチン、 ビンデシン のような植物アルカロイド、 夕モキシフェン、 デキサメタソンなどのホルモン剤 等の抗癌剤 [臨床腫瘍学 （日本臨床腫瘍研究会編 1 9 9 6年 癌と化学療法社 ) ] 、 またはハイドロコ一チゾン、 プレドニゾンなどのステロイ ド剤、 ァスピリ ン、 インドメタシンなどの非ステロイ ド剤、 金チォマレート、 ぺニシラミンなど の免疫調節剤、 サイクロフォスフアミ ド、 ァザチォプリンなどの免疫抑制剤、 マ レイン酸クロルフエ二ラミン、 クレマシチンのような抗ヒス夕ミン剤等の抗炎症 剤 [炎症と抗炎症療法 昭和 5 7年 医歯薬出版株式会社] などがあげられる。 例えば、 ダウノマイシンと抗体を結合させる方法としては、 グル夕一ルアルデヒ ドを介してダウノマイシンと抗体のアミノ基間を結合させる方法、 水溶性カルボ ジィミドを介してダウノマイシンのァミノ基と抗体のカルボキシル基を結合させ る方法等があげられる。 Examples of low molecular drugs include alkylating agents such as Nitrogen 'mustard and cyclophosphamide, antagonists such as 5-fluorouracil and methotrexate, and antibiotics such as daunomycin, bleomycin, mitomycin C, daunorubicin, and doxorubicin. Substances, plant alkaloids such as vincristine, vinblastine, vindesine, hormonal agents such as evening moxifen, dexamethasone [Clinical Oncology (Japanese Society for Clinical Oncology, 1996, Cancer and Chemotherapy)], or steroids such as hydrocotisone and prednisone, non-steroids such as aspirin and indomethacin; Immunomodulators such as gold thiomalate and penicillamine, immunosuppressants such as cyclophosphamide and azathioprine, and anti-inflammatory agents such as antihistamines such as chlorpheniramine maleate and clemacitin [Inflammation and anti-inflammatory Therapy 1977, Itoyaku Shuppan Publishing Co., Ltd.]. For example, as a method for binding daunomycin to an antibody, a method for binding between daunomycin and the amino group of the antibody via gludealdehyde, and a method for binding the amino group of daunomycin to the carboxyl group of the antibody via water-soluble carbodiimide And the like.
蛋白質としては、 免疫担当細胞を活性化するサイ トカインが好適であり、 例え ば、 hIL-2、 hGM-CSF, ヒトマクロファージコロニー剌激因子 （以下、 hM- CSFと表 記する） 、 ヒトイン夕一ロイキン 1 2 (以下、 hIL-12と表記する） 等があげられ る。 また、 癌細胞を直接障害するため、 リシンやジフテリア毒素などの毒素を用 いることができる。 例えば、 蛋白質との融合抗体ついては、 抗体または抗体断片 をコ一ドする cDNAに蛋白質をコードする cDNAを連結させ、 融合抗体をコ一ドする DNAを構築し、 該 DNAを原核生物あるいは真核生物用発現ベクターに挿入し、 該発 現べクタ一を原核生物あるレヽは真核生物へ導入することにより発現させ、 融合抗 体を作製することができる。  Suitable proteins include cytokines that activate immunocompetent cells, such as hIL-2, hGM-CSF, human macrophage colony stimulating factor (hereinafter abbreviated as hM-CSF), Leukin 12 (hereinafter referred to as hIL-12) and the like. Also, toxins such as ricin and diphtheria toxin can be used to directly damage cancer cells. For example, for a fusion antibody with a protein, a cDNA encoding the protein is linked to a cDNA encoding the antibody or antibody fragment, a DNA encoding the fusion antibody is constructed, and the DNA is converted to a prokaryotic or eukaryotic organism. The expression vector is inserted into an expression vector, and the expression vector is introduced into a prokaryotic cell by introducing it into a eukaryote, whereby a fusion antibody can be prepared.
抗 GM2ヒト化抗体の誘導体の具体例としては、抗体の H鎖と hIL- 2の融合蛋白質が 配列番号 1記載のアミノ酸配列を有し、 抗体の L鎖 V領域が配列番号 2記載のァミノ 酸配列を有する、 抗 GM2CDR^植抗体 KM8969と hIL- 2との融合蛋白質である KM8969 一 ML-2があげられる。  Specific examples of the derivative of the humanized anti-GM2 antibody include a fusion protein of the H chain and hIL-2 of the antibody having the amino acid sequence of SEQ ID NO: 1, and the V region of the L chain of the antibody having the amino acid sequence of SEQ ID NO: 2. KM8969-ML-2, which is a fusion protein of the anti-GM2CDR ^ -planted antibody KM8969 and hIL-2 having the sequence.
以下に、 GM2に特異的に反応するヒト化抗体の作製方法、 GM2に特異的に反応す る抗体断片の作製方法および GM2に特異的に反応する抗体の誘導体の作製方法に ついて説明する。  Hereinafter, a method for producing a humanized antibody specifically reacting with GM2, a method for producing an antibody fragment specifically reacting with GM2, and a method for producing a derivative of an antibody specifically reacting with GM2 will be described.
1 . ヒト化抗体の作製  1. Preparation of humanized antibody
( 1 ) ヒト化抗体発現用ベクターの構築  (1) Construction of humanized antibody expression vector
ヒト化抗体発現用ベクターとは、 ヒト抗体の CHおよび CLをコードする遺伝子が 組み込まれた動物細胞用発現ベクターであり、 動物細胞用発現べクタ一にヒト抗 体の CHおよび CLをコ一ドする遺伝子をそれぞれクローニングすることにより構築 することができる。ヒト抗体の C領域は任意のヒト抗体の CHおよび CLであることが でき、 例えば、 ヒ卜抗体の H鎖の IgGlサブクラスの C領域（以下、 hCァ 1と表記する ) およびヒト抗体の L鎖の クラスの C領域 （以下、 hC/cと表記する） 等があげら れる。 ヒト抗体の CHおよび CLをコードする遺伝子としてはェキソンとイントロン から成る染色体 DNAを用いることができ、 また、 cDNAを用いることもできる。動物 細胞用発現べクタ一としては、ヒト抗体の C領域をコードする遺伝子を細 λみ発現 できるものであればいかなるものでも用いることができる。 例えば、 pAGE107 [サ ィトテクノロジー (Cytotechnology), 3, 133 (1990)]、 pAGE103 [ジャーナル 'ォ ブ ·バイオケミストリー (J. Biochem. ) , 101, 1307 ( 1987)]、 pHSG274 [ジーン (Gene), 27, 223 ( 1984)]、 pKCR [プロシ一ディングス ·ォブ ·ザ ·ナショナル · アカデミー.ォブ ·サイエンス（Proc. Natl. Acad. Sci. U. S.A. ), 78, 1527 ( 1981 )] 、 pSGl ?d2-4 [サイ トテクノロジ一 (Cytotechnology), 4, 173 ( 1990)]等があげら れる。 動物細胞用発現べク夕一に用いるプロモ一夕一とェンハンサーとしては、 SV40の初期プロモー夕一とェンハンサ一 [ジャーナル'ォブ'バイオケミストリ一 (J. Biochem. ), 101, 1307 ( 1987)]、 モロニ一マウス白血病ウィルスの LTRプロモ 一夕一とェンハンサ一 [バイオケミカル 'アンド ·バイオフィジカル'リサーチ · コミュニケーションズ (Biochem. Biophys. Res. Co腿 un. )， 149, 960 ( 1987)]、 免疫グロブリン H鎖のプロモーター [セル (Cell ), 41, 479 ( 1985)]とェンハンサ 一 [セル (Cell )， ， 717 ( 1983)]等があげられる。 A humanized antibody expression vector is an expression vector for animal cells into which genes encoding human antibody CH and CL have been incorporated. It can be constructed by cloning genes coding for body CH and CL, respectively. The C region of a human antibody can be CH and CL of any human antibody. For example, the C region of the IgGl subclass of the H chain of a human antibody (hereinafter referred to as hCα1) and the L chain of a human antibody Class C region (hereinafter referred to as hC / c). As the genes encoding CH and CL of the human antibody, chromosomal DNA consisting of exons and introns can be used, and cDNA can also be used. As the expression vector for animal cells, any expression vector can be used as long as it can express the gene encoding the C region of the human antibody. For example, pAGE107 [Cytotechnology, 3, 133 (1990)], pAGE103 [Journal of Biochemistry (J. Biochem.), 101, 1307 (1987)], pHSG274 [Gene , 27, 223 (1984)], pKCR [Procedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 78, 1527 (1981)], pSGl? d2-4 [Cytotechnology, 4, 173 (1990)]. Promoters and enhancers used for expression vectors for animal cells include the early promoters and enhancers of SV40 [Journal of Ob 'Biochem. (J. Biochem.), 101, 1307 (1987) Moroni monoleukemia virus LTR promoter Ichiyuichi and Enhansa I [Biochemical 'and Biophysical' Research Communications (Biochem. Biophys. Res. Co. thigh un.), 149, 960 (1987)], Immunization Examples include the globulin H chain promoter [Cell, 41, 479 (1985)] and Enhansa I [Cell, 717 (1983)].
ヒト化抗体発現用べクタ一は、 抗体 H鎖および 1鎖が別々のベクター上に存在す るタイプ或いは同一のベクター上に存在するタイプ （以下、 タンデム型と表記す る） のどちらでも用いることができるが、 ヒト化抗体発現ベクターの構築の容易 さ、 動物細胞への導入の容易さ、 動物細胞内での抗体 H鎖および 1鎖の発現量のバ ランスが均衡する等の点からタンデム型のヒト化抗体発現用べク夕一の方が好ま しい [ジャーナル'ォブ'ィムノロジカル'メソヅズ (J. Immunol. Methods), 167, 271 ( 1994)]。  The vector for expression of humanized antibody may be of the type in which the antibody H chain and 1 chain are present on separate vectors or of the type present on the same vector (hereinafter referred to as tandem type). Tandem type because of the ease of construction of a humanized antibody expression vector, ease of introduction into animal cells, and balanced expression of antibody H chain and single chain in animal cells. For expression of a humanized antibody, one is more preferred [Journal of Immunological Methods], J. Immunol. Methods, 167, 271 (1994).
構築したヒト化抗体発現用べクタ一は、ヒト型キメラ抗体およびヒト型 CDR^植 抗体の動物細胞での発現に使用することができる。  The constructed humanized antibody expression vector can be used for expression of a human chimeric antibody and a human CDR ^ -planted antibody in animal cells.
( 2 ) ヒト以外の動物の抗体の V領域をコードする cDNAの取得 ヒト以外の動物の抗体、 例えば、 マウス抗体の VHおよび TLをコードする cDNAは 以下の様にして取得することができる。 (2) Acquisition of cDNA encoding V region of non-human animal antibody CDNAs encoding non-human animal antibodies, for example, VH and TL of mouse antibodies, can be obtained as follows.
マウス抗体を産生するハイプリ ドーマ細胞より mRNAを抽出し、 cDNAを合成する。 合成した cDNAをファージ或いはプラスミド等のベクターにクロ一ニングして cDNA ライブラリ一を作製する。 該ライブラリ一より、 マウス抗体の C領域部分或いは V 領域部分をプローブとして用い、 VHをコードする cDNAを有する組換えファージ或 Vヽは組換えプラスミドおよび VLをコードする cDNAを有する組換えファージ或いは 組換えプラスミドをそれぞれ単離する。 組換えファージ或いは組換えプラスミ ド 上の目的とするマウス抗体の VHおよび Lの全塩基配列を決定し、 塩基配列より VH およひ 7Lの全アミノ酸配列を推定する。  MRNA is extracted from hybridoma cells producing mouse antibodies, and cDNA is synthesized. The synthesized cDNA is cloned into a vector such as a phage or a plasmid to prepare a cDNA library. From the library, a recombinant phage having a cDNA encoding VH or a recombinant phage or a recombinant phage having a cDNA encoding VL using the C region or V region of a mouse antibody as a probe. Each of the replacement plasmids is isolated. The entire nucleotide sequence of VH and L of the target mouse antibody on the recombinant phage or recombinant plasmid is determined, and the entire amino acid sequence of VH and 7L is estimated from the nucleotide sequence.
ヒト以外の動物としては、 マウス、 ラット、 ハムスター、 ラビット等、 ハイブ リドーマ細胞を作製することが可能であれば、 いかなるものも用いることができ る。  As animals other than humans, any animal such as mouse, rat, hamster, and rabbit can be used as long as hybridoma cells can be produced.
ハイプリ ドーマ細胞から全 RNAを調製する方法としては、チォシアン酸グァニジ ンートリフルォロ酢酸セシウム法 [メソヅズ 'イン'ェンザィモロジ一 (Methods in Enzymol. ), 154, 3 ( 1987)]、 また全 RNAから mE を調製する方法としては、 オリ ゴ (dT)固定ィ匕セルロースカラム法 [モレキュラー 'クローニング：ァ 'ラボラトリ —— -マ二ユアノレ (Molecular Cloning: A Laboratory Manual ) , Cold Spring Harbor Lab. Press New York, 1989、 以下、 モレキュラー ·クローニング：ァ ·ラボラト リー ·マニュアルと表記する]等があげられる。 また、 ハイプリ ドーマ細胞から mRNAを調製するキヅトとしては、 Fast Track mRNA Isolation Kit (Invitrogen 社製） 、 Quick Prep mB Purification Kit (Pharmacia社製） 等があげられる。 cDNAの合成および cDNAライブラリー作製法としては、常法 [モレキュラー 'クロ 一二ング：ァ ·ラボラトリー 'マニュアル；カレント 'プロトコ一ルズ 'イン ' モレキュラー ·ノ ィォロジ一(Current Protocols in Molecular Biology), Supplement 1-34, 以下、 カレント 'プロトコ一ルズ 'イン 'モレキュラー 'バイ ォロジ一と表記する]、 或いは市販のキット、 例えば、 Super Script™ Plasmid System for cDNA Synthesis and Plasmid Cloning (GIBCO BRL社製） や ZAP-cDNA Synthesis Kit (Stratagene社製） を用いる方法等があげられる。  Methods for preparing total RNA from hybridoma cells include guanidine thiocyanate-cesium trifluoroacetate method [Methods in Enzymol., 154, 3 (1987)], and mE from total RNA. The method is as follows: Oligo (dT) immobilized cellulose column method [Molecular Cloning: A Laboratory Manual], Cold Spring Harbor Lab. Press New York, 1989, below. , Molecular cloning: a, laboratory, manual, etc.]. Examples of kits for preparing mRNA from hybridoma cells include Fast Track mRNA Isolation Kit (manufactured by Invitrogen), Quick Prep mB Purification Kit (manufactured by Pharmacia), and the like. Methods for cDNA synthesis and cDNA library preparation are routine methods [Molecular 'Clothing: A Laboratory' Manual; Current 'Protocols' in' Molecular Protocols in Molecular Biology, Supplement 1-34, hereinafter referred to as current 'protocols' in 'molecular' biologic) or a commercially available kit such as Super Script ™ Plasmid System for cDNA Synthesis and Plasmid Cloning (GIBCO BRL) or ZAP -A method using a cDNA Synthesis Kit (Stratagene) can be used.
cDNAライブラリ一の作製の際、 ハイプリドーマ細胞から抽出した mRNAを錄型と して合成した cDNAを組み込むべク夕一は、 該 cDNAを組み込めるベクターであれば いかなるものでも用いることができる。 例えば、 ZAP Express [ストラテジーズ (Strategies), 5, 58 ( 1992)]、 pBluescript I I SK( + ) [ヌクレイック 'ァシッズ . リサーチ (Nucleic Acids Research), 17, 9494 ( 1989)]、 Azap I I (Stratagene 社製）、 人 gtlO、 Agtll [DNA クローニング：ァ 'プラクティカル'アプローチ (DNA Cloning: A Practical Approach), I， 49 ( 1985)]、 Lambda BlueMid (Clontech 社製） 、 人 ExCell、 pT7T3 18U (Pharmacia社製） 、 pcD2 [モレキュラー 'アンド ' セルラー 'バイオロジー (Mol. Cell. Biol. ) , 3, 280 ( 1983)]および pUC18 [ジ一 ン（Gene) , 33, 103 ( 1985)]等が用いられる。 When preparing a cDNA library, mRNA extracted from hybridoma cells was Any vector can be used as a vector into which the cDNA synthesized in this manner can be incorporated, so long as the vector can incorporate the cDNA. For example, ZAP Express [Strategies, 5, 58 (1992)], pBluescript II SK (+) [Nucleic Acids Research, 17, 9494 (1989)], Azap II (Stratagene GtlO, Agtll [DNA Cloning: A Practical Approach], I, 49 (1985)], Lambda BlueMid (Clontech), ExCell, pT7T3 18U (Pharmacia) ), PcD2 [Molecular 'and' Cellular 'Biology (Mol. Cell. Biol.), 3, 280 (1983)], pUC18 [Gene, 33, 103 (1985)] and the like are used. .
ファージ或いはプラスミ ドベクターにより構築される cDNAライブラリーを導入 する大腸菌としては該 cDNAライブラリ一を導入、 発現および維持できるものであ ればいかなるものでも用いることができる。 例えば、 XLl-Blue MRF' [ストラテジ ーズ（Strategies), 5, 81 ( 1992)] 、 C600 [ジエネティックス（Genetics), 39, 440 ( 1954)]、 Y1088、 Y1090 [サイエンス（Science) , 222, 778 ( 1983)]、 NM522 [ジャー ナル 'ォブ 'モレキュラー 'バイオロジー (J. Mol . Biol. ), 166, 1 (1983)]、匿 [ ジャーナル 'ォブ 'モレキュラー'バイオロジー (J. Mol. Biol. ), 16, 118 ( 1966)] および JM105 [ジーン（Gene) , 38, 275 ( 1985)]等が用いられる。  As Escherichia coli into which a cDNA library constructed by a phage or plasmid vector is introduced, any Escherichia coli can be used as long as the cDNA library can be introduced, expressed and maintained. For example, XLl-Blue MRF '[Strategies, 5, 81 (1992)], C600 [Genetics, 39, 440 (1954)], Y1088, Y1090 [Science, 222, 778] (1983)], NM522 [Journal 'ob' Molecular 'Biology (J. Mol. Biol.), 166, 1 (1983)], concealed [journal' ob 'Molecular' Biology (J. Mol. Biol.), 16, 118 (1966)] and JM105 [Gene, 38, 275 (1985)].
cDNAライブラリーからのヒト以外の動物の抗体の VHおよび "VLをコ一ドする cDNA クローンの選択法としては、 ァイソトープ或いは蛍光標識したプロ一ブを用いた コロニー ·ハイブリダィゼーシヨン法或いはプラーク ·ハイブリダィゼ一シヨン 法 (モレキュラー ·クローニング：ァ ·ラボラトリー ·マニュアル）により選択す ることができる。 また、 プライマーを調製し、 mR NAから合成した cDNA或いは cDNAライブラリ一を錡型として、 Polymerase Chain Reaction (以下、 PCR法と表 記する；モレキュラー ·クロ一ニング：ァ ·ラボラトリ一'マニュアル；カレン ト ·プロトコ一ルズ 'イン 'モレキュラー ·バイオロジー） により VHおよび VLを コードする cDNAを調製することもできる。  Methods for selecting cDNA clones encoding VH and "VL" of non-human animal antibodies from a cDNA library include colony hybridization or plaque using an isotope or fluorescently labeled probe. · Hybridization method (molecular · cloning: laboratory · manual · manual) Also, prepare primers and use the cDNA or cDNA library synthesized from mRNA as type III to obtain the Polymerase Chain Reaction ( Hereinafter, it is referred to as PCR method; molecular-cloning: a laboratory-manual; current-protocols-in-molecular-biology) can be used to prepare cDNAs encoding VH and VL. .
上記方法により選択された cDNAを、 適当な制限酵素等で切断後、 pBluescript SK (-) (Stratagene社製）等のプラスミ ドにクローニングし、通常用いられる塩基 配列解析方法、 例えば、 サンガ一 （Sanger, F. ) らのジデォキシ法 [プロシ一ディ ングス ·ォブ 'ザ ·ナショナル ·アカデミー ·ォブ ·サイエンス（Proc. Natl. Acad. Sci . U. S.A. ) , 74, 5463 ( 1977)]等の反応を行い、 塩基配列自動分析装置、 例え ば、 A. L. F. D NAシークェンサ一 （Pharmacia社製） 等を用いて解析すること で該 cDNAの塩基配列を決定することができる。 The cDNA selected by the above method is digested with an appropriate restriction enzyme or the like, and then cloned into a plasmid such as pBluescript SK (-) (manufactured by Stratagene), and a commonly used nucleotide sequence analysis method, for example, Sangaichi (Sanger , F.) et al. [Procedings of the 'National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 74, 5463 (1977)], and analyzed using an automatic nucleotide sequence analyzer, for example, ALF DNA sequencer (Pharmacia) to obtain the nucleotide sequence of the cDNA. Can be determined.
決定した塩基配列から VHおよび 7Lの全アミノ酸配列を推定し、 既知の抗体の VH および 7Lの全ァミノ酸配列 [シーケンシズ'ォブ'プロテインズ'ォブ 'ィムノロジ カノレ-ィン夕レス卜 (Sequences oi Proteins oi Immunological Interest), US Dept. Health and Human Services, 1991、 以下、 シ一ケンシズ'ォブ'プロテインズ'ォ ブ'ィムノロジカル 'イン夕レストと表記する]と比較することにより、 取得した cDNAが分泌シグナル配列を含む抗体の VHおよび VLの完全なァミノ酸配列をコード しているかを確認することができる。  The entire amino acid sequence of VH and 7L was deduced from the determined nucleotide sequence, and the amino acid sequence of VH and 7L of a known antibody was sequenced [Sequences of the proteins] Proteins of the antibodies. oi Proteins oi Immunological Interest), US Dept. Health and Human Services, 1991, hereinafter referred to as “sequences” of “proteins” of “immunological”. Encodes the complete amino acid sequence of VH and VL of the antibody including the secretory signal sequence.
( 3 ) ヒト以外の動物の抗体の V領域のァミノ酸配列の麟斤  (3) The amino acid sequence of the V region of the antibody of a non-human animal
分泌シグナル配列を含む抗体の VHおよび VLの完全なァミノ酸配列に関しては、 既知の抗体の VHおよび VLの全アミノ酸配列（シ一ケンシズ 'ォブ 'プロティンズ 'ォ ブ'ィムノロジカル 'イン夕レスト）と比較することにより、分泌シグナル配列の長 さおよび ·Ν末端アミノ酸配列を推定でき、更にはそれらが属するサブグループを知 ることができる。 また、 VHおよひ の各 CDRのアミノ酸配列についても、既知の抗 体の VHおよび 7Lのァミノ酸配列（シーケンシズ'ォブ'プロテインズ'ォブ ·ィムノ ロジカル 'イン夕レスト）と比較することによって見出すことができる。  For the complete amino acid sequence 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 "ob" "proteins" "ob" immunological "" By comparing with, the length of the secretory signal sequence and the amino acid sequence at the Ν-terminal can be estimated, and the subgroup to which they belong can be known. Also, compare the amino acid sequences of VH and each CDR of known amino acids with VH and 7L amino acid sequences of known antibodies (sequences of proteins, proteins of immunological and in rest). Can be found by:
( 4 ) ヒト型キメラ抗体発現ベクターの構築  (4) Construction of human-type chimeric antibody expression vector
本項 1の （ 1 ) に記載のヒト化抗体発現用べクタ一のヒト抗体の CHび CLをコ一 ドする遺伝子の上流に、 ヒト以外の動物の抗体の VHおよひ" VLをコードする cDNAを クローニングし、 ヒト型キメラ抗体発現べクタ一を構築することができる。 例え ば、 ヒト以外の動物の抗体の VHおよび 7Lをコードする cDNAを、 ヒト以外の動物の 抗体 VHおよび VLの 3，末端側の塩基配列とヒト抗体の CHおよび CLの 5，末端側の塩基 配列とから成り、かつ適当な制限酵素の認識配列を両端に有する合 S¾)NAとそれぞ れ連結し、 それぞれを本項 1の （1 ) に記載のヒト化抗体発現用ベクターのヒト 抗体の CHおよび CLをコードする遺伝子の上流にそれらが適切な形で発現する様に クロ一ニングし、 ヒト型キメラ抗体発現べクタ一を構築することができる。  The VH and "VL" of a non-human animal antibody are coded upstream of the gene encoding CH and CL of the human antibody of the humanized antibody expression vector described in 1 (1) of this section. For example, a human chimeric antibody expression vector can be constructed by cloning the cDNA encoding the VH and 7L of a non-human animal antibody with the VH and VL of a non-human animal antibody. 3, consisting of the base sequence on the terminal side and the base sequence on the terminal side of CH and CL of the human antibody and having a recognition sequence for an appropriate restriction enzyme at both ends. The humanized chimeric antibody is cloned so that it can be expressed in an appropriate form upstream of the genes encoding CH and CL of the human antibody of the humanized antibody expression vector according to 1 (1) of this section. An expression vector can be constructed.
( 5 ) ヒト型 CDR^植抗体の V領域をコードする cDNAの構築  (5) Construction of cDNA encoding V region of human CDR ^ plant antibody
ヒト型 CD脇植抗体の VHおよひ 7Lをコ一ドする cDNAは、以下の様にして構築する ことができる。まず、 目的のヒト以外の動物の抗体の VHおよび 7Lの CDRのアミノ酸 配列を移植するヒト抗体の VHおよび VLのフレームワーク領域 （以下、 FRと表記す る） のアミノ酸配列を選択する。 ヒト抗体の VHおよひ 7Lの FRのアミノ酸配列とし ては、 ヒト抗体由来のものであれば、 いかなるものでも用いることができる。 例 えば、 Protein Data Bank等のデ一夕べ一スに登録されているヒト抗体の VHおよび VLの FRのアミノ酸配列、 ヒト抗体の VHおよび VLの FRの各サブグループの共通アミ ノ酸酉己列（シーケンシズ ·ォブ 'プロテインズ 'ォブ ·ィムノロジカル 'イン夕レスト )等があげられるが、 その中でも、 十分な活性を有するヒト型 CDR^植抗体を作製 するためには、 目的のヒト以外の動物の抗体の VHおよび 7Lの FRのアミノ酸配列と できるだけ高い相同性（少なくとも 60%以上）を有するアミノ酸配列を選択するこ とが望ましい。 次に、 選択したヒト抗体の VHおよび VLの FRのアミノ酸配列に目的 のヒト以外の動物の抗体の VHおよび "VLの CDRのァミノ酸配列を移植し、 ヒト型 CDR 移植抗体の VHおよび 7Lのアミノ酸配列を設計する。 設計したアミノ酸配列を抗体 の遺伝子の塩基配列に見られるコドンの使用頻度（シーケンシズ ·ォブ ·プロティ ンズ'ォブ'ィムノロジカル'ィン夕レスト）を考慮して DNA配列に変換し、 ヒト型 CDR^植抗体の VHおよび1 VLのァミノ酸配列をコードする DNA配列を設計する。 設計 した DNA配列に基づき、 100塩基前後の長さから成る数本の合 figDNAを合成し、それ らを用いて PCR法を行う。この場合、 PCRでの反応効率および合成可能な DNAの長さ から、 H鎖、 L鎖とも 6本の合 ¾¾DNAを設計することが好ましい。 CDNA encoding VH and 7L of human CD side plant antibody is constructed as follows be able to. First, the amino acid sequences of the framework regions (hereinafter referred to as FR) of the VH and VL of the human antibody to which the amino acid sequences of the VH and 7L CDRs of the antibody of the target non-human animal are transplanted are selected. As the amino acid sequence of human antibody VH and 7L FR, any amino acid sequence can be used as long as it is derived from a human antibody. For example, FR amino acid sequences of VH and VL of human antibodies registered in the Database of Protein Data Bank, etc., and common amino acid sequences of FR subgroups of VH and VL of human antibodies (Sequences of 'Proteins', of 'Immological', and 'Inrest Rest'). Among them, in order to produce human-type CDR ^ -planted antibodies having sufficient activity, It is desirable to select an amino acid sequence having the highest possible homology (at least 60% or more) with the amino acid sequence of the VH of the animal antibody and the FR of 7L. Next, the amino acid sequences of the target non-human animal antibody VH and "VL CDR" were grafted to the VH and VL FR amino acid sequences of the selected human antibody, and the human CDR-grafted antibody VH and 7L Designing the amino acid sequence The designed amino acid sequence is converted into a DNA sequence in consideration of the codon usage (sequences of proteins, proteins, proteins, immunologicals, etc.) found in the nucleotide sequence of the antibody gene. Convert and design a DNA sequence encoding the amino acid sequence of VH and 1 VL of the human CDR ^ plant antibody Based on the designed DNA sequence, synthesize several synthetic DNAs of about 100 bases in length. In this case, it is preferable to design a total of six DNAs for both the H chain and the L chain in view of the reaction efficiency in the PCR and the length of DNA that can be synthesized.
また、両端に位置する合成 DNAの 5'末端に適当な制限酵素の認識配列を導入する ことで、 ^：頁 1の （1 ) で構築したヒト化抗体発現用ベクターに容易にクロー二 ングすることができる。 PCR後、 増幅産物を pBluescript SK (-) (Stratagene社製 ) 等のプラスミドにクローニングし、 本項 1の （2 ) に記載の方法により、 塩基 配列を決定し、所望のヒト型 CDR^植抗体の VHおよび VLのアミノ酸配列をコ一ドす る DNA配列を有するプラスミ ドを取得する。  Also, by introducing an appropriate restriction enzyme recognition sequence into the 5 'end of the synthetic DNA located at both ends, ^: easily cloned into the humanized antibody expression vector constructed in (1) on page 1 be able to. After PCR, the amplified product is cloned into a plasmid such as pBluescript SK (-) (Stratagene), and the nucleotide sequence is determined by the method described in (2) of this section 1; A plasmid having a DNA sequence encoding the amino acid sequences of VH and VL is obtained.
( 6 ) ヒト型 CDR^植抗体の V領域のァミノ酸配列の改変  (6) Amino acid sequence modification of V region of human CDR ^ -planted antibody
ヒト型 CDR^植抗体は、 目的のヒト以外の動物の抗体の VHおよび VLの CDRのみを ヒト抗体の VHおよび VLの FRに移植しただけでは、 その 結合活性は元のヒト以 外の動物の抗体に比べて低下してしまうことが知られている [バイオテクノロジ -(BI0/TECHN0L0GY), 9， 266 ( 1991 )]。 この原因としては、 元のヒト以外の動物 の抗体の VHおよび "VLでは、 CDRのみならず、 FRのいくつかのァミノ酸残基が直接的 或いは間接的に抗原結合活性に関与している。そして、 ヒト抗体の FRへの CDRの移 植によりそれらァミノ酸残基が、 ヒト抗体の FRの異なるァミノ酸残基へと変化す るため、 結合活性が低下すると考えられている。 この問題を解決するため、 ヒト型 CD 植抗体では、 ヒト抗体の VHおよび 7Lの FRのアミノ酸配列の中で、直接 f¾lとの結合に関与しているァミノ酸残基、 CDRのァミノ酸残基と相互作用するァ ミノ酸残基、 抗体の立 造を維持し、 間接的に ί¾1との結合に関与しているァ ミノ酸残基を同定し、 それらを元のヒト以外の動物の抗体のアミノ酸残基に改変 し、低下した ί¾結合活性を上昇させることが行われている [バイオテクノロジー (BIO/TECHNOLOGY) , 9, 266 ( 1991 )]。 ヒト型 CDR^植抗体の作製においては、 それ ら 結合活性に関わる FRのアミノ酸残基を如何に効率よく同定するかが、 最も 重要な点であり、そのために X»線結晶解析 [ジャーナル'ォブ'モレキュラー'バイオ ロジ一 (J. Mol . Biol . ), 112, 535 ( 1977) ]或いはコンビユー夕一モデリング [プ 口ティン.エンジニアリング (Protein Engineering) , 7, 1501 ( 1994) ]等による抗 体の立體造の構築および謹が行われている。これら抗体の立體造の情報は、 ヒト型 CDR^植抗体の作製に多くの有益な情報をもたらして来たが、その一方、あ らゆる抗体に適応可能なヒト型 CDR^fi抗体の作製法は未だ確立されておらず、現 状ではそれぞれの抗体について数種の改変体を作製し、 それぞれの抗原結合活性 との相関を検討する等の種々の試行錯誤が必要である。 The human CDR ^ -grafted antibody has a binding activity of only the non-human animal antibody VH and VL CDRs transplanted into the human antibody VH and VL FR, and the binding activity of the original non-human animal It is known to be lower than antibodies [Biotechnology-(BI0 / TECHN0L0GY), 9, 266 (1991)]. This can be caused by the original non-human animal In the VH and "VL" antibodies, not only the CDRs but also some amino acid residues of the FR are directly or indirectly involved in the antigen-binding activity. It is thought that the transplantation changes these amino acid residues into amino acid residues having different FRs in the human antibody, resulting in a decrease in the binding activity. Amino acid residues that are directly involved in binding to f¾l, amino acid residues that interact with the amino acid residues of CDR in the amino acid sequence of human antibody VH and 7L FR, and antibody construction To identify amino acid residues that are indirectly involved in binding to ί¾1, modify them to amino acid residues of the original non-human animal antibody, and increase the reduced ί¾ binding activity [Biotechnology (BIO / TECHNOLOGY), 9, 266 (1991)] In the production of human CDR ^ -planted antibodies, the most important point is how to efficiently identify the amino acid residues of FR involved in the binding activity. Crystallographic analysis [J. Mol. Biol. Biol. (J. Mol. Biol.), 112, 535 (1977)] or Yuichi modeling of combi [Protein Engineering, 7, 1501 (1994) )], Etc., and the construction of antibodies has been carried out.The information on the structure of these antibodies has provided a great deal of useful information for the production of human CDR ^ -planted antibodies. On the other hand, a method for preparing a human CDR ^ fi antibody applicable to all antibodies has not been established yet, and at present, several variants of each antibody have been prepared, and their respective antigen-binding activities have been determined. Various trials and errors, such as examining the correlation, are required.
ヒト抗体の VHおよび VLの FRのアミノ酸残基の改変は、改変用合 β¾)ΝΑを用いて本 項 1の （5 ) に記載の PCR法を行うことにより、 達成できる。 PCR後の増幅産物に ついて本項 1の （2 ) に記載の方法により、 塩基配列を決定し、 目的の改変が施 されたことを確認する。  The modification of the amino acid residues of FRs of VH and VL of a human antibody can be achieved by performing the PCR method described in (5) of this section 1 using the modification compound β¾) ΝΑ. The nucleotide sequence of the amplified product after PCR is determined by the method described in (2) of this section 1 to confirm that the desired modification has been performed.
( 7 ) ヒト型 CDR^植抗体発現べク夕一の構築  (7) Construction of human CDR ^ plant antibody expression vector
頁 1の （ 1 ) に記載のヒト化抗体発現用べクタ一のヒト抗体の CHおよび CLを コードする遺伝子の上流に、 お頁 1の （5 ) および （6 ) で構築したヒト型 CDR 移植抗体の VHおよひ 7Lをコ一ドする cDNAをクロ一ニングし、ヒト型 CDR^植抗体発 現ぺク夕一を構築することができる。  The human CDR graft constructed in (5) and (6) on page 1 upstream of the genes encoding CH and CL of the human antibody in the vector for expression of the humanized antibody described in (1) on page 1 The cDNA encoding VH and 7L of the antibody can be cloned to construct a human CDR-transplanted antibody expression vector.
例えば、 お頁 1の（5 )および（6 )でヒト型 CDR^植抗体の VHおよひ TLを構築 する際に用レ、る合成 DNAのうち、 両端に位置する合成 DNAの 5'末端に適当な制限酵 素の認識配列を導入することで、 *1頁 1の （1 ) に記載のヒト化抗体発現用べク ターのヒト抗体の CHおよび CLをコ一ドする遺伝子の上流にそれらが適切な形で発 現する様にクローニングし、ヒト型 011 植抗体発現べク夕一を構築することがで さる。 For example, of the synthetic DNAs used for constructing VH and TL of the human CDR ^ -planted antibody in (5) and (6) on page 1, the 5 'end of the synthetic DNA located at both ends is used. Suitable restriction yeast By introducing the elemental recognition sequence, the appropriate form can be placed upstream of the genes encoding the CH and CL of the human antibody in the humanized antibody expression vector described in (1) on page 1 * 1. It is possible to construct a human-type 011-planted antibody expression vector by cloning to express in step (1).
( 8 ) ヒト化抗体の一過性発現  (8) Transient expression of humanized antibody
作製した多種類のヒト化抗体の 結合活性を効率的に評価するために、 1の （4 ) および （7 ) に記載のヒト化抗体発現べクタ一、 或いはそれらを改変 した発現べクタ一を用いてヒト化抗体の一過性発現を行うことができる。 発現べ クタ一を導入する宿主細胞としては、ヒト化抗体を発現できる宿主細胞であれば、 いかなる細胞でも用いることができるが、 その発現量の高さから、 COS- 7細胞 （ ATCC CRL1651)が一般に用いられる [メソッズ 'ィン*ヌクレイヅク ·ァシッズ-リサ —チ (Methods in Nucleic Acids Res. ), CRC press, 283 ( 1991 )]。 COS-7細胞へ の発現ベクターの導入法としては、 DEAE—デキストラン法 [メソッズ 'ィン'ヌクレ ィック ·ァシッズ，リサーチ (Methods in Nucleic Acids Res. ), CRC press, 283 ( 1991 )]、 リポフエクシヨン法 [プロシ一ディングス 'ォブ 'ザ'ナショナル'ァカデ ミ一'ォブ'サイエンス（Proc. Natl. Acad. Sci . U. S.A. ), 84, 7413 ( 1987)]等が あげられる。 YB2/0細胞で発現させたヒト化抗体 ttADCC活性が高まるので好ましい 発現ベクターの導入後、 培養上清中のヒト化抗体の発現量および抗原結合活性 は酵素免疫抗体法 [以下、 ELISA法と表記する；アンティボディズ：ァ 'ラボラトリ ——·マ二ユアノレ (Antibodies: A Laboratory Manual ) , Cold Spring Harbor Laboratory, Chapter 14, 1988、 モノクロ一ナル 'アンティボディズ：プリンシプ ノレズ-アンド-プラクティス (Monoclonal Antibodies: Principles and Practice), Academic Press Limited, 1996]等により測定できる。  In order to efficiently evaluate the binding activities of the various humanized antibodies thus prepared, the humanized antibody expression vector described in 1 (4) and (7) or an expression vector obtained by modifying them was used. Can be used to effect transient expression of a humanized antibody. As the host cell into which the expression vector is introduced, any cell can be used as long as it can express a humanized antibody. However, due to its high expression level, COS-7 cells (ATCC CRL1651) can be used. Commonly used [Methods in Nucleic Acids Res., CRC press, 283 (1991)]. Methods for introducing an expression vector into COS-7 cells include the DEAE-dextran method [Methods in Nucleic Acids Res., CRC press, 283 (1991)], Lipofexion method Proc. 'Ob' The 'National' Academia 'Ob' Science (Proc. Natl. Acad. Sci. USA), 84, 7413 (1987)]. Humanized antibody expressed in YB2 / 0 cells TTADCC activity is preferable because it increases. After introduction of the expression vector, the expression level of humanized antibody and antigen-binding activity in the culture supernatant are measured by enzyme-linked immunosorbent assay [hereinafter referred to as ELISA method]. Antibodies: A 'Laboratory-Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Chapter 14, 1988, Monochrome' Antibodies: Principle Norez and Practice : Principles and Practice), Academic Press Limited, 1996].
( 9 ) ヒト化抗体の安定発現  (9) Stable expression of humanized antibody
本項 1の （4 ) および （7 ) に記載のヒト化抗体発現ベクターを適当な宿主細 胞に導入することによりヒト化抗体を安定に発現する形質転換株を得ることがで さる。  By introducing the humanized antibody expression vector described in (4) and (7) of this section 1 into a suitable host cell, a transformant that stably expresses the humanized antibody can be obtained.
宿主細胞への発現べク夕一の導入法としては、エレクトロボレ一シヨン法 [特開 平 2- 257891、 サイ トテクノロジ一 (Cytotechnology), 3 , 133 (1990)]等があげら れる。 Examples of a method for introducing an expression vector into a host cell include an electrolysis method [Japanese Patent Laid-Open No. 257891/1990, Cytotechnology, 3, 133 (1990)] and the like. It is.
ヒト化抗体発現べクタ一を導入する宿主細胞としては、 ヒト化抗体を発現させ ることができる宿主細胞であれば、 いかなる細胞でも用いることができる。 例え ばヽ マウス SP2/0- Agl4細胞 (ATCC CRL1581) 、 マウス P3X63- Ag8.653細胞 (ATCC CRL1580)、 ジヒドロ葉酸還元酵素遺伝子（以下、 dhfrと表記する）が欠損した CH0 細胞 [プロシ一デイングス 'ォブ 'ザ'ナショナル'アカデミー'ォブ 'サイェン ス（Proc. Natl. Acad. Sci. U. S.A. ), 77, 4216 (1980)]、 ラット  As the host cell into which the humanized antibody expression vector is introduced, any cell can be used as long as it can express the humanized antibody. For example, mouse SP2 / 0-Agl4 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CH0 cells deficient in the dihydrofolate reductase gene (hereinafter referred to as dhfr) [Proceding's 'The' National 'Academy' Ob 'Science (Proc. Natl. Acad. Sci. USA), 77, 4216 (1980)], rat
YB2/3HL.P2.G11.16Ag.20細胞 （ATCC CRL1662、 以下、 YB2/0細胞と表記する）等が あげられる。 YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL1662, hereinafter referred to as YB2 / 0 cells) and the like.
発現ベクターの導入後、 ヒト化抗体を安定に発現する形質転 j«は、 特開平 2- 257891に開示されている方法に従い、 G418 sulfate (以下、 G418と表記する： SIGMA 社製） 等の薬剤を含む動物細胞培養用培地で培養することにより選択できる。 動 物細胞培養用培地としては、 RPMI1640培地（日水製辦土製）、 GIT培地（日本製薬 社製） 、 EX-CELL302培地 （JEH社製） 、 IMDM培地 (GIBCO BRL社製） 、 Hybridoma- SFM培地 （GIBC0 BRL社製） 、 またはこれら培地に牛胎児血清 （以下、 FBSと表記す る） 等の各種添加物を添加した培地等を用いることができる。 得られた形質転換 株を培地中で培養することで培養上清中にヒト化抗体を発現蓄積させることがで きる。培養上清中のヒト化抗体の発現量および抗原結合活性は ELISA法等により測 定できる。 また、 形質転 «は、 特開平 2- 257891に開示されている方法に従い、 dhfr増幅系等を利用してヒト化抗体の発現量を上昇させることができる。  After the introduction of the expression vector, the transformant which stably expresses the humanized antibody can be transformed with a drug such as G418 sulfate (hereinafter referred to as G418: manufactured by SIGMA) according to the method disclosed in JP-A-2-2577891. Can be selected by culturing in an animal cell culture medium containing As culture media for animal cells, RPMI1640 medium (Nissui-made), GIT medium (Nippon Pharmaceutical), EX-CELL302 medium (JEH), IMDM medium (GIBCO BRL), Hybridoma-SFM A medium (GIBC0 BRL) or a medium to which various additives such as fetal calf serum (hereinafter referred to as FBS) are added to these mediums can be used. By culturing the obtained transformant in a medium, the humanized antibody can be expressed and accumulated in the culture supernatant. The expression level and antigen-binding activity of the humanized antibody in the culture supernatant can be measured by ELISA or the like. In the transformation, the expression level of the humanized antibody can be increased using a dhfr amplification system or the like according to the method disclosed in JP-A-2-2577891.
ヒト化抗体は、形質転■の培養上清よりプロティン Aカラムを用いて精製する ことができる [アンティボディズ：ァ 'ラボラトリー'マニュアル、モノクローナル •アンティボディズ：プリンシプルズ 'アンド 'プラクティス（Monoclonal  The humanized antibody can be purified from the culture supernatant of the transformant using a protein A column. [Antibodies: Laboratory Manual, Monoclonal • Antibodies: Principles 'and' Practice (Monoclonal
Antibodies: Principles and Practice;, Academic Press Limited, 1996、 以下、 モノクローナル ·アンティボディズ ( 1996)と表記する]。 また、 その他に通常、 蛋 白質の精製で用いられる精製方法を使用することができる。 例えば、 ゲル濾過、 イオン交換クロマトグラフィ一および限外濾過等を組み合わせて行い、 精製する ことができる。 精製したヒト化抗体の H鎖、 L鎖或いは抗体分子全体の分子量は、 ポリアクリルアミドゲル電気泳動 [以下、 SDS- PAGEと表記する：ネィチヤ一 (Nature), 227， 680 ( 1970)]やウエスタンプロッティ ァ 'ラボラトリ一'マニュアル、 モノクローナル 'アンティボディズ（1996)]等で測 定することができる。 Antibodies: Principles and Practice ;, Academic Press Limited, 1996; hereinafter, referred to as monoclonal antibodies (1996)]. In addition, a purification method usually used for protein purification can be used. For example, purification can be performed by a combination of gel filtration, ion exchange chromatography and ultrafiltration. The molecular weight of the purified humanized antibody H-chain, L-chain or the whole antibody molecule can be determined by polyacrylamide gel electrophoresis [hereinafter referred to as SDS-PAGE: Nature, 227, 680 (1970)] or Western plotty. A) It can be measured by 'Laboratory I' manual, Monoclonal 'Antibody's (1996)].
( 1 0 ) ヒト化抗体の活性評価  (10) Activity evaluation of humanized antibody
精製したヒト化抗体の ί¾1との結合活性、 培^ ¾細«に対する結合活性は EL I SA法および虽光抗体法 [キャンサー 'ィムノロジ一'ィムノセラピー ( Cancer Immunol. I腿 unother. ), 36, 373 ( 1993)]等により測定できる。 ί¾陽性培 ¾ 細 «に対する細胞障害活性は、 CDC活性、 ADCC活性等を測定し、評価することが できる [キヤンサ一'ィムノロジ一'ィムノセラピー（Cancer Immunol .  The binding activity of the purified humanized antibody to ί¾1 and the binding activity to culture media were determined by ELISA and photoantibodies [Cancer 'Immunology I' Immunotherapy (Cancer Immunol. I Thigh unother.), 36, 373 (1993)]. Cytotoxic activity against ί¾positive cells can be evaluated by measuring CDC activity, ADCC activity and the like [Cancer Immunology 1 (Cancer Immunol.
I腿 unother. ), 36, 373 ( 1993)]。 I thigh unother.), 36, 373 (1993)].
2 . 抗体断片の作製 2. Preparation of antibody fragments
抗体断片は、 *J頁 1に記載のヒト化抗体または、 ハイプリドーマが産生する抗 体およびヒト抗体を元に遺伝子工学的手法あるいは蛋白質化学的手法により、 作 製することができる。 抗体断片としては、 Fab、 F(ab，）2、 Fab'、 scFv、 dsFv、 CDR を含むぺプチド等があげられる。 これらの抗体断片は蛋白質化学または遺伝子ェ 学の手法を用いて作製することができる。 Antibody fragments can be produced by genetic engineering or protein chemical techniques based on the humanized antibodies described on page J * 1 or antibodies produced by hybridomas and human antibodies. Examples of the antibody fragment include Fab, F (ab,) 2 , Fab ′, scFv, dsFv, peptides including CDR, and the like. These antibody fragments can be prepared using protein chemistry or genetics techniques.
( 1 ) Fabの作製  (1) Fab production
Fabは、 IgGを蛋白質分解酵素パパインで処理することにより、 作製することが できる。 パパインの処理後は、 元の抗体がプロテイン A結合性を有する IgGサブク ラスであれば、 プロテイン Aカラムに通すことで、 IgG分子や Fc断片と分離し、 均 一な Fabとして回収することができる [モノクローナル ·アンティボディズ：プリ ンシプゾレズ ·アンド ·プラクティス (Monoclonal Antibodies: Principles and Practice) ， third edition, 1995、以下、 モノクローナル ·アンティボディズ 第 3版と表記する]。プロテイン A結合性を持たない IgGサブクラスの抗体の場合には 、イオン交換クロマトグラフィ一により、 Fabは低塩濃度で溶出される画分中に回 収することができる （モノクローナル ·アンティポディズ 第 3版）。 また、 Fab は、 本項 1の （2 )および（5 ) に記載の抗体 V領域をコードする DNAを、 Fab発現 用ベクターにクローニングし、 Fab発現ベクターを作製し、該発現ベクターを宿主 に導入することにより発現し、作製することができる。 Fab発現用べク夕一として は、 Fab用の DNAを組み込み発現できるものであればいかなるものも用いることが できる。例えば、 PIT106 [サイエンス（Science), 240, 1041 ( 1988)]等があげられ る。 Fab can be prepared by treating IgG with the protease, papain. After papain treatment, if the original antibody is an IgG subclass that has protein A binding properties, it can be separated from IgG molecules and Fc fragments by passing through a protein A column and recovered as a uniform Fab. [Monoclonal Antibodies: Principles and Practice, third edition, 1995, hereinafter referred to as "Monoclonal Antibodies: Third Edition"]. In the case of an IgG subclass antibody that does not have protein A binding, Fab can be recovered in a fraction eluted at a low salt concentration by ion exchange chromatography (monoclonal antipodes 3rd edition) ). Further, Fab is prepared by cloning a DNA encoding the antibody V region described in (1) (2) and (5) of this section into a Fab expression vector, producing a Fab expression vector, and introducing the expression vector into a host. By doing so, it can be expressed and produced. Any Fab expression vector can be used as long as it can incorporate and express Fab DNA. An example is PIT106 [Science, 240, 1041 (1988)]. You.
宿主細胞としては真核生物、 原核生物などいかなるものでも用いることができ るが、 大腸菌が好ましい。  Any host cell such as eukaryotes and prokaryotes can be used, but Escherichia coli is preferred.
Fab発現べク夕一を適当な宿主細胞に導入し、封入体あるいはべリブラズマ層に Fabを生成蓄積させることができる。封入体からは、通常蛋白質で用いられるリフ オールデイング法により、活性のある Fabとすることができ、 また、 ペリプラズマ 層に発現させた場合は、培養上清中に活性を持った Fabが漏れ出る。 リフォールデ ィング後あるいは培養上清からは、 を結合させたカラムを用いることにより、 均一な Fabを精製することができる [アンティポディ 'エンジニアリング， ァ 'プ ラクティカゾレ 'ガイ ド (Antibody Engineering, A Practical Guide) , W. H. Freeman and Company, 1992]。  The Fab expression vector can be introduced into an appropriate host cell to produce and accumulate Fab in the inclusion body or Veriplasma layer. From the inclusion body, an active Fab can be converted to the active Fab by the refolding method usually used for proteins, and when expressed in the periplasm layer, the active Fab leaks into the culture supernatant. Get out. After refolding or from the culture supernatant, it is possible to purify a uniform Fab by using a column to which is bound [Antipod Engineering 'A' Practical Kasole 'Guide (Antibody Engineering, A Practical Guide) , WH Freeman and Company, 1992].
( 2 ) F(ab， )2の作製 (2) Preparation of F (ab,) 2
F(ab' )2は、 IgGを蛋白質分解酵素ペプシンで処理することにより、 作製するこ とができる。 ペプシンの処理後は、 Fabと同様の精製操作により、 均一な F(ab' )2 として回収することができる （モノクローナル 'アンティボディズ 第 3版） 。 また、後述する *ί貝 2の（3 )に記載の Fab'を o-PDMやビスマレイミ ドへキサン等 のようなマレイミドで処理し、 チォェ一テル結合させる方法、 DTNBで処理し、 ジ スルフィ ド結合させる方法によっても作製することができる [アンティボディ 'ェ ンジニァリング， ァ 'プラクティカル 'アプローチ （Antibody Engineering, A Practical Approach) , IRL PRESS, 1996、 以下、 アンティボディ 'エンジニアリ ングと表記する]。 F (ab ') 2 can be produced by treating IgG with the protease pepsin. After treatment with pepsin, it can be recovered as uniform F (ab ') 2 by the same purification procedure as Fab (monoclonal' Antibodies 3rd edition). Further, a method of treating Fab ′ described in (3) of * ί shell 2 described later with o-PDM or a maleimide such as bismaleimide hexane and the like to form a thioether bond, treatment with DTNB, and disulfide It can also be prepared by a method of bonding [Antibody Engineering, A Practical Approach], IRL PRESS, 1996, hereinafter referred to as Antibody 'Engineering'.
( 3 ) Fab'の作製  (3) Preparation of Fab '
Fab'は、 本項 2の （2 ) に記載の F(ab，）2をジチオスレィ トール等の還元剤で処 理して得ることができる。 また、 Fab 'は、 ffl lの （2 ) および （5 ) に記載の 抗体 V領域をコードする DNAを、 Fab'発現用べク夕一にクローニングし、 Fab，発現 ベクタ一を作製し、 該発現べクタ一を宿主細胞に導入することにより発現し、 作 製することができる。 Fab ′ can be obtained by treating F (ab,) 2 described in (2) of this section 2 with a reducing agent such as dithiothreitol. In addition, Fab ′ was cloned into a vector for Fab ′ expression using a DNA encoding the antibody V region described in (2) and (5) of ffll to prepare Fab and an expression vector. It can be expressed and produced by introducing the expression vector into a host cell.
Fab'発現用ベクターとしては、 Fab'用の DNAを組み込み発現できるものであれば いかなるものも用いることができる。 例えば、 pAK19 [バイオテクノロジ一 (BI0/TECHN0L0GY), 10， 163 ( 1992)]等があげられる。 宿主細胞としては、 真核生物、 原核生物などいかなるものも用いることができ るが、 大腸菌が好ましい。 As the Fab 'expression vector, any vector can be used as long as it can incorporate and express Fab' DNA. For example, pAK19 [Biotechnology I (BI0 / TECHN0L0GY), 10, 163 (1992)] and the like can be mentioned. As the host cell, any cells such as eukaryotes and prokaryotes can be used, and Escherichia coli is preferred.
Fab'発現べクタ一を適当な宿主細胞に導入し、 封入体あるいはペリプラズマ層 に Fabを生成蓄積させることができる。封入体からは、通常蛋白質で用いられるリ フォールデイング法により、 活性のある Fab'とすることができ、 また、 ペリブラ ズマ層に発現させた場合は、 リゾチームによる部分消化、 浸透圧ショック、 ソニ ケ一シヨン等の処理により菌を破砕し、 菌体外へ回収させることができる。 リフ オールディング後ぁるいは菌の破碎液からは、プロテイン Gカラム等を用いること により、 均一な Fab'を精製することができる （アンティボディ 'エンジニアリン グ） 。  The Fab 'expression vector can be introduced into an appropriate host cell to produce and accumulate Fab in the inclusion body or periplasm layer. From the inclusion body, active Fab 'can be obtained by the refolding method usually used for proteins, and when expressed in the periplasma layer, partial digestion with lysozyme, osmotic shock, sonication Bacteria can be crushed by treatment with one shot or the like and collected outside the cells. After the refolding, uniform Fab 'can be purified from the lysate of the fungi by using a protein G column or the like (Antibody' Engineering ').
( 4 ) scFvの作製  (4) Preparation of scFv
scFvは、 お頁 1の （2 ) および （5 ) に記載の抗体 V領域をコードする DNAを、 scFv発現用ベクターにクローニングし、 scFv発現べクタ一を作製し、 該発現べク 夕一を宿主細胞に導入することにより発現し、 作製することができる。  For scFv, DNA encoding the antibody V region described in (2) and (5) on page 1 is cloned into a scFv expression vector to prepare an scFv expression vector, and the expression vector is It can be expressed and produced by introducing it into host cells.
scFv発現用べク夕一としては、 scFvの DNAを組み込み発現できるものであれば ヽ かなるものも用いることができる。例えば、 pCANTAB5E (Pharmac ia社）、 pHFA[Hum. Antibody Hybridoma, 5, 48 ( 1994) ]等があげられる。  Any scFv expression vector can be used as long as it can incorporate and express scFv DNA. For example, pCANTAB5E (Pharmacia), pHFA [Hum. Antibody Hybridoma, 5, 48 (1994)] and the like can be mentioned.
宿主細胞としては、 真核生物、 原核生物などいかなるものも用いることができ るが、 大腸菌が好ましい。  As the host cell, any cells such as eukaryotes and prokaryotes can be used, and Escherichia coli is preferred.
scFv発現べク夕一を適当な宿主細胞に導入し、 ヘルパーファージを感染させる ことで、 ファ一ジ表面に scFvがファージ表面蛋白質と融合した形で発現するファ ージを得ることができる。 また、 scFv発現べクタ一を導入した大腸菌の封入体あ るいはペリプラズマ層に scFvを生成蓄積させることができる。 封入体からは、 通 常蛋白質で用いられるリフォールディング法により、 活性のある scFvとすること ができ、 また、 ペリプラズマ層に発現させた場合は、 リゾチームによる部分消化、 浸透圧ショック、 ソニケーシヨン等の処理により菌を破碎し、 菌体外へ回収する ことができる。 リフォールデイング後あるいは菌の破砕液からは、 陽イオン交換 クロマトグラフィー等を用いることにより、 均一な scFvを精製することができる By introducing the scFv expression vector into an appropriate host cell and infecting it with a helper phage, a phage that expresses the scFv on the phage surface in a form fused with the phage surface protein can be obtained. In addition, scFv can be produced and accumulated in the inclusion body of Escherichia coli or the periplasm layer into which the scFv expression vector has been introduced. The inclusion body can be converted into an active scFv by the refolding method usually used for proteins, and when expressed in the periplasmic layer, it can be used for partial digestion with lysozyme, osmotic shock, sonication, etc. The bacteria can be crushed by the treatment and collected outside the cells. Uniform scFv can be purified after refolding or from the lysate of bacteria by using cation exchange chromatography, etc.
(アンティボディ ·ェンジ二' (Antibody
( 5 ) dsFvの作製 dsFvは以下のように作製することができる。 まず、 本項 1の （2 ) および （5 )に記載の抗体 VHおよび VLをコードする DNAの適当な位置に変異を導入し、コード するアミノ酸残基がシスティンに置換された DNAを作製する。 作製した各 DNAを dsFv発現用べクタ一にクローニングし、 VHおよび L発現ベクターを作製し、 該発 現べク夕一を宿主細胞に導入することにより発現し、 作製することができる。 dsFv発現用べクタ一としては、 dsFv用の DNAを組み込み発現できるものであれば いかなるものも用いることができる。例えば、 pULI9 [プロティン 'エンジニアリン グ (Protein Engineering), 7, 697 ( 1994)]等があげられる。 (5) Preparation of dsFv dsFv can be prepared as follows. First, a mutation is introduced into an appropriate position of the DNA encoding the antibodies VH and VL described in (2) and (5) of this section 1 to prepare a DNA in which the encoding amino acid residue is substituted with cysteine. Each of the prepared DNAs is cloned into a dsFv expression vector, VH and L expression vectors are prepared, and the expression vector is introduced into a host cell to be expressed and prepared. As the vector for dsFv expression, any vector can be used as long as it can incorporate and express DNA for dsFv. An example is pULI9 [Protein Engineering, 7, 697 (1994)].
宿主細胞としては、 真核生物、 原核生物などいかなるものも用いることができ るが、 大腸菌が好ましい。  As the host cell, any cells such as eukaryotes and prokaryotes can be used, and Escherichia coli is preferred.
VHおよび 発現べクタ一を適当な宿主細胞に導入し、 封入体あるいはペリブラ ズマ層に dsFvを生成蓄積させることができる。 封入体あるいはペリブラズマ層か ら VHおよび 7Lを得、 混合し、 通常蛋白質で用いられるリフォールデイング法によ り、 活性のある dsFvとすることができる。 リフォールデイング後は、 イオン交換 クロマトグラフィーおよびゲル濾過等により、さらに精製することができる [プロ ティン.エンジニアリング (Protein Engineering), 7, 697 ( 1994)]。  The VH and expression vector can be introduced into a suitable host cell to produce and accumulate dsFv in inclusion bodies or periplasmic layers. VH and 7L can be obtained from the inclusion body or periplasma layer, mixed, and converted into active dsFv by the refolding method usually used for proteins. After refolding, it can be further purified by ion exchange chromatography, gel filtration, etc. [Protein Engineering, 7, 697 (1994)].
( 6 ) CDRペプチドの作製  (6) Preparation of CDR peptide
CDRを含むぺプチドは、 Fmoc法あるいは tBoc法等の化学合成法によつて作製する ことができる。 また、 CDRを含むペプチドをコードする DNAを作製し、作製した DNA を適当な発現用ベクターにクローニングし、 CDRペプチド発現ベクターを作製する ことができる。 発現用べクタ一としては、 CDRペプチドをコードする DNAを組み込 み発現できるものであればいかなるものも用いることができる。 例えば、 pLEX ( Invitrogen社） 、 pAX4a+ (Invitrogen社） 等があげられる。  The peptide containing CDR can be prepared by a chemical synthesis method such as the Fmoc method or the tBoc method. In addition, a DNA encoding a peptide containing CDR can be prepared, and the prepared DNA can be cloned into an appropriate expression vector to prepare a CDR peptide expression vector. Any expression vector can be used as long as it can incorporate and express the DNA encoding the CDR peptide. For example, pLEX (Invitrogen), pAX4a + (Invitrogen) and the like can be mentioned.
宿主細胞としては、 真核生物、 原核生物などいかなるものも用いることができ るが、 大腸菌が好ましい。  As the host cell, any cells such as eukaryotes and prokaryotes can be used, and Escherichia coli is preferred.
発現べク夕一を適当な宿主細胞に導入し、封入体あるいはべリブラズマ層に CDR ベプチドを生成蓄積させることができる。封入体あるいはべリプラズマ層から CDR ペプチドを得、 イオン交換クロマトグラフィーおよびゲル濾過等により、 精製す ることができる [プロテイン 'エンジニアリング (Protein Engineering), 7, 697 (腸)]。 ( 7 ) 抗体断片の活性評価 The expression vector can be introduced into a suitable host cell to produce and accumulate the CDR peptide in the inclusion body or Veriplasma layer. CDR peptides can be obtained from inclusion bodies or Veriplasma layers and purified by ion-exchange chromatography and gel filtration, etc. [Protein Engineering, 7, 697 (intestine)]. (7) Evaluation of antibody fragment activity
得られた抗体断片の ί¾ との結合活性、培 « 細 «に対する結合活性は ELISA 法および蛍光抗体法 [キャンサー 'ィムノロジ一'ィムノセラピー (Cancer  The binding activity of the obtained antibody fragment to 、 and the binding activity to culture media were determined by ELISA and immunofluorescence [Cancer 'Immunology-1' immunotherapy (Cancer).
Immunol. , Immunother. ), 36, 373 (1993)]等により測定できる。 Immunol., Immunother.), 36, 373 (1993)].
( 8 ) 抗体断片の使用方法  (8) How to use antibody fragments
本発明の抗体断片は、ヒト由来の培 細 JKに発現している GM2と特異的に結 合するため、 肺癌、 悪性黒^ 1、 flil腫瘍、 神経芽細 «等のヒト癌等の診断、 治 療において有用であると考えられる。抗体断片は、 IgG分子に比べ、分子量が小さ いため、 ヒト体内において速い標的移行性を示すことが期待される。  Since the antibody fragment of the present invention specifically binds to GM2 expressed in human-derived cultured JK, it can be used for diagnosis of human cancers such as lung cancer, malignant black ^ 1, flil tumor, and neuroblastoma. It may be useful in treatment. Since antibody fragments have a smaller molecular weight than IgG molecules, they are expected to show rapid target transfer in the human body.
本発明の抗体断片は、 単独で投与することも可能ではあるが、 通常は薬理学的 に許容される一つあるいはそれ以上の担体と一緒に混合し、 製剤学の技術分野に おいてよく知られる任意の方法により製造した医薬製剤として提供するのが望ま しい。  Although the antibody fragment of the present invention can be administered alone, it is usually mixed with one or more pharmacologically acceptable carriers and is well known in the technical field of pharmaceuticals. It is desirable to provide it as a pharmaceutical preparation produced by any method.
投与経路は、 治療に際して最も効果的なものを使用するのが望ましく、 経口投 与、 または口腔内、 気道内、 直腸内、 皮下、 筋肉内および静脈内等の非経口投与 をあげることができ、 蛋白質製剤の場合、 望ましくは静脈内投与をあげることが できる。  It is desirable to use the most effective route for treatment, including oral administration or parenteral administration such as buccal, respiratory, rectal, subcutaneous, intramuscular and intravenous administration. In the case of a protein preparation, intravenous administration can be preferably mentioned.
投与形態としては、 噴霧剤、 カプセル剤、 錠剤、 顆粒剤、 シロップ剤、 乳剤、 座剤、 注射剤、 軟膏、 テープ剤等があげられる。  Dosage forms include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like.
経口投与に適当な製剤としては、 乳剤、 シロップ剤、 カプセル剤、 錠剤、 散剤、 顆粒剤等があげられる。  Formulations suitable for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like.
？ L剤およびシロヅ Uのような液体調製物は、 水、 ショ糖、 ソルビトール、 果 糖等の糖類、ポリエチレングリコール、プロピレングリコ一ル等のグリコ一ル類、 ごま油、 ォリーブ油、 大豆油等の油類、 p—ヒドロキシ安息香酸エステル類等の 防腐剤、 ストロベリーフレーバー、 ペパーミント等のフレーバー類等を添加剤と して用いて製造できる。  ? Liquid preparations such as L-agents and syrup U include water, sugars such as sucrose, sorbitol, fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil, and soybean oil. And preservatives such as p-hydroxybenzoic acid esters, and flavors such as strawberry flavor and peppermint as additives.
カプセル剤、 錠剤、 散剤、 顆粒剤等は、 乳糖、 ブドウ糖、 ショ糖、 マンニト一 ル等の賦形剤、 デンプン、 アルギン酸ナトリウム等の崩壊剤、 ステアリン酸マグ ネシゥム、 タルク等の滑沢剤、 ポリビニルアルコール、 ヒドロキシプロピルセル ロース、 ゼラチン等の結合剤、 脂肪酸エステル等の界面活性剤、 グリセリン等の 可塑剤等を添加剤として用いて製造できる。 Capsules, tablets, powders, granules, etc. are excipients such as lactose, glucose, sucrose, mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate, talc, polyvinyl Binders such as alcohol, hydroxypropyl cellulose and gelatin, surfactants such as fatty acid esters, and glycerin It can be produced using a plasticizer or the like as an additive.
非経口投与に適当な製剤としては、 注射剤、 座剤、 噴霧剤等があげられる。 注射剤は、 塩溶液、 ブドウ糖溶液、 あるいは両者の混合物からなる担体等を用 いて調製される。  Formulations suitable for parenteral administration include injections, suppositories, sprays and the like. An injection is prepared using a carrier comprising a salt solution, a glucose solution, or a mixture of both.
座剤はカカオ脂、 水素化脂肪またはカルボン酸等の担体を用いて調製される。 また、 噴霧剤は該抗体断片そのもの、 ないしは受容者の口腔および気道粘膜を 刺激せず、 かつ該抗体断片を微細な粒子として分散させ吸収を容易にさせる担体 等を用いて調製される。  Suppositories are prepared using carriers such as cocoa butter, hydrogenated fats or carboxylic acids. Sprays are prepared using the antibody fragment itself or a carrier that does not irritate the oral and respiratory mucosa of the recipient and disperses the antibody fragment as fine particles to facilitate absorption.
担体として具体的には乳糖、 グリセリン等が例示される。 該抗体断片および用 いる担体の性質により、 エアロゾル、 ドライパウダー等の製剤が可能である。 ま た、 これらの非経口剤においても経口剤で添加剤として例示した成分を添カ卩する こともできる。  Specific examples of the carrier include lactose and glycerin. Formulations such as aerosols and dry powders can be made depending on the properties of the antibody fragment and the carrier used. In these parenteral preparations, the components exemplified as additives in the oral preparation can be added.
投与量または投与回数は、 目的とする治療効果、 投与方法、 治療期間、 年齢、 体重等により異なるが、 通常成人 1日当たり 10 g/kg〜8mg/kgである。  The dosage or frequency of administration varies depending on the desired therapeutic effect, administration method, treatment period, age, body weight, etc., but is usually 10 g / kg to 8 mg / kg per adult per day.
3 . 抗体の誘導体の作製〜ヒト化抗体とサイトカインとの融合蛋白質の作製3. Preparation of Antibody Derivatives-Preparation of Fusion Protein of Humanized Antibody and Cytokine
( 1 ) ヒト化抗体とサイトカインとの融合蛋白質をコードする遺伝子の構築 サイトカインをコードする遺伝子を適当な合 fi¾DNAを介して、 ヒト化抗体の HII 或いは L鎖をコ一ドする遺伝子の 5'末端或いは 3'末端に連結することにより、ヒト 化抗体とサイトカインとの融合蛋白質をコードする遺伝子を構築することができ る。 また、 サイトカインをコードする遺伝子を PCR法で増幅する際に、増幅用ブラ イマ一の 5'末端に適当な制限酵素の認識配列を導入し、 ヒト化抗体の H鎖或いは L 鎖をコードする遺伝子の 5'末端或いは 3'末端に連結することにより、 ヒト化抗体 とサイトカインとの融合蛋白質をコードする遺伝子を構築することができる。 サ ィトカインをコードする遺伝子は、染色体 DNA、 cDNAのいずれも用いることができ る。 構築したヒト化抗体とサイトカインの融合蛋白質をコードする遺伝子につい ては、 頁 1の （2 ) に記載の方法により、 塩基配列を決定し、 目的の配列であ ることを確認する。 (1) Construction of gene encoding fusion protein of humanized antibody and cytokine 5'-end of gene encoding HII or L chain of humanized antibody through appropriate synthetic fi¾DNA Alternatively, a gene encoding a fusion protein of a humanized antibody and a cytokine can be constructed by linking to the 3 'end. In addition, when a gene encoding a cytokine is amplified by PCR, a recognition sequence for an appropriate restriction enzyme is introduced into the 5 'end of the amplification primer, and the gene encoding the H chain or L chain of the humanized antibody is obtained. By linking to the 5 ′ end or 3 ′ end of the above, a gene encoding a fusion protein of a humanized antibody and a cytokine can be constructed. As the gene encoding a cytokine, either chromosomal DNA or cDNA can be used. The nucleotide sequence of the constructed gene encoding the fusion protein of the humanized antibody and cytokine is determined by the method described in (1) on page 1 to confirm that it is the desired sequence.
( 2 ) ヒト化抗体とサイ トカインとの融合蛋白質の発現ベクターの構築  (2) Construction of expression vector for fusion protein of humanized antibody and cytokine
の （4 ) および （7 ) に記載のヒト化抗体発現べクタ一上のヒト化抗体 の H鎖或いは L鎖をコードする遺伝子の一部またはすベてを、 本項 3の （1 ) に記 載のヒト化抗体とサイ トカインとの融合蛋白質をコードする遺伝子と置換するこ とによって、 ヒト化抗体とサイトカインとの融合蛋白質の発現ベクターを構築す ることができる。 例えば、 ヒト化抗体の H鎖の C末端にサイトカインが融合した融 合蛋白質を作製する場合は、 « 3の （1 ) において、 ヒト化抗体の CHをコード する遺伝子の 3'末端にサイトカインをコードする遺伝子を連結してヒ卜化抗体の CHとサイ トカインとの融合蛋白質をコードする遺伝子を構築し、 該遺伝子と: W 1の （4 ) および （7 ) に記載のヒト化抗体発現ベクター上のヒト化抗体の CHを コードする遺伝子を置換することにより、発現ベクターを構築することができる。Part or all of the gene encoding the H chain or L chain of the humanized antibody on the humanized antibody expression vector described in (4) and (7) of (3) above is described in (1) of this item 3. Record By substituting the gene encoding the fusion protein between the humanized antibody and cytokine described above, an expression vector for the fusion protein of the humanized antibody and cytokine can be constructed. For example, when preparing a fusion protein in which a cytokine is fused to the C-terminus of the H chain of a humanized antibody, in (3) of (3), the cytokine is encoded at the 3 'end of the gene encoding CH of the humanized antibody. And a gene encoding a fusion protein of CH and cytokine of the humanized antibody is constructed by connecting the gene to the humanized antibody expression vector described in (4) and (7) of W1. An expression vector can be constructed by substituting the gene encoding CH of the humanized antibody in (1).
( 3 ) ヒト化抗体とサイト力インとの融合蛋白質の安定発現 (3) Stable expression of fusion protein between humanized antibody and cytokin
本項 3の （2 ) に記載のヒト化抗体とサイトカインとの融合蛋白質の発現べク 夕一を用いて *J頁 1の （9 ) に記載した方法に従い、 ヒト化抗体とサイ トカイン との融合蛋白質の安定発現を行うことにより、 ヒト化抗体とサイ トカインとの融 合蛋白質を安定に発現する形質転 ί«を得、 その培養上清からヒト化抗体とサイ トカインとの融合蛋白質を精製し、 その分子量等を^ =斤することができる。  Using the expression vector of the fusion protein of the humanized antibody and the cytokine described in (2) of this section 3 * According to the method described in (9) of page 1 By performing stable expression of the fusion protein, a transformant that stably expresses the fusion protein between the humanized antibody and cytokine is obtained, and the fusion protein between the humanized antibody and cytokine is purified from the culture supernatant. And its molecular weight can be increased.
( 4 ) ヒト化抗体とサイト力インとの融合蛋白質の活性 fffi  (4) Activity of fusion protein between humanized antibody and cytokinin fffi
精製したヒト化抗体とサイトカインとの融合蛋白質の活性のうち、 ヒト化抗体 部分の活性、すなわち との結合活性、培 «^細«に対する結合活性は ELISA 法および蛍光抗体法等により測定できる。 また、 抗原陽性培養癌細胞株に対する 細胞障害活性は、 CDC活性、 ADCC活性等を測定し、 評価することができる。 一方、 サイ トカイン部分の活性は、 例えば、 該サイ トカインに対して濃度依存的な増殖 を示す培養細] «の増殖支持活性を指標に評価することができる [プロシ一ディ ングス ·ォブ 'ザ'ナショナル ·アカデミー'ォブ 'サイエンス（Proc. Natl. Acad. Sci. U. S.A. ) , 91, 9626 ( 1994)]。  Among the activities of the fusion protein of the purified humanized antibody and cytokine, the activity of the humanized antibody portion, that is, the binding activity to and the binding activity to culture medium can be measured by an ELISA method, a fluorescent antibody method, or the like. In addition, cytotoxic activity against antigen-positive cultured cancer cell lines can be evaluated by measuring CDC activity, ADCC activity and the like. On the other hand, the activity of the cytokine portion can be evaluated, for example, using the growth supporting activity of a culture cell exhibiting a concentration-dependent growth for the cytokine as an index. 'National Academy' of Science (Proc. Natl. Acad. Sci. USA), 91, 9626 (1994)].
ヒト化抗体とサイトカインとの融合蛋白質は、例えば、 GM2を発現している培養 マウス癌細 «を移植した野生型マウスに投与することで、 その ί¾®瘍効果を評 価することができ、 また、 ヒト化抗体単独、 サイト力イン単独或いはヒト化抗体 とサイトカインの同時投与と比較することにより、 生体内におけるより強い抗腫 瘍効果を評価することができる [キヤンサ一'ィムノロジ一'ィムノセラピー (Cancer Immunol. , Innnunother. ), 42, 88 ( 1996)]0 A fusion protein of a humanized antibody and a cytokine can be evaluated for its tumor effect by, for example, administering it to a wild-type mouse transplanted with a cultured mouse cancer cell expressing GM2. By comparing with humanized antibody alone, cytodynamic antibody alone or co-administration of humanized antibody and cytokine, stronger anti-tumor effect in vivo can be evaluated [Cancer-Immunology-Immunotherapy (Cancer) Immunol., Innnunother.), 42, 88 (1996)] 0
( 5 ) ヒト化抗体とサイト力インとの融合蛋白質の使用方法 本発明のヒト化抗体とサイ トカインとの融合蛋白質は、 ヒト由来の培養癌細胞 株に発現している GM2と特異的に結合し、 かつ CDC活性および ADCC活性等の細胞障 害活性を示すため、 肺癌、 悪性黒色腫、 脳腫瘍、 神経芽細 ¾等のヒト癌等の診 断、 治療において有用であると考えられる。 (5) How to use fusion protein between humanized antibody and cytokin The fusion protein of the humanized antibody of the present invention and cytokine specifically binds to GM2 expressed in a human-derived cultured cancer cell line and exhibits cytotoxic activities such as CDC activity and ADCC activity. It is considered to be useful in the diagnosis and treatment of human cancers such as lung cancer, malignant melanoma, brain tumors, and neuroblasts.
ヒト化抗体は、 ヒト以外の動物の抗体に比べ、 ヒト抗体のアミノ酸配列に由来 する部分がほとんどであるため、 ヒト体内において強い抗腫瘍効果を示し、 かつ 免疫原性を示さず、 その効果が長期間にわたり持続することが期待され、 更に、 融合させたサイ トカイン部分の活性により、 癌の近傍で免疫担当細胞を活性化で きることから、 ヒト化抗体単独、 サイト力イン単独或いはヒト化抗体とサイ ト力 インの同時投与等に比べ、 より強い抗腫瘍効果が期待され、 また、 サイト力イン の全身投与に比べ、 副作用の低減が期待される。  Humanized antibodies have a strong antitumor effect in the human body and do not show immunogenicity because most of the portion derived from the amino acid sequence of the human antibody compared to antibodies from non-human animals. It is expected to last for a long time, and furthermore, the activity of the fused cytokine can activate immunocompetent cells in the vicinity of the cancer. The anti-tumor effect is expected to be stronger than that of the simultaneous administration of site force, and the side effects are expected to be reduced compared to the systemic administration of site force.
本発明のヒト化抗体とサイ トカインとの融合蛋白質は、 単独で投与することも 可能ではあるが、 通常は薬理学的に許容される一つあるいはそれ以上の担体と一 緒に混合し、 製剤学の技術分野においてよく知られる任意の方法により製造した 医薬製剤として提供するのが望ましい。  Although the fusion protein of the humanized antibody of the present invention and cytokines can be administered alone, it is usually mixed with one or more pharmacologically acceptable carriers to prepare a preparation. It is desirable to provide it as a pharmaceutical preparation manufactured by any method well known in the technical field of science.
投与^ ¾は、 治療に際して最も効果的なものを使用するのが望ましく、 経口投 与、 または口腔内、 気道内、 直腸内、 皮下、 筋肉内および静脈内等の非経口投与 をあげることができ、 蛋白質製剤の場合、 望ましくは静脈内投与をあげることが できる。  It is desirable to use the most effective one for treatment, and it can be oral administration or parenteral administration such as buccal, respiratory, rectal, subcutaneous, intramuscular and intravenous. In the case of a protein preparation, intravenous administration can be preferably used.
投与形態としては、 噴霧剤、 カプセル剤、 錠剤、 顆粒剤、 シロップ剤、 乳剤、 座剤、 剤、 軟膏、 テー U等があげられる。  Dosage forms include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, agents, ointments, tablets and the like.
経口投与に適当な製剤としては、 乳剤、 シロップ剤、 カプセル剤、 錠剤、 散剤、 顆粒剤等があげられる。  Formulations suitable for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like.
乳剤およびシロッ 7¾のような液体調製物は、 水、 ショ糖、 ソルビトール、 果 糖等の糖類、ポリエチレングリコ一ル、 プロピレングリコール等のグリコ一ル類、 ごま油、 オリ一ブ油、 大豆油等の油類、 p—ヒドロキシ安息香酸エステル類等の 防腐剤、 ストロベリーフレーバー、 ペパーミント等のフレーバー類等を添加剤と して用いて製造できる。  Emulsions and liquid preparations such as syrup can be obtained from sugars such as water, sucrose, sorbitol, fructose, glycols such as polyethylene glycol and propylene glycol, sesame oil, olive oil, soybean oil, etc. It can be produced using preservatives such as oils and p-hydroxybenzoic acid esters, and flavors such as strawberry flavor and peppermint as additives.
カプセル剤、 錠剤、 散剤、 顆粒剤等は、 乳糖、 ブドウ糖、 ショ糖、 マンニト一 ル等の賦形剤、 デンプン、 アルギン酸ナトリウム等の崩壊剤、 ステアリン酸マグ ネシゥム、 タルク等の滑沢剤、 ポリビニルアルコール、 ヒドロキシプロビルセル ロース、 ゼラチン等の結合剤、 脂肪酸エステル等の界面活性剤、 グリセリン等の 可塑剤等を添加剤として用いて製造できる。 Capsules, tablets, powders, granules, etc. are excipients such as lactose, glucose, sucrose, mannitol, disintegrants such as starch, sodium alginate, mag stearate It can be manufactured using lubricants such as nesidum and talc, binders such as polyvinyl alcohol, hydroxypropyl cellulose and gelatin, surfactants such as fatty acid esters, and plasticizers such as glycerin as additives.
非経口投与に適当な製剤としては、 注射剤、 座剤、 噴霧剤等があげられる。 翅剤は、 塩溶液、 ブドウ糖溶液、 あるいは両者の混合物からなる担体等を用 いて調製される。  Formulations suitable for parenteral administration include injections, suppositories, sprays and the like. The wing preparation is prepared using a carrier comprising a salt solution, a glucose solution, or a mixture of both.
座剤はカカオ脂、 水素化脂肪またはカルボン酸等の担体を用いて調製される。 また、 噴霧剤は該融合蛋白質そのもの、 ないしは受容者の口腔および気道粘膜 を刺激せず、 かつ該融合蛋白質を微細な粒子として分散させ吸収を容易にさせる 担体等を用いて調製される。  Suppositories are prepared using carriers such as cocoa butter, hydrogenated fats or carboxylic acids. A spray is prepared using the fusion protein itself or a carrier which does not irritate the oral and respiratory tract mucosa of the recipient and disperses the fusion protein as fine particles to facilitate absorption.
担体として具体的には乳糖、 グリセリン等が例示される。 該融合蛋白質および 用いる担体の性質により、 エアロゾル、 ドライパウダー等の製剤が可能である。 また、 これらの非経口剤においても経口剤で添加剤として例示した成分を添カロす ることもできる。  Specific examples of the carrier include lactose and glycerin. Depending on the properties of the fusion protein and the carrier used, preparations such as aerosols and dry powders can be made. In addition, in these parenteral preparations, the components exemplified as additives in the oral preparation can be added.
投与量または投与回数は、 目的とする治療効果、 投与方法、 治療期間、 年齢、 体重等により異なるが、 通常成人 1日当たり lO zg/k Smg/kgである。 図面の簡単な説明  The dose or frequency of administration varies depending on the desired therapeutic effect, administration method, treatment period, age, body weight, etc., but is usually lO zg / k Smg / kg per adult per day. BRIEF DESCRIPTION OF THE FIGURES
第 1図はプラスミド pBSA-Bの造成工程を示した図である。 FIG. 1 is a diagram showing a construction process of plasmid pBSA-B.
第 2図はプラスミド pBS ihCァ卜 IL- 2の造成工程を示した図である。 FIG. 2 is a view showing a construction process of plasmid pBS ihCat IL-2.
第 3図はプラスミド pBShCァ卜 IL- 2の造成工程を示した図である。 FIG. 3 is a diagram showing a construction process of plasmid pBShCIL-2.
第 4図はプラスミド pKANTEX8969-hIL2の造成工程を示した図である。 FIG. 4 is a view showing a construction process of plasmid pKANTEX8969-hIL2.
第 5図は精製した抗 GM2CD職植抗体 KM8969と精製した融合蛋白質 M8969— hIL- 2 の SDS-PAGE (4〜15%グラジェントゲルを使用） の電気泳動パターンを示した図で ある。左側が非還元条件、右側が還元条件でそれぞれ電気泳動を行った図である。 レーン 1が低分子量マ一力一、 2か 1M8969、 3が IM8969—hIL-2、 4が低分子量マーカ 一、 5 «8969, 6か 1M8969— hIL-2の泳動パターンをそれぞれ示す。 FIG. 5 is a diagram showing an SDS-PAGE (using a 4 to 15% gradient gel) electrophoresis pattern of the purified anti-GM2CD antibody KM8969 and the purified fusion protein M8969-hIL-2. The left side shows the results of electrophoresis under non-reducing conditions, and the right side shows electrophoresis under reducing conditions. Lane 1 shows the migration patterns of low molecular weight proteins, 2 or 1 M8969, 3 shows IM8969-hIL-2, 4 shows low molecular weight markers 1 and 5 «8969, 6 or 1M8969-hIL-2, respectively.
第 6図は精製した抗 GM2CD職植抗体 KM8969と精製した融合蛋白質 KM8969— hi 2 の GM2との結合活性を抗体濃度を変ィ匕させて測定した図である。 縦軸は GM2との結 合活性、横軸は抗体濃度をそれぞれ示す。〇が KM8969、 參が KM8969— hIL-2の活性 をそれぞれ示す。 FIG. 6 is a graph showing the binding activity of the purified anti-GM2CD artificial antibody KM8969 and the purified fusion protein KM8969-hi 2 to GM2 measured by varying the antibody concentration. The vertical axis shows the binding activity to GM2, and the horizontal axis shows the antibody concentration. 〇 is KM8969, reference is KM8969—hIL-2 activity Are respectively shown.
第 7図は hIL- 2と精製した融合蛋白質 KM8969— hIL- 2の hIL- 2依存性細胞 CTLL- 2に 対する増殖支持活性を各蛋白質の濃度を変ィ匕させて測定した図である。 縦軸は増 殖支持活性、 横軸は蛋白質濃度をそれぞれ示す。 〇が hIL-2、 參が KM8969— hIL-2 の活{生をそれぞれ示す。 FIG. 7 shows the results of measuring the growth supporting activity of hIL-2 and the purified fusion protein KM8969-hIL-2 on hIL-2-dependent cell CTLL-2 by varying the concentration of each protein. The vertical axis shows the growth supporting activity, and the horizontal axis shows the protein concentration. 〇 indicates hIL-2 and reference indicates KM8969—activity of hIL-2.
第 8図は精製した抗 GM2CDR^植抗体 KM8969と精製した融合蛋白質 KM8969-hIL-2 によるヒトエフヱク夕一細胞の活性化とそれに伴う細胞障害活性を測定した図で ある。 縦軸は細胞障害活性、 横軸は用いた蛋白質をそれぞれ示す。 口が KM8969、 園が KM8969— hIL-2の活性をそれぞれ示す。 発明を するための最良の形態 FIG. 8 is a graph showing the activation of human EFX-1 cells by the purified anti-GM2CDR ^ -implanted antibody KM8969 and the purified fusion protein KM8969-hIL-2 and the resulting cytotoxicity. The vertical axis indicates the cytotoxic activity, and the horizontal axis indicates the protein used. The mouth shows the activity of KM8969, and the garden shows the activity of KM8969-hIL-2. BEST MODE FOR CARRYING OUT THE INVENTION
以下に、 本発明の実施例を示すが、 これにより本発明の範囲が限定されるもの ではない。 Hereinafter, examples of the present invention will be described, but the scope of the present invention is not limited thereto.
m i . 抗 GM2ヒ卜化抗体とサイト力インとの融合蛋白質の作製  Preparation of fusion protein between anti-GM2 humanized antibody and cytokinin
抗 GM2ヒト化抗体とサイトカインとの融合蛋白質の具体例として、抗 GM2CDR^植 抗体 KM8969と hIL- 2の融合蛋白質である KM8969— hIL- 2を以下の様にして作製し、 その活性評価を行った。  As a specific example of a fusion protein of a humanized anti-GM2 antibody and a cytokine, KM8969-hIL-2, a fusion protein of the anti-GM2 CDR ^ plant antibody KM8969 and hIL-2, was prepared as follows and its activity was evaluated. Was.
1 . hCァ 1と hIL_2との融合蛋白質をコードする cDNAの構築  1. Construction of cDNA encoding fusion protein between hCα1 and hIL_2
( 1 ) hCァ 1の cDNAの 3，末端の約 65塩基と成熟型 hIL-2の完全長 cDNAから成る cMA を有するプラスミ ド pBSAhCァ卜 IL-2の構築  (1) Construction of plasmid pBSAhCat IL-2 having cMA consisting of the full-length cDNA of mature hIL-2 and about 65 bases at the 3 and terminus of hCa1 cDNA
プラスミド pBluescript SK (-) (Stratagene社製）の 3^gを 10^1の lOmMトリス- 塩酸 (pH7.5)、 10mM塩化マグネシウムおよび lmM DTTから成る緩衝液に加え、 更 に 10単位の制限酵素 Apal (宝酒造社製）を加えて 37°Cで 1時間反応させた。該反応 液をエタノール TOし、 ΙΟχζΙの 20 Μトリス-塩酸（pH8.5)、 lOmM塩化マグネシゥ ム、 lmM DTTおよび lOOmM塩ィ匕カリウムから成る緩衝液に加え、 更に 10単位の制限 酵素 Ba il (宝酒造社製） を加えて 30°Cで 1時間反応させた。 該反応液をァガ口一 スゲル電気泳動にて分画し、 QIAquick Gel Extraction Kit (QIAGEN社製） を用い て添付の説明書に従い、 約 2.95kbの Apal-BamHI断片を約 2 g回収した。  Add 3 ^ g of plasmid pBluescript SK (-) (Stratagene) to a buffer consisting of 10 ^ 1 lOmM Tris-HCl (pH7.5), 10mM magnesium chloride and lmM DTT, and add 10 units of restriction enzyme. Apal (Takara Shuzo) was added and reacted at 37 ° C for 1 hour. The reaction solution was subjected to ethanol TO, added to a buffer solution consisting of 20% Tris-hydrochloric acid (pH 8.5), 10 mM magnesium chloride, lmM DTT and 100 mM potassium chloride, and further 10 units of restriction enzyme Bail ( (Takara Shuzo) and reacted at 30 ° C for 1 hour. The reaction solution was fractionated by agarose gel electrophoresis, and about 2 g of an Apal-BamHI fragment of about 2.95 kb was recovered using a QIAquick Gel Extraction Kit (manufactured by QIAGEN) according to the attached instruction.
次に、 配列番号 3、 4に記載の塩基配列をそれぞれ有する合卿 NAを自動 DNA合成 機 （380A、 Applied Biosystems社製） を用いて合成した。 得られた合 fi¾DNAの 0.3 〃gずつを 15 z lの滅菌水に加え、 65°Cで 5分間加熱した。 該反応液を室温にて 30 分間放置した後、 2^ 1の 10倍緩衝液 [500IDMトリス-塩酸 (pH7.6 )、 lOOmM塩ィ匕マグネ シゥム、 50mM DTT]と 2 z lの lOmM ATPを加え、 更に 10単位の T4 Polynucleotide Kinase (宝酒造社製） を加えて 37°Cで 30分間反応させ、 5'末端をリン酸ィ匕した。 次に、 上記で得られたプラスミ ド pBluescript SK ( -)由来の Apal-BajnHI断片 0. 1 gとリン酸ィ匕合 fi¾ A0.05〃gを全量 10 1の滅菌水に加え、 DNA ligation Kit Ver.2 (宝酒造社製） を用いて使用説明書に従い、 連結した。 この様にして得られ た組換えプラスミド DNA溶液を用いて大腸菌 DH5ひ株 （Stratagene社製） を形質転 換し、 第 1図に示したプラスミド pBSA-Bを得た。 得られたプラスミドの 10 /gを用 い、 AutoRead Sequencing Kit (Pharmacia社製） に添付の説明書に従って反応後、 A. L.F . DNA Sequencer (Pharmacia社製） により電気泳動し、 塩基配列を決定した 結果、 目的の DNAがクローニングされたプラスミ ドが得られたことを ¾t した。 次に、 上記で得られたプラスミド pBSA-Bの 3 gを 10 1の 50mMトリス-塩酸 ( PH7.5) 、 lOOmMJ 化ナトリウム、 lOmM塩化マグネシウムおよび ImM DTTから成る緩 衝液に加え、 更に 10単位の制限酵素 EcoRI (宝酒造社製） を加えて 37°Cで 1時間反 応させた。該反応液をエタノール沈殿し、 10〃1の 33mMトリス -酢酸（pH7.9)、 66mM 酢酸カリウム、 lOniMS酸マグネシウム、 0.5mM DTTおよび 100 /g/ml BSAから成る 緩衝液に加え、更に 10単位の制限酵素 Smal (宝酒造社製）を加えて 30°Cで 1時間反 応させた。 該反応液をァガロースゲル電気泳動にて分画し、 QIAquick Gel Extraction Kit (QIAGEN社製） を用いて、 寸の説明書に従い、 約 3.00kbの EcoRI- Smal断片を約 2^g回収した。 Next, Shiro NA having the nucleotide sequence of SEQ ID NO: 3 or 4 was synthesized using an automatic DNA synthesizer (380A, manufactured by Applied Biosystems). 0.3 of the obtained fi¾DNA 〃G each was added to 15 zl of sterilized water, and heated at 65 ° C for 5 minutes. The reaction solution was left at room temperature for 30 minutes, and 2 × 1 10-fold buffer [500 IDM Tris-hydrochloric acid (pH7.6), 100 mM Shiridani Magnesium, 50 mM DTT] and 2 zl lOmM ATP were added. Further, 10 units of T4 Polynucleotide Kinase (manufactured by Takara Shuzo Co., Ltd.) was added and reacted at 37 ° C. for 30 minutes to phosphorylate the 5 ′ end. Next, 0.1 g of the Apal-BajnHI fragment derived from the plasmid pBluescript SK (-) obtained above and 0.05 g of phosphoric acid conjugated fi¾A were added to a total of 101 sterile water, and the DNA ligation Kit was used. Ver.2 (Takara Shuzo) was connected according to the instruction manual. Escherichia coli DH5 strain (manufactured by Stratagene) was transformed using the recombinant plasmid DNA solution obtained in this manner to obtain a plasmid pBSA-B shown in FIG. Using 10 / g of the obtained plasmid, it was reacted with the AutoRead Sequencing Kit (Pharmacia) according to the attached instructions, followed by electrophoresis using ALF. DNA Sequencer (Pharmacia), and the nucleotide sequence was determined. It was confirmed that a plasmid in which the target DNA was cloned was obtained. Next, 3 g of the plasmid pBSA-B obtained above was added to a buffer consisting of 101 50 mM Tris-HCl (PH7.5), 100 mM sodium chloride, 100 mM magnesium chloride and ImM DTT, and an additional 10 units of the buffer were added. The restriction enzyme EcoRI (Takara Shuzo) was added and reacted at 37 ° C for 1 hour. The reaction solution was precipitated with ethanol, and added to 10〃1 of a buffer solution consisting of 33 mM Tris-acetic acid (pH 7.9), 66 mM potassium acetate, magnesium lOniMS, 0.5 mM DTT and 100 / g / ml BSA. The restriction enzyme Smal (Takara Shuzo) was added thereto and reacted at 30 ° C for 1 hour. The reaction solution was fractionated by agarose gel electrophoresis, and about 2 ^ g of an EcoRI-Smal fragment of about 3.00 kb was recovered using a QIAquick Gel Extraction Kit (manufactured by QIAGEN) according to the specifications.
次に、 成熟型 hIL-2の完全長 cDNAを含むプラスミ ド pILL4 [ァグリカルチュラル' アンド'バイオロジカル 'ケミストリ一 (Agri Biol . Chem. ) , 51 , 1135 ( 1987) ] を铸型として、以下に示す PCRを行った。プラスミ ド pILL4の lngを 100 z lの反応液 (1倍濃度の Ex Taq buffer (宝酒造社製)、 200 /M dNTPsヽ 1.0 /M revlプライマー (配列番号 5 )、 1.0〃M fw2プライマ一 (配列番号 6 )および 2.5単位の TaXaRa Ex Taq DNA polymerase (宝酒造社製)） に加え、 100〃1の鉱油で覆い、 DNAサ一マルサイク ラー （PJ480、 PERKIN ELMER社製） にセットし、 94°Cにて 3分間反応後、 96°Cにて 30秒間、 55°Cにて 1分間、 72°Cにて 1分間のサイクルを 30サイクル行い、 最後に 72 °Cにて 7分間の反応を行った。該反応液をエタノール沈殿し、 30 1の 50mMトリス- 塩酸 (pH7.5)、 lOOmM塩化ナトリウム、 lOmM塩化マグネシウムおよび ImMDTTから 成る緩衝液に加え、 更に 10単位の制限酵素 EcoRI (宝酒造社製） を加えて 37°Cで 1 時間反応させた。該反応液をエタノール沈殿し、 10 1の 33mMトリス-酢酸（pH7.9 ) 、 66mM酢酸カリウム、 lOmM酢酸マグネシウム、 0.5m DTTおよび 100 /g/mlBSA から成る緩衝液に加え、 更に 10単位の制限酵素 Smal (宝酒造社製） を加えて 30°C で 1時間反応させた。 該反応液をァガロースゲル電気泳動にて分画し、 QIAquick Gel Extraction Kit (QIAGEN社製） を用いて添付の説明書に従い、 約 0.41kbの EcoRI-Smal断片を約 1 zg回収した。 Next, plasmid pILL4 [Agricultural 'and' biological 'Chemistry (Agri Biol. Chem.), 51, 1135 (1987)] containing the mature full-length cDNA of hIL-2 PCR was performed as shown in the following. Lng of plasmid pILL4 was added to a 100 zl reaction solution (1x concentration of Ex Taq buffer (Takara Shuzo), 200 / M dNTPs ヽ 1.0 / M revl primer (SEQ ID NO: 5), 1.0〃M fw2 primer (SEQ ID NO: 5) 6) and 2.5 units of TaXaRa Ex Taq DNA polymerase (Takara Shuzo Co., Ltd.)), cover with 100〃1 mineral oil, set in a DNA thermal cycler (PJ480, PERKIN ELMER), and set at 94 ° C. After reacting for 3 minutes, 30 cycles of 96 ° C. for 30 seconds, 55 ° C. for 1 minute, 72 ° C. for 1 minute were performed 30 times, and finally, the reaction was performed at 72 ° C. for 7 minutes. The reaction solution was precipitated with ethanol, and 30 1 of 50 mM Tris- In addition to a buffer solution consisting of hydrochloric acid (pH 7.5), 100 mM sodium chloride, 100 mM magnesium chloride and ImMDTT, 10 units of a restriction enzyme EcoRI (Takara Shuzo Co., Ltd.) was further added and reacted at 37 ° C for 1 hour. The reaction was precipitated with ethanol and added to 10 1 of a buffer consisting of 33 mM Tris-acetic acid (pH 7.9), 66 mM potassium acetate, 10 mM magnesium acetate, 0.5 mM DTT and 100 / g / ml BSA. The enzyme Smal (Takara Shuzo) was added and reacted at 30 ° C for 1 hour. The reaction solution was fractionated by agarose gel electrophoresis, and about 1 zg of an EcoRI-Smal fragment of about 0.41 kb was recovered using a QIAquick Gel Extraction Kit (manufactured by QIAGEN) according to the attached instruction.
次に、 上記で得られたプラスミド pBSA-Bの EcoRI- Smal断片 0.1 gと成熟型 hlL- 2の完全長 cDNAの PCR産物の EcoRI- Smal断片 0.1〃gを全量 10〃1の滅菌水に加え、 DNA ligation Kit Ver.2 (宝酒造社製） を用いて使用説明書に従い、 連結した。 この様にして得られた組換えプラスミ ド DNA溶液を用いて大腸菌 DH5ひ株を形質転 換した。 形質転 »の 10個のクローンより各プラスミド DNAを調製し、 AutoRead Sequencing Kit (Pharmacia社製） に添付の説明書に従って反応後、 A.L.F. DNA Sequencer (Pharmacia社製） により電気泳動し、 挿入された cDNAの塩基配列を決 定した結果、 目的の塩基配列を有する第 2図に示したプラスミド pBSAhCァ卜 IL-2 を得た。  Next, 0.1 g of the EcoRI-Smal fragment of the plasmid pBSA-B obtained above and 0.1 g of the EcoRI-Smal fragment of the PCR product of the full-length cDNA of mature hlL-2 were added to a total of 10-1 sterile water. The DNA was ligated using DNA Ligation Kit Ver.2 (Takara Shuzo) according to the instruction manual. Using the recombinant plasmid DNA solution thus obtained, Escherichia coli DH5 strain was transformed. Each plasmid DNA was prepared from 10 clones of the transformation », followed by reaction with the AutoRead Sequencing Kit (Pharmacia) according to the attached instructions, followed by electrophoresis using ALF DNA Sequencer (Pharmacia), and the inserted cDNA. As a result of determining the nucleotide sequence, plasmid pBSAhCat IL-2 shown in FIG. 2 having the target nucleotide sequence was obtained.
( 2 ) hCァ 1の完全長 cDNAと成熟型 hIL- 2の完全長 cDNAから成る cDNAを有するブラ スミ ド pBShCァ卜 IL-2の構築  (2) Construction of plasmid pBShCat IL-2 containing cDNA consisting of full-length cDNA of hCα1 and full-length cDNA of mature hIL-2
まず、 特開平 10-257893に記載のプラスミド pBShCァ 1の 3 gを 10〃1の 10 トリ ス-塩酸 (pH7.5) 、 lOmM塩化マグネシウムおよび ImM DTTから成る緩衝液に加え、 更に 10単位の制限酵素 Apal (宝酒造社製）を加えて 37°Cで 1時間反応させた。該反 応液をエタノール燃し、 10〃1の 50mMトリス-塩酸 (pH7.5)、 lOmM塩化マグネシ ゥム、 lmM DTTおよび lOOmM塩化ナトリゥムから成る緩衝液に加え、 更に 10単位の 制限酵素 EcoT22I (宝酒造社製） を加えて 37 Cで 1時間反応させた。 該反応液をァ ガロースゲル電気泳動にて分画し、 QIAquick Gel Extraction Kit (QIAGEN社製） を用いて添付の説明書に従い、 約 0.92kbの ApaI-EcoT22I断片を約 l g回収した。 次に、上記実施例 1の 1項（ 1 )で得られたプラスミド pBSAhCァ卜 IL- 2の 3 g を 10 1の lOmMトリス-塩酸 (pH7.5) 、 10mM^化マグネシウムおよび lmM DTTから 成る緩衝液に加え、 更に 10単位の制限酵素 Apal (宝酒造社製） を加えて 37°Cで 1 時間反応させた。該反応液をエタノール沈殿し、 10〃1の 50mMトリス-塩酸 (pH7.5 ) 、 10mM塩化マグネシウム、 ImM DTTおよび lOOmM塩化ナトリウムから成る緩衝液 に加え、 更に 10単位の制限酵素 EcoT22I (宝酒造社製） を加えて 37°Cで 1時間反応 させた。 該反応液をァガロースゲル電気泳動にて分画し、 QIAquick Gel First, 3 g of the plasmid pBShC1 described in JP-A-10-257893 was added to a buffer solution consisting of 10〃1 of 10-tris-hydrochloric acid (pH 7.5), 10 mM magnesium chloride and ImM DTT. The restriction enzyme Apal (manufactured by Takara Shuzo) was added and reacted at 37 ° C for 1 hour. The reaction solution was burned with ethanol, added to 10〃1 of a buffer solution consisting of 50 mM Tris-HCl (pH 7.5), 10 mM magnesium chloride, lmM DTT and 100 mM sodium chloride, and further added 10 units of restriction enzyme EcoT22I ( (Takara Shuzo) and reacted at 37 C for 1 hour. The reaction solution was fractionated by agarose gel electrophoresis, and about lg of an ApaI-EcoT22I fragment of about 0.92 kb was recovered using a QIAquick Gel Extraction Kit (manufactured by QIAGEN) according to the attached instruction. Next, 3 g of the plasmid pBSAhCat IL-2 obtained in paragraph (1) of Example 1 above was composed of 10 1 lOmM Tris-HCl (pH 7.5), 10 mM magnesium chloride, and lmM DTT. Add 10 units of restriction enzyme Apal (Takara Shuzo) to the buffer at 37 ° C. Allowed to react for hours. The reaction solution was precipitated with ethanol, added to a buffer solution consisting of 10〃1 of 50 mM Tris-HCl (pH 7.5), 10 mM magnesium chloride, ImM DTT and 100 mM sodium chloride, and further added 10 units of restriction enzyme EcoT22I (Takara Shuzo Co., Ltd.). ) Was added and reacted at 37 ° C for 1 hour. The reaction solution was fractionated by agarose gel electrophoresis, and QIAquick Gel
Extraction Kit (QIAGEN社製） を用いて添付の説明書に従い、 約 3.40kbの Apal-Using an Extraction Kit (manufactured by QIAGEN), according to the attached instructions, the Apal-
EcoT22I断片を約 2〃g回収した。 About 2 μg of the EcoT22I fragment was recovered.
次に、 上記で得られたプラスミ ド pBShCァ 1の ApaI-EcoT22I断片 0.1 gとプラス ミ ド pBS厶 hCァ卜 IL- 2の ApaI-EcoT22I断片 0.1 zgを全量 10 1の滅菌水に加え、 DNA ligation Kit Ver.2 (宝酒造社製） を用いて使用説明書に従い、 連結した。 この 様にして得られた組換えプラスミド DNA溶液を用いて大腸菌 DH5 o:株を形質転換し、 第 3図に示したプラスミド pBShCァ卜 IL- 2を得た。得られたプラスミ ドの 10〃gを用 い、 AutoRead Sequencing Kit (Pharmacia社製） に添付の説明書に従って反応後、 A.L.F. DNA Sequencer (Pharmacia社製） により電気泳動し、 揷入された cDNAの塩 基配列を決定した結果、 目的の塩基配列を有するプラスミ ドが得られたことを確 し/こ。  Next, 0.1 g of the ApaI-EcoT22I fragment of the plasmid pBShCα1 obtained above and 0.1 zg of the ApaI-EcoT22I fragment of the plasmid pBS muhCat IL-2 were added to 10 1 of sterile water, and the DNA was added. Ligation Kit Ver.2 (Takara Shuzo Co., Ltd.) was used in accordance with the instruction manual. Escherichia coli DH5o: strain was transformed using the recombinant plasmid DNA solution obtained in this manner to obtain plasmid pBShCatIL-2 shown in FIG. After using 10 µg of the obtained plasmid and reacting with the AutoRead Sequencing Kit (Pharmacia) according to the attached instructions, electrophoresis was performed using an ALF DNA Sequencer (Pharmacia), and the salt of the inserted cDNA was analyzed. As a result of determining the base sequence, confirm that a plasmid having the desired base sequence was obtained.
2 . KM8969— hIL-2の動物細胞を用いた安定発現  2. KM8969—Stable expression of hIL-2 in animal cells
( 1 ) KM8969— hIL-2の安定発現べクタ一の構築  (1) KM8969—Construction of hIL-2 stable expression vector
特開平 10- 257893に記載の抗 GM2CDR^植抗体 KM8969の安定発現べク夕― pKANTEX796HM2Lm-28No.1と上記 例 1の 1項 （ 2 ) で得られた hCァ 1と hIL- 2と の融合蛋白質をコードする cDNAを有するプラスミド pBShCァ卜 IL- 2を用いて KM8969— hIL-2の安定発現べクタ一を以下の様にして構築した。  Fusion of stable expression vector of anti-GM2CDR ^ plant antibody KM8969 described in JP-A-10-257893-pKANTEX796HM2Lm-28No.1 with hCα1 and hIL-2 obtained in Section 1 (2) of Example 1 above A stable expression vector of KM8969-hIL-2 was constructed as follows using plasmid pBShCat IL-2 having cDNA encoding the protein.
特閧平 10- 257893に記載のプラスミド p ANTEX796HM2Lm- 28No. lの 3〃gを 10 1の lOmMトリス-塩酸 (pH7.5)、 10mM塩化マグネシウムおよび lmMDTTから成る緩衝液 に加え、更に 10単位の制限酵素 Apal (宝酒造社製）を加えて 37°Cで 1時間反応させ た。該反応液をエタノール «し、 10 /1の 2幢トリス-塩酸（pH8.5)、 10mM塩ィ匕 マグネシウム、 ImM DTTおよび ΙΟΟιΜ塩ィ匕カリウムから成る緩衝液に加え、 更に 10 単位の制限酵素 BamHI (宝酒造社製） を加えて 30°Cで 1時間反応させた。 該反応液 をァガロースゲル電気泳動にて分画し、 QIAquick Gel Extraction Kit (QIAGEN 社製）を用いて添付の説明書に従い、約 12.57kbの Apal-BamHI断片を約 2 g回収し た。 次に、 実施例 1の 1項 （2 ) で得られたプラスミド pBShCァ卜 IL-2の 3 zgを 10 1の 10mMトリス-塩酸 (ρΗ7.5) 、 ΙΟιηΜ塩化マグネシウムおよび ImM DTTから成る 緩衝液に加え、 更に 10単位の制限酵素 Apal (宝酒造社製）を加えて 37°Cで 1時間反 応させた。該反応液をエタノールミ し、 10〃1の 20m トリス-塩酸 (pH8.5) , lOmM 塩化マグネシウム、 ImM DTTおよび lOOmM塩ィ匕カリウムから成る緩衝液に加え、 更 に 10単位の制限酵素 Ba il (宝酒造社製） を加えて 30°Cで 1時間反応させた。 該反 応液をァガロースゲル電気泳動にて分画し、 QIAquick Gel Extraction Kit (QIAGEN 社製） を用いて添付の説明書に従い、 約 1.45kbの Apal- BamHI断片を約 2/ g回収し た。 3 〃g of plasmid pANTEX796HM2Lm-28No.l described in 10-257893 is added to a buffer consisting of 101 lOmM Tris-HCl (pH 7.5), 10 mM magnesium chloride and lmMDTT, and an additional 10 units The restriction enzyme Apal (Takara Shuzo) was added and reacted at 37 ° C for 1 hour. The reaction solution is added to ethanol, added to a buffer containing 10/1 2 tris-hydrochloric acid (pH 8.5), 10 mM magnesium salt, ImM DTT and potassium salt, and further 10 units of restriction enzyme. BamHI (manufactured by Takara Shuzo) was added and reacted at 30 ° C. for 1 hour. The reaction solution was fractionated by agarose gel electrophoresis, and about 2 g of an Apal-BamHI fragment of about 12.57 kb was recovered using a QIAquick Gel Extraction Kit (manufactured by QIAGEN) according to the attached instruction. Next, 3 zg of the plasmid pBShCat IL-2 obtained in section 1 (2) of Example 1 was buffered with 10 1 of 10 mM Tris-hydrochloride (ρΗ7.5), ΙΟιηΜ magnesium chloride and ImM DTT. In addition, 10 units of the restriction enzyme Apal (Takara Shuzo) was further added and reacted at 37 ° C for 1 hour. The reaction mixture was diluted with ethanol and added to a buffer consisting of 10〃1 of 20m Tris-hydrochloric acid (pH 8.5), lOmM magnesium chloride, ImM DTT and lOOmM potassium chloride, and further 10 units of restriction enzyme Bail (Takara Shuzo) and reacted at 30 ° C. for 1 hour. The reaction solution was fractionated by agarose gel electrophoresis, and about 2 / g of an Apal-BamHI fragment of about 1.45 kb was recovered using a QIAquick Gel Extraction Kit (manufactured by QIAGEN) according to the attached instruction.
次に、 上記で得られたプラスミ ド pKANTEX796HM2Lm- 28No. l由来の Apal- BamHI断 片 0.1 gとプラスミド pBShCァ卜 IL- 2由来の Apal- BajnHI断片 0.1〃gを全量 10〃1の 滅菌水に加え、 DNA ligation Kit Ver.2 (宝酒造社製） を用いて使用説明書に従 い、 連結した。 この様にして得られた組換えプラスミド DNA溶液を用いて大腸菌 DH5ひ株を形質転換し、 第 4図に示した KM8969— hIL- 2の安定発現べクタ一 pKANTEX8969-hIL2を得た。 得られたプラスミドの 10 zgを用い、 AutoRead Sequencing Kit (Pharmacia社製） に添付の説明書に従って反応後、 A.L.F. DNA Sequencer (Pharmacia社製） により電気泳動し、 塩基配列を決定した結果、 目的 の DNAがクローニングされたプラスミ ドが得られたことを確認した。  Next, 0.1 g of the Apal-BamHI fragment derived from the plasmid pKANTEX796HM2Lm-28No.l obtained above and 0.1 g of the Apal-BajnHI fragment derived from the plasmid pBShCatIL-2 in a total amount of 10-1 sterile water. In addition, ligation was performed using DNA ligation Kit Ver.2 (Takara Shuzo) according to the instruction manual. Escherichia coli DH5 strain was transformed using the recombinant plasmid DNA solution obtained in this manner to obtain a stable expression vector pKANTEX8969-hIL2 of KM8969-hIL-2 shown in FIG. Using 10 zg of the obtained plasmid, it was reacted with the AutoRead Sequencing Kit (Pharmacia) according to the attached instructions, followed by electrophoresis with ALF DNA Sequencer (Pharmacia), and the base sequence was determined. It was confirmed that a plasmid cloned was obtained.
( 2 ) KM8969— hIL- 2の動物細胞での発現  (2) KM8969—expression of hIL-2 in animal cells
上記実施例 1の 2項 （1 ) で得られた KM8969— hIL- 2の安定発現べクタ一 p ANTEX8969-hIL2を用いて KM8969— hIL-2の動物細胞での発現を以下の様にして 行った。  The expression of KM8969-hIL-2 in animal cells was carried out as follows using pANTEX8969-hIL2, which is a stable expression vector of KM8969-hIL-2 obtained in paragraph (2) of Example 1 above. Was.
発現べクタ一 p ANTEX8969-hIL2の 4〃gを 4x l06細胞のラットミエローマ細胞株 YB2/0細胞 (ATCC CRL1662) へエレクトロポレーシヨン法 [サイ トテクノロジ一 (Cytotechnology), 3, 133 ( 1990)]により導入後、 40mlの RPMI640-FBS(10) ( FBS(GIBC0 BRL社製）を 10%含む RPMI1640培地） に懸濁し、 96ウェルマイク口タイ夕 一プレート （ffi¾ベークライ ト社製） に 200〃1/ゥエルずつ分注した。 5%C02イン キュベ一夕一内で 37°C、 24時間培養した後、 G418を 0.5mg/mlになる様に添加して 1 〜2週間培養した。 G418耐性を示す形質転賺のコロニーが出現し、コンフルェン トになったゥエルより培養上清を回収し、 上清中の抗 GM2CDR^植抗体 KM8969に由 来する GM2結合活性を実施例 1の 3項に示す ELISA法により測定した。 Expression base Kuta one p ANTEX8969-hIL2 of 4〃G 4x l0 6 cells of the rat myeloma cell line YB2 / 0 cells (ATCC CRL1662) to electroporation Chillon method [site technology one (Cytotechnology), 3, 133 ( 1990 )], Suspended in 40 ml of RPMI640-FBS (10) (RPI1640 medium containing 10% FBS (GIBC0 BRL)), and placed in a 96-well mic-mouthed Thai plate (Fiffi-Baclyte) for 200 μl. 〃1 / ゥ was dispensed. 37 ° C, after 24 hours at 5% C0 2 incuba- Isseki within one and cultured two weeks was added so as to become the G418 to 0.5 mg / ml. G418-resistant transformant colonies appeared, and the culture supernatant was recovered from the confluent wells. The anti-GM2CDR ^ planted antibody KM8969 in the supernatant was used to recover the culture supernatant. The resulting GM2 binding activity was measured by the ELISA method described in section 3 of Example 1.
培養上清中に抗 GM2CDR^植抗体 KM8969に由来する GM2結合活性が認められたゥ エルの形質転換株については、 dhfr増幅系を利用して抗体発現量を増加させる目 的で、 G418を 0.5mg/ml、 dhfr産物のジヒドロ葉酸還元酵素 （以下、 DHFRと表記す る）の阻害剤であるメソトレキセ一ト （以下、 MTXと表記する： SIGMA社製）を 50nM 含む RPMI1640- FBS( 10)培地に 1〜2 105細胞/ mlになる様に懸濁し、 24ゥエルプレ ート （Greiner社製） に 2mlずつ分注した。 5%C02インキュベーター内で 37°Cで 1〜2 週間培養して、 50nM MTX耐性を示す形質転搬朱を誘導した。 形質転 ί«がゥエル にコンフルェントになった時点で培養上清中の抗 GM2CDR^植抗体 ΚΜ8969に由来す る GM2結合活性を実施例 1の 3項に示す ELISA法により測定した。 培養上清中に抗 GM2CDR^植抗体 KM8969に由来する GM結合活性が認められたゥエルの形質転換株 については、 上記と同様の方法により、 MTX濃度を 100nM、 200nMと順次上昇させ、 最終的に G418を 0.5mg/ml、 MTXを 200nMの濃度で含む RPMI1640- FBS( IO)培地で増殖 可能かつ、 KM8969— hIL- 2を高発現する形質転 ί«を得た。得られた形質転«に ついては、 2回の限界希釈法による単一細胞ィ匕（クローン化） を行い、 最終的に約 6〃g/106細胞 /24hrの発現量を示す形質転換細胞クローン KM8969hIL2を得た。なお 、 KM8969WL2は平成 11年 7月 22日付で、工業技術院生命工学工業技術研究所（日本 国茨城県つくば巿東 1丁目 1番 3号） に FERM BP- 6792として寄託されている。 ( 3 ) KM8969— hIL- 2の培養上清からの精製 In a transformant of a well in which GM2 binding activity derived from the anti-GM2CDR ^ -planted antibody KM8969 was observed in the culture supernatant, G418 was added to 0.5 g of G418 in order to increase the amount of antibody expression using the dhfr amplification system. RPMI1640-FBS (10) medium containing 50 nM methotrexate (hereinafter referred to as MTX: SIGMA), an inhibitor of dihydrofolate reductase (hereinafter referred to as DHFR) of mg / ml, dhfr product The cells were suspended at a concentration of 1-210 5 cells / ml, and dispensed in 2 ml portions into 24-well plates (manufactured by Greiner). After culturing at 37 ° C. for 1 to 2 weeks in a 5% CO 2 incubator, a transgenic carrier showing 50 nM MTX resistance was induced. When the transformation became confluent with the wells, the GM2 binding activity derived from the anti-GM2CDR ^ planted antibody # 8969 in the culture supernatant was measured by the ELISA method described in Example 1, section 3. For the transformed strain of Peer in which GM-binding activity derived from the anti-GM2CDR ^ plant antibody KM8969 was found in the culture supernatant, the MTX concentration was increased to 100 nM and 200 nM in the same manner as above, and finally A transformant capable of growing on an RPMI1640-FBS (IO) medium containing G418 at a concentration of 0.5 mg / ml and MTX at a concentration of 200 nM and highly expressing KM8969-hIL-2 was obtained. The obtained transformant was subjected to single cell dilution (cloning) by the limiting dilution method twice, and finally a transformed cell clone showing an expression level of about 6 μg / 10 6 cells / 24 hr KM8969hIL2 was obtained. KM8969WL2 was deposited as FERM BP-6792 on July 22, 1999 with the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology (1-3-1 Tsukuba East, Ibaraki, Japan). (3) Purification of KM8969—hIL-2 from culture supernatant
上記実施例 1の 2項（ 2 )で得られた M8969— hIL- 2を発現する形質転換細胞ク ローン KM8969hIL2を G418を 0.5mg/ml、 MTXを 200nMの濃度で含む GIT培地（日本製薬 社製） に l〜2 x l05細胞 /mlとなる様に懸濁し、 175cm2フラスコ （Greiner社製） に 200mlずつ分注した。 5%C02インキュベータ一内で 37°Cで 5〜7日間培養し、 コンフ ルェントになった時点で培養上清を回収した。 培養上清約 4Lより Prosep- A ( Bioprocessin社製） カラムを用いて、 添付の説明書に従い、 約 30mgの KM8969— hIL- 2を精製した。 得られた KM8969_hIL- 2および抗 6 12 011 植抗体 KM8969の約 4 jiigを、 公知の方法 [ネイチヤー (Nature ) , 227, 680 ( 1970) ]に従って電気泳動し、 分子量および製精度を調べた。 その結果を第 5図に示した。 第 5図に示した様に、 精製した KM8969— hIL-2は、 非還元条件下では分子量は約 180Kdであり、 還元条件 下では約 65Kdと約 25Kdの 2本のバンドが認められた。 これらの分子量は、 KM8969 - 一 hIL-2の H鎖と hIL-2およひ 1鎖の cDNAの塩基配列から推定される分子量 （H鎖と hIL- 2 :約 65Kd、 L鎖：約 23Kd、 分子全体：約 176Kd) とほぽ一致し、 抗体分子とし ての構造は、 hIL-2の融合後においても保たれていることが確認された。 The transformed cell clone expressing M8969-hIL-2 obtained in Section 2 (2) of Example 1 above, a GIT medium containing KM8969hIL2 at a concentration of 0.5 mg / ml G418 and MTX at a concentration of 200 nM (manufactured by Nippon Pharmaceutical Co., Ltd.) ) the suspension so as to be l~2 x l0 5 cells / ml, aliquoted 200ml min to 175cm 2 flasks (Greiner Co.). In 5% C0 2 incubator within one and cultured at 37 ° C 5 to 7 days, the culture supernatant was recovered when they became configurator Ruento. About 30 mg of KM8969-hIL-2 was purified from about 4 L of the culture supernatant using a Prosep-A (manufactured by Bioprocessin) column according to the attached instructions. About 4 jiig of the obtained KM8969_hIL-2 and anti-6121201 plant antibody KM8969 were subjected to electrophoresis according to a known method [Nature, 227, 680 (1970)], and the molecular weight and production accuracy were examined. The results are shown in FIG. As shown in FIG. 5, purified KM8969-hIL-2 had a molecular weight of about 180 Kd under non-reducing conditions, and two bands of about 65 Kd and about 25 Kd were observed under reducing conditions. These molecular weights are KM8969- The molecular weights estimated from the base sequences of the H chain of hIL-2 and the cDNA of hIL-2 and one chain (H chain and hIL-2: about 65 Kd, L chain: about 23 Kd, whole molecule: about 176 Kd) It was almost identical, and it was confirmed that the structure as an antibody molecule was maintained after fusion of hIL-2.
3 . 抗体の各種ガングリオシドに対する結合活性の測定 （ELISA法）  3. Measurement of binding activity of antibody to various gangliosides (ELISA method)
抗体の各種ガングリオシドに対する結合活性は以下の様にして測定した。 2nmolの各種ガングリオシドを のジパルミ トイルフォスファチジルコリン The binding activity of the antibody to various gangliosides was measured as follows. Dipalmitoyl phosphatidylcholine with 2 nmol of various gangliosides
(SIGMA社製） と 5 zgのコレステロール（SIGMA社製） とを含む 2mlのエタノール溶 液に溶解した。 該溶液の 20 1 (20pmol/ゥエルとなる） または該溶液をエタノー ルで希釈した溶液の 20〃1を 96ゥエルの ELISA用のプレート（Greiner社製）の各ゥ エルにそれぞれ分注し、 風乾後、 1%BSAを含む PBS (以下、 1%BSA-PBSと表記する） を 100〃1/ゥエルで加え、室温で 1時間反応させて残存する活性基をプロックした。 1%BSA- PBSを捨て、形質転換株の培養上清或いは精製した抗体の各種希釈溶液を 50 ゥエルで加え、 室温で 1時間反応させた。 反応後、 各ゥエルを 0.05%Tween20 を含む PBS (以下、 Tween- PBSと表記する）で洗浄後、 1%BSA- PBSで 3000倍に希釈し たペルォキシダ一ゼ標識ャギ抗ヒト IgG(H&L)抗体溶液（American Qualex社製） を 二次抗体溶液として、 50 /1/ゥエルで加え、 室温で 1時間反応させた。 反応後、 Tween-PBSで洗浄後、 ^了8基質液[2,2， -アジノ -ビス（3-ェチルベンゾチアゾリン- 6-スルホン酸）アンモニゥムの 0.55gを 1Lの 0.1Mクェン酉 衝液 (pH4,2)に溶解し、 使用直前に過酸化水素を l〃l/mlで添加した溶液]を 50〃1/ゥエルで加えて発色さ せ、 415nmの吸體 （以下、 0D415と表記する） を測定した。 (SIGMA) and 5 zg of cholesterol (SIGMA) were dissolved in 2 ml of ethanol solution. 20 1 (20 pmol / well) of this solution or 20-1 of a solution obtained by diluting this solution with ethanol is dispensed into each well of a 96-well ELISA plate (manufactured by Greiner), and air-dried. Thereafter, PBS containing 1% BSA (hereinafter referred to as 1% BSA-PBS) was added at 100〃 / ゥ, and the mixture was reacted at room temperature for 1 hour to block the remaining active groups. The 1% BSA-PBS was discarded, and the culture supernatant of the transformant or various dilutions of the purified antibody were added at 50 μl and reacted at room temperature for 1 hour. After the reaction, wash each well with PBS containing 0.05% Tween20 (hereinafter referred to as Tween-PBS), and then dilute 3000 times with 1% BSA-PBS to peroxidase-labeled goat anti-human IgG (H & L). An antibody solution (manufactured by American Qualex) was added as a secondary antibody solution at 50/1 / well, and reacted at room temperature for 1 hour. After the reaction, after washing with Tween-PBS, 0.55 g of the substrate solution [2,2, -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) ammonium is added to 1 L of 0.1 M quenched solution ( pH4,2), and add a solution of hydrogen peroxide at l / l / ml immediately before use] at 50〃1 / ゥ to develop a color, and absorb at 415 nm (hereinafter referred to as 0D415) Was measured.
4 . KM8969— hIL- 2の活性評価  4. KM8969—hIL-2 activity evaluation
( 1 ) KM8969_hIL-2の GM2に対する反応性 （ELISA法）  (1) Reactivity of KM8969_hIL-2 to GM2 (ELISA method)
精製した KM8969— hIL- 2の GM2 (DIA-IATR0N社製） に対する反応性を実施例 1の 3項に記載の方法に従い、 測定した。 第 6図は、 ELISA用のプレートの各ゥエルに 吸着させる GM2の量を 20pmol/ゥエルに固定し、 添加する抗 GM2CDR^植抗体 KM8969 および KM8969— hIL-2の濃度を変化させて反応性を検討した結果である。 第 6図に 示した様に、 KM8969— hIL-2は抗 GM2CDR^植抗体 KM8969と同等以上の GM2に対する 結合活性を有していることが示された。また、 ELISA用のプレートの各ゥエルに吸 着させるガングリオシドの種類を変えて （吸着量： 20pmol/ゥエル）、 一定濃度 ( 10 zg/ml )の抗 GM2CDR^植抗体 KM8969および KM8969— hIL-2の反応性を検討した結 果、 KM8969— hIL- 2は抗 GM2CDR^植抗体 KM8969と同様に GM2に対して強く結合する ことを確認した。 The reactivity of purified KM8969-hIL-2 to GM2 (DIA-IATR0N) was measured according to the method described in Example 1, section 3. Figure 6 shows that the amount of GM2 adsorbed to each well of the ELISA plate was fixed at 20 pmol / well, and the reactivity was examined by changing the concentration of the added anti-GM2CDR ^ plant antibody KM8969 and KM8969-hIL-2. This is the result. As shown in FIG. 6, KM8969-hIL-2 was shown to have a binding activity to GM2 equal to or higher than that of the anti-GM2CDR ^ plant antibody KM8969. Also, by changing the type of ganglioside adsorbed to each well of the ELISA plate (adsorption amount: 20 pmol / well), a fixed concentration (10 zg / ml) of anti-GM2CDR ^ planted antibodies KM8969 and KM8969-hIL-2 The result of considering the reactivity As a result, it was confirmed that KM8969-hIL-2 strongly binds to GM2 similarly to the anti-GM2CDR ^ -planted antibody KM8969.
( 2 ) KM8969— hIL-2の hIL- 2活性の評価  (2) Evaluation of hIL-2 activity of KM8969-hIL-2
精製した KM8969— hIL-2の hIL-2としての活性を以下に示す方法に従い、 測定し た。 hIL- 2に対して濃度依存的な増殖を示すマウス T細胞株 CTLL- 2 (ATCC TIB214 )を 2 X 105細胞/ mlの濃度で RPMI1640-FBS( 10)培地に懸濁し、 96ウェルマイク口夕 イタ—プレート （肢ベ一クライト社製） に 50〃1/ゥエルずつ分注した。 各ゥェ ルに hIL- 2 (R&D SYSTEMS社製）或いは精製した KM8969— hIL-2を RPMI1640- FBS( IO) 培地で各種濃度に希釈した溶液の 50〃1を加え、 5 C02ィンキュベー夕一内で 37°C で 30時間培養した。 培養後、 Cell Counting Kit (同仁化学研究所製） を用いて使 用説明書に従い、 生細胞数を測定した。 その結果を第 7図に示した。 第 7図に示し た様に、 KM8969— hIL- 2は hIL- 2と同程度の CTLL- 2細胞の増殖支持活性を示した。 以上の結果は、 KM8969— hIL-2の hIL- 2としての活性は抗 G 2CDR^植抗体 KM8969と の融合後においても保たれていることを示している。 The activity of purified KM8969-hIL-2 as hIL-2 was measured according to the method described below. A mouse T cell line CTLL-2 (ATCC TIB214) showing concentration-dependent growth for hIL-2 was suspended in RPMI1640-FBS (10) medium at a concentration of 2 × 10 5 cells / ml, Evening Dispensed 50〃1 / ゥ l into an iterative plate (manufactured by Limestone Bright). The 50〃1 solution diluted to various concentrations added to each © E Le hIL- 2 (R & D Ltd. SYSTEMS Inc.) or were purified KM8969- the hIL-2 RPMI1640- FBS (IO) medium, 5 C0 2 Inkyube evening one And incubated at 37 ° C for 30 hours. After the culture, the number of viable cells was measured using a Cell Counting Kit (manufactured by Dojindo Laboratories) according to the instruction manual. The results are shown in FIG. As shown in FIG. 7, KM8969-hIL-2 exhibited the activity of supporting the growth of CTLL-2 cells at a level similar to that of hIL-2. The above results indicate that the activity of KM8969-hIL-2 as hIL-2 is maintained even after fusion with the anti-G2CDR ^ -implanted antibody KM8969.
( 3 ) KM8969-hIL- 2によるヒトエフェクター細胞の活性化と細胞障害活性の増強 KM8969-hIL-2の hIL-2部分によるヒトェフエクタ一細胞の活性化と、それに伴う 細胞障害活性の増強を in vitroで評価するため、 以下に示す方法に従い、 細胞障 害活性を測定した。  (3) Activation of human effector cells and enhancement of cytotoxicity by KM8969-hIL-2 Activation of human Effecta cells by the hIL-2 part of KM8969-hIL-2 and concomitant enhancement of cytotoxicity in vitro In order to evaluate the above, the cytotoxic activity was measured according to the method described below.
a. 標的細胞溶液の調製 a. Preparation of target cell solution
RPMI1640- FBS(IO)培地で培養したヒト小細翻市癌培養細胞株 SBC-3 (JCRB 0818 ) の l x lO6細胞を調製し、 TO性物質である N 51Cr04を 1.85MBq当量加えて 37°Cで 1時間反応させ、 細胞を放!^識した。 反応後、 RPMI1640- FBS( IO)培地で懸濁お よび遠心分離操作により 3回洗浄し、 培地に再懸濁し、 4°Cで 30分間氷中に放置し て放射性物質を自然解離させた。 遠心分離後、 RPMI1640- FBS( IO)培地を 5ml加え、 2 x l05細胞/ mlに調製し、 標的細胞溶液とした。 RPMI1640- FBS and lx lO 6 cells of human small Hosokoboshi City cancer cell cultured in (IO) Medium strain SBC-3 (JCRB 0818) was prepared, the N 51 Cr0 4 is a TO substance added 1.85MBq eq The cells were allowed to react at 37 ° C for 1 hour to release the cells. After the reaction, the suspension was washed with RPMI1640-FBS (IO) medium three times by suspending and centrifuging, resuspended in the medium, and left on ice at 4 ° C for 30 minutes to allow natural dissociation of radioactive substances. After centrifugation, 5 ml of RPMI1640-FBS (IO) medium was added to adjust to 2 × 10 5 cells / ml, and used as a target cell solution.
b. ヒトエフェクター細胞溶液の調製 b. Preparation of human effector cell solution
健常人静脈血 50mlを採取し、 へパリンナトリウム （武田薬品工業社製） 0.5ml を加え穏やかに混ぜた。 これを Polymorphprep (Nycomed Pharaa AS社製） を用い て使用説明書に従い、 遠心分離（1500〜1800g、 30分間） して 球層を分離した RPMI1640-FBS( 10)培地で 3回遠心分離 （1500〜； I800g、 5分間） して洗浄後、 培 地を用いて 5 x lO5細胞/ mlの各密度に再懸濁し、ヒトエフヱクタ一細胞溶液とした c. ヒトエフェクター細胞の活性ィ匕 50 ml of venous blood of a healthy person was collected, and 0.5 ml of heparin sodium (manufactured by Takeda Pharmaceutical Company Limited) was added and mixed gently. This was centrifuged (1500 to 1800 g, 30 minutes) using Polymorphprep (Nycomed Pharaa AS) according to the instruction manual, and centrifuged three times with RPMI1640-FBS (10) medium from which the sphere layer was separated (1500 to 1500 g). ; 800g for 5 minutes) Using the earth resuspended in the density of 5 x lO 5 cells / ml, c. Human effector cells of the active I spoon which was Hitoefuwekuta single cell solution
96ゥエル U字底プレート （Falcon社製） の各ゥエルに b.で調製したエフェク夕 一細胞溶液 100^1を分注した （5 X 104細胞/ゥエル） 。 さらに KM8969或いは KM8969- hIL- 2を各最終濃度 ll. lnMとなる様に 50^1ずつ添カ卩し、 37°C, 5%C02イン キュベー夕一内で 24時間反応させた。 To each well of a 96-well U-shaped bottom plate (manufactured by Falcon), 100 1001 of the effector cell solution prepared in b. Was dispensed (5 × 10 4 cells / well). Further KM8969 or KM8969- hIL- 2 was添Ka卩each final concentration ll. As a lnM by 50 ^ 1, and reacted for 24 hours at 37 ° C, 5% C0 2 in Kyube evening within one.
d. 細胞障害活性の測定 d. Measurement of cytotoxic activity
で用意したプレートの各ゥエルに a.で調製した標的細胞溶液の 50 1 (1 X 104 細胞/ゥエル） を添加した。 この時、 エフェクター細胞と標的細胞の比は 5 : 1とな る。 37°Cで 4時間反応後、 プレートを遠心分離し、 上清の51 Cr量をァ-カウン夕一 にて測定した。 自然解離51 Cr量は、 エフェクター細胞溶液、 抗体溶液の代わりに 培地のみを用いて上記と同様の操作を行い、 上清の51 Cr量を測定することにより 求めた。 全解離51 Cr量は、 抗体溶液の代わりに培地のみを、 エフヱクタ一細胞溶 液の代わりに 5規¾ 酸化ナトリゥムを添加し、上記と同様の操作を行い、上清の 51Cr量を測定することにより求めた。 細胞障害活性は下式により求めた。 検体上清中の51 Cr量 -自然解離51 Cr量 To each well of the plate prepared in the above, 50 1 (1 × 10 4 cells / well) of the target cell solution prepared in a. Was added. At this time, the ratio of effector cells to target cells is 5: 1. After reacting at 37 ° C. for 4 hours, the plate was centrifuged, and the amount of 51 Cr in the supernatant was measured at a county. The amount of spontaneously dissociated 51 Cr was determined by performing the same operation as above using only the medium instead of the effector cell solution and the antibody solution, and measuring the amount of 51 Cr in the supernatant. For the total amount of dissociated 51 Cr, add only the medium instead of the antibody solution, and add 5N sodium oxide instead of the effector-cell solution, and perform the same operation as above to measure the amount of 51 Cr in the supernatant. I asked for it. The cytotoxic activity was determined by the following equation. Amount of 51 Cr in sample supernatant - spontaneously dissociated 51 Cr amount
ADCC活性 （％) = X 100  ADCC activity (%) = X 100
全解離51 Crft "自然解離51 Cr量 その結果を第 8図に示した。第 8図に示した様に、 KM8969— hIL-2は KM8969よりも 強い細胞障害活性を誘導することが明らかとなった。本結果は、 KM8969— hIL- 2を 用いてヒトエフェクター細胞を活性化でき、 細胞障害活性が増強されることを示 したものであり、 ヒ卜への臨床応用においても同様の効果が期待できることを示 唆する結果である。 産業上の利用可能性 Total dissociation 51 Crft "Amount of spontaneous dissociation 51 Cr The results are shown in Fig. 8. As shown in Fig. 8, it was revealed that KM8969-hIL-2 induces stronger cytotoxic activity than KM8969. This result indicates that human effector cells can be activated using KM8969-hIL-2, and that the cytotoxic activity is enhanced, and similar effects are expected in clinical application to humans. The result suggests that it can be used.
本発明により、 GM2に特異的に反応する抗体の誘導体が提供される。  According to the present invention, a derivative of an antibody that specifically reacts with GM2 is provided.
「配列表フリーテキスト」 配列番号 3—人工配列の説明：合^ DNA 配列番号 4一人工配列の説明：合 S¾DNA 配列番号 5—人工配列の説明：合 SgDNA 配列番号 6—人工配列の説明：合 figDNA "Sequence List Free Text" SEQ ID No. 3—Description of Artificial Sequence: Synthetic DNA SEQ ID No. 4—Description of Artificial Sequence: Synthetic S¾DNA SEQ ID No. 5—Description of Artificial Sequence: Synthetic SgDNA SEQ ID NO.

Claims

請 求 の 範 囲 The scope of the claims
1 ガングリオシド GM2に特異的に反応するモノクローナル抗体またはその抗 体断片と、 ¾1ί性同位元素、 蛋白質または低分子の薬剤とを結合させた抗体の誘 導体。 1 Ganglioside A derivative of an antibody in which a monoclonal antibody or an antibody fragment that specifically reacts with GM2 is bound to a {1} isotope, protein or small molecule drug.
2 ガングリオシド GM2に特異的に反応するモノクローナル抗体が、ハイプリ ド —マが産生する抗体、 ヒト化抗体およびヒト抗体から選ばれる抗体である、 請求 項 1記載の抗体の誘導体。  2. The antibody derivative according to claim 1, wherein the monoclonal antibody specifically reacting with ganglioside GM2 is an antibody selected from an antibody produced by a hybridoma, a humanized antibody and a human antibody.
3 モノクローナル抗体の Η鎖 V領域の CDR1、 CDR2および CDR3がそれぞれ配列番 号 9、 1 0および 1 1で示されるアミノ酸配列を含む請求項 1または 2記載の抗 体の誘導体。  3. The derivative of the antibody according to claim 1, wherein CDR1, CDR2, and CDR3 of the V region of the monoclonal antibody comprise the amino acid sequences represented by SEQ ID NOs: 9, 10, and 11, respectively.
4 モノクロ一ナル抗体の L鎖 V領域の CDR1、 CDR2および CDR3がそれぞれ配列番 号 1 2、 1 3および 1 4で示されるアミノ酸配列を含む請求項 1または 2記載の 抗体の誘導体。  4. The antibody derivative according to claim 1, wherein CDR1, CDR2, and CDR3 of the V region of the L chain of the monoclonal antibody include the amino acid sequences represented by SEQ ID NOS: 12, 13, and 14, respectively.
5 モノクローナル抗体の重鎖 （H鎖） 可変領域 （V領域） の CDR1、 CDR2および CDR3がそれぞれ配列番号 9、 1 0および 1 1、 軽鎖 （L鎖） V領域の CDR1、 CDR2お よび CDR3がそれぞれ配列番号 1 2、 1 3および 1 4で示されるァミノ酸配列を含 む請求項 1または 2記載の抗体の誘導体。  5 CDR1, CDR2, and CDR3 of the heavy chain (H chain) variable region (V region) of the monoclonal antibody are SEQ ID NOs: 9, 10, and 11, respectively. Light chain (L chain) CDR1, CDR2, and CDR3 of the V region are 3. The derivative of the antibody according to claim 1, which comprises an amino acid sequence represented by SEQ ID NOS: 12, 13, and 14, respectively.
6 ハイプリドーマが産生する抗体が KM796 (FERM BP-3340) である、 請求項 2 記載の抗体の誘導体。  6. The antibody derivative according to claim 2, wherein the antibody produced by the hybridoma is KM796 (FERM BP-3340).
7 ヒト化抗体が、 ヒト型キヌラ抗体またはヒト型 CDR^植抗体である、請求項 2記載の抗体の誘導体。  7. The derivative of the antibody according to claim 2, wherein the humanized antibody is a human quinula antibody or a human CDR ^ -planted antibody.
8 ヒト型キメラ抗体が、ガングリオシド GM2に対するハイプリドーマが産生す るモノクローナル抗体の H鎖 V領域および L鎖 V領域のァミノ酸配列を含む、 請求項 7記載のヒト型キメラ抗体の誘導体。  8. The derivative of the human chimeric antibody according to claim 7, wherein the human chimeric antibody comprises the amino acid sequences of the H chain V region and L chain V region of a monoclonal antibody produced by a hybridoma against ganglioside GM2.
9 ヒト型キメラ抗体が、ガングリオシド GM2に対するハイプリドーマが産生す るモノクロ一ナル抗体の H鎖 V領域および L鎖 V領域、ならびにヒト抗体の H鎖定常領 域（C領域）およひ "L鎖 C領域のアミノ酸配列を含む、請求項 7記載のヒト型キメラ 抗体の誘導体。  9 The human chimeric antibody is composed of the H chain V region and L chain V region of the monoclonal antibody produced by the hybridoma against ganglioside GM2, and the H chain constant region (C region) and the “L chain” of the human antibody. 8. The derivative of the human chimeric antibody according to claim 7, comprising the amino acid sequence of the C region.
1 0 HHV領域が配列番号 7で示されるアミノ酸配列を含む、請求項 8または 9記 載のヒト型キメラ抗体の誘導体。 10.The method according to claim 8, wherein the 10 HHV region comprises the amino acid sequence represented by SEQ ID NO: 7. A derivative of the above-mentioned human chimeric antibody.
1 1 L鎖 V領域が配列番号 8で示されるァミノ酸配列を含む、請求項 8または 9記 載のヒト型キメラ抗体の誘導体。  10. The derivative of the human chimeric antibody according to claim 8, wherein the 11 L chain V region comprises the amino acid sequence represented by SEQ ID NO: 8.
1 2 H鎖 V領域が配列番号 7で示されるアミノ酸配列を含み、 L鎖 V領域が配列番号 8で示されるアミノ酸配列を含む請求項 8または 9記載のヒト型キヌラ抗体の誘  The human quinula antibody of claim 8 or 9, wherein the 12 H chain V region comprises the amino acid sequence represented by SEQ ID NO: 7, and the L chain V region comprises the amino acid sequence represented by SEQ ID NO: 8.
1 3 H鎖 V領域が配列番号 7で示されるアミノ酸配列を含み、 L鎖 V領域が配列番号 8で示されるァミノ酸配列を含む請求項 8または 9記載のヒト型キメラ抗体 KM966 の誘導体。 10. The human chimeric antibody KM966 derivative according to claim 8 or 9, wherein the 13 H chain V region comprises the amino acid sequence represented by SEQ ID NO: 7, and the L chain V region comprises the amino acid sequence represented by SEQ ID NO: 8.
1 4 ヒト型 CDR^植抗体が、ガングリオシド GM2に対するモノクロ一ナル抗体の H 鎖 V領域および "L鎖 V領域の CDRのァミノ酸配列を含む、 請求項 7記載のヒト型 CDR 移植抗体の誘導体。  14. The human CDR-grafted antibody derivative according to claim 7, wherein the human CDR-transplanted antibody comprises the amino acid sequences of the CDRs of the H chain V region and the "L chain V region" of the monoclonal antibody against ganglioside GM2.
1 5 ヒト型 CDR^植抗体が、ガングリオシド GM2に対するモノクロ一ナル抗体の H 鎖 V領域および 1鎖 V領域の CDRとヒト抗体の H鎖 V領域およひ 1鎖 V領域のフレームヮ ーク領域（FR)のアミノ酸配列を含む、請求項 7記載のヒト型 CDR^植抗体の誘導 体。  15 The human CDR ^ -grafted antibody is composed of the CDRs of the H chain V region and 1 chain V region of the monoclonal antibody against ganglioside GM2 and the framework regions of the H chain V region and 1 chain V region of the human antibody ( 8. The derivative of the human CDR ^ -planted antibody according to claim 7, which comprises the amino acid sequence of FR).
1 6 ヒト型 CDR^植抗体が、ガングリオシド GM2に対するモノクローナル抗体の H 鎖 V領域およひ 1鎖 V領域の CDR、 ヒト抗体の H鎖 V領域および 1鎖 V領域の FR、 ならび にヒト抗体の H鎖 C領域およひ 1鎖 C領域のアミノ酸配列を含む、 請求項 7記載のヒ ト型 CD 植抗体の誘導体。  16 The human CDR ^ -grafted antibody is capable of detecting the CDRs of the H chain V region and 1 chain V region of the monoclonal antibody against ganglioside GM2, the FRs of the H chain V region and 1 chain V region of the human antibody, and the human antibody. 8. The derivative of the human CD-grafted antibody according to claim 7, which comprises the amino acid sequences of the H chain C region and the 1 chain C region.
1 7 抗体の H鎖 V領域の CDR 1、 CDR2および CDR 3がそれぞれ配列番号 9、 10および 11で示されるアミノ酸配列を含む、 請求項 1 4〜 1 6のいずれか 1項に記載のヒ 卜型 CD 植抗体の誘導体 c  The human according to any one of claims 14 to 16, wherein CDR1, CDR2, and CDR3 of the H chain V region of the 17 antibody comprise the amino acid sequences represented by SEQ ID NOS: 9, 10, and 11, respectively. Derivative of type CD implanted antibody c
1 8 抗体の L鎖 V領域の CDR1、 CDR2および CDR3がそれぞれ配列番号 12、 13および 14で示されるアミノ酸配列を含む、 請求項 1 4〜1 6のいずれか 1項に記載のヒ ト型 C贿植抗体の誘導体。  The human type C according to any one of claims 14 to 16, wherein CDR1, CDR2, and CDR3 of the L chain V region of the 18 antibody comprise the amino acid sequences represented by SEQ ID NOs: 12, 13, and 14, respectively.誘導 体 Derivatives of planted antibodies.
1 9 抗体の H鎖 V領域の CDR1、 CDR2および CDR3がそれそれ配列番号 9、 10および 11 で示されるアミノ酸配列を含み、 L鎖 V領域の CDR1、 CDR2および CDR3がそれぞれ配 列番号 12、 13および 14で示されるアミノ酸配列を含む、 請求項 1 4 ~ 1 6のいず れか 1項に記載のヒト型 CDR^植抗体の誘導体。 2 0 抗体の H鎖 V領域が配列番号 15で示されるアミノ酸配列を含む請求項 1 4〜19 The CDR1, CDR2, and CDR3 of the H chain V region of the antibody each include the amino acid sequences represented by SEQ ID NOs: 9, 10, and 11, and the CDR1, CDR2, and CDR3 of the L chain V region correspond to SEQ ID NOs: 12, 13, respectively. 17. The derivative of the human CDR ^ -planted antibody according to any one of claims 14 to 16, comprising the amino acid sequence of The H chain V region of the 20 antibody comprises the amino acid sequence represented by SEQ ID NO: 15.
1 6のいずれか 1項に記載のヒト型 CDR^植抗体の誘導体。 16. The derivative of the human CDR ^ -planted antibody according to any one of 16.
2 1 抗体の L鎖 V領域が配列番号 16で示されるアミノ酸配列を含む請求項 1 4〜 21.The L chain V region of the 21 antibody comprises the amino acid sequence of SEQ ID NO: 16.
1 6のいずれか 1項に記載のヒト型 CDR^植抗体の誘導体。 16. The derivative of the human CDR ^ -planted antibody according to any one of 16.
2 2 抗体の H鎖 V領域が配列番号 15で示されるアミノ酸配列を含み、 L鎖 V領域が 配列番号 16で示されるアミノ酸配列を含む請求項 1 4 - 1 6のいずれか 1項に記 載のヒト型 CDR^植抗体の誘導体。  22.The antibody according to any one of claims 14 to 16, wherein the H chain V region of the antibody comprises the amino acid sequence represented by SEQ ID NO: 15, and the L chain V region comprises the amino acid sequence represented by SEQ ID NO: 16. Derivative of human CDR ^ plant antibody.
2 3 抗体の H鎖 V領域が配列番号 15で示されるアミノ酸配列を含み、 L鎖 V領域が 配列番号 16で示されるアミノ酸配列を含む請求項 1 4 - 1 6のいずれか 1項に記 載のヒト型 CDR^植抗体 KM8969の誘導体。  23.The antibody according to any one of claims 14 to 16, wherein the H chain V region of the antibody comprises the amino acid sequence represented by SEQ ID NO: 15, and the L chain V region comprises the amino acid sequence represented by SEQ ID NO: 16. Derivative of human type CDR ^ plant antibody KM8969.
2 4 抗体断片が、 Fab、 Fab，、 F(ab，）2、 一本鎖抗体 (scFv) 、 ジスルフィ ド安 定化 V領域断片 （dsFv) および CDRを含むペプチドから選ばれる抗体断片である請 求項 1記載の抗体断片の誘導体。 24 The antibody fragment is an antibody fragment selected from Fab, Fab, F (ab,) 2 , a single-chain antibody (scFv), a disulfide-stabilized V region fragment (dsFv), and a peptide including CDR. A derivative of the antibody fragment according to claim 1.
2 5 抗体断片が、ガングリオシド GM2に対するハイプリドーマが産生するモノク 口一ナル抗体の H鎖 V領域および 1鎖 V領域のアミノ酸配列を含む、 請求項 2 4記載 の抗体断片の誘導体。  25. The derivative of the antibody fragment according to claim 24, wherein the antibody fragment comprises the amino acid sequences of the H chain V region and the 1 chain V region of a monoclonal antibody produced by a hybridoma against ganglioside GM2.
2 6 抗体断片が、ガングリオシド GM2に対するハイプリ ドーマが産生するモノク 口一ナル抗体の H鎖 V領域および L鎖領 ならびにヒト抗体の H鎖 C領域および "L 鎖 C領域のァミノ酸配列を含む、 請求項 2 4記載の抗体断片の誘導体。  26 The antibody fragment comprises the H chain V region and L chain region of a monoclonal antibody produced by a hybridoma to ganglioside GM2, and the amino acid sequences of the H chain C region and "L chain C region" of a human antibody. Item 24. A derivative of the antibody fragment according to item 24.
2 7 抗体断片が、 抗体の H鎖 V領域が配列番号 7で示されるアミノ酸配列を含む、 請求項 2 5または 2 6記載の抗体断片の誘導体。  27. The antibody fragment derivative according to claim 25, wherein the antibody fragment has an H chain V region of the antibody comprising the amino acid sequence represented by SEQ ID NO: 7.
2 8 抗体断片が、 抗体の L鎖 V領域が配列番号 8で示されるアミノ酸配列を含む、 請求項 2 5または 2 6記載の抗体断片の誘導体。  27. The antibody fragment derivative according to claim 25 or 26, wherein the antibody fragment comprises an L chain V region of the antibody comprising the amino acid sequence represented by SEQ ID NO: 8.
2 9 抗体断片が、 抗体の職 V領域が配列番号 7で示されるアミノ酸配列を含み、 抗体の L鎖 V領域が配列番号 8で示されるアミノ酸配列を含む、請求項 2 5または 2 6記載の抗体断片の誘導体。  29.An antibody fragment according to claim 25 or 26, wherein the antibody V region comprises the amino acid sequence represented by SEQ ID NO: 7, and the L chain V region of the antibody comprises the amino acid sequence represented by SEQ ID NO: 8. Derivatives of antibody fragments.
3 0 抗体断片が、 抗体の H鎖 V領域が配列番号 7で示されるアミノ酸配列を含み、 抗体の L鎖 V領域が配列番号 8で示されるアミノ酸配列を含む、請求項 2 5記載のハ イブリドーマが産生する抗体 KM796の抗体断片の誘導体。  The hybridoma according to claim 25, wherein the 30 antibody fragment has an H chain V region of the antibody comprising the amino acid sequence represented by SEQ ID NO: 7, and an L chain V region of the antibody comprising the amino acid sequence represented by SEQ ID NO: 8. A derivative of the antibody fragment of antibody KM796 produced by.
3 1 抗体断片が、 抗体の H鎖 V領域が配列番号 7で示されるアミノ酸配列を含み、 抗体の L鎖 V領域が配列番号 8で示されるアミノ酸配列を含む、請求項 2 6記載のヒ ト型キメラ抗体 KM966の抗体断片の誘導体。 31 antibody fragment, wherein the H chain V region of the antibody comprises the amino acid sequence of SEQ ID NO: 7, 27. The derivative of the human chimeric antibody KM966 antibody fragment according to claim 26, wherein the L chain V region of the antibody comprises the amino acid sequence represented by SEQ ID NO: 8.
3 2 抗体断片が、 ガングリオシド GM2に対するヒト型 CDR^植抗体の H鎖 V領域お よひ 1鎖 V領域のァミノ酸配列を含む、 請求項 2 4記載の抗体断片の誘導体。 3 3 抗体断片が、 ガングリオシド GM2に対するヒト型 CDR^植抗体の H鎖 V領域お よび L鎖 V領域、 ならびにヒト抗体の H鎖 C領域および 1鎖 C領域のアミノ酸配列を含 む、 請求項 2 4記載の抗体断片の誘導体。  32. The derivative of the antibody fragment according to claim 24, wherein the 32 antibody fragment comprises the amino acid sequence of the H chain V region and the 1 chain V region of a human CDR ^ -planted antibody against ganglioside GM2. 33. The antibody fragment comprising the amino acid sequences of the H chain V region and L chain V region of a human CDR ^ -planted antibody to ganglioside GM2, and the H chain C region and 1 chain C region of a human antibody. A derivative of the antibody fragment according to 4.
3 4 抗体断片が、抗体の H鎖 V領域の CDM、 CDR2および CDR3がそれぞれ配列番号 9 、 10および 11で示されるアミノ酸配列を含む、 請求項 3 2または 3 3記載の抗体 断片の誘導体。  34. The derivative of the antibody fragment according to claim 32 or 33, wherein the 34 antibody fragment has the CDM, CDR2 and CDR3 of the V region of the H chain of the antibody comprising the amino acid sequences represented by SEQ ID NOS: 9, 10 and 11, respectively.
3 5 抗体断片が、 抗体の L鎖 V領域の CDR1、 CDR2および CDR3がそれぞれ配列番号 12、 13および 14で示されるアミノ酸配列を含む、 請求項 3 2または 3 3記載の抗 体断片の誘導体。  35. The derivative of the antibody fragment according to claim 32 or 33, wherein the antibody fragment comprises CDR1, CDR2 and CDR3 of the L chain V region of the antibody comprising the amino acid sequences represented by SEQ ID NOS: 12, 13 and 14, respectively.
3 6 抗体断片が、抗体の H鎖 V領域の CDR1、 CDR2および CDR3がそれぞれ配列番号 9 、 10および 11で示されるアミノ酸配列を含み、 L鎖 V領域の CDR1、 CDR2および CDR3 がそれぞれ配列番号 12、 13および 14で示されるアミノ酸配列を含む、 請求項 3 2 または 3 3記載の抗体断片の誘導体。  36 antibody fragment, the CDR1, CDR2, and CDR3 of the H chain V region of the antibody include the amino acid sequences represented by SEQ ID NOS: 9, 10, and 11, respectively, and the CDR1, CDR2, and CDR3 of the L chain V region correspond to SEQ ID NO: 12, respectively. 34. The derivative of the antibody fragment according to claim 32, which comprises the amino acid sequence represented by any one of SEQ ID NOs: 13, 13 and 14.
3 7 抗体断片が、抗体の H鎖 V領域が配列番号 15で示されるアミノ酸配列を含む、 請求項 3 2または 3 3記載の抗体断片の誘導体。  37. The derivative of the antibody fragment according to claim 32, wherein the antibody fragment comprises the amino acid sequence represented by SEQ ID NO: 15 in the H chain V region of the antibody.
3 8 抗体断片が、抗体の L鎖 V領域が配列番号 16で示されるアミノ酸配列を含む、 請求項 3 2または 3 3記載の抗体断片の誘導体。  34. The antibody fragment derivative according to claim 32, wherein the antibody fragment comprises an amino acid sequence represented by SEQ ID NO: 16 in the L chain V region of the antibody.
3 9 抗体断片が、 抗体の H鎖 V領域が配列番号 15、 抗体の L鎖 V領域が配列番号 16 で示されるアミノ酸配列を含む、請求項 3 2または 3 3記載の抗体断片の誘導体。 39. The antibody fragment derivative according to claim 32 or 33, wherein the antibody fragment comprises the amino acid sequence represented by SEQ ID NO: 15 in the H chain V region of the antibody and SEQ ID NO: 16 in the L chain V region of the antibody.
4 0 抗体断片が、抗体の H鎖 V領域が配列番号 15で示されるアミノ酸配列を含み、 抗体の L鎖 V領域が配列番号 16で示されるアミノ酸配列を含む、 請求項 3 2または40.The antibody fragment, wherein the H chain V region of the antibody comprises the amino acid sequence represented by SEQ ID NO: 15, and the L chain V region of the antibody comprises the amino acid sequence represented by SEQ ID NO: 16.
3 3記載のヒト型 CDR^植抗体 KM8969の抗体断片の誘導体。 33 A derivative of the antibody fragment of the human CDR ^ plant antibody KM8969 according to 3.
4 1 蛋白質がサイトカインである、 請求項 1記載のモノクローナル抗体の誘導 体またはその抗体断片の誘導体。  4. The derivative of the monoclonal antibody or the derivative of the antibody fragment thereof according to claim 1, wherein the 41 protein is a cytokine.
4 2 サイ トカインがヒトインターロイキン 2 (hIL-2) である請求項 4 1記載の モノクロ一ナル抗体の誘導体またはその抗体断片の誘導体。 4 3 抗体の H鎖 V領域と hIL- 2との融合蛋白質が配列番号 1記載のァミノ酸配列 を有し、 抗体の L鎖 V領域が配列番号 2記載のアミノ酸配列を有する請求項 1記載 の抗体の誘導体。 42. The derivative of the monoclonal antibody or the derivative of the antibody fragment thereof according to claim 41, wherein the 42 cytokine is human interleukin 2 (hIL-2). 43.A fusion protein of the H chain V region and hIL-2 of the antibody has the amino acid sequence of SEQ ID NO: 1, and the L chain V region of the antibody has the amino acid sequence of SEQ ID NO: 2. Derivatives of antibodies.
4 4 抗体の誘導体が、 ヒト型 CDR^植抗体 KM8969と hIL- 2とからなる請求項 4 1 〜 4 3のいずれか 1項に記載の抗体の誘導体。  44. The antibody derivative according to any one of claims 41 to 43, wherein the derivative of the antibody comprises the human CDR ^ -planted antibody KM8969 and hIL-2.
4 5 抗体の誘導体が、 ヒト型 CDR^植抗体 KM8969と hIL- 2とからなる請求項 4 1 45. The method according to claim 41, wherein the derivative of the antibody comprises the human CDR ^ -planted antibody KM8969 and hIL-2.
〜4 3のいずれか 1項に記載の抗体の誘導体 KM8969- hIL-2。 The derivative KM8969-hIL-2 of the antibody according to any one of to 43.
4 6 請求項 1〜4 5のいずれか 1項に記載のガングリオシド GM に特異的に反 応するモノクロ一ナル抗体の誘導体またはその抗体断片の誘導体をコードする  46 Encodes a monoclonal antibody derivative or antibody fragment derivative thereof that specifically reacts with ganglioside GM according to any one of claims 1 to 45.
4 7 請求項 4 6記載の DNAを含有する組換えベクター。 47. A recombinant vector containing the DNA according to claim 46.
4 8 請求項 4 7記載の組換えベクターを宿主細胞に導入して得られる形質転換 株。  48 A transformant obtained by introducing the recombinant vector according to claim 47 into a host cell.
4 9 請求項 4 5記載の誘導体を生産する形質転換 ¾KM8969hIL2 (FERM BP-6792 49 Transformation producing the derivative according to claim 45 ¾KM8969hIL2 (FERM BP-6792
) o ) o
5 0 請求項 4 8または 4 9記載の形質転賺を培地に培養し、 培養物中に請求 項 1〜4 5のいずれか 1項に記載のモノクローナル抗体の誘導体またはその抗体 断片の誘導体を生成蓄積させ、 該培養物から該抗体の誘導体またはその抗体断片 の誘導体を採取することを特徴とする製造方法。  50.The transformant described in claim 48 or 49 is cultured in a medium to produce a derivative of the monoclonal antibody or a derivative of the antibody fragment thereof according to any one of claims 1 to 45 in the culture. A production method, comprising accumulating and collecting a derivative of the antibody or a derivative of an antibody fragment thereof from the culture.
5 1 ガングリオシド GM2に特異的に反応する抗体断片。  5 1 Ganglioside An antibody fragment that specifically reacts with GM2.
5 2 抗体断片が、 Fab、 Fab，、 F(ab，）2、 一本鎖抗体 (scFv) 、 ジスルフィ ド安 定化 V領域断片 （dsFv) および CDRを含むペプチドから選ばれる抗体断片である請 求項 5 1 3載の抗体断片。 52 The antibody fragment is an antibody fragment selected from Fab, Fab, F (ab,) 2 , a single-chain antibody (scFv), a disulfide-stabilized V region fragment (dsFv), and a peptide containing CDR. The antibody fragment of claim 5 13.
5 3 抗体断片が、ガングリオシド GM2に対するハイプリドーマが産生するモノク ローナル抗体の H鎖 V領域および "L鎖 V領域のアミノ酸配列を含む、 請求項 5 1記載 の抗体断片。  53. The antibody fragment according to claim 51, wherein the antibody fragment comprises the amino acid sequences of the H chain V region and the "L chain V region" of a monoclonal antibody produced by a hybridoma against ganglioside GM2.
5 4 抗体断片が、ガングリオシド GM2に対するハイプリドーマが産生するモノク 口一ナル抗体の H鎖 V領域およひ "L鎖 V領域、 ならびにヒト抗体の H鎖 C領域およひ 1 鎖 C領域のアミノ酸配列を含む、 請求項 5 1記載の抗体断片。  5 4 The antibody fragment is composed of the H chain V region and L chain V region of the monoclonal antibody produced by the hybridoma against ganglioside GM2, and the amino acids of the H chain C region and 1 chain C region of the human antibody. The antibody fragment of claim 51, comprising a sequence.
5 5 抗体断片が、 抗体の H鎖 V領域が配列番号 7で示されるアミノ酸配列を含む、 請求項 5 3または 5 4記載の抗体断片。 55 antibody fragment, wherein the H chain V region of the antibody comprises the amino acid sequence represented by SEQ ID NO: 7, The antibody fragment according to claim 53 or 54.
5 6 抗体断片が、 抗体の L鎖 V領域が配列番号 8で示されるアミノ酸配列を含む、 請求項 5 3または 5 4記載の抗体断片。  56. The antibody fragment according to claim 53 or 54, wherein the antibody fragment comprises an L chain V region of the antibody comprising the amino acid sequence represented by SEQ ID NO: 8.
5 7 抗体断片が、 抗体の H$ 領域が配列番号 7で示されるアミノ酸配列を含み、 抗体の L鎖 V領域が配列番号 8で示されるァミノ酸配列を含む、請求項 5 3または 5 4記載の抗体断片。  57.The antibody fragment, wherein the H $ region of the antibody comprises the amino acid sequence represented by SEQ ID NO: 7, and the L chain V region of the antibody comprises the amino acid sequence represented by SEQ ID NO: 8. Antibody fragment.
5 8 抗体断片が、 抗体の H鎖 V領域が配列番号 7で示されるアミノ酸配列を含み、 抗体の L鎖 V領域が配列番号 8で示されるァミノ酸配列を含む、請求項 5 3記載のノ、 ィブリドーマが産生する抗体M796の抗体断片。  58. The antibody according to claim 53, wherein the antibody fragment has an H chain V region of the antibody comprising the amino acid sequence represented by SEQ ID NO: 7, and an L chain V region of the antibody comprising the amino acid sequence represented by SEQ ID NO: 8. An antibody fragment of antibody M796 produced by hybridomas.
5 9 抗体断片が、 抗体の H 領域が配列番号 7で示されるアミノ酸配列を含み、 抗体の L鎖 V領域が配列番号 8で示されるアミノ酸配列を含む、請求項 5 3または 5 4記載のヒト型キメラ抗体 KM966の抗体断片。  59.The human of claim 53 or 54, wherein the antibody fragment has an H region of the antibody comprising the amino acid sequence of SEQ ID NO: 7, and an L chain V region of the antibody comprising the amino acid sequence of SEQ ID NO: 8. An antibody fragment of the chimeric antibody KM966.
6 0 抗体断片が、 ガングリオシド GM2に対するヒト型 CDR^植抗体の H鎖 V領域お よび "L鎖 V領域のアミノ酸配列を含む、 請求項 5 2記載の抗体断片。  The antibody fragment according to claim 52, wherein the 60 antibody fragment comprises the amino acid sequences of the H chain V region and the "L chain V region" of a human CDR ^ -planted antibody against ganglioside GM2.
6 1 抗体断片が、 ガングリオシド GM2に対するヒト型 CDR^植抗体の H鎖 V領域お よび L鎖 V領域、 ならびにヒト抗体の H鎖 C領域および 1鎖 C領域のアミノ酸配列を含 む、 請求項 5 2記載の抗体断片。  61. The antibody fragment comprising the amino acid sequences of the H chain V region and L chain V region of a human CDR ^ -planted antibody against ganglioside GM2, and the H chain C region and 1 chain C region of a human antibody. 2. The antibody fragment according to 2.
6 2 抗体断片が、抗体の H鎖 V領域の CDR1、 CDR2および CDR3がそれぞれ配列番号 9 、 10および 11で示されるアミノ酸配列を含む、 請求項 6 0または 6 1記載の抗体 断片。  62. The antibody fragment according to claim 60, wherein CDR1, CDR2, and CDR3 of the V region of the H chain of the antibody include the amino acid sequences represented by SEQ ID NOs: 9, 10, and 11, respectively.
6 3 抗体断片が、 抗体の L鎖 V領域の CDR1、 CDR2および CDR3がそれぞれ配列番号 12、 13および 14で示されるアミノ酸配列を含む、 請求項 6 0または 6 1記載の抗 体断片。  63. The antibody fragment according to claim 60, wherein CDR1, CDR2, and CDR3 of the L chain V region of the antibody include the amino acid sequences represented by SEQ ID NOs: 12, 13, and 14, respectively.
6 4 抗体断片が、抗体の H鎖 V領域の CDR1、 CDR2および CDR3がそれぞれ配列番号 9 、 10および 11で示されるアミノ酸配列を含み、 L鎖 V領域の CDR1、 CDR2および CDR3 がそれぞれ配列番号 12、 13および 14で示されるアミノ酸配列を含む、 請求項 6 0 または 6 1記載の抗体断片。  64 antibody fragment, wherein the CDR1, CDR2, and CDR3 of the H chain V region of the antibody include the amino acid sequences represented by SEQ ID NOs: 9, 10, and 11, respectively, and the CDR1, CDR2, and CDR3 of the L chain V region correspond to SEQ ID NO: 12, respectively. 63. The antibody fragment according to claim 60 or 61, comprising the amino acid sequence represented by any one of SEQ ID NOS: 13, 14
6 5 抗体断片が、抗体の職 V領域が配列番号 15で示されるアミノ酸配列を含む、 請求項 6 0または 6 1記載の抗体断片。  65. The antibody fragment according to claim 60 or 61, wherein the antibody fragment has an amino acid sequence represented by SEQ ID NO: 15 in the antibody V region.
6 6 抗体断片が、抗体の L鎖 V領域が配列番号 16で示されるアミノ酸配列を含む、 請求項 6 0または 6 1記載の抗体断片。 66 antibody fragment, the L chain V region of the antibody comprises the amino acid sequence of SEQ ID NO: 16, The antibody fragment according to claim 60 or 61.
6 7 抗体断片が、抗体の H鎖 V領域が配列番号 15で示されるァミノ酸配列を含み、 抗体の L鎖 V領域が配列番号 16で示されるアミノ酸配列を含む、 請求項 6 0または 6 1記載の抗体断片。  67. The antibody fragment, wherein the H chain V region of the antibody comprises an amino acid sequence represented by SEQ ID NO: 15, and the L chain V region of the antibody comprises an amino acid sequence represented by SEQ ID NO: 16. The antibody fragment of the above.
6 8 抗体断片が、抗体の H鎖 V領域が配列番号 15で示されるアミノ酸配列を含み、 抗体の L鎖 V領域が配列番号 16で示されるアミノ酸配列を含む、 請求項 6 0または 6 1記載のヒト型 CDR^植抗体 KM8969の抗体断片。  68.The antibody fragment according to claim 60, wherein the H chain V region of the antibody comprises the amino acid sequence represented by SEQ ID NO: 15, and the L chain V region of the antibody comprises the amino acid sequence represented by SEQ ID NO: 16. Antibody fragment of human CDR ^ plant antibody KM8969.
6 9 請求項 5 1〜6 8のいずれか 1項に記載のガングリオシド G 2に特異的に 反応する抗体断片をコードする DNA。  69. A DNA encoding an antibody fragment that specifically reacts with ganglioside G2 according to any one of claims 51 to 68.
7 0 請求項 6 9記載の DNAを含有する組換えベクター。  70 A recombinant vector containing the DNA of claim 69.
7 1 請求項 7 0記載の組換えベクターを宿主細胞に導入して得られる形質転換 株 o  71 1 A transformed strain obtained by introducing the recombinant vector according to claim 70 into a host cell.o
7 2 請求項 7 1記載の形質転換株を培地に培養し、 培養物中に請求項 5 2〜 6 8のいずれか 1項に記載の抗体断片を生成蓄積させ、 該培養物から抗体断片を採 取することを特徴とする抗体断片の製造方法。  72.The transformed strain according to claim 71 is cultured in a medium, the antibody fragment according to any one of claims 52 to 68 is produced and accumulated in the culture, and the antibody fragment is isolated from the culture. A method for producing an antibody fragment, which comprises collecting the antibody fragment.
7 3 請求項 1〜4 5記載のモノクロ一ナル抗体の誘導体およびその抗体断片の 誘導体、 ならびに請求項 5 1 - 6 8記載の抗体断片から選ばれる少なくとも 1種 からなる医薬。  73. A medicament comprising the derivative of the monoclonal antibody according to claims 1-45 and a derivative of the antibody fragment thereof, and a drug comprising at least one selected from the antibody fragment according to claim 51-68.
7 4 請求項 1〜4 5記載のモノクローナル抗体の誘導体およびその抗体断片の 誘導体、 ならびに請求項 5 1〜6 8記載の抗体断片から選ばれる少なくとも 1種 を有効成分として含有する癌の治療薬。  74. A therapeutic agent for cancer, comprising as an active ingredient at least one selected from the derivatives of the monoclonal antibody according to claims 1-45 and derivatives of the antibody fragment thereof, and the antibody fragment according to claim 51-68.
7 5 請求項 1〜4 5記載のモノクローナル抗体の誘導体およびその抗体断片の 誘導体、 ならびに請求項 5 1〜6 8記載の抗体断片から選ばれる少なくとも 1種 を有効成分として含有する癌の診断薬。  75. A diagnostic agent for cancer, comprising as an active ingredient at least one selected from the derivatives of the monoclonal antibody according to claims 1-45 and derivatives of the antibody fragment thereof, and the antibody fragment according to claims 51-68.
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