WO2001023431A1 - Derives d'anticorps contre le ganglioside gm2 - Google Patents

Derives d'anticorps contre le ganglioside gm2 Download PDF

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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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English (en)
Japanese (ja)
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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/fr

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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.

Abstract

Cette invention se rapporte à des dérivés d'un anticorps contre le ganglioside GM2 (appelé ici GM2); à des fragments de cet anticorps contre le GM2; et à des diagnostics et des remèdes du cancer utilisant ces dérivés et ces fragments d'anticorps.
PCT/JP2000/006775 1999-09-30 2000-09-29 Derives d'anticorps contre le ganglioside gm2 WO2001023431A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8257703B2 (en) 2003-07-15 2012-09-04 Chugai Seiyaku Kabushiki Kaisha Anti-ganglioside antibodies and compositions
US8920797B2 (en) 2003-10-09 2014-12-30 Chugai Seiyaku Kabushiki Kaisha Highly concentrated stabilized IgM solution
JP5651893B2 (ja) * 2004-06-07 2015-01-14 協和発酵キリン株式会社 抗ｐｅｒｐ抗体
JP6077745B2 (ja) * 2009-09-30 2017-02-08 富士フイルムＲｉファーマ株式会社 抗ポドプラニン抗体、及び抗ポドプラニン抗体を含む医薬組成物
WO2018181207A1 (fr) * 2017-03-27 2018-10-04 ノイルイミューン・バイオテック株式会社 Récepteur antigénique chimérique

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EP0598998A2 (fr) * 1992-09-07 1994-06-01 Kyowa Hakko Kogyo Co., Ltd Anticorps humanisés réagissant avec le ganglioside GM2
EP0882794A2 (fr) * 1997-03-19 1998-12-09 Kyowa Hakko Kogyo Co., Ltd. Anticorps ant-ganglioside gm2 comprenant des CDR d'origine humaine

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JPH041138A (ja) * 1990-04-13 1992-01-06 Kyowa Hakko Kogyo Co Ltd 抗腫瘍剤
EP0598998A2 (fr) * 1992-09-07 1994-06-01 Kyowa Hakko Kogyo Co., Ltd Anticorps humanisés réagissant avec le ganglioside GM2
EP0882794A2 (fr) * 1997-03-19 1998-12-09 Kyowa Hakko Kogyo Co., Ltd. Anticorps ant-ganglioside gm2 comprenant des CDR d'origine humaine

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8257703B2 (en) 2003-07-15 2012-09-04 Chugai Seiyaku Kabushiki Kaisha Anti-ganglioside antibodies and compositions
US8920797B2 (en) 2003-10-09 2014-12-30 Chugai Seiyaku Kabushiki Kaisha Highly concentrated stabilized IgM solution
JP5651893B2 (ja) * 2004-06-07 2015-01-14 協和発酵キリン株式会社 抗ｐｅｒｐ抗体
JP6077745B2 (ja) * 2009-09-30 2017-02-08 富士フイルムＲｉファーマ株式会社 抗ポドプラニン抗体、及び抗ポドプラニン抗体を含む医薬組成物
WO2018181207A1 (fr) * 2017-03-27 2018-10-04 ノイルイミューン・バイオテック株式会社 Récepteur antigénique chimérique
JPWO2018181207A1 (ja) * 2017-03-27 2020-02-27 ノイルイミューン・バイオテック株式会社 キメラ抗原受容体
EP3604344A4 (fr) * 2017-03-27 2021-01-06 Noile-Immune Biotech, Inc. Récepteur antigénique chimérique
US20210122831A1 (en) * 2017-03-27 2021-04-29 Noile-Immune Biotech, Inc. Chimeric antigen receptor
TWI753141B (zh) * 2017-03-27 2022-01-21 日商諾伊爾免疫生物科技股份有限公司 嵌合抗原受體
AU2018242408B2 (en) * 2017-03-27 2022-08-04 Noile-Immune Biotech, Inc. Chimeric antigen receptor

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