WO2012074097A1 - Anti-cd33 antibody - Google Patents

Anti-cd33 antibody Download PDF

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Publication number
WO2012074097A1
WO2012074097A1 PCT/JP2011/077924 JP2011077924W WO2012074097A1 WO 2012074097 A1 WO2012074097 A1 WO 2012074097A1 JP 2011077924 W JP2011077924 W JP 2011077924W WO 2012074097 A1 WO2012074097 A1 WO 2012074097A1
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amino acid
seq
acid sequence
antibody
sequence represented
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PCT/JP2011/077924
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French (fr)
Japanese (ja)
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丹羽　倫平
知明 中川
拓也 村上
麗夫 久保田
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協和発酵キリン株式会社
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to a monoclonal antibody or fragment thereof that binds to the human CD33 extracellular region with high affinity and exhibits high antibody-dependent cellular cytotoxicity (hereinafter sometimes referred to as ADCC activity).
  • ADCC activity antibody-dependent cellular cytotoxicity
  • the present invention relates to a therapeutic agent and a diagnostic agent using the fragment.
  • CD 33 Cluster of differentiation (hereinafter sometimes referred to as CD) 33 is a glycoprotein having a molecular weight of about 67,000 expressed in myeloid progenitor cells, monocytes and granulocytes.
  • CD33 is a type I transmembrane protein, and there are two immunoglobulin superfamily domains (IgV and IgC2 in order from the N-terminal toward the cell membrane) outside the cell. Three cysteine residues exist in each domain. Form intradomain and interdomain disulfide bonds. There are a total of five N-linked glycosylation sequences in the CD33 extracellular region.
  • CD33 is known to bind to sialic acid via the IgV region, and is also called as Siglec-3 or sialoadhesin, but the details of its function are unknown (Non-patent Document 1).
  • CD33 also known as gp67, sialoadhesin, siglec-3, was cloned in 1988 (Non-Patent Document 2), and the DNA sequence, amino acid sequence and three-dimensional structure of CD33 have been published on public databases. For example, it can be referred to from an accession number such as P20138 (SWISSPROT) or M23197 (EMBL).
  • Non-patent Document 3 L4F3 and L1B2 established for the first time as monoclonal antibodies that bind to CD33 (hereinafter sometimes referred to as mAb) were reported in 1983 (Non-patent Document 3). Since then, many monoclonal antibodies against CD33 (hereinafter sometimes referred to as anti-CD33 mAb) have been reported. Many of the reported anti-CD33 mAbs are known to recognize the same or adjacent regions (Non-patent Document 1).
  • CD33 is highly expressed in most acute myelogenous leukemia (acute myelogenous leukemia, hereinafter sometimes referred to as AML) cells compared to normal cells, whereas it is not expressed in hematopoietic stem cells.
  • CD33 mAb is being clinically applied with the aim of treating AML.
  • gemtuzumab ozogamicin, a conjugate of P67.6, an anti-CD33 mAb, and a calicheamicin derivative has been used clinically as an AML therapeutic.
  • anti-CD33 mAb developed as a cancer therapeutic agent examples include gemtumumab ozogamicin (P67.6) (Non-patent Document 4), lintuzumab (HuM195) (Non-patent Document 5), and the like. These antibodies are antibodies that exert their medicinal effects by specifically damaging CD33-expressing cells that are target cells. Gemtumumab ozogamicin has the property of being bound into the cell after binding to the CD33 antigen on the cell surface. Utilizing this property, calicheamicin derivatives are sent into the cell and have cytotoxic activity in the cell. It is considered that ADCC activity is the main medicinal mechanism of lintuzumab (Non-patent Document 6).
  • the ADCC activity is an activity that activates immune cells (such as natural killer cells) by damaging the target cells by binding an antibody bound to an antigen on the target cells to the Fc receptor of immune cells through its Fc region.
  • immune cells such as natural killer cells
  • CDC activity complement-dependent injury activity of these antibodies
  • CDC activity is an activity in which an antibody bound to an antigen on a target cell activates a series of cascades (complement activation pathways) composed of complement-related proteins in the blood and damages the target cell.
  • cascades complement activation pathways
  • migration and activation of immune cells can be induced by protein fragments generated by complement activation.
  • a human antibody of IgG type has a consensus sequence in which one N-linked sugar chain binds to its constant region.
  • an antibody having a consensus sequence to which an N-linked sugar chain binds also in the variable region changes the sugar chain binding, making it difficult to stably supply a uniform antibody as a pharmaceutical product.
  • it is essential for the binding between proteins.
  • LFA-3 lymphocyte function-associated antigen 3
  • CD2 CD2
  • antibody There is a possibility that the binding property of the antibody to the antigen is changed by binding of the sugar chain to the variable region which is the binding site of
  • Non-patent Document 8 In the binding between an antibody and an Fc receptor, the importance of a sugar chain bound to the second domain of the hinge region and constant region of the antibody has been suggested (Non-patent Document 8). It is known that galactose is added to the non-reducing end of the N-glycoside-linked complex sugar chain bound to the Fc region of the antibody molecule and fucose is added to N-acetylglucosamine at the reducing end. In particular, an antibody in which fucose is not bound to N-acetylglucosamine at the reducing end of a sugar chain has been reported to exhibit high ADCC activity (Non-patent Document 10 and Patent Document 9). 1).
  • antibodies generally used as pharmaceuticals are produced using gene recombination techniques, and are produced using animal cells such as CHO cells derived from Chinese hamster ovary tissue as host cells.
  • animal cells such as CHO cells derived from Chinese hamster ovary tissue as host cells.
  • the sugar chain structure varies depending on the host cell.
  • CDC activity varies depending on the antibody subclass. CDC activity is high in human IgG1 and IgG3, and its strength is generally in the order of IgG3 ⁇ IgG1 >> IgG2 ⁇ IgG4. Furthermore, it is known that the CDC activity can be further increased as compared with IgG3 by exchanging the CH2 domain of the constant region of human IgG1 with the CH2 domain of IgG3 (Patent Document 3).
  • An object of the present invention is to provide a monoclonal antibody or fragment thereof that binds to the human CD33 extracellular region with high affinity and exhibits high ADCC activity, DNA encoding the antibody or the fragment, a vector containing the DNA, and a vector comprising the vector It is to provide a transformant obtained by introduction, a method for producing the antibody or the fragment using the transformant, and a therapeutic agent and a diagnostic agent containing the antibody or the fragment.
  • the present inventors obtained mouse and rat monoclonal antibodies that bind to the human CD33 extracellular region with high affinity, and the complementarity determining region (hereinafter sometimes referred to as VH) of the antibody heavy chain variable region (hereinafter sometimes referred to as VH).
  • VH complementarity determining region
  • VL antibody heavy chain variable region
  • the present invention is as follows. 1. (I) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 52, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 99, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 54, and SEQ ID NO: 55 A monoclonal antibody that binds to human CD33, comprising a light chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 56, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 56, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 57, or A fragment of the antibody, (Ii) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 52, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 100, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 54, and SEQ ID NO: 55 A monoclonal antibody that binds to
  • CDR1, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 56 and a light chain variable region comprising a CDR3 comprising an amino acid sequence shown in SEQ ID NO: 57, a fragment of a monoclonal antibody or the antibody that binds human CD33.
  • a monoclonal antibody that binds to human CD33 comprising a CDR1 comprising an amino acid sequence, a CDR2 comprising an amino acid sequence represented by SEQ ID NO: 56, and a light chain variable region having a CDR3 comprising an amino acid sequence represented by SEQ ID NO: 57 fragment.
  • the monoclonal antibody or the fragment of the antibody according to item 2 comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 86 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 85. 4).
  • the monoclonal antibody or fragment of the antibody according to item 2 comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 87 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 85. 5.
  • a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36, and SEQ ID NO: 37
  • a monoclonal antibody that binds to human CD33 comprising a light chain variable region comprising CDR1, comprising the amino acid sequence represented by SEQ ID NO: 38, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 38, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 39;
  • a fragment of the antibody (Ii) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 40, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 41, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 42, and SEQ ID NO: 43
  • a monoclonal antibody that binds to human CD33 comprising a light chain
  • Dissociation constant K D for human CD33 is 4.0 ⁇ 10 -9 M or less, a monoclonal antibody or fragment of the antibody according to any one of the preceding 1 7.
  • Dissociation constant K D for human CD33 is 2.0 ⁇ 10 -9 M or less, a monoclonal antibody or fragment of the antibody according to [8. 10.
  • a recombinant vector comprising DNA encoding a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 89 and a recombinant vector comprising DNA encoding a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 90
  • a recombinant vector comprising a DNA encoding a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 89 and a recombinant vector comprising a DNA encoding a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 91 20.
  • the transformant according to the item 18 or 19 is cultured in a medium, and the monoclonal antibody or the fragment of the antibody according to any one of the items 1 to 14 is produced and accumulated in the culture, and the monoclonal antibody is produced from the culture.
  • the method for producing a monoclonal antibody or a fragment of the antibody according to any one of items 1 to 14, wherein a fragment of the antibody is collected.
  • 21. 15 A pharmaceutical composition comprising the monoclonal antibody or the antibody fragment according to any one of items 1 to 14 as an active ingredient.
  • 22. 15 A therapeutic agent for a disease involving CD33 positive cells, comprising the monoclonal antibody or the antibody fragment according to any one of items 1 to 14 as an active ingredient. 23. 23.
  • the therapeutic agent according to item 22 above, wherein the disease involving CD33 positive cells is cancer, autoimmune disease or allergic disease.
  • the cancer is blood cancer, breast cancer, uterine cancer, colon cancer, esophageal cancer, stomach cancer, ovarian cancer, lung cancer, kidney cancer, rectal cancer, thyroid cancer, cervical cancer, small intestine cancer, prostate cancer or pancreatic cancer.
  • the blood cancer is acute myeloid leukemia, anaplastic large cell lymphoma, acute lymphocytic leukemia, myelodysplastic syndrome, multiple myeloma, Hodgkin lymphoma, or non-Hodgkin lymphoma.
  • a method for treating a disease involving CD33-positive cells comprising a step of administering the monoclonal antibody or the antibody fragment according to any one of items 1 to 14 to a mammal. 27.
  • the disease involving CD33 positive cells is cancer, autoimmune disease or allergic disease. 28.
  • the cancer is blood cancer, breast cancer, uterine cancer, colon cancer, esophageal cancer, stomach cancer, ovarian cancer, lung cancer, kidney cancer, rectal cancer, thyroid cancer, cervical cancer, small intestine cancer, prostate cancer or pancreatic cancer. The method described in 1. 29. 29.
  • the blood cancer is acute myeloid leukemia, anaplastic large cell lymphoma, acute lymphocytic leukemia, myelodysplastic syndrome, multiple myeloma, Hodgkin lymphoma, or non-Hodgkin lymphoma.
  • the monoclonal antibody or fragment of the antibody according to any one of 1 to 14 above for treating a disease involving CD33 positive cells.
  • 31. 31. The use according to item 30 above, wherein the disease involving CD33 positive cells is cancer, autoimmune disease or allergic disease. 32.
  • the cancer is blood cancer, breast cancer, uterine cancer, colon cancer, esophageal cancer, stomach cancer, ovarian cancer, lung cancer, kidney cancer, rectal cancer, thyroid cancer, cervical cancer, small intestine cancer, prostate cancer or pancreatic cancer.
  • the blood cancer is acute myeloid leukemia, anaplastic large cell lymphoma, acute lymphocytic leukemia, myelodysplastic syndrome, multiple myeloma, Hodgkin lymphoma or non-Hodgkin lymphoma.
  • the monoclonal antibody of the present invention binds to the human CD33 extracellular region with high affinity and exhibits high ADCC activity. Therefore, the present invention relates to the antibody or the fragment, the DNA encoding the antibody or the fragment, the vector containing the DNA, the transformant obtained by introducing the vector, the antibody using the transformant or the A method for producing a fragment, and a therapeutic agent and a diagnostic agent containing the antibody or the fragment can be provided.
  • FIG. 1A shows a schematic diagram of human CD33 expression vector construction.
  • FIG. 1B shows a schematic diagram of cynomolgus monkey CD33 expression vector construction.
  • FIG. 1C shows a schematic diagram of chimpanzee CD33 expression vector construction.
  • FIG. 2 shows a schematic diagram of the construction of a histidine-tagged soluble human CD33 expression vector.
  • FIG. 3 shows the evaluation (FCM) of rat and mouse anti-CD33 monoclonal antibodies.
  • FIG. 4 shows the evaluation (ELISA) of rat and mouse anti-CD33 monoclonal antibodies.
  • FIG. 5A shows a schematic diagram of construction of an anti-CD33 chimeric antibody expression vector.
  • FIG. 5B shows a schematic diagram of construction of an anti-CD33 chimeric antibody expression vector.
  • FIG. 5A shows a schematic diagram of construction of an anti-CD33 chimeric antibody expression vector.
  • FIG. 5B shows a schematic diagram of construction of an anti-CD33 chimeric antibody expression vector.
  • FIG. 6 shows the evaluation (FCM) of the anti-CD33 chimeric antibody.
  • FIG. 7 shows the evaluation (ELISA) of anti-CD33 chimeric antibody.
  • FIG. 8 shows the evaluation (ADCC) of anti-CD33 chimeric antibody.
  • FIG. 9 shows the evaluation (CDC) of anti-CD33 chimeric antibody.
  • FIG. 10 shows the binding amount (FIG. 10A) and residual rate (FIG. 10B) of the antibody on the cell membrane of the anti-CD33 chimeric antibody.
  • FIG. 11 shows a schematic diagram of HM195-1, -113F antibody expression vector construction.
  • FIG. 12 shows the anti-CD33 humanized antibody evaluation (ADCC).
  • FIG. 13 shows the amount of antibody bound on the cell membrane of the anti-CD33 humanized antibody (FIG.
  • FIG. 14 shows the cell removal activity of anti-CD33 humanized antibody in human peripheral blood.
  • FIG. 15 shows the reactivity of anti-CD33 humanized antibodies to human, cynomolgus monkey and chimpanzee CD33.
  • the present invention relates to a monoclonal antibody or a fragment thereof that binds to the extracellular region of human CD33 with high affinity and has high ADCC activity (hereinafter simply referred to as “CD33” means human CD33).
  • CD33 means human CD33.
  • CD33 in the present invention one or more amino acids in the polypeptide having the amino acid sequence represented by SEQ ID NO: 3 or P20148 (SWISSPROT) or the amino acid sequence represented by SEQ ID NO: 3 or EMBL accession number M23197 are deleted or substituted.
  • a polypeptide having an amino acid sequence having the homology of (2) most preferably a polypeptide having an amino acid sequence having a homology of 95% or more and having the function of CD33.
  • a polypeptide having an amino acid sequence in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 3 or P20148 can be obtained by site-directed mutagenesis [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997), Nucleic 64 Proc. Natl. Acad. Sci. USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci.
  • the number of amino acids to be deleted, substituted or added is not particularly limited, but is preferably 1 to several tens, for example 1 to 20, more preferably 1 to several, for example 1 to 5 amino acids. It is.
  • Examples of the gene encoding CD33 include the nucleotide sequence represented by SEQ ID NO: 4 or EMBL accession number M23197.
  • DNA having the base sequence represented by SEQ ID NO: 4 or EMBL accession number M23197 was used as a probe, and colony hybridization, plaque hybridization, Southern blot -It means a hybridizable DNA obtained by a hybridization method or a DNA microarray method.
  • 0.7 to 1.0 mol / L of sodium chloride is present using a DNA or DNA derived from a hybridized colony or plaque, or a filter or slide glass on which a PCR product or oligo DNA having the sequence is immobilized. Hybridization at 65 ° C.
  • the DNA capable of hybridizing is DNA having at least 60% homology with the base sequence represented by SEQ ID NO: 4 or EMBL accession number M23197, preferably DNA having 80% homology or more, more preferably 95 % Of DNA having a homology of at least%.
  • a polymorphism of the gene is often observed.
  • a gene in which a small-scale variation is caused in the nucleotide sequence due to such polymorphism in the gene used in the present invention is also included in the gene encoding CD33 of the present invention.
  • the numerical value of homology in the present invention may be a numerical value calculated using a homology search program known to those skilled in the art unless otherwise specified, but the base sequence may be BLAST [J. Mol. Biol. , 215, 403 (1990)], for amino acid sequences such as numerical values calculated using default parameters, BLAST2 [Nucleic Acids Res. , 25, 3389 (1997), Genome Res. , 7, 649 (1997), http: // www. ncbi. nlm. nih. gov / Education / BLASTinfo / information3. numerical values calculated using default parameters in [html].
  • the default parameters are 5 if G (Cost to open gap) is a base sequence, 11 if it is an amino acid sequence, 2 if -E (Cost to extend gap) is a base sequence, and 1 if it is an amino acid sequence.
  • -Q (Penalty for nucleotide mismatch) is -3
  • -r (reward for nucleotide match) is 1
  • -e (expect value) is 10
  • 11 residues when -W (wordsize) is a base sequence
  • -y [Dropoff (X) for blast extensions in bits] is 20 if blastn, 7 for programs other than blastn
  • -X X dropoff value f If the r aligned alignment in bits) is 15 and -Z (final X dropoff value for gapd alignment in bits) is blastn, it is 25 for programs other than blastn (http: // www. gov / blast / html / blastcgihelp.html).
  • a polypeptide comprising a partial sequence of the amino acid sequence represented by SEQ ID NO: 3 or P20148 can be prepared by methods known to those skilled in the art. For example, it can be produced by culturing a transformant in which a part of DNA encoding the amino acid sequence represented by SEQ ID NO: 3 has been deleted and an expression vector containing the DNA is introduced. Further, based on the polypeptide or DNA produced by the above method, one or more amino acids are deleted or substituted in the partial sequence of the amino acid sequence represented by SEQ ID NO: 3 or P20148 (SWISSPROT) by the same method as above. Alternatively, a polypeptide having an added amino acid sequence can be obtained.
  • a polypeptide comprising a partial sequence of the amino acid sequence represented by SEQ ID NO: 3 or P20148 (SWISSPROT), or one or more amino acids in the partial sequence of the amino acid sequence represented by SEQ ID NO: 3 or P20138 (SWISSPROT) is deleted or substituted
  • a polypeptide having an added amino acid sequence can also be produced by a chemical synthesis method such as a fluorenylmethyloxycarbonyl (Fmoc) method or a t-butyloxycarbonyl (tBoc) method.
  • the amino acid sequence of the polypeptide represented by SEQ ID NO: 3 is converted into a known transmembrane region prediction program SOSUI (http://bp.nuap.nagoya-u.ac.jp / SOSUI / SOSUI_submit.), TMHMM ver. 2 (http://www.cbs.dtu.dk/services/TMHMM-2.0/) or ExPASy Proteomics Server (http://Ca.expasy.org/) It is done. Specifically, the 242nd from the N-terminal which is an extracellular domain predicted in SOSUI is mentioned. The extracellular domain has two immunoglobulin superfamily domains, and both domains are included.
  • SOSUI transmembrane region prediction program
  • the extracellular region of CD33 in the present invention is any as long as the extracellular region of CD33 having the amino acid sequence represented by SEQ ID NO: 3 or P20148 (SWISSPROT) has a structure equivalent to the structure that can be taken in the natural state. It may be a structure.
  • the structure that the extracellular region of CD33 can take in the natural state refers to the natural three-dimensional structure of CD33 expressed on the cell membrane.
  • CD33 The details of the function of CD33 are unknown. Examples of the function of CD33 in the present invention include expression in the differentiation stage of myeloid cells, adhesion to sialic acid-containing cells such as CD34 positive stem cells, and involvement in the control of cell differentiation. Further, since CD33 is a sialic acid binding protein, the activity of binding to sialic acid molecules and the intracellular signal transduction function or the motility of cells due to the binding with sialic acid molecules can also be mentioned as functions of CD33.
  • the binding of the antibody of the present invention or a fragment thereof to the extracellular region of CD33 is known for cells expressing CD33 using a radioimmunoassay using a solid phase sandwich method or an enzyme immunoassay (ELISA). It can be confirmed by an immunological detection method, preferably a method capable of examining the binding ability of a cell expressing a specific antigen and an antibody to the specific antigen, such as a fluorescent cell staining method.
  • a fluorescent antibody staining method using a FMAT8100HTS system (Applied Biosystems) or the like [Cancer Immunol. Immunother. , 36, 373 (1993)], fluorescent cell staining using flow cytometry, or surface plasmon resonance using a Biacore system (manufactured by GE Healthcare).
  • known immunological detection methods [Monoclonal Antibodies-Principles and practices, Third edition, Academic Press (1996), Antibodies-A Laboratory Manual, ColdHordrology, ColdHordrology, ColdHorman, ColdHordrology, ColdHorman, ColdHorman (1987)] etc. can also be confirmed.
  • the cells expressing CD33 may be any cells as long as they express CD33.
  • cells naturally existing in the human body cell lines established from cells naturally existing in the human body, or genetic recombination Examples include cells obtained by technology.
  • cells that naturally exist in the human body include normal monocytes, granulocytes, or myeloid cells, as well as myeloid leukemia, for example, cells in which the polypeptide is expressed in AML patients.
  • normal monocytes granulocytes, or myeloid cells
  • myeloid leukemia for example, cells in which the polypeptide is expressed in AML patients.
  • tumor cells obtained by biopsy or the like cells expressing CD33 can be mentioned.
  • CD33 is expressed among the cell lines obtained by stocking cells expressing CD33 obtained from the above cancer patients.
  • Cell lines For example, human myeloid leukemia cell line NB-4 [DSMZ number: ACC207] or HL-60 [ATCC number: CCL-240], which are cell lines established from humans, can be mentioned.
  • the cells obtained by gene recombination techniques include, for example, cells expressing CD33 obtained by introducing an expression vector containing cDNA encoding CD33 into insect cells or animal cells. Etc.
  • the antibody of the present invention is a monoclonal antibody against human CD33 that binds to the extracellular region of human CD33 and has high ADCC activity, and a fragment of the antibody, and preferably has high affinity for human CD33. Having high affinity for human CD33 means that the dissociation constant for human CD33 is low.
  • K D Dissociation constant for human CD33 antibodies of the present invention
  • K D Dissociation constant for human CD33 antibodies of the present invention
  • dissociation constant K D for human CD33 4.0 ⁇ 10 -9 M or less, can bind to the extracellular region of CD33 with sufficient affinity as a therapeutic antibody, and high ADC activity and antitumor activity Can have.
  • the dissociation constant, K D is 4 ⁇ 10 -9 M about affinity, since the show a therapeutic effect, the antibodies of the invention, higher therapeutic effects Can be shown.
  • Affinity is measured by reaction kinetic analysis, and can be measured using, for example, Biacore T100 (manufactured by GE Healthcare Bioscience).
  • the antibody of the present invention is characterized by very slow dissociation from the antigen. Since the dissociation is slow, or the dissociation is slow and the incorporation into the cell is slow, there is an advantage that the amount of antibody bound to the cell surface and the residual ratio are high.
  • examples of the monoclonal antibody include an antibody produced by a hybridoma or a recombinant antibody produced by a transformant transformed with an expression vector containing an antibody gene.
  • a monoclonal antibody is an antibody that is secreted by an antibody-producing cell of a single clone, recognizes only one epitope (also referred to as an antigenic determinant), and has a uniform amino acid sequence (primary structure) constituting the monoclonal antibody. .
  • Epitopes include a single amino acid sequence that is recognized and bound by a monoclonal antibody, a three-dimensional structure composed of amino acid sequences, an amino acid sequence linked with sugar chains, and a three-dimensional structure composed of amino acid sequences combined with sugar chains.
  • the epitope that is recognized and bound by the monoclonal antibody of the present invention is preferably an epitope consisting of a plurality of amino acid residues contained in the extracellular region of CD33, the 18th to 259th amino acid sequences of the amino acid sequence shown in SEQ ID NO: 3. More preferably, an epitope consisting of a plurality of amino acid residues contained in an immunoglobulin domain far from the cell membrane of the extracellular region of CD33, or a plurality of amino acid residues contained in an immunoglobulin domain close to the cell membrane of the extracellular region of CD33
  • slow dissociation means that the value of the antibody dissociation rate constant kd calculated in Biacore T100 shows a smaller value.
  • the dissociation rate constant kd is measured using, for example, Biacore T100 (manufactured by GE Healthcare Bioscience), and can be calculated using attached software Biacore T100 evaluation software (manufactured by Biacore).
  • an antibody having ADCC activity refers to a known measurement method [Cancer Immunol. Immunother. , 36, 373 (1993)], which is an antibody having detectable ADCC activity.
  • an antibody having CDC activity refers to the CDC activity of a plurality of antibodies at the same time against CD33-expressing cells using a known CDC measurement method [Cancer Immunol. Immunother. , 36, 373 (1993)], which is an antibody having detectable CDC activity.
  • the monoclonal antibody of the present invention specifically includes a monoclonal antibody that binds to the same epitope as that on human CD33 to which any one of the following antibodies (i) to (v) binds: Examples thereof include a monoclonal antibody that competes with any one of the antibodies (1) to (v) and binds to human CD33, and an antibody having the same reactivity as any one of the following antibodies (i) to (v).
  • VH of an antibody is an amino acid sequence represented by SEQ ID NO: 132
  • VL of an antibody is an amino acid sequence represented by SEQ ID NO: 137.
  • VH of an antibody is represented by SEQ ID NO: 133
  • VL of the antibody is the amino acid sequence represented by SEQ ID NO: 138.
  • the VH of the monoclonal antibody (iii) antibody is the amino acid sequence represented by SEQ ID NO: 134
  • the VL of the antibody is SEQ ID NO:
  • the monoclonal antibody (v) of the monoclonal antibody (iv) antibody having the amino acid sequence represented by 139 is the amino acid sequence represented by SEQ ID NO: 135, and the monoclonal antibody (v )
  • An amino acid sequence in which the VH of the antibody is an amino acid sequence represented by SEQ ID NO: 136 and the VL of the antibody is represented by SEQ ID NO: 141
  • the monoclonal antibody of the present invention includes a monoclonal antibody that binds to the same epitope on human CD33 to which any one of the following antibodies (i) to (v) binds, and the following (i) to (v) Examples thereof include a monoclonal antibody that competes with any one of the antibodies and binds to human CD33, and an antibody having reactivity equivalent to any one of the following antibodies (i) to (v).
  • VH of an antibody is an amino acid sequence represented by SEQ ID NO: 103
  • VL of an antibody is an amino acid sequence represented by SEQ ID NO: 95.
  • VH of an antibody is represented by SEQ ID NO: 104
  • VL of the antibody is the amino acid sequence represented by SEQ ID NO: 95
  • the VH of the monoclonal antibody (iii) antibody is the amino acid sequence represented by SEQ ID NO: 105
  • the VL of the antibody is SEQ ID NO:
  • the monoclonal antibody (v) of the monoclonal antibody (iv) having the amino acid sequence represented by 95 is the amino acid sequence represented by SEQ ID NO: 93
  • the VH of the antibody is the amino acid sequence represented by SEQ ID NO: 106
  • the VL of the antibody is the amino acid sequence represented by SEQ ID NO: 95
  • the monoclonal antibodies of the present invention include the following monoclonal antibodies that bind to human CD33 (i) to (v).
  • a monoclonal antibody that binds to human CD33 comprising a light chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 38, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 38, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 39.
  • a monoclonal antibody that binds to human CD33 comprising a light chain variable region comprising CDR3 comprising an amino acid sequence
  • a CDR1 comprising an amino acid sequence represented by SEQ ID NO: 58
  • a CDR2 comprising an amino acid sequence represented by SEQ ID NO: 59
  • a sequence A heavy chain variable region comprising CDR3 comprising the amino acid sequence represented by number 60
  • CDR1 comprising the amino acid sequence represented by SEQ ID NO: 61
  • CDR2 comprising the amino acid sequence represented by SEQ ID NO: 62
  • an amino acid sequence represented by SEQ ID NO: 63 A human CD comprising a light chain variable region comprising CDR3 comprising Monoclonal antibody binding to 33
  • the monoclonal antibody of the present invention comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 52, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 53, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 54.
  • An antibody modified so as not to have a consensus sequence for binding an N-linked sugar chain to the V region of a monoclonal antibody that binds to human CD33 (hereinafter sometimes referred to as a modified antibody) is also included.
  • CDR1 and CDR3 of VH of the antibody include the amino acid sequences shown by SEQ ID NOs: 52 and 54, respectively, and CDR1, CDR2 and CDR3 of VL of the antibody are respectively And monoclonal antibodies comprising the amino acid sequences shown in SEQ ID NOs: 55, 56 and 57.
  • the VH CDR2 of the above antibody is the CDR2 of the modified antibody VH, for example, a modification in which the third Asn in the amino acid sequence represented by SEQ ID NO: 53 is substituted with another amino acid residue, and the fifth CDR2 containing an amino acid sequence in which at least one modification is introduced among the modifications that substitute Ser in the other amino acid residues.
  • Specific examples include the following amino acid sequences (1) to (5).
  • the hybridoma is prepared by using, for example, cells expressing the above-mentioned CD33 as an antigen, inducing antibody-producing cells having antigen specificity from an animal immunized with the antigen, and further fusing the antibody-producing cells and myeloma cells.
  • the anti-CD33 monoclonal antibody can be obtained by culturing the hybridoma or administering the hybridoma to an animal to cause the animal to undergo ascites cancer, and separating and purifying the culture medium or ascites.
  • Any animal can be used as the animal to immunize with the antigen as long as it can produce a hybridoma, but mice, rats, hamsters, chickens, rabbits, cows and the like are preferably used.
  • An antibody produced by a hybridoma prepared by obtaining a cell capable of producing an antibody from such an animal, immunizing the cell in vitro, and then fusing it with a myeloma cell is also an antibody of the present invention. Is included.
  • Examples of the recombinant antibody in the present invention include antibodies produced by genetic recombination, such as human chimeric antibodies, human CDR-grafted antibodies (sometimes referred to as humanized antibodies), human antibodies or antibody fragments. .
  • a recombinant antibody having characteristics of a monoclonal antibody, low antigenicity, and extended blood half-life is preferable as a therapeutic agent.
  • the recombinant antibody include those obtained by modifying the monoclonal antibody of the present invention using a gene recombination technique.
  • the human chimeric antibody is sometimes referred to as VH and VL of a non-human animal antibody, heavy chain constant region of human antibody (hereinafter sometimes referred to as CH) and light chain constant region (hereinafter referred to as CL). ).
  • the human chimeric antibody of the present invention obtains cDNAs encoding VH and VL from a hybridoma that specifically recognizes CD33 and produces a monoclonal antibody that binds to the extracellular region of CD33.
  • a human chimeric antibody expression vector can be constructed by inserting into an expression vector for animal cells having a gene encoding CL, and introduced into animal cells for expression and production.
  • the CH of the human chimeric antibody may be any as long as it belongs to human immunoglobulin (hereinafter sometimes referred to as hIg), but preferably the hIgG class is used, and hIgG1, which belongs to the hIgG class, Any of the subclasses such as hIgG2, hIgG3 or hIgG4 can be used.
  • the CL of the human chimeric antibody may be any as long as it belongs to hIg, and those of ⁇ class or ⁇ class can be used.
  • the chimeric antibody of the present invention is the same as the chimeric antibody that binds to the extracellular region of CD33 and the epitope present on the extracellular region of CD33 to which the chimeric antibody binds by competing with the monoclonal antibody described above. Mention may be made of chimeric antibodies that bind to an epitope.
  • the human CDR-grafted antibody refers to an antibody obtained by grafting the VH and VL CDR amino acid sequences of non-human animal antibodies to appropriate positions of the human antibody VH and VL.
  • the human CDR-grafted antibody of the present invention specifically recognizes V33 of a non-human animal antibody produced from a hybridoma that specifically recognizes CD33 and produces a monoclonal antibody of a non-human animal that binds to the extracellular region.
  • a cDNA encoding the V region in which the amino acid sequence of the CDR of the VL was grafted to the VH and VL framework regions of the human antibody (hereinafter sometimes referred to as FR) was constructed, and the CH and CL of the human antibody were
  • a human CDR-grafted antibody expression vector can be constructed by inserting it into an expression vector for animal cells having a gene to be encoded, and introduced into animal cells for expression and production.
  • the CH of the human CDR-grafted antibody may be any as long as it belongs to hIg, but preferably the hIgG class is used, and any subclass such as hIgG1, hIgG2, hIgG3, or hIgG4 belonging to the hIgG class is used. be able to.
  • the CL of the human CDR-grafted antibody may be any CL as long as it belongs to hIg, and those of ⁇ class or ⁇ class can be used.
  • the VH of the antibody has the amino acid sequence represented by SEQ ID NO: 84 or the 25th Ser in the amino acid sequence represented by SEQ ID NO: 84, 27th Gly, 28th Ser, 29th Val, 30th Ser, 40th Gln, 45th Gly, 46th Leu, 49th Ile, 72nd Val, 93th Val,
  • An amino acid sequence in which at least one amino acid residue selected from the 95th Tyr, the 97th Ala and the 106th Thr is substituted with another amino acid residue, and / or the VL of the antibody is SEQ ID NO: 85; Or the second Ile in the amino acid sequence represented by SEQ ID NO: 85, the 8th Pro, the 11th Leu, the 15th
  • a humanized antibody including an amino acid sequence in which at least one amino acid residue selected from Pro and 90th Val is substituted with another amino acid residue, but the number of modifications to be introduced is not limited .
  • the VH of the antibody is the 25th Ser, 27th Gly, 28th Ser, 29th Val, 30th Ser, 40th Gln, 45th Gly in the amino acid sequence represented by SEQ ID NO: 84, At least one amino acid residue selected from 46th Leu, 49th Ile, 72nd Val, 93th Val, 95th Tyr, 97th Ala and 106th Thr is another amino acid residue
  • a humanized antibody comprising an amino acid sequence substituted with.
  • the following (1) to (3) are preferable.
  • the humanized antibody comprising the amino acid sequence in which the No. Ala is substituted with another amino acid residue (2) 27th Gly, 28th Ser, 29 in the amino acid sequence in which the VH of the antibody is represented by SEQ ID NO: 84
  • the VH of the humanized antibody (3) antibody comprising the amino acid sequence in which the No.
  • Val, the 30th Ser, the 72nd Val, and the 97th Ala are substituted with other amino acid residues is represented by SEQ ID NO: 84
  • Humanized antibody comprising an amino acid sequence in which the 27th Gly and the 72nd Val in the amino acid sequence are substituted with other amino acid residues
  • the amino acid sequence of the VH of the obtained antibody is, for example, the 25th Ser in the amino acid sequence represented by SEQ ID NO: 84, the 27th Gly in the Tyr, and the 28th in the amino acid sequence.
  • An amino acid sequence into which one modification has been introduced is mentioned.
  • amino acid sequence of VH into which 14 modifications have been introduced include, for example, 25th Ser in the amino acid sequence represented by SEQ ID NO: 84, Thr, 27th Gly in Tyr, 28 The th Ser, Thr, the 29th Val to Ile, the 30th Ser to Thr, the 40th Gln to Lys, the 45th Gly to Arg, the 46th Leu to Met, and the 49th An amino acid sequence in which Ile is replaced by Met, 72nd Val is replaced by Arg, 93rd Val is replaced by Thr, 95th Tyr is replaced by Phe, 97th Ala is replaced by Thr, and 106th Thr is replaced by Val. Can be mentioned.
  • amino acid sequence of VH into which 13 modifications have been introduced include the following amino acid sequences (1) to (14).
  • the 27th Gly in the amino acid sequence represented by SEQ ID NO: 84 is Tyr, the 28th Ser is Thr, the 29th Val is Ile, the 30th Ser is Thr, and the 40th Gln To Lys, 45th Gly to Arg, 46th Leu to Met, 49th Ile to Met, 72nd Val to Arg, 93rd Val to Thr, 95th Tyr to Phe
  • amino acid sequence represented by SEQ ID NO: 84 the 97th Ala is replaced with Thr and the 106th Thr is replaced with Val.
  • the 25th Ser in the amino acid sequence represented by SEQ ID NO: 84 is Thr, and the 28th Ser is Thr. 29th Val to Ile, 30th Ser to Thr, 40th Gln to Lys, 45th Gly to Arg, 46th Leu to M t, 49th Ile to Met, 72nd Val to Arg, 93rd Val to Thr, 95th Tyr to Phe, 97th Ala to Thr, and 106th Thr to Val (3)
  • the 25th Ser in Thr, the 27th Gly in Tyr, the 29th Val in Ile, and the 30th Ser in Thr 40th Gln to Lys 45th Gly to Arg, 46th Leu to Met, 49th Ile to Met, 72nd Val to Arg, 93th Val to Thr, 95
  • the 25th Ser in Thr is Thr
  • the 25th Ser is set to Thr, the 27th Gly to Tyr, the 28th Ser to Thr, the 29th Val to Ile, the 30th Ser to Thr, 45th Gly to Arg, 46th Leu to Met, 49th Ile to Met, 72nd Val to Arg, 93rd Val to Thr, 95th Tyr to Phe, 97th Amino acid sequence in which Ala is replaced with Thr, and Thr at 106th is replaced with Val (7)
  • the 25th Ser in the amino acid sequence represented by SEQ ID NO: 84 is hr, 27th Gly to Tyr, 28th Ser to Thr, 29th Val to Ile, 30th Ser to Thr, 40th Gln to Lys, 46th Leu to Met
  • the 49th Ile is replaced by Met
  • the 72nd Val is replaced by Arg
  • the 93rd Val is replaced by Thr
  • the 95th Tyr is replaced by Phe
  • the 97th Ala is replaced by Thr
  • the 25th Ser in the amino acid sequence represented by SEQ ID NO: 84 is Thr, the 27th Gly is Tyr, and the 28th Ser Thr, 29th Val to Ile, 30th Ser to Thr, 40th Gln to Lys, 45th Gly to Arg, 46th Leu to Met, 49th Ile to Met
  • the 41st Gly is Arg
  • the 46th Leu is Met
  • the 49th Ile is Met
  • the 72nd Val is Arg
  • the 93rd Val is Thr.
  • amino acid sequence of VH into which 10 modifications have been introduced include the following amino acid sequences (1) to (3).
  • the 25th Ser in the amino acid sequence represented by SEQ ID NO: 84 is Thr
  • the 27th Gly is Tyr
  • the 28th Ser is Thr
  • the 29th Val is Ile
  • the 30th Ser Is replaced with Thr, 72nd Val with Arg, 93rd Val with Thr, 95th Tyr with Phe, 97th Ala with Thr, and 106th Thr with Val.
  • the 27th Gly is Tyr
  • the 28th Ser is Thr
  • the 29th Val is Ile
  • the 30th Ser is Thr
  • the 40th Gln is Lys.
  • amino acid sequence of VH into which 8 modifications have been introduced is, for example, 28th Ser in the amino acid sequence represented by SEQ ID NO: 84, Thr, 29th Val in Ile, 30
  • An amino acid in which the th Ser is replaced with Thr, the 40th Gln with Lys, the 45th Gly with Arg, the 46th Leu with Met, the 49th Ile with Met, and the 97th Ala with Thr examples include sequences
  • amino acid sequence of VH into which seven modifications have been introduced include, for example, the 27th Gly in the amino acid sequence represented by SEQ ID NO: 84 as Tyr, the 40th Gln as Lys, and 45 Examples include amino acid sequences in which the first Gly is replaced by Arg, the 46th Leu is replaced by Met, the 49th Ile is replaced by Met, the 72nd Val is replaced by Arg, and the 97th Ala is replaced by Thr.
  • the amino acid sequence of VH into which six modifications are introduced is, for example, the 27th Gly in the amino acid sequence represented by SEQ ID NO: 84 as Tyr, the 28th Ser as Thr, 29 Examples include amino acid sequences in which the 1st Val is replaced with Ile, the 30th Ser with Thr, the 72nd Val with Arg, and the 97th Ala with Thr.
  • the amino acid sequence of VH into which five modifications are introduced is, for example, that the 40th Gln in the amino acid sequence represented by SEQ ID NO: 84 is Lys, the 45th Gly is Arg, Examples include amino acid sequences in which the first Leu is replaced by Met, the 49th Ile is replaced by Met, and the 97th Ala is replaced by Thr.
  • amino acid sequence of VH into which four modifications have been introduced include the following amino acid sequences (1) to (6).
  • An amino acid sequence in which the 40th Gln in the amino acid sequence represented by SEQ ID NO: 84 is substituted with Lys, the 45th Gly with Arg, the 46th Leu with Met, and the 49th Ile with Met (2) An amino acid sequence in which the 27th Gly in the amino acid sequence represented by SEQ ID NO: 84 is substituted with Tyr, the 28th Ser with Thr, the 29th Val with Ile, and the 30th Ser with Thr.
  • amino acid sequence in which 97th Ala is replaced with Thr In the amino acid sequence represented by SEQ ID NO: 84, 40th Gln is Lys, 46th Leu is Met, 49th Ile is Met, In the amino acid sequence represented by SEQ ID NO: 84, the 45th Gly is Arg, the 46th Leu is Met, the 49th Ile is Met, and the 97th Ala is replaced with Thr. And amino acid sequence in which 97th Ala is replaced with Thr
  • amino acid sequence of VH into which two modifications have been introduced include the following amino acid sequences (1) to (12).
  • the 27th amino acid in the amino acid sequence represented by SEQ ID NO: 84 In the amino acid sequence represented by SEQ ID NO: 84, the 40th Gln in the amino acid sequence represented by SEQ ID NO: 84 was replaced with Lys, and the 45th Gly was replaced with Arg.
  • Amino acid sequence (4) In the amino acid sequence represented by SEQ ID NO: 84, the 40th Gln in the amino acid sequence was replaced with Lys, and the 46th Leu was replaced with Met (5) in the amino acid sequence represented by SEQ ID NO: 84 An amino acid sequence in which the 40th Gln is replaced with Lys and the 49th Ile is replaced with Met (6) 4 in the amino acid sequence represented by SEQ ID NO: 84 The amino acid sequence in which the Ginth Gln is replaced with Lys and the 97th Ala is replaced with Thr (7) The 45th Gly in the amino acid sequence represented by SEQ ID NO: 84 is replaced with Arg, and the 46th Leu is replaced with Met Amino acid sequence (8) In the amino acid sequence represented by SEQ ID NO: 84, the 45th Gly in the amino acid sequence represented by Arg and the 49th Ile substituted with Met (9) In the amino acid sequence represented by SEQ ID NO: 84 In the amino acid sequence represented by SEQ ID NO: 84, the 45
  • the 46th Leu in the amino acid sequence represented by SEQ ID NO: 84 was designated as Met, and the 49th Ile was designated as Met.
  • the amino acid sequence of VH into which one modification is introduced is, for example, an amino acid sequence in which the 25th Ser in the amino acid sequence represented by SEQ ID NO: 84 is replaced with Thr, and the 27th Gly is Amino acid sequence substituted with Tyr, amino acid sequence substituted with 28th Ser with Thr, amino acid sequence substituted with 29th Val with Ile, amino acid sequence substituted with 30th Ser with Thr, 40th Gln with Lys Substituted amino acid sequence, amino acid sequence in which the 45th Gly was replaced with Arg, amino acid sequence in which the 46th Leu was replaced with Met, amino acid sequence in which the 49th Ile was replaced with Met, and the 72nd Val was replaced with Arg Amino acid sequence, amino acid sequence in which 93rd Val is replaced with Thr, 95th Tyr is placed in Phe Amino acid sequence, the 97th Ala amino acid sequence is substituted with Thr, or the amino acid sequence obtained by replacing 106 th Thr in Val, and the like.
  • amino acid residues selected from the second Ile, the eighth Pro, the eleventh Leu, the fifteenth Pro, and the 90th Val in the amino acid sequence of SEQ ID NO: 85 are humanized antibodies comprising amino acid sequences substituted with other amino acid residues.
  • a humanized antibody comprising an amino acid sequence in which the second Ile, the 11th Leu, the 15th Pro, and the 90th Val in the amino acid sequence of SEQ ID NO: 85 are substituted with other amino acid residues ( 2) A humanized antibody comprising an amino acid sequence in which the second Ile, 15th Pro, and 90th Val in the amino acid sequence of SEQ ID NO: 85 are substituted with other amino acid residues. (3) A humanized antibody comprising an amino acid sequence in which the second Ile, 11th Leu, and 15th Pro in the amino acid sequence of SEQ ID NO: 85 are substituted with other amino acid residues.
  • a humanized antibody comprising an amino acid sequence in which the second Ile and 15th Pro in the amino acid sequence of SEQ ID NO: 85 are substituted with other amino acid residues ( 4) A humanized antibody comprising an amino acid sequence in which the second Ile and 15th Pro in the amino acid sequence of SEQ ID NO: 85 are substituted with other amino acid residues
  • the amino acid sequence of the antibody VL obtained as a result of the above amino acid modification is as follows.
  • the second Ile in the amino acid sequence of SEQ ID NO: 85 is Val
  • the 8th Pro is Leu
  • the 11th Leu is Gln
  • 15 An amino acid sequence in which at least one modification selected from the modification in which the 1st Pro is replaced with Leu and the 90th Val is replaced with Leu is introduced.
  • the amino acid sequence of VL into which five modifications are introduced for example, the second Ile in the amino acid sequence of SEQ ID NO: 85 is Val, the 8th Pro is Leu, and the 11th Leu Amino acid sequence in which is replaced with Gln, 15th Pro is replaced with Leu, and 90th Val is replaced with Leu.
  • amino acid sequence of VL into which four modifications have been introduced include the following amino acid sequences (1) to (5).
  • SEQ ID NO: 85 The amino acid sequence of SEQ ID NO: 85 in which the second Ile is replaced with Val, the 8th Pro is replaced with Leu, the 15th Pro is replaced with Leu, and the 90th Val is replaced with Leu.
  • the second Ile is replaced with Val
  • the 8th Pro is replaced with Leu
  • the 11th Leu is replaced with Gln
  • the 90th Val is replaced with Leu.
  • amino acid sequence of VL into which three modifications have been introduced include the following amino acid sequences (1) to (3).
  • Amino acid sequence in which the second Ile in the amino acid sequence of SEQ ID NO: 85 is replaced with Val, the 8th Pro is replaced with Leu, and the 15th Pro is replaced with Leu (2) in the amino acid sequence of SEQ ID NO: 85 The amino acid sequence in which the second Ile is replaced with Val, the 11th Leu is replaced with Gln, and the 15th Pro is replaced with Leu.
  • the second Ile in the amino acid sequence of SEQ ID NO: 85 is Val
  • the 15th Amino acid sequence in which Pro is replaced with Leu and 90th Val is replaced with Leu
  • amino acid sequence of VL into which two modifications are introduced include the following amino acid sequences (1) to (7).
  • the second Ile in the amino acid sequence of SEQ ID NO: 85 is Val.
  • the amino acid sequence in which the second Ile in the amino acid sequence is substituted with Val and the 90th Val is substituted with Leu (5)
  • the 8th Pro in the amino acid sequence of SEQ ID NO: 85 is Leu
  • the 15th Pro is Leu
  • the 11th Leu in Gln and the 15th Pro in Leu was the 15 th Pro amino acid sequence (7) in the amino acid sequence of SEQ ID NO: 85 to Leu, and 90 amino acid sequence Val has been substituted with Leu
  • the amino acid sequence of VL into which one modification is introduced is, for example, an amino acid sequence in which the second Ile in the amino acid sequence of SEQ ID NO: 85 is substituted with Val, and the eighth Pro is substituted with Leu.
  • a human antibody originally refers to an antibody that naturally exists in the human body, but a human antibody phage library and a human antibody-producing transgene prepared by recent advances in genetic engineering, cell engineering, and developmental engineering techniques. Also included are antibodies obtained from transgenic animals.
  • the antibody naturally present in the human body can be cultured by, for example, isolating human peripheral blood lymphocytes, infecting and immortalizing EB virus, etc., and cloning the lymphocytes that produce the antibody.
  • the antibody can be further purified.
  • the human antibody phage library is a library in which antibody fragments such as Fab or scFv are expressed on the phage surface by inserting antibody genes prepared from human B cells into the phage genes. From the library, phages expressing an antibody fragment having a desired antigen-binding activity on the surface can be collected using the binding activity to the substrate on which the antigen is immobilized as an index. The antibody fragment can be further converted into a human antibody molecule comprising two complete heavy chains and two complete light chains by genetic engineering techniques.
  • a human antibody-producing transgenic animal means an animal in which a human antibody gene is incorporated into cells.
  • a human antibody-producing transgenic mouse can be produced by introducing a human antibody gene into a mouse ES cell, transplanting the ES cell into an early mouse embryo, and generating it.
  • human antibody-producing hybridomas are obtained and cultured using normal hybridoma production methods performed in non-human animals to produce human antibodies in the culture supernatant. It can be produced by accumulating.
  • the number of amino acids to be deleted, substituted, inserted and / or added is one or more, and the number is not particularly limited, but site-specific mutagenesis [Molecular Cloning, 2nd Edition, Cold Spring Harbor Laboratory Press (1989), Current protocols in Molecular Biology, John Wiley & Sons (1987-1997), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci. USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci USA, 82, 488 (1985)], etc., and the number that can be deleted, substituted, or added.
  • the number is 1 to several tens, preferably 1 to 20, more preferably 1 to 10, and still more preferably 1 to 5.
  • deletion, substitution, insertion or addition of one or more amino acid residues in the amino acid sequence of the above antibody indicates the following. That is, it means that there is a deletion, substitution, insertion or addition of one or a plurality of amino acid residues in any one and a plurality of amino acid sequences in the same sequence.
  • deletion, substitution, insertion or addition may occur simultaneously, and the amino acid residue to be substituted, inserted or added may be either a natural type or a non-natural type.
  • natural amino acid residues include L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L -Methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, or L-cysteine.
  • amino acid residues contained in the same group can be substituted for each other.
  • Group A leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, O-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine
  • Group B aspartic acid, glutamic acid, isoaspartic acid, Isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid
  • Group C asparagine, glutamine
  • D lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid
  • Group E proline, 3 -Hydroxyproline, 4-hydroxyproline
  • Group F serine, threonine, homoserine
  • antibody fragments include, for example, peptides including Fab, F (ab ′) 2 , Fab ′, scFv, diabody, dsFv and CDR.
  • Fab is a fragment obtained by treating IgG with papain, a proteolytic enzyme (cleaved at the 224th amino acid residue of the H chain), about half of the N chain side of the H chain and the entire L chain are disulfides. It is an antibody fragment having an antigen binding activity with a molecular weight of about 50,000 bound by binding.
  • the Fab of the present invention can be obtained by treating a monoclonal antibody that specifically recognizes CD33 and binds to the extracellular region with papain.
  • a Fab may be produced by inserting a DNA encoding the Fab of the antibody into a prokaryotic expression vector or a eukaryotic expression vector, and expressing the vector by introducing the vector into a prokaryotic or eukaryotic organism. it can.
  • F (ab ′) 2 was obtained by decomposing the lower part of two disulfide bonds in the hinge region of IgG with pepsin, a proteolytic enzyme, and was constructed by binding two Fab regions at the hinge portion. This fragment has an antigen binding activity with a molecular weight of about 100,000.
  • the F (ab ′) 2 of the present invention can be obtained by treating a monoclonal antibody specifically recognizing CD33 and binding to its extracellular region with pepsin.
  • Fab ′ described below can be produced by thioether bond or disulfide bond.
  • Fab ′ is an antibody fragment having an antigen-binding activity having a molecular weight of about 50,000, which is obtained by cleaving the disulfide bond in the hinge region of F (ab ′) 2 .
  • Fab ′ of the present invention can be obtained by treating F (ab ′) 2 that specifically recognizes CD33 of the present invention and binds to the extracellular region with a reducing agent such as dithiothreitol.
  • Fab ′ may be produced by inserting a DNA encoding a Fab ′ fragment into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryotic or eukaryotic organism to express the Fab ′ fragment. it can.
  • scFv is 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), and has antigen-binding activity. It is an antibody fragment having
  • the scFv of the present invention specifically recognizes the CD33 of the present invention and obtains cDNA encoding the VH and VL of a monoclonal antibody that binds to the extracellular region, constructs a DNA encoding scFv, Can be expressed and produced by inserting the expression vector into a prokaryotic expression vector or eukaryotic expression vector and introducing the expression vector into a prokaryotic or eukaryotic organism.
  • Diabody is an antibody fragment obtained by dimerizing scFv and is an antibody fragment having a bivalent antigen-binding activity.
  • the bivalent antigen binding activity can be the same, or one can be a different antigen binding activity.
  • the diabody of the present invention obtains cDNA encoding VH and VL of a monoclonal antibody that specifically recognizes CD33 of the present invention and binds to the extracellular region, and converts the DNA encoding scFv into the amino acid sequence of the peptide linker. Is constructed so that its length is 8 residues or less, and the DNA is inserted into a prokaryotic expression vector or a eukaryotic expression vector, and the expression vector is introduced into a prokaryotic or eukaryotic expression. And can be manufactured.
  • DsFv refers to a polypeptide in which one amino acid residue in each of VH and VL is substituted with a cysteine residue and bonded via a disulfide bond between the cysteine residues.
  • the amino acid residue to be substituted for the cysteine residue can be selected based on the three-dimensional structure prediction of the antibody according to a known method [Protein Engineering, 7, 697 (1994)].
  • the dsFv of the present invention specifically recognizes the CD33 of the present invention and obtains cDNA encoding the VH and VL of the monoclonal antibody that binds to the extracellular region, constructs a DNA encoding dsFv, Can be expressed and produced by inserting the expression vector into a prokaryotic expression vector or eukaryotic expression vector and introducing the expression vector into a prokaryotic or eukaryotic organism.
  • the peptide containing CDR is configured to contain at least one region of CDR of VH or VL.
  • Peptides containing multiple CDRs can be linked directly or via a suitable peptide linker.
  • the peptide comprising the CDR of the present invention constructs DNA encoding the CDRs of VH and VL of the monoclonal antibody that specifically recognizes CD33 of the present invention and binds to the extracellular region, and uses the DNA for prokaryotes It can be expressed and produced by inserting into an expression vector or eukaryotic expression vector and introducing the expression vector into a prokaryotic or eukaryotic organism.
  • the peptide containing CDR can also be manufactured by chemical synthesis methods, such as Fmoc method or tBoc method.
  • the monoclonal antibody of the present invention specifically recognizes the extracellular region of CD33 of the present invention and binds to the extracellular region with a radioisotope, a low molecular drug, a high molecular drug, It includes a derivative of an antibody in which a protein or an antibody drug is chemically or genetically bound.
  • the derivative of the antibody in the present invention specifically recognizes the extracellular region of CD33 of the present invention and binds to the extracellular region, or the N-terminal side or C-terminal of the H chain or L chain thereof.
  • an appropriate substituent or side chain in the antibody or fragment thereof, or a sugar chain in the monoclonal antibody or fragment thereof, etc., radioisotope, low molecular weight drug, high molecular weight drug, immunostimulant, protein or It can be produced by combining antibody drugs and the like by chemical methods [Introduction to Antibody Engineering, Jinshokan (1994)].
  • a DNA encoding a monoclonal antibody or a fragment thereof that specifically recognizes the extracellular region of CD33 of the present invention and binds to the extracellular region is linked to a DNA encoding a protein or antibody drug to be bound. And then inserted into an expression vector, introduced into an appropriate host cell, and expressed by a genetic engineering technique.
  • radioisotope examples include 131 I, 125 I, 90 Y, 64 Cu, 99 Tc, 77 Lu, and 211 At.
  • the radioisotope can be directly bound to the antibody by the chloramine T method or the like. Further, a substance that chelates a radioisotope may be bound to the antibody.
  • the chelating agent include 1-isothiocyanate benzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA).
  • low molecular weight drugs examples include alkylating agents, nitrosourea agents, antimetabolites, antibiotics, plant alkaloids, topoisomerase inhibitors, hormone therapy agents, hormone antagonists, aromatase inhibitors, P glycoprotein inhibitors, platinum Anticancer agents such as complex derivatives, M phase inhibitors or kinase inhibitors [Clinical Oncology, Cancer and Chemotherapy (1996)], steroidal agents such as hydrocortisone or prednisone, non-steroidal agents such as aspirin or indomethacin, gold thiomalate or Anti-inflammatory agents such as penicillamine, immunosuppressive agents such as cyclophosphamide or azathioprine, or antihistamines such as chlorpheniramine maleate or clemacitine [Inflammation and anti-inflammatory therapy, Ishiyaku Publishing Co., Ltd. ( (1982) And the like.
  • anticancer agents include amifostine (ethiol), cisplatin, dacarbazine (DTIC), dactinomycin, mechlorethamine (nitrogen mustard), streptozocin, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), doxorubicin (Adriamycin), epirubicin, gemcitabine (gemzar), daunorubicin, procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil, fluorouracil, vinblastine, vincristine, bleomycin, daunomycin, pepromycin, estramustine, paclitaxel, paclitaxel, paclitaxel Taxotea), aldesleukin, asparaginase, buoy Ruphan, carboplatin, oxaliplatin, nedaplatin, cladribine
  • Examples of a method for binding a low molecular weight drug and an antibody include, for example, a method of binding a drug and an amino group of an antibody via glutaraldehyde, or a drug amino group and an antibody carboxyl group via a water-soluble carbodiimide. And the like.
  • polymer drug examples include polyethylene glycol (hereinafter sometimes referred to as PEG), albumin, dextran, polyoxyethylene, styrene maleic acid copolymer, polyvinyl pyrrolidone, pyran copolymer, or hydroxypropyl methacrylamide. It is done.
  • PEG polyethylene glycol
  • albumin dextran
  • polyoxyethylene polyoxyethylene
  • styrene maleic acid copolymer polyvinyl pyrrolidone
  • pyran copolymer hydroxypropyl methacrylamide
  • PEGylation modifying reagent examples include a modifying agent for ⁇ -amino group of lysine (Japanese Unexamined Patent Publication No. 61-178926), a modifying agent for carboxyl group of aspartic acid and glutamic acid (Japanese Unexamined Patent Publication No. Sho 56). No. -23587), or a modifier for guanidino group of arginine (Japanese Patent Laid-Open No. 2-117920).
  • the immunostimulant may be a natural product known as an immunoadjuvant, and specific examples thereof include ⁇ (1 ⁇ 3) glucan (lentinan, schizophyllan), ⁇ galactosylceramide and the like. It is done.
  • proteins include cytokines or growth factors that activate immunocompetent cells such as NK cells, macrophages, or neutrophils, or toxin proteins.
  • cytokines or growth factors examples include interferon (hereinafter referred to as INF) - ⁇ , INF- ⁇ , INF- ⁇ , interleukin (hereinafter referred to as IL) -2, IL-12, IL-15, IL- 18, IL-21, IL-23, granulocyte colony stimulating factor (G-CSF), granulocyte / macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF) and the like.
  • toxin protein examples include ricin and diphtheria toxin, and also include protein toxins in which mutations are introduced into the protein in order to regulate toxicity.
  • Examples of the antibody drug include an antigen against which apoptosis is induced by antibody binding, an antigen associated with tumor pathogenesis or an antigen that regulates immune function, and an antibody against an antigen involved in angiogenesis of a lesion site.
  • Antigens that induce apoptosis by antibody binding include, for example, CD4, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b , CD80 (B7.1), CD81, CD82, CD83, CDw84, CD85, CD86 (B7.2), human leukocyte antigen (HLA) -Class II, or Epidermal Growth Factor Receptor (EGFR).
  • antigens involved in tumor pathogenesis or antibodies that regulate immune functions include CD4, CD40, CD40 ligand, B7 family molecules (CD80, CD86, CD274, B7-DC, B7-H2, B7-H3, Or B7-H4), a ligand of a B7 family molecule (CD28, CTLA-4, ICOS, PD-1, or BTLA), OX-40, OX-40 ligand, CD137, a tumor necrosis factor (TNF) receptor family molecule ( DR4, DR5, TNFR1, or TNFR2), TNF-related apoptosis-inducing ligand receptor (TRAIL) family molecule, TRAIL family molecule receptor family (TRAIL-R1, RAIL-R2, TRAIL-R3, or TRAIL-R4), receptor activator of nuclear factor kappa B ligand (RANK), RANK ligand, CD25, folate receptor 4, cytokine [IL-1 ⁇ , IL-1 ⁇ , IL-4, IL
  • antigens for antibodies that inhibit angiogenesis of a lesion site include, for example, vascular electrical growth factor (VEGF), anangiopoietin, fibroblast growth factor (FGF), EGF, and platelet-derived GF. IGF), erythropoietin (EPO), TGF ⁇ , IL-8, Ephilin, SDF-1, or a receptor thereof.
  • VEGF vascular electrical growth factor
  • FGF fibroblast growth factor
  • EGF fibroblast growth factor
  • IGF erythropoietin
  • TGF ⁇ IL-8
  • Ephilin Ephilin
  • SDF-1 or a receptor thereof.
  • a fusion antibody of a monoclonal antibody or a fragment thereof that binds to CD33 and a protein or antibody drug is linked to a cDNA encoding the monoclonal antibody or the fragment, and a cDNA encoding the protein or the antibody drug is linked to encode the fusion antibody.
  • the fusion antibody can be produced by constructing a DNA to be expressed, inserting the DNA into a prokaryotic or eukaryotic expression vector, and introducing the expression vector into a prokaryotic or eukaryotic organism.
  • the extracellular region of CD33 of the present invention is specifically recognized and binds to the extracellular region.
  • the drug that binds to the monoclonal antibody or a fragment thereof include a label used in a usual immunological detection or measurement method.
  • the label examples include an enzyme such as alkaline phosphatase, peroxidase or luciferase, a luminescent substance such as acridinium ester or lophine, or a fluorescent substance such as fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (RITC).
  • an enzyme such as alkaline phosphatase, peroxidase or luciferase, a luminescent substance such as acridinium ester or lophine, or a fluorescent substance such as fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (RITC).
  • FITC fluorescein isothiocyanate
  • RITC tetramethylrhodamine isothiocyanate
  • the present invention also relates to a therapeutic agent for a disease involving CD33-positive cells that specifically recognize the extracellular region of CD33 and contain a monoclonal antibody or fragment thereof that binds to the extracellular region as an active ingredient.
  • the disease involving CD33-positive cells may be any disease involving cells expressing CD33, and examples thereof include cancer, autoimmune diseases, and allergic diseases.
  • CD33 positive cells examples include monocytes, macrophages, granulocytes and the like which are CD33 positive cells. Since monocytes and neutrophils expressing CD33 are cells involved in the removal of foreign substances such as microorganisms, diseases involving foreign body phagocytosis can be mentioned.
  • cancer examples include blood cancer, breast cancer, uterine cancer, colon cancer, esophageal cancer, stomach cancer, ovarian cancer, lung cancer, kidney cancer, rectal cancer, thyroid cancer, cervical cancer, small intestine cancer, prostate cancer or pancreatic cancer.
  • hematological cancer, esophageal cancer, stomach cancer, colon cancer, liver cancer or prostate cancer can be mentioned.
  • hematological cancers include AML, anaplastic large cell lymphoma (ALCL), acute lymphocytic leukemia (ALL), myelodysplastic syndrome, MD, and other myelodysplastic syndromes. , Multiple myeloma, Hodgkin lymphoma, or non-Hodgkin lymphoma.
  • autoimmune disease examples include rheumatoid arthritis, psoriasis, Crohn's disease, ankylosing spondylitis, multiple sclerosis, type I diabetes, hepatitis, myocarditis, Sjogren's syndrome, or rejection after transplantation.
  • allergic diseases include acute or chronic airway hypersensitivity, bronchial asthma, atopic dermatitis, or allergic rhinitis.
  • the therapeutic agent of the present invention contains the above-described monoclonal antibody of the present invention or a fragment thereof as an active ingredient.
  • the therapeutic agent containing the antibody of the present invention or a fragment thereof may contain only the antibody or the fragment as an active ingredient, but usually together with one or more pharmacologically acceptable carriers. It is desirable to mix and provide as a pharmaceutical formulation produced by any method known in the art of pharmaceutical sciences.
  • administration route that is most effective for treatment, and examples include oral administration, oral administration, intratracheal, rectal, subcutaneous, intramuscular or intravenous administration, preferably Intravenous administration is mentioned.
  • administration forms include sprays, capsules, tablets, powders, granules, syrups, emulsions, suppositories, injections, ointments, or tapes.
  • the dose or frequency of administration varies depending on the intended therapeutic effect, administration method, treatment period, age, weight, etc., but is usually 10 ⁇ g / kg to 20 mg / kg per day for an adult.
  • the present invention relates to a method for immunological detection or measurement of CD33, which contains as an active ingredient a monoclonal antibody or a fragment thereof that specifically recognizes the extracellular region of CD33 and binds to the extracellular region.
  • the present invention relates to a reagent for immunological detection or measurement, an immunological detection or measurement method for cells expressing CD33, and a diagnostic agent for a disease involving CD33-positive cells.
  • any known method may be used as a method for detecting or measuring the amount of CD33.
  • Examples include immunological detection or measurement methods.
  • the immunological detection or measurement method is a method of detecting or measuring the amount of antibody or the amount of antigen using a labeled antigen or antibody.
  • immunological detection or measurement methods include radiolabeled immunoassay (RIA), enzyme immunoassay (EIA or ELISA), fluorescence immunoassay (FIA), luminescence immunoassay (Western immunoassay), Western Examples include blotting or physicochemical techniques.
  • a disease associated with CD33 can be diagnosed.
  • Suitable immunological detection methods can be used for detection of cells expressing the polypeptide, but immunoprecipitation, fluorescent cell staining, immunohistochemical staining, immunohistological staining, etc. Preferably used. Moreover, fluorescent antibody staining methods such as FMAT8100HTS system (Applied Biosystems) can also be used.
  • Examples of biological samples to be detected or measured for CD33 in the present invention include CD33 expression such as tissue cells, bone marrow cells, bone marrow fluid, blood, plasma, serum, pancreatic juice, urine, stool, tissue fluid, or culture fluid.
  • the cell is not particularly limited as long as it may contain the cells.
  • the diagnostic agent containing the monoclonal antibody or fragment thereof of the present invention may contain a reagent for conducting an antigen-antibody reaction and a reagent for detecting the reaction, depending on the target diagnostic method.
  • a reagent for performing the antigen-antibody reaction include a buffer or a salt.
  • the reagent for detection include a labeled secondary antibody that recognizes the monoclonal antibody or a fragment thereof, or a reagent used for usual immunological detection or measurement methods such as a substrate corresponding to the label.
  • the antibody production method, disease treatment method, and disease diagnosis method of the present invention will be specifically described below.
  • Method for Producing Monoclonal Antibody Preparation of antigen Cells expressing CD33 or CD33 as an antigen are expressed in E. coli, yeast, insect cells, or animal cells using an expression vector containing cDNA encoding CD33 full length or a partial length thereof. It can be obtained by introducing into the above.
  • CD33 can be purified and obtained from cultured human tumor cells, human tissues, etc. that express CD33 in large amounts. Further, the cultured tumor cells or the tissue can be used as an antigen as it is.
  • a synthetic peptide having a partial sequence of CD33 can be prepared by a chemical synthesis method such as the Fmoc method or the tBoc method and used as an antigen.
  • CD33 used in the present invention is described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) or Current Protocols in MolecularJoule7, 198
  • the DNA encoding CD33 can be expressed in a host cell and produced by the following method.
  • a recombinant vector is prepared by inserting a full-length cDNA containing a portion encoding CD33 downstream of a promoter of an appropriate expression vector.
  • a DNA fragment of an appropriate length containing a polypeptide-encoding portion prepared based on the full-length cDNA may be used.
  • a transformant producing a polypeptide can be obtained by introducing the obtained recombinant vector into a host cell suitable for the recombinant vector.
  • Any expression vector can be used as long as it can autonomously replicate in the host cell to be used or can be integrated into the chromosome, and contains an appropriate promoter at a position where DNA encoding the polypeptide can be transcribed. Can do.
  • any microorganism that belongs to the genus Escherichia such as Escherichia coli, yeast, insect cells, or animal cells can be used so long as it can express the target gene.
  • the recombinant vector When a prokaryote such as E. coli is used as a host cell, the recombinant vector is capable of autonomous replication in a prokaryote and at the same time contains a promoter, a ribosome binding sequence, DNA containing a CD33-encoding portion, and a transcription termination sequence. It is preferable that it is a vector containing.
  • the recombinant vector does not necessarily require a transcription termination sequence, but it is preferable to place the transcription termination sequence immediately below the structural gene.
  • the recombinant vector may contain a gene that controls the promoter.
  • the recombinant vector it is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence (also referred to as SD sequence), which is a ribosome binding sequence, and the start codon is adjusted to an appropriate distance (eg, 6 to 18 bases).
  • SD sequence also referred to as SD sequence
  • start codon is adjusted to an appropriate distance (eg, 6 to 18 bases).
  • the base sequence of the DNA encoding CD33 can be substituted so that the codon is optimal for expression in the host, thereby improving the production rate of the target CD33.
  • Any expression vector can be used as long as it can function in the host cell to be used.
  • pBTrp2, pBTac1, pBTac2 above, Roche Diagnostics
  • pKK233-2 Pharmacia
  • pSE280 Invitrogen
  • pGEMEX-1 Promega
  • pQE-8 Qiagen
  • PKYP10 Japanese Unexamined Patent Publication No. 58-110600
  • pKYP200 [Agricultural Biological Chemistry, 48, 669 (1984)]
  • pLSA1 Agric. Biol. Chem. , 53, 277 (1989)]
  • pGEL1 Proc. Natl. Acad. Sci.
  • any promoter can be used as long as it can function in the host cell to be used.
  • promoters derived from Escherichia coli or phage such as trp promoter (Ptrp), lac promoter, PL promoter, PR promoter, or T7 promoter can be mentioned.
  • trp promoter Ptrp
  • lac promoter PL promoter
  • PR promoter PR promoter
  • T7 promoter T7 promoter
  • artificially designed and modified promoters such as a tandem promoter, tac promoter, lacT7 promoter, or let I promoter in which two Ptrps are connected in series can also be used.
  • Examples of host cells include E. coli XL1-Blue, E. coli XL2-Blue, E. coli DH1, E. coli MC1000, E. coli KY3276, E. coli W1485, E. coli JM109, E. coli HB101, E. coli No. 49, E. coli W3110, E. coli NY49, or E. coli DH5 ⁇ .
  • Any method can be used for introducing a recombinant vector into a host cell as long as it is a method for introducing DNA into the host cell to be used.
  • a method using calcium ions Proc. Natl. Acad. Sci. USA, 69, 2110 (1972), Gene, 17, 107 (1982), Molecular & General Genetics, 168, 111 (1979)].
  • any expression vector can be used as long as it can function in animal cells.
  • Any promoter can be used as long as it can function in animal cells. Examples include cytomegalovirus (CMV) immediate early (IE) gene promoter, SV40 early promoter, retroviral promoter, metallothionein promoter, heat shock promoter, SR ⁇ promoter, or Moloney murine leukemia virus promoter or enhancer. . In addition, an enhancer of human CMV IE gene may be used together with a promoter.
  • CMV cytomegalovirus
  • IE immediate early
  • SV40 early promoter SV40 early promoter
  • retroviral promoter metallothionein promoter
  • heat shock promoter metallothionein promoter
  • SR ⁇ promoter heat shock promoter
  • Moloney murine leukemia virus promoter or enhancer Moloney murine leukemia virus promoter or enhancer.
  • an enhancer of human CMV IE gene may be used together with a promoter.
  • host cells examples include human leukemia cells Namalwa cells, monkey cells COS cells, Chinese hamster ovary cells CHO cells (Journal of Experimental Medicine, 108, 945 (1958); Proc. Natl. Acad. Sci. USA, 60, 1275 (1968); Genetics, 55, 513 (1968); Chromoma, 41, 129 (1973); Methods in Cell Science, 18, 115 (1996); Radiation Research, 148, 260 (1997); Proc. Natl. Sci.USA, 77, 4216 (1980); Proc.Natl.Acad.Sci., 60,127 (1968); Cell, 6, 121 (1975); Molecular Cell Genetics, Appendix I, II (pp.
  • any method for introducing a recombinant vector into a host cell any method can be used as long as it introduces DNA into animal cells.
  • electroporation method [Cytotechnology, 3, 133 (1990)]
  • calcium phosphate method Japanese Patent Laid-Open No. 2-227075
  • lipofection method Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)].
  • CD33 Culturing a transformant derived from a microorganism or animal cell having a recombinant vector into which DNA encoding CD33 obtained as described above is incorporated in a medium, and producing and accumulating the CD33 in the culture, By collecting from the culture, CD33 can be produced.
  • the method of culturing the transformant in a medium can be performed according to a usual method used for culturing a host.
  • CD33 When expressed in cells derived from eukaryotes, CD33 with an added sugar or sugar chain can be obtained.
  • an inducer may be added to the medium as necessary.
  • cultivating a microorganism transformed with a recombinant vector using the lac promoter cultivate a microorganism transformed with isopropyl- ⁇ -D-thiogalactopyranoside or the like using a recombinant vector using the trp promoter.
  • indole acrylic acid or the like may be added to the medium.
  • Examples of a medium for culturing a transformant obtained using an animal cell as a host include, for example, a commonly used RPMI 1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], Eagle's MEM medium [Science]. , 122, 501 (1952)], Dulbecco's modified MEM medium [Virology, 8, 396 (1959)], 199 medium [Proc. Soc. Exp. Biol. Med. 73, 1 (1950)], Iscove's Modified Dulbecco's Medium (IMDM) medium, or a medium obtained by adding fetal bovine serum (FBS) or the like to these mediums.
  • RPMI 1640 medium The Journal of the American Medical Association, 199, 519 (1967)]
  • Eagle's MEM medium Science].
  • 122, 501 (1952) Dulbecco's modified MEM medium
  • 199 medium Proc. Soc. Exp. Biol. Med. 73, 1 (1950)]
  • the culture is usually carried out for 1 to 7 days under conditions such as pH 6 to 8, 30 to 40 ° C., and 5% CO 2 .
  • Examples of the production method of CD33 include a method of producing in a host cell, a method of secreting it outside the host cell, and a method of producing it on the host cell outer membrane.
  • the host cell to be used and the structure of CD33 to be produced are exemplified. By changing, an appropriate method can be selected.
  • CD33 When CD33 is produced in the host cell or on the host cell outer membrane, the method of Paulson et al. [J. Biol. Chem. , 264, 17619 (1989)], the method of Lowe et al. [Proc. Natl. Acad. Sci. USA, 86, 8227 (1989), Genes Develop. , 4, 1288 (1990)], and actively secreting CD33 outside the host cell by using the method described in Japanese Patent Application Laid-Open No. 05-336963 or International Publication No. 94/23021. Can do.
  • the production amount of CD33 can be increased by using a gene amplification system using a dihydrofolate reductase gene or the like (Japanese Patent Laid-Open No. 2-227075).
  • the obtained CD33 can be isolated and purified as follows, for example.
  • the cells When CD33 is expressed in a dissolved state in the cells, the cells are collected by centrifugation after culturing, suspended in an aqueous buffer solution, and then used with an ultrasonic crusher, a French press, a Manton Gaurin homogenizer, or a dynomill. The cells are disrupted to obtain a cell-free extract.
  • an ordinary protein isolation and purification method that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, diethylamino Anion exchange chromatography using a resin such as ethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Chemical), cation exchange chromatography using a resin such as S-Sepharose FF (manufactured by Pharmacia) , Methods such as hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, or electrophoresis such as isoelectric focusing Can be used alone or in combination to obtain purified preparations. Can.
  • CD33 When CD33 is expressed by forming an insoluble substance in the cell, the cell is recovered and crushed in the same manner as described above, and centrifuged to collect the insoluble substance of CD33 as a precipitate fraction.
  • the recovered insoluble matter of CD33 is solubilized with a protein denaturant.
  • the solubilized solution is diluted or dialyzed to return the CD33 to a normal three-dimensional structure, and then a purified polypeptide preparation can be obtained by the same isolation and purification method as described above.
  • the derivative such as CD33 or a modified sugar thereof can be recovered from the culture supernatant.
  • a soluble fraction can be obtained by treating the culture by a method such as centrifugation as described above, and a purified preparation can be obtained from the soluble fraction by using the same isolation and purification method as described above. it can.
  • Purification can be easily performed by adding an appropriate peptide to CD33.
  • an appropriate peptide for example, by adding the Fc region of immunoglobulin to CD33 and using CD33-Fc fusion protein secreted extracellularly using protein A or protein G, a purified preparation can be obtained.
  • CD33 used in the present invention can also be produced by a chemical synthesis method such as the Fmoc method or the tBoc method.
  • chemical synthesis using peptide synthesizers such as Advanced Chemtech, Perkin Elmer, Pharmacia, Protein Technology Instrument, Synthecel-Vega, Perceptive, or Shimadzu It can also be synthesized.
  • Immunization is performed by administering the antigen subcutaneously, intravenously or intraperitoneally to the animal together with an appropriate adjuvant such as Freund's complete adjuvant or aluminum hydroxide gel and pertussis vaccine.
  • an appropriate adjuvant such as Freund's complete adjuvant or aluminum hydroxide gel and pertussis vaccine.
  • the antigen is a partial peptide
  • a conjugate with a carrier protein such as BSA (bovine serum albumin) or KLH (Keyhole limpet hemocyanin) is prepared and used as an immunogen.
  • BSA bovine serum albumin
  • KLH Keyhole limpet hemocyanin
  • the antigen is administered 5 to 10 times every 1 to 2 weeks after the first administration. Three to seven days after each administration, blood is collected from the fundus venous plexus, and the antibody titer of the serum is measured using an enzyme immunoassay [Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988)]. An animal whose serum shows a sufficient antibody titer against the antigen used for immunization is used as a source of antibody producing cells for fusion.
  • tissues containing antibody-producing cells such as the spleen are removed from the immunized animal, and antibody-producing cells are collected.
  • the spleen is shredded and loosened, and then centrifuged, and the erythrocytes are removed to obtain antibody producing cells for fusion.
  • myeloma cells cell lines obtained from mice are used.
  • myeloma cell line P3-X63Ag8-U1 P3-U1 [Current Topics in Microbiology and Immunology, 18, 1 (1978)]
  • P3-NS1 / 1-Ag41 P3-NS1 / 1-Ag41
  • SP2 / 0-Ag14 SP2 / 0-Ag14
  • SP-2 SP2 / 0-Ag14
  • P3-X63-Ag8653 6
  • J. Immunology, 123, 1548 (1979) or P3-X63-Ag8 (X63) [Nature, 256, 495 (1975)].
  • the myeloma cells are passaged in normal medium [RPMI 1640 medium supplemented with glutamine, 2-mercaptoethanol, gentamicin, FBS, and 8-azaguanine] and passaged to normal medium 3-4 days prior to cell fusion. On the day of fusion, secure a cell count of 2 ⁇ 10 7 or more.
  • a mixture of polyethylene glycol-1000 (PEG-1000), MEM medium and dimethyl sulfoxide is added at 37 ° C. with stirring.
  • the antibody-producing cells for fusion are gently suspended in a HAT medium [normal medium supplemented with hypoxanthine, thymidine, and aminopterin]. This suspension is cultured for 7-14 days at 37 ° C. in a 5% CO 2 incubator.
  • a part of the culture supernatant is extracted, and a cell group that reacts with an antigen containing CD33 and does not react with an antigen not containing CD33 is selected by a hybridoma selection method such as a binding assay described later.
  • cloning was repeated twice by the limiting dilution method (first time was HT medium (medium obtained by removing aminopterin from HAT medium), second time normal medium was used), and a stable and strong antibody titer was observed.
  • One is selected as a monoclonal antibody-producing hybridoma.
  • the monoclonal antibody-producing hybridoma obtained in (4) is cultured in an RPMI1640 medium supplemented with 10% FBS, the supernatant is removed by centrifugation, and the suspension is suspended in a Hybridoma SFM medium and cultured for 3 to 7 days. .
  • the obtained cell suspension is centrifuged, and purified using a protein A-column or protein G-column from the resulting supernatant, and the IgG fraction is collected to obtain a purified monoclonal antibody.
  • 5% Digo GF21 can also be added to the Hybridoma SFM medium.
  • the antibody subclass is determined by enzyme immunoassay using a sub-clustering kit.
  • the amount of protein is calculated from the Raleigh method or absorbance at 280 nm.
  • the monoclonal antibody is selected by a binding assay by the enzyme immunoassay shown below and a kinetic analysis by Biacore.
  • an antigen for example, a gene-transferred cell obtained by introducing an expression vector containing cDNA encoding CD33 obtained in (1) into Escherichia coli, yeast, insect cells or animal cells, Recombinant protein, or purified polypeptide or partial peptide obtained from human tissue.
  • the antigen is a partial peptide
  • a conjugate with a carrier protein such as BSA or KLH is prepared and used.
  • a test substance such as serum, hybridoma culture supernatant or purified monoclonal antibody is dispensed as the first antibody and allowed to react.
  • an anti-immunoglobulin antibody labeled with biotin, an enzyme, a chemiluminescent substance, a radiation compound or the like is dispensed and reacted as a second antibody.
  • a reaction is performed according to the labeling substance of the second antibody, and a monoclonal antibody that specifically reacts with the immunogen is selected.
  • a monoclonal antibody that competes with the anti-CD33 monoclonal antibody of the present invention and binds to CD33 can be obtained by adding a test antibody to the above-described binding assay system for reaction. That is, by screening for an antibody that inhibits the binding of the monoclonal antibody when the test antibody is added, a monoclonal antibody that competes with the acquired monoclonal antibody for binding to the CD33 extracellular region can be obtained.
  • the monoclonal antibody that binds to the extracellular region of CD33 of the present invention recognizes and binds to the same epitope as the epitope that identifies the epitope of the antibody obtained by the above-described binding assay system, A partial synthetic peptide of the identified epitope, or a synthetic peptide mimicking the three-dimensional structure of the epitope can be prepared and immunized.
  • human CD33 is immobilized on a sensor chip by, for example, an amine coupling method, and then purified monoclonal antibodies having a plurality of known concentrations are flowed to measure binding and dissociation.
  • the obtained data is subjected to kinetic analysis using a software that is included with the device, using a binding binding model, and various parameters are acquired.
  • a recombinant antibody expression vector is an animal cell expression vector in which DNAs encoding human antibodies CH and CL are incorporated, and the animal cell expression vector is human. It can be constructed by cloning DNAs encoding antibody CH and CL, respectively.
  • Any human antibody CH and CL can be used for the C region of a human antibody.
  • ⁇ 1 subclass CH and ⁇ class CL of human antibodies are used.
  • cDNA is used for DNA encoding CH and CL of human antibodies
  • chromosomal DNA consisting of exons and introns can also be used.
  • Any animal cell expression vector can be used as long as it can incorporate and express a gene encoding the C region of a human antibody.
  • promoters and enhancers include SV40 early promoter [J. Biochem. , 101, 1307 (1987)], Moloney murine leukemia virus LTR [Biochem. Biophys. Res. Commun. , 149, 960 (1987)], or an immunoglobulin heavy chain promoter [Cell, 41, 479 (1985)] and an enhancer [Cell, 33, 717 (1983)].
  • Recombinant antibody expression vectors balance the ease of construction of recombinant antibody expression vectors, the ease of introduction into animal cells, and the balance of expression levels of antibody H and L chains in animal cells.
  • a vector for expressing a recombinant antibody of a type (tandem type) in which the antibody H chain and L chain are present on the same vector [J. Immunol. Methods, 167, 271 (1994)]
  • pKANTEX93 International Publication No. 97/10354
  • pEE18 Hybridoma, 17, 559 (1998)
  • MRNA is extracted from hybridoma cells producing non-human 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.
  • Recombinant phages or recombinant plasmids having cDNA encoding VH or VL are isolated from the library using DNA encoding the C region portion or V region portion of the mouse antibody as a probe.
  • the entire base sequence of VH or VL of the target mouse antibody on the recombinant phage or recombinant plasmid is determined, respectively, and the total amino acid sequence of VH or VL is estimated from the base sequence.
  • non-human animal for producing a hybridoma cell producing a non-human antibody a mouse, rat, hamster, rabbit or the like is used, but any animal can be used as long as it can produce a hybridoma cell. .
  • RNA easy kit manufactured by Qiagen
  • oligo (dT) -immobilized cellulose column method For preparation of mRNA from total RNA, oligo (dT) -immobilized cellulose column method [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Press (1989)] or Oligo-dT> SurPiSpR> ⁇ SpiK> Use a kit such as (Takara Bio).
  • mRNA can be prepared from hybridoma cells using a kit such as Fast Track mRNA Isolation Kit (manufactured by Invitrogen) or QuickPrep mRNA Purification Kit (manufactured by Pharmacia).
  • any vector can be used as a vector into which cDNA synthesized using mRNA extracted from a hybridoma cell as a template is incorporated.
  • ZAP Express [Stratesies, 5, 58 (1992)], pBluescript II SK (+) [Nucleic Acids Research, 17, 9494 (1989)], ⁇ ZAPIII (manufactured by Stratagene), ⁇ gt10, Clgt11A: Clgt11 DNA Approach, I, 49 (1985)], Lambda BlueMid (Clontech), ⁇ ExCell, pT7T3-18U (Pharmacia), pcD2 [Mol. Cell. Biol. 3, 280 (1983)], or pUC18 [Gene, 33, 103 (1985)].
  • Escherichia coli into which a cDNA library constructed by a phage or plasmid vector is introduced can be used as long as the cDNA library can be introduced, expressed and maintained.
  • a method for selecting a cDNA clone encoding VH or VL of a non-human antibody from a cDNA library for example, a colony hybridization method using an isotope or fluorescently labeled probe, or a plaque hybridization method [Molecular Cloning] , A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)].
  • PCR method Polymerase Chain Reaction method
  • the selected cDNA is cleaved with an appropriate restriction enzyme and then cloned into a plasmid such as pBluescript SK (-) (Stratagene), and the nucleotide sequence of the cDNA is determined by a commonly used nucleotide sequence analysis method.
  • a base sequence analysis method include the dideoxy method [Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)], etc., followed by ABI PRISM 3700 (manufactured by PE Biosystems) or A.P. L. F. An automatic base sequence analyzer such as a DNA sequencer (Pharmacia) is used.
  • VH and VL complete amino acid sequences for example, BLAST method [J. Mol. Biol. , 215, 403 (1990)], and the like, it can be confirmed whether the complete amino acid sequences of VH and VL are novel.
  • Antibody V region in particular CDR, is an important region that regulates antibody binding to antigen. Therefore, substitution to any amino acid residue in the V region of an antibody, particularly in a CDR, may change the binding property of the antibody to an antigen. Therefore, when producing a V region that does not have the consensus sequence, it is necessary to modify the amino acid sequence so that the binding property of the antibody to the antigen does not change. The specific method is shown below.
  • a DNA sequence encoding the amino acid sequence of the antibody V region into which the mutation has been introduced is obtained. design. Based on the designed DNA sequence, several synthetic DNAs having a length of about 100 bases are synthesized, and PCR reaction is performed using them. In this case, preferably 6 synthetic DNAs are designed for both the H chain and the L chain from the reaction efficiency in the PCR reaction and the length of the synthesizable DNA.
  • a plasmid containing a cDNA having a designed DNA sequence may be prepared by introducing a mutation into DNA encoding DNA by PCR or the like and substituting it with the cDNA encoding the V region of the antibody from which the mutation was introduced. it can.
  • the non-human antibody VH or VL is encoded upstream of each gene encoding the human antibody CH or CL of the recombinant antibody expression vector obtained in (1).
  • a human chimeric antibody expression vector can be constructed by cloning each of the cDNAs.
  • the base sequence of the linking portion encodes an appropriate amino acid
  • VH and VL cDNAs designed to be appropriate restriction enzyme recognition sequences are prepared.
  • the prepared VH and VL cDNAs are expressed in an appropriate form upstream of each gene encoding the human antibody CH or CL of the human CDR-grafted antibody expression vector obtained in (1).
  • Each is cloned to construct a human chimeric antibody expression vector.
  • a cDNA encoding the non-human antibody VH or VL is amplified by a PCR method using a synthetic DNA having a recognition sequence of an appropriate restriction enzyme at both ends, and the recombinant antibody expression vector obtained in (1) Can also be cloned.
  • a cDNA encoding VH or VL of human CDR-grafted antibody can be constructed as follows.
  • the amino acid sequence of the VH or VL FR of the human antibody to be grafted with the VH or VL CDR amino acid sequence of the non-human antibody is selected. Any amino acid sequence can be used as long as it is derived from a human antibody.
  • the FR amino acid sequence of human antibodies registered in databases such as Protein Data Bank, or the common amino acid sequence of each subgroup of FRs of human antibodies [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)] are used.
  • an FR amino acid sequence having the highest homology (at least 60% or more) with the FR amino acid sequence of the VH or VL of the original antibody is selected.
  • the amino acid sequence of the CDR of the original antibody is transplanted to the amino acid sequence of VH or VL of the selected human antibody, respectively, and the amino acid sequence of VH or VL of the human CDR-grafted antibody is designed.
  • Frequency of codon usage of the designed amino acid sequence in the nucleotide sequence of the antibody gene [Sequences of Proteins of Immunological Interest, US Dept. Considering Health and Human Services (1991)]
  • the DNA sequence is converted into a DNA sequence, and the DNA sequence encoding the VH or VL amino acid sequence of the human CDR-grafted antibody is designed.
  • the human CDR-grafted antibody VH can be easily added to the human CDR-grafted antibody expression vector obtained in (1).
  • cDNA encoding VL can be cloned.
  • each H chain and L chain full-length synthetic DNA synthesized as one DNA based on the designed DNA sequence.
  • the amplified product is cloned into a plasmid such as pBluescript SK (-) (manufactured by Stratagene), the base sequence is determined by the same method as described in (2), and the desired human CDR is obtained.
  • a plasmid having a DNA sequence encoding the amino acid sequence of the VH or VL of the transplanted antibody is obtained.
  • a human CDR-grafted antibody can be obtained by transplanting only the non-human antibody VH and VL CDRs into the human antibody VH and VL FRs. The binding activity is reduced compared to the original non-human antibody [BIO / TECHNOLOGY, 9, 266 (1991)].
  • amino acid residues that are directly involved in antigen binding amino acid residues that interact with CDR amino acid residues, and Reduced by maintaining the conformation of the antibody, identifying amino acid residues that are indirectly involved in antigen binding, and substituting those amino acid residues with the amino acid residues of the original non-human antibody Antigen binding activity can be increased.
  • the amino acid residues of FR of human antibody VH and VL can be modified by performing the PCR reaction described in (4) using the synthetic DNA for modification.
  • the base sequence is determined by the method described in (2) and it is confirmed that the target modification has been performed.
  • a recognition sequence for an appropriate restriction enzyme at the 5 ′ end of the synthetic DNA located at both ends are introduced into the human CDR-grafted antibody expression vector obtained in (1), and cloned so as to be expressed in an appropriate form upstream of each gene encoding CH or CL of the human antibody.
  • any cell can be used as long as it can express the recombinant antibody.
  • COS-7 cells American Type Culture Collection (ATCC) number: CRL1651] [Methods in Nucleic Acids Res. , CRC press, 283 (1991)].
  • the expression amount and antigen binding activity of the recombinant antibody in the culture supernatant are measured by enzyme immunoantibody method [Monoclonal Antibodies-Principles and Practice, Third edition, Academic Press (1996), Antibodies-ALaboratory. Cold Spring Harbor Laboratory (1988), monoclonal antibody experiment manual, Kodansha Scientific (1987)] and the like.
  • Any host cell capable of expressing a recombinant antibody can be used as a host cell into which the recombinant antibody expression vector is introduced.
  • CHO-K1 ATCC CCL-61
  • DUkXB11 ATCC CCL-9096
  • Pro-5 ATCC CCL-1781
  • CHO-S Life Technologies, Cat # 11619
  • mice 20 also referred to as YB2 / 0
  • mouse myeloma cell NSO mouse myeloma cell SP2 / 0-Ag14
  • mouse P3X63-Ag8.653 cell ATCC number: CRL1580
  • dihydrofolate reductase gene (Hereinafter referred to as dhfr) deficient CHO cells [Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)], Lec13 [Somatic Cell and Molecular genetics, 12, 55 (1986)] that has acquired lectin resistance
  • CHO cells deficient in the ⁇ 1,6-fucose transferase gene International Publication No. 2005 / 035586, WO 02/31140
  • rat YB2 / 3HL. P2. G11.16 Ag. 20 cells (ATCC number: CRL1662) and the like.
  • a protein such as an enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose, a sugar in which the 1-position of fucose is ⁇ -bonded to the 6-position of N-acetylglucosamine at the reducing end of the N-glycoside-linked complex sugar chain
  • Host cells in which the activity of a protein such as an enzyme involved in chain modification or a protein involved in the transport of intracellular sugar nucleotide GDP-fucose to the Golgi apparatus is reduced or deleted, such as ⁇ 1,6-fucose transferase gene Deficient CHO cells (WO 2005/035586, WO 02/31140) and the like can be mentioned.
  • a transformant that stably expresses the recombinant antibody is selected by culturing in an animal cell culture medium containing a drug such as G418 sulfate (hereinafter referred to as G418) (Japan).
  • G418 sulfate hereinafter referred to as G418, (Japan).
  • animal cell culture medium examples include RPMI 1640 medium (manufactured by Invitrogen), GIT medium (manufactured by Nippon Pharmaceutical Co., Ltd.), EX-CELL301 medium (manufactured by JRH), IMDM medium (manufactured by Invitrogen), Hybridoma-SFM.
  • examples thereof include a medium (manufactured by Invitrogen) or a medium obtained by adding various additives such as FBS to these mediums. By culturing the obtained transformant in a medium, the recombinant antibody is expressed and accumulated in the culture supernatant.
  • the expression level and antigen binding activity of the recombinant antibody in the culture supernatant can be measured by ELISA method or the like.
  • the transformed strain can increase the expression level of the recombinant antibody using a DHFR amplification system (Japanese Patent Laid-Open No. 2-257891).
  • the recombinant antibody is purified from the culture supernatant of the transformant using a protein A-column [Monoclonal Antibodies-Principles and Practice, Third edition, Academic Press (1996), Antibodies-A LaboratoryLaboratoryLaboratoryLaboratoryLaboratory. (1988)].
  • methods used in protein purification such as gel filtration, ion exchange chromatography, and ultrafiltration can be combined.
  • the molecular weight of the purified recombinant antibody H chain, L chain, or whole antibody molecule is determined by polyacrylamide gel electrophoresis [Nature, 227, 680 (1970)] or Western blotting [Monoclonal Antibodies-Principles and practicies, Third]. edition, Academic Press (1996), Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988)].
  • the binding activity to the CD33-expressing cell line is measured using the above-described binding assay described in 1- (6a) and the surface plasmon resonance method using the Biacore system described in (6b). In addition, the fluorescent antibody method [Cancer Immunol. Immunother. , 36, 373 (1993)].
  • CDC activity or ADCC activity against an antigen-positive cultured cell line is measured by a known measurement method [Cancer Immunol. Immunother. , 36, 373 (1993)].
  • Method for Controlling Effector Activity of Antibody As a method for controlling the effector activity of the anti-CD33 monoclonal antibody of the present invention, an N-linked complex type sugar chain that binds to the 297th asparagine (Asn) of the Fc region of the antibody.
  • Methods for controlling the amount of fucose also referred to as core fucose
  • GlcNAc N-acetylglucosamine
  • the effector activity can be controlled using any method for the anti-CD33 monoclonal antibody of the present invention.
  • the effector activity refers to antibody-dependent activity caused through the Fc region of an antibody, and includes ADCC activity, CDC activity, or antibody-dependent phagocytosis by phagocytic cells such as macrophages or dendritic cells (Antibody-dependent phagocytosis). , ADP activity) and the like.
  • the effector activity of the antibody can be increased or decreased.
  • expressing the antibody using CHO cells deficient in the ⁇ 1,6-fucose transferase gene An antibody to which fucose is not bound can be obtained.
  • An antibody to which fucose is not bound has high ADCC activity.
  • the antibody is expressed using a host cell into which an ⁇ 1,6-fucose transferase gene has been introduced.
  • an antibody to which fucose is bound can be obtained.
  • An antibody to which fucose is bound has a lower ADCC activity than an antibody to which fucose is not bound.
  • ADCC activity or CDC activity can be increased or decreased by modifying amino acid residues in the Fc region of the antibody.
  • the CDC activity of an antibody can be increased by using the amino acid sequence of the Fc region described in US Patent Application Publication No. 2007/0148165.
  • ADCC activity or CDC activity can be increased or decreased.
  • an antibody with controlled effector activity of an antibody can be obtained by using the above method in combination with one antibody.
  • the monoclonal antibody or fragment thereof of the present invention can be used for the treatment of diseases involving CD33 positive cells.
  • the therapeutic agent containing the monoclonal antibody or fragment thereof of the present invention may contain only the antibody or fragment as an active ingredient, but usually together with one or more pharmacologically acceptable carriers. And prepared as a pharmaceutical preparation produced by a method known in the technical field of pharmaceutics.
  • Examples of the administration route include oral administration and parenteral administration such as intraoral, intratracheal, rectal, subcutaneous, intramuscular or intravenous.
  • Examples of the dosage form include sprays, capsules, tablets, powders, granules, syrups, emulsions, suppositories, injections, ointments, or tapes.
  • Suitable formulations for oral administration are emulsions, syrups, capsules, tablets, powders, or granules.
  • Liquid preparations such as emulsions or syrups include saccharides such as water, sucrose, sorbitol or fructose, glycols such as polyethylene glycol or propylene glycol, oils such as sesame oil, olive oil or soybean oil, p-hydroxybenzoic acid Manufactured using preservatives such as esters, or flavors such as strawberry flavor or peppermint as additives.
  • Capsules, tablets, powders or granules include excipients such as lactose, glucose, sucrose or mannitol, disintegrants such as starch or sodium alginate, lubricants such as magnesium stearate or talc, polyvinyl alcohol, hydroxy A binder such as propylcellulose or gelatin, a surfactant such as fatty acid ester, or a plasticizer such as glycerin is used as an additive.
  • preparations suitable for parenteral administration include injections, suppositories, and sprays.
  • Injection is manufactured using a carrier made of a salt solution, a glucose solution, or a mixture of both.
  • Suppositories are produced using a carrier such as cacao butter, hydrogenated fat or carboxylic acid.
  • the propellant is produced using a carrier that does not irritate the recipient's oral cavity and airway mucosa, and in which the monoclonal antibody of the present invention or a fragment thereof is dispersed as fine particles to facilitate absorption.
  • a carrier for example, lactose or glycerin is used. It can also be produced as an aerosol or dry powder.
  • Method of diagnosing disease using anti-CD33 monoclonal antibody or fragment thereof of the present invention By detecting or measuring cells expressing CD33 or CD33 using the monoclonal antibody or fragment thereof of the present invention, a disease associated with CD33 can be detected. Can be diagnosed.
  • the diagnosis of cancer which is one of the diseases associated with CD33, can be performed by detecting or measuring CD33 as follows, for example.
  • Diagnosis can be made by detecting CD33 expressed in cancer cells in a patient using an immunological technique such as a flow cytometer.
  • An immunological technique is a method for detecting or measuring the amount of antibody or the amount of antigen using a labeled antigen or antibody.
  • a radioactive substance-labeled immunoantibody method an enzyme immunoassay method, a fluorescence immunoassay method, a luminescence immunoassay method, a Western blot method, a physicochemical method, or the like can be given.
  • the radioactive substance-labeled immunoantibody method is, for example, reacting an antigen or a cell expressing the antigen with the antibody of the present invention or a fragment thereof, and further reacting with a radiolabeled anti-immunoglobulin antibody or a binding thereof, Measure with a scintillation counter.
  • an antigen or a cell expressing the antigen is reacted with the antibody of the present invention or a fragment thereof, and further, a labeled anti-immunoglobulin antibody or a binding fragment thereof is reacted, and then a coloring dye. Is measured with an absorptiometer. For example, a sandwich ELISA method is used.
  • a known enzyme label [enzyme immunoassay, Medical School (1987)] can be used.
  • enzyme immunoassay Medical School (1987)
  • alkaline phosphatase label, peroxidase label, luciferase label, biotin label or the like is used.
  • Sandwich ELISA is a method in which an antibody to be bound to a solid phase, an antigen to be detected or measured is trapped, and a second antibody is reacted with the trapped antigen.
  • two types of antibodies that recognize an antigen to be detected or measured or fragments thereof and having different antigen recognition sites are prepared, and the first antibody or fragment thereof is preliminarily placed on a plate (for example, 96 Next, the second antibody or a fragment thereof is labeled with a fluorescent substance such as FITC, an enzyme such as peroxidase, or biotin.
  • the label is obtained.
  • the detected monoclonal antibody or a fragment thereof is reacted, and a detection reaction corresponding to the labeling substance is performed.
  • the antigen concentration in the test sample is calculated from a calibration curve prepared by diluting antigens with known concentrations stepwise.
  • an antibody used in the sandwich ELISA method either a polyclonal antibody or a monoclonal antibody may be used, and an antibody fragment such as Fab, Fab ′, or F (ab) 2 may be used.
  • the combination of the two types of antibodies used in the sandwich ELISA method may be a combination of a monoclonal antibody that recognizes different epitopes or a fragment thereof, or a combination of a polyclonal antibody and a monoclonal antibody or a fragment thereof.
  • the fluorescence immunoassay is measured by the method described in the literature [Monoclonal Antibodies-Principles and practices, Third edition, Academic Press (1996), Monoclonal Antibody Experiment Manual, Kodansha Scientific (1987)].
  • a label used in the fluorescence immunoassay a fluorescent label known in the art [fluorescent antibody method, Soft Science (1983)] can be used.
  • FITC or RITC is used.
  • the luminescent immunoassay is measured by the method described in the literature [Bioluminescence and chemiluminescence, clinical examination 42, Yodogawa Shoten (1998)].
  • Examples of the label used in the luminescent immunoassay include known luminescent labels, such as acridinium ester or lophine.
  • an antigen or cells expressing the antigen are fractionated with SDS (sodium dodecyl sulfate) -PAGE [Antibodies-A Laboratory Manual Spring Spring Laboratory (1988)], and the gel is then polyvinylidene fluoride (PVDF).
  • SDS sodium dodecyl sulfate
  • PVDF polyvinylidene fluoride
  • a cell or tissue expressing a polypeptide containing the amino acid sequence represented by SEQ ID NO: 3 is lysed, and 0.1 to 30 ⁇ g of protein per lane is electrophoresed by SDS-PAGE under reducing conditions.
  • the migrated protein is transferred to a PVDF membrane and reacted with PBS containing 1 to 10% BSA (hereinafter sometimes referred to as BSA-PBS) at room temperature for 30 minutes to perform a blocking operation.
  • BSA-PBS PBS containing 1 to 10% BSA
  • the monoclonal antibody of the present invention was reacted, washed with PBS containing 0.05 to 0.1% Tween-20 (hereinafter sometimes referred to as Tween-PBS), and peroxidase-labeled goat anti-mouse IgG. For 2 hours at room temperature.
  • the physicochemical method is performed by, for example, forming an aggregate by binding the antigen CD33 and the monoclonal antibody of the present invention or a fragment thereof, and detecting the aggregate.
  • examples of the physicochemical method include a capillary method, a one-dimensional immunodiffusion method, an immunoturbidimetric method, or a latex immunoturbidimetric method [Proposal for Clinical Laboratory Methods, Kanbara Publishing (1998)].
  • Latex immunoturbidimetry is a method in which an antibody or antigen-sensitized carrier such as polystyrene latex having a particle size of about 0.1 to 1 ⁇ m is used to cause an antigen-antibody reaction with the corresponding antigen or antibody. Scattered light increases and transmitted light decreases. By detecting this change as absorbance or integrating sphere turbidity, the antigen concentration or the like in the test sample is measured.
  • an antibody or antigen-sensitized carrier such as polystyrene latex having a particle size of about 0.1 to 1 ⁇ m
  • a known immunological detection method can be used for the detection or measurement of cells expressing CD33.
  • immunoprecipitation method immune cell staining method, immunohistochemical staining method, fluorescent antibody staining method, etc. Is used.
  • a cell expressing CD33 or the like is reacted with the monoclonal antibody of the present invention or a fragment thereof, and then a carrier having a specific binding ability to immunoglobulin such as protein G-sepharose is added to the antigen-antibody complex. To settle.
  • a carrier having a specific binding ability to immunoglobulin such as protein G-sepharose is added to the antigen-antibody complex.
  • the following method can be used.
  • the above-described monoclonal antibody of the present invention or a fragment thereof is immobilized on a 96-well plate for ELISA, and then blocked with BSA-PBS.
  • the antibody is in an unpurified state, such as a hybridoma culture supernatant, anti-mouse immunoglobulin, anti-rat immunoglobulin, protein-A or protein-G is immobilized on a 96-well plate for ELISA in advance.
  • the hybridoma culture supernatant is dispensed and bound.
  • a lysate of cells and tissues expressing CD33 is reacted. Immunoprecipitates are extracted from the well-washed plate with SDS-PAGE sample buffer and detected by Western blotting as described above.
  • the immune cell staining method or the immunohistochemical staining method is a method in which cells or tissues expressing an antigen are treated with a surfactant or methanol in order to improve antibody passage, and then reacted with the monoclonal antibody of the present invention. And then reacting with a fluorescent label such as FITC, an enzyme label such as peroxidase or a biotin label, or an anti-immunoglobulin antibody or a binding fragment thereof, then the label is visualized and microscopically observed .
  • a fluorescent label such as FITC
  • an enzyme label such as peroxidase or a biotin label
  • a fluorescent antibody staining method in which a fluorescently labeled antibody is reacted with a cell and analyzed with a flow cytometer [Monoclonal Antibodies-Principles and Practice, Academic Press (1996), Monoclonal Antibody Experiment Manual, Kodansha Scientific Fick (1987)].
  • the monoclonal antibody of the present invention or a fragment thereof that binds to the extracellular region of CD33 can detect cells expressing the natural three-dimensional structure by fluorescent antibody staining.
  • the formed antibody-antigen complex and the free that is not involved in the formation of the antibody-antigen complex can be measured without separating the antibody or antigen.
  • Example 1 Production of CD33 transfectant (1) Preparation of CD33 expression vector From a vector in which the human CD33 gene is incorporated into the vector pCMV / SPORT6 (Open Biosystems, Clone No. 5217182, hereinafter sometimes referred to as pCMV / hCD33), human CD33 is prepared as follows. A vector incorporating the gene sequence was prepared.
  • the human CD33 gene was amplified by a normal PCR method using pCMV / hCD33 as a template and primers having the nucleotide sequences represented by SEQ ID NOs: 1 and 2.
  • the PCR product was purified by PCR Purification Kit (manufactured by QIAGEN) and treated with restriction enzymes with SacI and HindIII.
  • the vector pBluescript II sk (-) (Stratagene) (hereinafter sometimes referred to as pBS) is similarly treated with SacI and HindIII, and these two fragments are ligated high (TOYOBO). Used and ligated according to the attached instructions.
  • the obtained recombinant plasmid DNA solution was used to transform E. coli DH5 ⁇ strain (manufactured by TOYOBO).
  • a plasmid pBS / hCD33 into which the human CD33 gene has been inserted is obtained by preparing plasmid DNA from a clone of the transformant and confirming the size of the fragment excised by restriction enzyme treatment by agarose gel electrophoresis. did.
  • the pBS / hCD33 into which the human CD33 gene thus obtained was integrated and the animal cell expression vector pKANTEX93 (International Publication No. 97/10354) were subjected to restriction enzyme treatment with BsiWI and SpeI.
  • the reaction mixture was fractionated by agarose gel electrophoresis, and the BsiWI-SpeI fragment of hCD33 and the BsiWI-SpeI fragment of pKANTEX93 were recovered.
  • Plasmid DNA was prepared from the clone of the transformant and confirmed by comparing the size of the fragment excised by the restriction enzyme treatment by agarose gel electrophoresis.
  • the base sequence of the plasmid was analyzed using the BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (Applied Biosystems) according to the attached instructions, and then analyzed by the sequencer ABI PRISM 3700 of the company.
  • FIG. 1A A schematic diagram of vector construction is shown in FIG. 1A.
  • the amino acid sequence of human CD33 is shown in SEQ ID NO: 3
  • the nucleotide sequence of CD33 is shown in SEQ ID NO: 4.
  • An expression vector pKANTEX / cynoCD33 incorporating a cynomolgus monkey CD33 gene is a base represented by SEQ ID NOs: 115 and 116 from mRNA extracted from a cynomolgus monkey peripheral blood mononuclear cell (Peripheral blood mononuclear cell: hereinafter sometimes referred to as PBMC).
  • PBMC peripheral blood mononuclear cell
  • a human CD33 gene was amplified by a normal PCR method using a primer having a sequence, and prepared by the same method as described above.
  • a schematic diagram of vector construction is shown in FIG. 1B.
  • the base sequence confirmed by the above analysis is shown in SEQ ID NO: 118, and the amino acid sequence of cynomolgus CD33 predicted from the base sequence is shown in SEQ ID NO: 117.
  • the chimpanzee CD33 gene is a plasmid in which the base sequence represented by SEQ ID NO: 119 is designed based on the base sequence of the chimpanzee CD33 gene registered in the NCBI database as XM — 5128850.2, and the base sequence is incorporated into the cloning vector pUC53.
  • SEQ ID NO: 119 is designed based on the base sequence of the chimpanzee CD33 gene registered in the NCBI database as XM — 5128850.2, and the base sequence is incorporated into the cloning vector pUC53.
  • an expression vector pKANTEX / chimpCD33 incorporating the chimpanzee CD33 gene was prepared by the same method as described above.
  • a schematic diagram of vector construction is shown in FIG. 1C.
  • the amino acid sequence of chimpanzee CD33 is shown in SEQ ID NO: 120, and the base sequence of chimpanzee CD33 is shown in SEQ ID NO: 121.
  • CD33 expression vectors pKANTEX / hCD33, pKANTEX / cynoCD33, and pKANTEX / chimpCD33 prepared in (1) above were prepared by electroporation [Cytotechnology, 3, 133 (1990)]. Then, it was introduced into CHO / DG44 cells [Somatic Cell and Molecular Genetics, 12, 555 (1986)] as follows.
  • IMDM medium Invitrogen
  • A3 medium IMDM medium
  • Gentamin Nacalai Tesque, 50 ⁇ g / mL
  • 1 ⁇ HT supplement manufactured by Invitrogen was used in a subculture medium.
  • CHO / DG44 cells contain 137 nmol / L potassium chloride, 2.7 nmol / L sodium chloride, 8.1 mmol / L disodium monohydrogen phosphate, 1.5 nmol / L monosodium dihydrogen phosphate and 4 mmol / L magnesium chloride Suspend in a buffer solution (hereinafter sometimes referred to as K-PBS) to 8 ⁇ 10 6 cells / mL, and express 200 ⁇ L (1.6 ⁇ 10 6 cells) of the resulting cell suspension for each expression. Mixed with vector (8 ⁇ g).
  • K-PBS buffer solution
  • the mixture was transferred to a cuvette (distance between electrodes: 2 mm), and gene introduction was performed using GenePulser II (Bio-Rad) under conditions of a pulse voltage of 0.35 kV and an electric capacity of 250 ⁇ F.
  • GenePulser II Bio-Rad
  • the cell suspension in the cuvette was suspended in a cell culture vessel containing A3 medium, and cultured in a 37 ° C., 5% carbon dioxide incubator.
  • the cells were cultured in a medium supplemented with G418 (manufactured by Invitrogen, 0.5 mg / mL) to obtain a transformed cell line resistant to G418. Further, methotrexate-resistant clones were selected by adding methotrexate to the medium and gradually increasing the concentration.
  • G418 manufactured by Invitrogen, 0.5 mg / mL
  • Example 1 (3) these cells were fluorescently immunostained, and the fluorescence intensity was measured with a flow cytometer (manufactured by Beckman Coulter) to select a clone that highly expresses CD33.
  • a CD33-expressing cell line, a cynomolgus monkey CD33-expressing cell line, and a chimpanzee CD33-expressing cell line were obtained.
  • Example 2 Preparation of histidine-tagged soluble human CD33 (hCD33-His) (1) Preparation of hCD33-His expression vector Using plasmid pBS / hCD33 inserted with human CD33 gene obtained in Example 1 (1) as a template, A gene sequence (hereinafter referred to as a his sequence) encoding a 6 ⁇ histidine sequence (hereinafter sometimes referred to as a His sequence) by a normal PCR method using a primer having a base sequence represented by SEQ ID NOs: 1 and 5 The hCD33 gene (hereinafter sometimes referred to as hCD33-his) added to the 3 ′ end side was amplified.
  • a his sequence encoding a 6 ⁇ histidine sequence
  • hCD33-his The hCD33 gene (hereinafter sometimes referred to as hCD33-his) added to the 3 ′ end side was amplified.
  • the PCR product was purified by PCR Purification Kit and subjected to restriction enzyme treatment with BsiWI and SpeI.
  • the vector pKANTEX93 was similarly digested with BsiWI and SpeI.
  • the reaction solution was fractionated by agarose gel electrophoresis, and the BsiWI-SpeI fragment of hCD33-his and the BsiWI-SpeI fragment of pKANTEX93 were recovered. These two types of fragments were ligated using Ligation high according to the attached instructions.
  • Plasmid DNA was prepared from the clone of the transformant and confirmed by restriction enzyme treatment. Further, the plasmid was reacted using the BigDye Terminator Cycle Sequencing FS Ready Reaction Kit according to the attached instruction, and the base sequence was analyzed by the sequencer ABI PRISM 3700.
  • FIG. 6 A schematic diagram of vector construction is shown in FIG.
  • the amino acid sequence of hCD33-His is shown in SEQ ID NO: 6, and the nucleotide sequence of hCD33-his is shown in SEQ ID NO: 7.
  • the cells used were subcultured with a medium supplemented with 1 ⁇ HT supplement to A3 medium.
  • CHO / DG44 cells were suspended in K-PBS to 8 ⁇ 10 6 cells / mL, and 200 ⁇ L (1.6 ⁇ 10 6 cells) of the obtained cell suspension was used as an expression vector pKANTEX / hCD33-his (8 ⁇ g). ).
  • the mixture was transferred to a cuvette (distance between electrodes: 2 mm), and gene introduction was performed using GenePulser II under conditions of a pulse voltage of 0.35 kV and an electric capacity of 250 ⁇ F.
  • the cell suspension in the cuvette was suspended in a cell culture vessel containing A3 medium, and cultured in a 37 ° C., 5% carbon dioxide incubator.
  • the cells were cultured in a medium supplemented with G418 to obtain a transformed cell line having resistance to G418. Further, methotrexate-resistant clones were selected by adding methotrexate to the medium and gradually increasing the concentration. Further, according to the method (3) below, by comparing the concentration of hCD33-His in the culture supernatant of these cells, a clone producing high hCD33-His was selected, and an hCD33-His-producing cell line was obtained.
  • 1% BSA-PBS was discarded, and the cell culture supernatant containing hCD33-His was appropriately diluted on the plate, dispensed at 50 ⁇ L / well, and allowed to stand for 1 hour.
  • the plate was washed with 0.05% polyoxyethylene (20) sorbitan monolaurate (ICI trademark Tween 20 equivalent: manufactured by Wako Pure Chemical Industries, Ltd.) / PBS (hereinafter sometimes referred to as Tween / PBS).
  • a peroxidase-labeled mouse anti-Penta-His antibody manufactured by QIAGEN
  • QIAGEN peroxidase-labeled mouse anti-Penta-His antibody
  • the plate was washed with Tween / PBS, ABTS [2.2-azinobis (3-ethylbenzothiazole-6-sulfonic acid) ammonium] substrate solution [1 mmoL / L ABTS / 0.1 moL / L citrate buffer (pH 4. 2), 0.1% H 2 O 2 ] was added at 50 ⁇ L / well for color development, and 5% SDS (Sodium Lauryl Sulfate) solution was added at 50 ⁇ L / well to stop the reaction, and the sample wavelength was 415 nm and the reference wavelength was 490 nm. Absorbance (OD415-OD490) was measured using a plate reader (Emax microplate reader, Molecular Devices).
  • Example 3 Preparation of anti-CD33 monoclonal antibody (1) Preparation of immunogen Recombinant human Siglec-3 (CD33) / Fc Chimera (catalog number 1137-SL) manufactured by R & D Systems (hereinafter sometimes referred to as recombinant human CD33-Fc) The freeze-dried product, human CD33-expressing cell line obtained by the method described in Example 1, or human acute monocytic leukemia cell line THP-1 positive for CD33 was dissolved in PBS and used as an immunogen.
  • the human CD33-expressing cell line 1 ⁇ 10 7 cells prepared in (1) above was administered to SD rats (Japan SLC) four times in total, once per week, with pertussis vaccine 1 ⁇ 10 9 cells.
  • Partial blood was collected from the fundus vein of the rat or mouse, the serum antibody titer was confirmed by the enzyme immunoassay shown below, and the spleen was removed 3 days after the final immunization from the rat or mouse showing a sufficient antibody titer.
  • the spleen was shredded in MEM (Minimum Essential Medium) medium (manufactured by Nissui Pharmaceutical), ground and centrifuged (1200 rpm, 5 minutes).
  • Enzyme immunoassay binding ELISA
  • a 96-well ELISA plate (Greiner) was used for the assay.
  • Recombinant human CD33-Fc, anti-rat immunoglobulin antibody (DAKO) or anti-mouse immunoglobulin antibody (DAKO) was dispensed at 50 ⁇ L / well and allowed to stand overnight at 4 ° C. for adsorption.
  • the plate was washed with PBS, 1% bovine serum albumin (BSA) -PBS was added at 100 ⁇ L / well, and the plate was allowed to stand at room temperature for 1 hour to block remaining active groups.
  • BSA bovine serum albumin
  • 1% BSA-PBS was discarded, and immunized rat or mouse serum was appropriately diluted as a primary antibody on the plate, dispensed at 50 ⁇ L / well, and allowed to stand for 1 hour.
  • 50 ⁇ L / peroxidase-labeled goat anti-rat IgG (H + L chain) antibody (ZYMED) or peroxidase-labeled goat anti-mouse IgG ( ⁇ ) antibody manufactured by KPL was used as the secondary antibody.
  • the wells were added and left at room temperature for 1 hour.
  • the plate was washed with Tween-PBS, ABTS [2.2-azinobis (3-ethylbenzothiazole-6-sulfonic acid) ammonium] substrate solution [1 mmoL / L ABTS / 0.1 moL / L citrate buffer (pH 4. 2), 0.1% H 2 O 2 ] was added at 50 ⁇ L / well for color development, and 5% SDS (Sodium Lauryl Sulfate) solution was added at 50 ⁇ L / well to stop the reaction, and the sample wavelength was 415 nm and the reference wavelength was 490 nm. Absorbance (OD415-OD490) was measured using a plate reader (Emax microplate reader, manufactured by Molecular Devices).
  • mice 8-Azaguanine resistant mouse myeloma cell line P3X63Ag8U. 1 (P3-U1: purchased from ATCC) was cultured in RPMI 1640 supplemented with 10% fetal bovine serum (manufactured by Invitrogen), 2 ⁇ 10 7 cells or more were secured at the time of cell fusion, and used as a parent strain for cell fusion.
  • mice spleen cells obtained in (2) above and the myeloma cells obtained in (4) above were mixed at 10: 1 and centrifuged (1200 rpm, 5 minutes). After thoroughly disaggregating the cells of the obtained precipitate fraction, a mixed solution of 1 g of polyethylene glycol-1000 (PEG-1000), 1 mL of MEM medium, and 0.35 mL of dimethyl sulfoxide (DMSO) is stirred at 37 ° C. 0.5 mL per 1 ⁇ 10 8 mouse spleen cells was added, and 1 mL of MEM medium was added several times to the suspension every minute, and then the MEM medium was added to make the total volume 50 mL.
  • PEG-1000 polyethylene glycol-1000
  • MEM medium dimethyl sulfoxide
  • the suspension was centrifuged (900 rpm, 5 minutes), and the cells of the obtained precipitate fraction were loosely loosened. Medium gently added to 100 mL. The suspension was dispensed into a 96-well culture plate at 200 ⁇ L / well and cultured at 37 ° C. for 10-14 days in a 5% CO 2 incubator.
  • KM4071 refers to a hybridoma or a monoclonal antibody produced by the hybridoma (the same applies to other hybridomas).
  • Example 4 Activity evaluation of rat or mouse anti-CD33 monoclonal antibody against human CD33 (1) Reactivity with human CD33-positive cell line in FCM 1-5 ⁇ 10 5 cells of THP-1 with human IgG (Sigma) KM4071 to KM4075 and P67.6 [Biocomjugate Chem, 13, 47 (2002)] were appropriately diluted with FCM buffer to make a total volume of 100 ⁇ L. These cell suspensions were reacted for 60 minutes on ice and then washed twice with PBS.
  • FIG. 3 shows the average fluorescence intensity when the anti-CD33 monoclonal antibody obtained in Example 3 was reacted stepwise diluted from 3 ⁇ g / mL at a 4-fold dilution (hereinafter sometimes referred to as MFI). Indicates. As shown in FIG. 3, it was confirmed that KM4071 to KM4075 and p67.6 all bind to the CD33 positive cell line in a concentration-dependent manner.
  • FIG. 4 shows the results of using KM4071 to KM4075 and P67.6 obtained in Example 3 stepwise diluted from 2 ⁇ g / mL at a 5-fold dilution as the primary antibody.
  • the anti-mouse IgG antibody was solidified on a CM5 sensor chip (manufactured by GE Healthcare Bioscience) by the amine coupling method according to the attached protocol. Measurement samples (KM4071 to KM4075 and P67.6 were added to a chip on which an anti-mouse IgG antibody was solidified, and the sample was captured so as to be about 200 RU (resonance unit).
  • KM4071, 4073, 4074, and 4075 showed higher affinity for CD33-Fc than P67.6.
  • Example 5 Isolation and Analysis of cDNA Encoding Variable Region of Anti-CD33 Monoclonal Antibody (1) Preparation of mRNA from Anti-CD33 Monoclonal Antibody-Producing Hybridoma Cells From KM4071 to KM4075 described in Example 3, RNAeasy Mini kit (QIAGEN) Using Oligotex TM -dT30 ⁇ Super> mRNA Purification Kit (TaKaRa), mRNA was prepared from hybridoma 5 ⁇ 10 7 to 1 ⁇ 10 8 cells according to the attached instruction manual.
  • a universal primer A mix attached to the kit a primer specific to rat IgG1 (SEQ ID NO: 8), a primer specific to rat IgG2a (SEQ ID NO: 9), or a primer specific to mouse IgG1 (SEQ ID NO: 10) or PCR reaction using a primer specific to mouse IgG2a (SEQ ID NO: 11) to amplify the cDNA fragment of each antibody heavy chain variable region (hereinafter sometimes referred to as VH) did.
  • VH antibody heavy chain variable region
  • PCR was performed using a rat Ig ( ⁇ ) -specific primer (SEQ ID NO: 12) or a mouse Ig ( ⁇ ) -specific primer (SEQ ID NO: 13) instead of each antibody subclass-specific primer.
  • a cDNA fragment of the light chain variable region (hereinafter sometimes referred to as VL) was amplified.
  • VL light chain variable region
  • PCR was repeated 30 times, consisting of 5 cycles, 94 ° C for 15 seconds, 68 ° C for 30 seconds, 72 ° C for 3 minutes, and then reacted at 72 ° C for 10 minutes.
  • PCR was performed using PTC-200 DNA Engine (manufactured by BioRad).
  • the obtained PCR products were separated by agarose gel electrophoresis in order to clone and determine the base sequence, and the H chain and L chain were extracted using Gel Extraction Kit (manufactured by QIAGEN).
  • Target Clone Plus manufactured by TOYOBO was used for the extracted fragment, and after the dA addition reaction according to the attached instruction, VH or VL of each antibody was incorporated into the pTA2 vector (manufactured by TOYOBO).
  • Escherichia coli DH5 ⁇ strain was transformed according to the instruction manual attached to Competent Quick (manufactured by TOYOBO).
  • a plasmid was extracted from the obtained transformant using an automatic plasmid extractor (manufactured by Kurabo Industries), and reacted according to the attached instructions using BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (manufactured by PE Biosystems).
  • the base sequence of the PCR product cloned by the sequencer ABI PRISM 3700 was analyzed.
  • the base sequence obtained by removing the signal sequence from the base sequence shown in SEQ ID NOs: 14 to 18 is shown in SEQ ID NO: 122-126
  • the base sequence obtained by removing the signal sequence from the base sequences shown in SEQ ID NOs: 24-28 is shown in SEQ ID NO: 127 -131
  • the amino acid sequence obtained by removing the signal sequence from the amino acid sequence shown in SEQ ID NOs: 19-23 is shown in SEQ ID NO: 29-33
  • the numbers 137 to 141 are shown respectively.
  • each isolated cDNA is a full-length cDNA encoding KM4071 to KM4075 including a secretory signal sequence, and the KM4071 heavy chain is represented by SEQ ID NO: 19.
  • the 1st to 19th amino acid sequence of the sequence is the secretion signal sequence for the KM4071 L chain, the 1st to 19th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 29, and the KM4072 H chain is represented by SEQ ID NO: 20
  • the amino acid sequence from 1 to 18 of the amino acid sequence shown is the secretory signal sequence for the L chain of KM4072 and the amino acid sequence of 1 to 22 of the amino acid sequence shown in SEQ ID NO: 30 is the sequence for the H chain of KM4073 Ami shown in number 21
  • the 1st to 18th amino acid sequence of the acid sequence is the secretory signal sequence for the L chain of KM4073, the 1st to 20th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 31, and the SEQ ID NO: 22 for the H chain of KM4074
  • the secretory signal sequence for the KM4074 L chain is the secretory signal sequence for the KM4074 L chain, the amino acid sequence from 1 to 19 of the amino acid sequence shown in SEQ ID NO: 32, and the KM4075 H chain is It is clear that the 1st to 20th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 23 is the secretory signal sequence, and the 1st to 19th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 33 for the KM4075 L chain is the secretory signal sequence. became.
  • the CDRs of VH and VL of each monoclonal antibody were identified by comparing with the amino acid sequences of known antibodies.
  • amino acid sequences of CDR1, CDR2, and CDR3 of VH of KM4071 are shown in SEQ ID NOs: 34, 35, and 36, and the amino acid sequences of CDR1, CDR2, and CDR3 of VL are shown in SEQ ID NOs: 37, 38, and 39, respectively.
  • amino acid sequences of CDR1, CDR2 and CDR3 of VH of KM4072 are shown in SEQ ID NOs: 40, 41 and 42, and the amino acid sequences of CDR1, CDR2 and CDR3 of VL are shown in SEQ ID NOs: 43, 44 and 45, respectively.
  • amino acid sequences of CDR1, CDR2 and CDR3 of VH of KM4073 are shown in SEQ ID NOs: 46, 47 and 48, and the amino acid sequences of CDR1, CDR2 and CDR3 of VL are shown in SEQ ID NOs: 49, 50 and 51, respectively.
  • amino acid sequences of CDR1, CDR2, and CDR3 of VH of KM4074 are shown in SEQ ID NOs: 52, 53, and 54, and the amino acid sequences of CDR1, CDR2, and CDR3 of VL are shown in SEQ ID NOs: 55, 56, and 57, respectively.
  • amino acid sequences of CDR1, CDR2 and CDR3 of VH of KM4075 are shown in SEQ ID NOs: 58, 59 and 60, and the amino acid sequences of CDR1, CDR2 and CDR3 of VL are shown in SEQ ID NOs: 61, 62 and 63, respectively.
  • Example 6 Preparation of anti-CD33 chimeric antibody
  • the activity between antibodies of different species, or antibodies of different subclasses of the same species is the reactivity of the detection antibody. It cannot be compared because it is different. Therefore, a chimeric antibody in which the constant regions KM4071 to KM4075 established in Example 3 were replaced with human constant region sequences was prepared according to the following method. Thus, by aligning the constant region with human-derived sequences, various activities depending on the constant region can be measured and compared.
  • anti-CD33 chimeric antibody expression vector The chimeric antibody produced this time is a strong complement-dependent cytotoxic activity consisting of a mixed sequence of human IgG1 and human IgG3 (hereinafter sometimes referred to as CDC activity).
  • a chimeric antibody comprising a constant region containing hereinafter, an antibody containing the constant region may be referred to as a 113F antibody) and the variable region of the monoclonal antibody obtained in Example 5 (2).
  • high CDC type chimeric antibody expression vector PKTX93 / 113F (described in US Patent Application Publication No. 2007/0148165) containing the variable part of anti-CD20 antibody and obtained in Example 5 (2) were used.
  • an anti-CD33 chimeric antibody expression vector was constructed as follows.
  • the base sequence of the KM4071 VH primer is SEQ ID NOs: 64 and 65
  • the base sequence of the VL primer is SEQ ID NOs: 66 and 67
  • the base sequence of the KM4072 VH primer is SEQ ID NOs: 68 and 69
  • the base sequence of the VL primer is SEQ ID NO: 70 and 71
  • KM4073 VH primer base sequence is SEQ ID NO: 72 and 73
  • KM4074 VH primer base sequence is SEQ ID NO: 76 and 77
  • VL SEQ ID NOs: 78 and 79 are shown in SEQ ID NOs: 78 and 79
  • SEQ ID NOs: 80 and 81 are shown in the sequence of VH primers in KM 4075
  • SEQ ID NOS: 82 and 83 are used in the base sequences of VL primers.
  • VH of each antibody thus obtained was subjected to restriction enzyme treatment with ApaI (manufactured by New England Biolabs) and NotI (manufactured by New England Biolabs) to obtain a NotI-ApaI fragment of VH.
  • VL of each antibody was subjected to restriction enzyme treatment with BsiWI (manufactured by New England Biolabs) and EcoRI (manufactured by New England Biolabs) to obtain an EcoRI-BsiWI fragment of VL.
  • the vector PKTX93 / 113F was subjected to restriction enzyme treatment with NotI and ApaI, or EcoRI and BsiWI.
  • VH or VL fragment of each monoclonal antibody was ligated with a restriction enzyme-treated vector and Ligation high (manufactured by TOYOBO) according to the attached instructions, and the resulting recombinant plasmid DNA solution was used.
  • E. coli DH5 ⁇ strain manufactured by TOYOBO was transformed.
  • Each plasmid DNA was prepared from the clone of the transformant and confirmed by restriction enzyme treatment to obtain a plasmid into which the ApaI-NotI fragment of the target VH or the EcoRI-BSIWI fragment of VL was inserted.
  • the plasmid was reacted using the BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (manufactured by PE Biosystems) according to the attached instructions, and the base sequence was analyzed by the sequencer ABI PRISM 3700 of the company.
  • the plasmid was reacted using the BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (manufactured by PE Biosystems) according to the attached instructions, and the base sequence was analyzed by the sequencer ABI PRISM 3700 of the company.
  • an anti-CD33 chimeric antibody expression vector in which cDNAs encoding VH and VL of KM4071 to KM4075 were cloned was obtained.
  • a schematic diagram of vector construction is shown in FIG.
  • a CHO / DG44 cell line (hereinafter sometimes referred to as FUT8 knockout CHO cell or CHO / Ms704) in which the ⁇ 1,6-fucosyltransferase (FUT8) gene was double knocked out was used. It is known that fucose is not added to the core part of the N-linked complex sugar chain of the antibody expressed in this host cell line (WO 2002/31140).
  • the human IgG1 antibody to which fucose is not added has higher antibody-dependent cytotoxic activity (hereinafter sometimes referred to as ADCC activity) than the case where it is added.
  • ADCC activity antibody-dependent cytotoxic activity
  • an antibody to which fucose is not added may be referred to as a high ADCC type antibody.
  • Example 7 Evaluation of Activity of Anti-CD33 Chimeric Antibody
  • HM195 anti-CD33 antibody
  • HM195-P anti-CD33 antibody
  • HM195 type 113F high ADCC type antibody
  • HM195-113F high ADCC type antibody
  • Fc goat anti-human IgG
  • FCM buffer 30 ⁇ L of FITC-labeled goat anti-human IgG (Fc) antibody (manufactured by Acris Antibodies) diluted with FCM buffer was added to the cells and reacted on ice for 40 minutes. After washing with PBS three times, the suspension was suspended in FCM buffer and the fluorescence intensity was measured using a flow cytometer (manufactured by Beckman Coulter).
  • FIG. 6 shows the MFI when the KM4051 to KM4055 obtained in Example 6 are reacted stepwise diluted from 2 ⁇ g / mL at a 4-fold dilution at each concentration. It was confirmed that KM4051 to KM4055 and HM195-113F all bind to THP-1 in a concentration-dependent manner.
  • Peroxidase-labeled goat anti-human IgG (H + L chain) antibody (American Qualex) or peroxidase-labeled goat anti-human ⁇ chain (Southern Biotech) was used as the secondary antibody.
  • the results of recombinant human CD33-Fc and goat anti-human IgG (H + L) antibody are shown in FIGS. 7 (A) and (B), respectively.
  • Binding activity to recombinant human CD33-Fc in Biacore In order to analyze the binding activity of KM4051 to KM4055 and HM195-113F to hCD33-His, binding was performed using the surface plasmon resonance method (SPR method). Activity measurement was performed.
  • SPR method surface plasmon resonance method
  • Samples (KM4051 to KM4055 and HM195-113F) were added to the chip on which the anti-human IgG antibody was solidified, and captured to about 300 RU (resonance unit), and then diluted from 1.25 ⁇ g / mL in 5 steps
  • the hCD33-His was flowed over the chip at a rate of 30 ⁇ L / min, and sensorgrams at each concentration were obtained, analyzed using a 1: 1 binding model using analysis software attached to the device, and each antibody against human CD33
  • the association rate constant ka and the dissociation rate constant kd were calculated.
  • ADCC activity of anti-CD33 chimeric antibody against human hematological cancer cell line ADCC activity of KM4051 to KM4055, HM195-1, and HM195-113F was measured according to the following method.
  • PBMCs were separated from healthy human peripheral blood by the method described below.
  • 50 mL of healthy human peripheral blood was collected with a syringe containing 0.5 mL of heparin sodium injection N “Shimizu” (manufactured by Shimizu Pharmaceutical Co., Ltd.).
  • the collected peripheral blood was diluted by adding the same amount of physiological saline (manufactured by Otsuka Pharmaceutical) and stirred well.
  • Lymphoprep Axis-Shield
  • 10 mL of diluted peripheral blood is gently layered, and then 20 minutes at 2000 rpm, brake off, and room temperature. Keep in mind to separate the mononuclear cell layer.
  • the thus obtained mononuclear cell fraction was washed twice with an ADCC medium, adjusted to an optimal concentration with the same medium, and used as an effector cell solution.
  • ADCC activity was determined by the following formula.
  • ADCC activity (%) ⁇ ([Absorbance of specimen]-[Absorbance of spontaneous release of target cell]-[Absorption of spontaneous release of effector cell]) / ([Absorbance of total release of target cell]-[Absorbance of spontaneous release of target cell] ]) X 100
  • the results are shown in FIG.
  • the antibody concentration was diluted 10-fold in steps from 1 ⁇ g / mL.
  • KM4051 to KM4055, HM195-1, and HM195-113F showed antibody concentration-dependent ADCC activity against NB-4.
  • KM4051 to KM4055 showed stronger ADCC activity than HM195-113F.
  • the CD33 transfectant was washed with RPMI 1640 medium (Wako Pure Chemical Industries, Ltd.) containing 10% fetal bovine serum (FBS). .
  • the target cell solution is added to a 96-well flat bottom plate (manufactured by Sumitomo Bakelite Co., Ltd.) so that the number of target cell solutions is 5 ⁇ 10 4 per well, and the anti-CD33 chimeric antibody solution prepared to an appropriate concentration and the final concentration is diluted 4-fold.
  • Human complement manufactured by SIGMA was added to prepare 100 ⁇ L per well.
  • reaction well containing no antibody (0% Lysis well) was prepared as a control when CDC was not induced, and a reaction well containing no cells (100% Lysis well) was prepared as a control when CDC was induced.
  • the reaction was performed in a 37 ° C. incubator for 3 hours.
  • Antibody binding amount on cell membrane of CD33-expressing cell line of anti-CD33 chimeric antibody The amount of antibody binding over time of anti-CD33 chimeric antibody and HM195-113F on the cell membrane of THP-1 was measured according to the following method. .
  • Diluted mouse serum (CEDARLANE LABORATORIES) was added to 1-5 ⁇ 10 5 THP-1 for blocking, and KM4051-KM4055 and HM195-113F shown in Example 8 were appropriately diluted with 10% FCS-RPMI. was added to make a total volume of 100 ⁇ L.
  • FIG. 10 ((A) and (B) in FIG. 10 show the amount of antibody binding and the antibody remaining rate, respectively).
  • KM4051 to KM4055 and HM195-113F decreased the amount of binding on the cell surface in a time-dependent manner.
  • KM4051 to KM4055 exceeded both HM195-113F and the amount of antibody bound to the cell surface and the residual ratio at each time.
  • Example 8 Production of HM195 IgG1-type and 113F-type antibodies (1) Production of HM195-1 and HM195-113F expression vectors Human antibodies that specifically recognize human CD33 based on the sequence information described in US Pat. No. 7,022,500 Genes encoding HM195 VH and VL were constructed by the method described in Example 6 (1) and incorporated into vector pKANTEX93 and vector PKTX93 / 113F. A schematic diagram of vector construction is shown in FIG.
  • HM195-1 and HM195-113F Expression of HM195-1 and HM195-113F in animal cells
  • the vector obtained in (1) above was used and the method described in Example 6 (2).
  • animal cells to express HM195-1 CHO / DG44 cells and CHO / Ms704 cells were used to express normal and non-fucose-added antibodies.
  • CHO / Ms704 cells were used as animal cells that express HM195-113F.
  • Example 9 Preparation of anti-CD33 chimeric antibody having no consensus sequence for binding N-linked sugar chain to variable region
  • Anti-CD33 chimeric antibody having no consensus sequence for binding N-linked sugar chain to variable region (hereinafter referred to as modified anti-antibody) CD33 chimeric antibody) was prepared as described below in Example 3 based on KM4074.
  • the antibody had a consensus sequence to which an N-linked sugar chain was bound in the amino acid sequence of CDR2 of VH represented by SEQ ID NO: 53.
  • the consensus sequence to which the N-linked sugar chain contained in VH of KM4074 binds is SEQ ID NO: 22 Is a sequence consisting of 71st Asn, 72nd Ser, and 73rd Ser of the amino acid sequence shown in FIG.
  • CDR2 has the amino acid sequence shown in SEQ ID NO: 53.
  • the consensus sequence to which the N-linked sugar chain binds is a sequence composed of the third Asn, the fourth Ser, and the fifth Ser.
  • the consensus sequence to which the N-linked sugar chain binds is that the fourth amino acid is arbitrary in the amino acid sequence represented by SEQ ID NO: 53, and therefore the third Asn and the fifth Ser were used as candidate modification residues.
  • Existing antibody sequences [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)] and the amino acid modification and side chain structure in consideration of the amino acid modification and the third Asn are considered as Tyr, Ser, or Gln.
  • the amino acid modification which substitutes either of these, or the amino acid modification which substitutes 5th Ser to either Ala or Gly was selected.
  • ver.3 in which the third Asn is replaced with Tyr. 1 and 3 where Asn is replaced with Ser.
  • the second and third Asn were replaced with Gln ver. 3 and 5 where Ser was replaced with Ala. 4th and 5th Ser were replaced with Gly.
  • Five VHs of 5 types of modified CD33 chimeric antibodies were designed.
  • Ver. 1 to ver. The amino acid sequences of each VH of 5 are shown in SEQ ID NOs: 103, 104, 105, 93, and 106, respectively.
  • the amino acid sequences of CDR2 of VH are shown in SEQ ID NOs: 99, 100, 101, 96 and 102, respectively.
  • ver. Antibody with 1 is simply ver. Sometimes referred to as 1.
  • the VH of the antibody is an amino acid sequence represented by any one of SEQ ID NOs: 103, 104, 105, 93, and 106
  • the VL of the antibody is SEQ ID NO: 95 (represented by SEQ ID NO: 32).
  • a modified CD33 chimeric antibody having an amino acid sequence represented by the amino acid sequence minus the signal sequence was designed.
  • the obtained vector was analyzed using the BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (manufactured by PE Biosystems) according to the attached instructions, and the base sequence of the PCR product cloned by its sequencer ABI PRISM3700 was analyzed. It was confirmed that a modified antibody expression vector having been modified was obtained.
  • Example 10 Activity evaluation of the modified CD33 chimeric antibody The activity of the KM4054 obtained in Example 6 and the modified CD33 chimeric antibody prepared in Example 9 were evaluated.
  • modified CD33 chimeric antibody ver. 1 to 5 have an affinity equivalent to that of KM4054. 4 was named KM4084.
  • amino acid sequences of CDR1, CDR2 and CDR3 of VH of KM4084 are shown in SEQ ID NOs: 52, 96 and 54, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 of VL are shown in SEQ ID NOs: 55, 56 and 57, respectively.
  • nucleotide sequence of the DNA encoding the amino acid sequence of KM4084 represented by SEQ ID NO: 93 is represented by SEQ ID NO: 92
  • nucleotide sequence of the DNA encoding the amino acid sequence of VL of KM4084 represented by SEQ ID NO: 95 is sequenced. The number 94 is shown.
  • Example 11 Preparation of anti-CD33 humanized antibody (1) Design of amino acid sequences of VH and VL of anti-CD33 humanized antibody The amino acid sequence of VH of anti-CD33 humanized antibody was designed as follows. First, in order to transplant the amino acid sequence of CDR1 to 3 of VH of KM4084 (SEQ ID NO: 52, 96, 54), the amino acid sequence of the VH framework region of human antibody (hereinafter sometimes referred to as FR) Selected.
  • FR the amino acid sequence of the VH framework region of human antibody
  • the homology of HSGI, HSGII, and HSGIII was 50.6%, 67.1%, and 54.1%, respectively. Therefore, the FR amino acid sequence of KM4084VH had the highest homology with subgroup II.
  • the amino acid sequence of CDRs 1 to 3 (SEQ ID NOs: 52, 96, and 54) of KM4084VH was transplanted to the appropriate position of the FR amino acid sequence of the consensus sequence of VH subgroup II of the human antibody.
  • the amino acid sequence HV0 (SEQ ID NO: 84) of VH of the KM4084 humanized antibody was designed.
  • the amino acid sequence of VL of the KM4084 humanized antibody was designed as follows. In order to graft the amino acid sequence of CDR1-3 of VL of KM4084 (SEQ ID NOs: 55-57), the amino acid sequence of FR of VL of human antibody was selected. Kabat et al. Classify VL of various known human antibodies into subgroups (HSG I-IV) based on their amino acid sequence homology, and report common sequences for each subgroup [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)]. Therefore, a homology search was performed between the FR amino acid sequence of the consensus sequence of subgroups I to IV of human antibody VL and the FR amino acid sequence of KM4084VL.
  • the homology of HSGI, HSGII, HSGIII, and HSGIV was 60.0%, 77.5%, 65.0%, and 65.0%, respectively. Therefore, the amino acid sequence of FR of KM4084VL had the highest homology with subgroup II.
  • the amino acid sequence of CDRs 1 to 3 (SEQ ID NOs: 55 to 57) of KM4084VL was transplanted to the appropriate position of the FR amino acid sequence of the consensus sequence of subgroup II of the VL of the human antibody.
  • the 4th Leu and 109th Leu in the amino acid sequence of KM4084VL are the most frequently used amino acid residues at the site corresponding to the amino acid sequence of human antibody FR mentioned by Kabat et al. Although it is not a group, since it is an amino acid residue that is used at a relatively high frequency, the amino acid residue recognized in the amino acid sequence of KM4084 is used. In this way, the amino acid sequence LV0 (SEQ ID NO: 85) of the VL of the KM4084 humanized antibody was designed.
  • VH amino acid sequence HV0 and VL amino acid sequence LV0 of the KM4084 humanized antibody designed above are sequences in which only the amino acid sequence of the CDR of KM4084 is grafted to the FR amino acid sequence of the selected human antibody.
  • the binding activity often decreases only by transplanting the amino acid sequence of the CDR of the mouse antibody to the FR of the human antibody.
  • FR amino acid residues that are thought to affect the binding activity were identified as follows.
  • HV0LV0 the three-dimensional structure of the antibody V region consisting of the VH amino acid sequence HV0 and the VL amino acid sequence LV0 of the KM4084 humanized antibody designed above. It was constructed. Discovery Studio (manufactured by Accelrys Co., Ltd.) was used according to the attached instruction manual for preparing the three-dimensional structure coordinates and displaying the three-dimensional structure.
  • the computer model of the three-dimensional structure of the V region of KM4084 was also constructed in the same manner. Furthermore, in the amino acid sequence of HV0LV0 VH and VL FR, an amino acid residue different from KM4084 is selected, an amino acid sequence modified to KM4084 amino acid residue is prepared, and a three-dimensional structure model is constructed in the same manner. did. The three-dimensional structures of the V regions of these prepared KM4084, HV0LV0 and the variant were compared, and amino acid residues predicted to affect the binding activity of the antibody were identified.
  • the amino acid residues of FR of HV0LV0 are 25th Ser, 27th in HV0.
  • Gly, 28th Ser, 29th Val, 30th Ser, 40th Gln, 45th Gly, 46th Leu, 49th Ile, 72nd Val, 93th Val, 95th Tyr, 97th Ala, and 106th Thr were selected for LV0 as 2nd Ile, 8th Pro, 11th Leu, 15th Pro, and 90th Val, respectively.
  • At least one amino acid sequence was modified to an amino acid residue present at the same site of KM4084, and VH and VL of humanized antibodies having various modifications were designed.
  • the 25th Ser of the amino acid sequence of SEQ ID NO: 84 is Thr
  • the 27th Gly is Tyr
  • the 28th Ser is Thr
  • the 29th Val is Ile
  • the 30th Ser to Thr 40th Gln to Lys, 45th Gly to Arg, 46th Leu to Met, 49th Ile to Met, 72nd Val to Arg, 93th Val
  • At least one of the amino acid modifications was introduced into Thr, replacing 95th Tyr with Phe, 97th Ala with Thr, and 106th Thr with Val.
  • the second Ile of the amino acid sequence of SEQ ID NO: 85 is Val
  • the 8th Pro is Leu
  • the 11th Leu is Gln
  • the 15th Pro is Leu
  • the 90th Val At least one modification among the amino acid modifications in which is substituted for Leu was introduced.
  • HV0LV0, HV7LV0, and HV14LV0 were each designed as an antibody V region of an anti-CD33 humanized antibody in which at least one amino acid residue was present in the FR of HV0LV0.
  • the amino acid sequences of the heavy chain variable regions HV7 and HV14 are shown in SEQ ID NOs: 86 and 87, respectively.
  • the DNA encoding the variable region amino acid sequence of the anti-CD33 humanized antibody is the codon used in the DNA encoding the amino acid sequences of KM4084VH and KM4084VL (SEQ ID NOs: 92 and 94). When amino acid modification is carried out using a codon, it was prepared using codons frequently used in mammalian cells.
  • the DNA sequences encoding the amino acid sequences of HV0 and LV0 of the anti-CD33 humanized antibody are shown in SEQ ID NOs: 88 and 89, respectively, and the DNA sequences encoding the amino acid sequences of the variable regions HV7 and HV14 subjected to amino acid modification are The numbers 90 and 91 are shown respectively.
  • DNA encoding each variable region was prepared and inserted into the antibody expression vector pKANTEX93 to prepare an anti-CD33 humanized antibody expression vector. That is, according to the method of Example 6 (2) and (3), the prepared anti-CD33 humanized antibody expression vector is introduced into animal cells to express the humanized antibody as a high ADCC type antibody, and anti-CD33 having hIgG1 as CH. Humanized antibodies HV0LV0, HV7LV0, and HV14LV0 were generated.
  • Binding activity with hCD33-His in Biacore In order to analyze the binding activity of each anti-CD33 humanized antibody obtained in (2) above with respect to hCD33-His, the surface plasmon resonance method (SPR method) is used. The binding activity was measured by the same method as in Example 7- (3).
  • Example 12 Evaluation of activity of anti-CD33 humanized antibody
  • KM8084 which is a high ADCC type antibody of HV7LV0 of KM4084 produced in Example 11 and HM195-1, HM1955-P produced in Example 8 The activity was evaluated.
  • a negative control antibody a high ADCC type antibody (anti-DNP) against dinitrophenol (DNP) was used.
  • the antibody-free sample was shown as -Ab.
  • Example 7 (1) Evaluation of ADCC activity against human blood cancer cell line It was performed according to the method described in Example 7 (4).
  • target cells THP-1, NB-4, human erythroleukemia cell line TF-1, and human acute megakaryocytic leukemia cell line CMK11-5 were used.
  • the antibody concentration was diluted 4-fold stepwise from 0.5 ⁇ g / mL. The results are shown in FIG.
  • KM8084, HM195-1, and HM195-P exhibited an antibody concentration-dependent ADCC activity against each cell line. KM8084 showed stronger ADCC activity than HM195-P.
  • FIG. 13 [(A) and (B) of FIG. 13 show the amount of antibody binding and the antibody remaining rate, respectively].
  • KM8084 and HM195-P decreased the amount of binding on the cell surface in a time-dependent manner.
  • KM8084 exceeded both HM195-P and the amount of antibody bound to the cell surface and the residual rate at each time.
  • NB-4 and TF-1 were fluorescently labeled according to the instructions attached to PKH26 Red Fluorescent Cell Linker Kit (manufactured by Sigma) and diluted with RPMI 1640 (manufactured by Invitrogen).
  • Healthy human peripheral blood containing 1% of heparin sodium injection N (manufactured by Shimizu Pharmaceutical Co., Ltd.) in a 24-well plate (manufactured by NUNC) was dispensed at 500 ⁇ L / well.
  • 50 ⁇ L of each antibody diluted with RPMI1640 and 50 ⁇ L of the fluorescently labeled cell solution were added to make a total volume of 600 ⁇ L, and the reaction was carried out with shaking and stirring at 37 ° C. for 16 hours in a 5% CO 2 incubator.
  • the tube was washed twice with 1% BSA-PBS, suspended in 500 ⁇ L of 1% BSA-PBS, and the number of FB and the number of fluorescently labeled cells were measured using a flow cytometer (manufactured by Beckman Coulter). . From the obtained results, the number of fluorescently labeled cells per 1000 FBs was calculated.
  • HM195-P which is a high ADCC type antibody of the same antibody, showed about 10 times higher activity than HM195-1.
  • KM8084 exhibited a cell removal activity 100 times or more stronger than HM195-1, and several tens of times stronger than HM195-P.
  • This evaluation system in which leukemia cells are added to human peripheral blood is a system that models the environment in the blood of an acute myeloid leukemia (AML) patient, and is more compared to the ADCC evaluation system of Example 12 (1). It is thought to reflect clinical efficacy. For this reason, it is suggested that KM8084 exhibits an effect higher than that of HM195 for blood cancer such as acute myeloid leukemia and myelodysplastic syndrome accompanying bone marrow cell lesions.
  • Example 7 (4) Reactivity to human, cynomolgus monkey and chimpanzee CD33
  • the reaction was performed according to the method described in Example 7 (1).
  • the cells used were the human CD33-expressing cell line, cynomolgus monkey CD33-expressing cell line, and chimpanzee CD33-expressing cell line prepared in Example 1.
  • primary antibodies KM8084, HM195-1, and HM195-P were diluted serially.
  • a FITC-labeled goat anti-human IgG (Fc) antibody manufactured by Acris Antibodies was used as the secondary antibody.
  • FIG. 15 shows the MFI when KM8084, HM195-1, and HM195-P are reacted stepwise diluted from 50 ⁇ g / mL at 10-fold dilutions.
  • KM8084, HM195-1, and HM195-P all reacted with human CD33-expressing cell lines in a concentration-dependent manner, and there was no difference in the strength of reactivity.
  • KM8084 reacted in a concentration-dependent manner to cynomolgus monkey CD33-expressing cell line and chimpanzee CD33-expressing cell line, but HM195-1 and HM195-P hardly reacted.
  • SEQ ID NO: 1 nucleotide sequence of primer for preparing human CD33 expression vector
  • SEQ ID NO: 2 nucleotide sequence of primer for preparing human CD33 expression vector
  • SEQ ID NO: 3 amino acid sequence of human CD33
  • SEQ ID NO: 4 nucleotide sequence of human CD33 gene
  • SEQ ID NO: 5 Nucleotide sequence of primer for preparing hCD33-His expression vector
  • SEQ ID NO: 6 amino acid sequence of hCD33-His
  • SEQ ID NO: 7 nucleotide sequence of hCD33-his sequence
  • SEQ ID NO: 8 nucleotide sequence of rat IgG1-specific primer
  • SEQ ID NO: 9 rat Base sequence of IgG2a-specific primer
  • SEQ ID NO: 10 Base sequence of mouse IgG1-specific primer
  • SEQ ID NO: 11 Base sequence of mouse IgG2a-specific primer
  • SEQ ID NO: 12 Base sequence of rat Ig ( ⁇ )
  • VH amino acid sequence SEQ ID NO: 107 VH CDR2 amino acid modified antibody ver. 1.
  • Base sequence of primer for preparation (sense strand) SEQ ID NO: 108: VH CDR2 amino acid-modified antibody ver. 1
  • Base sequence of primer for preparation (antisense strand) SEQ ID NO: 109: VH CDR2 amino acid modified antibody ver. 2
  • Base sequence of primer for preparation (sense strand) SEQ ID NO: 110: VH CDR2 amino acid modified antibody ver.
  • Base sequence of primer for preparation (sense strand) SEQ ID NO: 112: VH CDR2 amino acid modified antibody ver.
  • Base sequence of primer for preparation (antisense strand) SEQ ID NO: 113: VH CDR2 amino acid modified antibody ver.
  • Base sequence of primer for preparation (sense strand) SEQ ID NO: 114: VH CDR2 amino acid modified antibody ver.

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Abstract

Provided are: a monoclonal antibody or a fragment thereof, said monoclonal antibody being capable of binding to the extracellular region of a human CD33 cell at a high affinity and exerting a high ADCC activity; a DNA encoding said antibody or fragment; a vector containing said DNA; a transformant obtained by transferring said vector; a method for producing the aforesaid antibody or fragment using said transformant; and a therapeutic agent and a diagnostic agent using the aforesaid antibody or fragment.

Description

抗ＣＤ３３抗体Anti-CD33 antibody
　本発明は、高いアフィニティーでヒトＣＤ３３細胞外領域に結合し、かつ高い抗体依存的細胞傷害活性（ａｎｔｉｂｏｄｙ－ｄｅｐｅｎｄｅｎｔ　ｃｅｌｌｕｌａｒ　ｃｙｔｏｔｏｘｉｃｉｔｙ、以下ＡＤＣＣ活性と表記することがある）を発揮するモノクローナル抗体またはその断片、該抗体を産生するハイブリドーマ、該抗体をコードするＤＮＡ、該ＤＮＡを含むベクター、該ベクターを導入して得られる形質転換体、該ハイブリドーマまたは該形質転換体を用いる抗体または該断片の製造方法、抗体または該断片を用いる治療剤および診断剤に関する。 The present invention relates to a monoclonal antibody or fragment thereof that binds to the human CD33 extracellular region with high affinity and exhibits high antibody-dependent cellular cytotoxicity (hereinafter sometimes referred to as ADCC activity). Hybridoma producing the antibody, DNA encoding the antibody, a vector containing the DNA, a transformant obtained by introducing the vector, a method for producing an antibody or fragment using the hybridoma or the transformant, antibody Alternatively, the present invention relates to a therapeutic agent and a diagnostic agent using the fragment.
　Ｃｌｕｓｔｅｒ　ｏｆ　ｄｉｆｆｅｒｅｎｔｉａｔｉｏｎ（以下、ＣＤと表記することがある）３３は、骨髄系前駆細胞、単球および顆粒球に発現する、分子量約６．７万の糖タンパク質である。ＣＤ３３はＩ型膜貫通タンパク質であり、細胞外に２つのイムノグロブリンスーパーファミリードメイン（Ｎ末より細胞膜側に向かって、順にＩｇＶ、ＩｇＣ２）が存在し、各ドメインに存在するそれぞれ３つのシステイン残基がドメイン内およびドメイン間ジスルフィド結合を形成している。ＣＤ３３細胞外領域には合計５箇所のＮ結合型糖鎖付加配列が存在する。ＣＤ３３はＩｇＶ領域を介してシアル酸と結合することが知られ、別名シグレック―３、シアロアドヘジンとも呼ばれるが、その機能の詳細については不明である（非特許文献１）。 Cluster of differentiation (hereinafter sometimes referred to as CD) 33 is a glycoprotein having a molecular weight of about 67,000 expressed in myeloid progenitor cells, monocytes and granulocytes. CD33 is a type I transmembrane protein, and there are two immunoglobulin superfamily domains (IgV and IgC2 in order from the N-terminal toward the cell membrane) outside the cell. Three cysteine residues exist in each domain. Form intradomain and interdomain disulfide bonds. There are a total of five N-linked glycosylation sequences in the CD33 extracellular region. CD33 is known to bind to sialic acid via the IgV region, and is also called as Siglec-3 or sialoadhesin, but the details of its function are unknown (Non-patent Document 1).
　ＣＤ３３は、別名ｇｐ６７、シアロアドヘジン、シグレック－３としても知られ、１９８８年に遺伝子がクローニングされ（非特許文献２）、ＣＤ３３のＤＮＡ配列、アミノ酸配列および立体構造は、公的データベース上に公開されており、例えば、Ｐ２０１３８（ＳＷＩＳＳＰＲＯＴ）、Ｍ２３１９７（ＥＭＢＬ）等のアクセッション番号から参照可能である。 CD33, also known as gp67, sialoadhesin, siglec-3, was cloned in 1988 (Non-Patent Document 2), and the DNA sequence, amino acid sequence and three-dimensional structure of CD33 have been published on public databases. For example, it can be referred to from an accession number such as P20138 (SWISSPROT) or M23197 (EMBL).
　ＣＤ３３に結合するモノクローナル抗体（以下、ｍＡｂと表記することがある）として、初めて確立されたＬ４Ｆ３、Ｌ１Ｂ２は１９８３年に報告されている（非特許文献３）。以後、ＣＤ３３に対する多くのモノクローナル抗体（以下、抗ＣＤ３３ｍＡｂと表記することがある）が報告されている。報告されている抗ＣＤ３３ｍＡｂの多くは、同じまたは隣接した領域を認識することが知られている（非特許文献１）。 L4F3 and L1B2 established for the first time as monoclonal antibodies that bind to CD33 (hereinafter sometimes referred to as mAb) were reported in 1983 (Non-patent Document 3). Since then, many monoclonal antibodies against CD33 (hereinafter sometimes referred to as anti-CD33 mAb) have been reported. Many of the reported anti-CD33 mAbs are known to recognize the same or adjacent regions (Non-patent Document 1).
　ＣＤ３３は大部分の急性骨髄性白血病（ａｃｕｔｅ　ｍｙｅｌｏｇｅｎｏｕｓ　ｌｅｕｋｅｍｉａ、以下ＡＭＬと表記することがある）細胞において正常細胞に比べて高い発現が認められ、一方、造血幹細胞においては発現していないことから、抗ＣＤ３３ｍＡｂはＡＭＬ治療薬を目指し臨床応用が進められている。例えば、抗ＣＤ３３ｍＡｂであるＰ６７．６とカリケアマイシン誘導体の結合物であるゲムツズマブオゾガマイシンはＡＭＬ治療薬として臨床において使用されている。 CD33 is highly expressed in most acute myelogenous leukemia (acute myelogenous leukemia, hereinafter sometimes referred to as AML) cells compared to normal cells, whereas it is not expressed in hematopoietic stem cells. CD33 mAb is being clinically applied with the aim of treating AML. For example, gemtuzumab ozogamicin, a conjugate of P67.6, an anti-CD33 mAb, and a calicheamicin derivative has been used clinically as an AML therapeutic.
　癌治療薬として開発されている抗ＣＤ３３ｍＡｂには、例えば、ｇｅｍｔｕｚｕｍａｂ　ｏｚｏｇａｍｉｃｉｎ（Ｐ６７．６）（非特許文献４）、ｌｉｎｔｕｚｕｍａｂ（ＨｕＭ１９５）（非特許文献５）などがある。これらの抗体は、標的細胞であるＣＤ３３発現細胞を特異的に傷害することにより薬効を発揮する抗体である。ｇｅｍｔｕｚｕｍａｂ　ｏｚｏｇａｍｉｃｉｎは細胞表面上のＣＤ３３抗原に結合した後、細胞内に取り込まれる性質を持ち、この性質を利用してカリケアマイシン誘導体を細胞内に送り込み、細胞内において細胞傷害活性を持つ。ｌｉｎｔｕｚｕｍａｂの薬効メカニズムはＡＤＣＣ活性が主であると考えられる（非特許文献６）。 Examples of anti-CD33 mAb developed as a cancer therapeutic agent include gemtumumab ozogamicin (P67.6) (Non-patent Document 4), lintuzumab (HuM195) (Non-patent Document 5), and the like. These antibodies are antibodies that exert their medicinal effects by specifically damaging CD33-expressing cells that are target cells. Gemtumumab ozogamicin has the property of being bound into the cell after binding to the CD33 antigen on the cell surface. Utilizing this property, calicheamicin derivatives are sent into the cell and have cytotoxic activity in the cell. It is considered that ADCC activity is the main medicinal mechanism of lintuzumab (Non-patent Document 6).
　ＡＤＣＣ活性とは標的細胞上の抗原に結合した抗体がそのＦｃ領域により免疫細胞のＦｃ受容体と結合することで免疫細胞（ナチュラルキラー細胞など）を活性化し、標的細胞を傷害する活性である。一方、これらの抗体の補体依存性傷害活性（以下、ＣＤＣ活性と表記することがある）は、抗原発現量が高い場合にのみ発現する（非特許文献６）。 The ADCC activity is an activity that activates immune cells (such as natural killer cells) by damaging the target cells by binding an antibody bound to an antigen on the target cells to the Fc receptor of immune cells through its Fc region. On the other hand, the complement-dependent injury activity of these antibodies (hereinafter sometimes referred to as CDC activity) is expressed only when the antigen expression level is high (Non-patent Document 6).
　ＣＤＣ活性とは標的細胞上の抗原に結合した抗体が血液中の補体関連タンパク質群からなる一連のカスケード（補体活性化経路）を活性化し、標的細胞を傷害する活性である。また、補体の活性化により生じるタンパク質断片により免疫細胞の遊走および活性化を誘導することができる。 CDC activity is an activity in which an antibody bound to an antigen on a target cell activates a series of cascades (complement activation pathways) composed of complement-related proteins in the blood and damages the target cell. In addition, migration and activation of immune cells can be induced by protein fragments generated by complement activation.
　通常ＩｇＧ型のヒト抗体には、その定常領域に１箇所のＮ結合型糖鎖が結合するコンセンサス配列を有している。しかし、可変領域中にもＮ結合型糖鎖が結合するコンセンサス配列を有している抗体は、糖鎖の結合が変化し、医薬品として均一な抗体の安定供給が困難となる。さらには、蛋白質同士の結合に必須である場合もあり、例えば、ＬＦＡ－３（ｌｙｍｐｈｏｃｙｔｅ　ｆｕｎｃｔｉｏｎ－ａｓｓｏｃｉａｔｅｄ　ａｎｔｉｇｅｎ　３）ではＣＤ２との結合にＮ結合型糖鎖が必要であると報告されており、抗体の結合部位である可変領域に糖鎖が結合することにより、抗体の抗原への結合性が変化してしまう可能性が考えられる（非特許文献７）。 Usually, a human antibody of IgG type has a consensus sequence in which one N-linked sugar chain binds to its constant region. However, an antibody having a consensus sequence to which an N-linked sugar chain binds also in the variable region changes the sugar chain binding, making it difficult to stably supply a uniform antibody as a pharmaceutical product. Furthermore, in some cases, it is essential for the binding between proteins. For example, LFA-3 (lymphocyte function-associated antigen 3) has been reported to require an N-linked sugar chain for binding to CD2, and antibody There is a possibility that the binding property of the antibody to the antigen is changed by binding of the sugar chain to the variable region which is the binding site of (Non-patent Document 7).
　抗体とＦｃ受容体との結合においては、抗体のヒンジ領域及び定常領域の２番目のドメインに結合している糖鎖の重要性が示唆されている（非特許文献８）。抗体分子のＦｃ領域に結合しているＮ－グリコシド結合複合型糖鎖の非還元末端へのガラクトースの付加、および還元末端のＮ－アセチルグルコサミンへのフコースの付加に関しては多様性があることが知られており（非特許文献９）、特に糖鎖の還元末端のＮ－アセチルグルコサミンにフコースが結合していない抗体は、高いＡＤＣＣ活性を示すことが報告されている（非特許文献１０、特許文献１）。 In the binding between an antibody and an Fc receptor, the importance of a sugar chain bound to the second domain of the hinge region and constant region of the antibody has been suggested (Non-patent Document 8). It is known that galactose is added to the non-reducing end of the N-glycoside-linked complex sugar chain bound to the Fc region of the antibody molecule and fucose is added to N-acetylglucosamine at the reducing end. In particular, an antibody in which fucose is not bound to N-acetylglucosamine at the reducing end of a sugar chain has been reported to exhibit high ADCC activity (Non-patent Document 10 and Patent Document 9). 1).
　一般に医薬品として利用される抗体の多くは、遺伝子組換え技術を用いて作製され、動物細胞、例えば、チャイニーズ・ハムスター卵巣組織由来のＣＨＯ細胞などを宿主細胞として製造されているが、発現させた抗体の糖鎖構造は宿主細胞によって異なる。 Many of the antibodies generally used as pharmaceuticals are produced using gene recombination techniques, and are produced using animal cells such as CHO cells derived from Chinese hamster ovary tissue as host cells. The sugar chain structure varies depending on the host cell.
　抗体生産細胞内のα１，６－フコシルトランスフェラーゼ、ＧＤＰ－マンノース４，６－デヒドラターゼまたはＧＤＰ－４－ケト－６－デオキシ－Ｄ－マンノース－３，５－エピメラーゼの活性を低下または欠失することにより、抗体分子のＦｃ領域に結合するＮ－グリコシド結合複合型糖鎖の還元末端のＮ－アセチルグルコサミンにフコースが結合していない糖鎖の割合を増加させることが出来る（特許文献２）。 By reducing or eliminating the activity of α1,6-fucosyltransferase, GDP-mannose 4,6-dehydratase or GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase in antibody-producing cells It is possible to increase the proportion of sugar chains in which fucose is not bound to N-acetylglucosamine at the reducing end of the N-glycoside-linked complex-type sugar chain that binds to the Fc region of the antibody molecule (Patent Document 2).
　抗体のサブクラスにより、ＣＤＣ活性が異なることが知られている。ＣＤＣ活性はヒトＩｇＧ１およびＩｇＧ３において高く、その強さは一般的にＩｇＧ３≧ＩｇＧ１＞＞ＩｇＧ２≒ＩｇＧ４の序列となる。さらに、ヒトＩｇＧ１の定常領域のＣＨ２ドメインをＩｇＧ３のＣＨ２ドメインと交換することによりＣＤＣ活性がＩｇＧ３よりもさらに高まることが出来ることが知られている（特許文献３）。 It is known that CDC activity varies depending on the antibody subclass. CDC activity is high in human IgG1 and IgG3, and its strength is generally in the order of IgG3 ≧ IgG1 >> IgG2≈IgG4. Furthermore, it is known that the CDC activity can be further increased as compared with IgG3 by exchanging the CH2 domain of the constant region of human IgG1 with the CH2 domain of IgG3 (Patent Document 3).
国際公開第００／６１７３９号International Publication No. 00/61739 国際公開第０２／３１１４０号International Publication No. 02/31140 米国特許出願公開第２００７／０１４８１６５号明細書US Patent Application Publication No. 2007/0148165
　本発明の課題は、高いアフィニティーでヒトＣＤ３３細胞外領域に結合し、かつ高いＡＤＣＣ活性を発揮するモノクローナル抗体またはその断片、該抗体または該断片をコードするＤＮＡ、該ＤＮＡを含むベクター、該ベクターを導入して得られる形質転換体、該形質転換体を用いる該抗体または該断片の製造方法、並びに該抗体または該断片を含む治療剤および診断剤を提供することにある。 An object of the present invention is to provide a monoclonal antibody or fragment thereof that binds to the human CD33 extracellular region with high affinity and exhibits high ADCC activity, DNA encoding the antibody or the fragment, a vector containing the DNA, and a vector comprising the vector It is to provide a transformant obtained by introduction, a method for producing the antibody or the fragment using the transformant, and a therapeutic agent and a diagnostic agent containing the antibody or the fragment.
　本発明者らは、高いアフィニティーでヒトＣＤ３３細胞外領域に結合するマウスおよびラットモノクローナル抗体を取得し、該抗体の重鎖可変領域（以下、ＶＨと表記することがある）の相補性決定領域（Ｃｏｍｐｌｅｍｅｎｔａｒｉｔｙ　Ｄｅｔｅｒｍｉｎｉｎｇ　Ｒｅｇｉｏｎ、以下、ＣＤＲと表記することがある）１～３および軽鎖可変領域（以下、ＶＬと表記することがある）のＣＤＲ１～３を有する抗ＣＤ３３ヒト化抗体、ならびに該ＣＤＲの一部のアミノ酸残基を適宜置換した抗ＣＤ３３ヒト化抗体を作製し、本発明を完成するに至った。 The present inventors obtained mouse and rat monoclonal antibodies that bind to the human CD33 extracellular region with high affinity, and the complementarity determining region (hereinafter sometimes referred to as VH) of the antibody heavy chain variable region (hereinafter sometimes referred to as VH). Complementarity Determining Region (hereinafter, sometimes referred to as CDR) 1-3 and anti-CD33 humanized antibody having CDR 1-3 of light chain variable region (hereinafter sometimes referred to as VL), and one of the CDRs An anti-CD33 humanized antibody in which amino acid residues at certain parts are appropriately substituted was prepared, and the present invention was completed.
　すなわち、本発明は以下のとおりである。
１．（ｉ）配列番号５２で示されるアミノ酸配列を含むＣＤＲ１、配列番号９９で示されるアミノ酸配列を含むＣＤＲ２、および配列番号５４で示されるアミノ酸配列を含むＣＤＲ３を含む重鎖可変領域、並びに配列番号５５で示されるアミノ酸配列を含むＣＤＲ１、配列番号５６で示されるアミノ酸配列を含むＣＤＲ２、および配列番号５７で示されるアミノ酸配列を含むＣＤＲ３を含む軽鎖可変領域を含む、ヒトＣＤ３３に結合するモノクローナル抗体または該抗体の断片、
（ｉｉ）配列番号５２で示されるアミノ酸配列を含むＣＤＲ１、配列番号１００で示されるアミノ酸配列を含むＣＤＲ２、および配列番号５４で示されるアミノ酸配列を含むＣＤＲ３を含む重鎖可変領域、並びに配列番号５５で示されるアミノ酸配列を含むＣＤＲ１、配列番号５６で示されるアミノ酸配列を含むＣＤＲ２、および配列番号５７で示されるアミノ酸配列を含むＣＤＲ３を含む軽鎖可変領域を含む、ヒトＣＤ３３に結合するモノクローナル抗体または該抗体の断片、
（ｉｉｉ）配列番号５２で示されるアミノ酸配列を含むＣＤＲ１、配列番号１０１で示されるアミノ酸配列を含むＣＤＲ２、および配列番号５４で示されるアミノ酸配列を含むＣＤＲ３を含む重鎖可変領域、並びに配列番号５５で示されるアミノ酸配列を含むＣＤＲ１、配列番号５６で示されるアミノ酸配列を含むＣＤＲ２、および配列番号５７で示されるアミノ酸配列を含むＣＤＲ３を含む軽鎖可変領域を含む、ヒトＣＤ３３に結合するモノクローナル抗体または該抗体の断片、または
（ｉｖ）配列番号５２で示されるアミノ酸配列を含むＣＤＲ１、配列番号１０２で示されるアミノ酸配列を含むＣＤＲ２、および配列番号５４で示されるアミノ酸配列を含むＣＤＲ３を含む重鎖可変領域、並びに配列番号５５で示されるアミノ酸配列を含むＣＤＲ１、配列番号５６で示されるアミノ酸配列を含むＣＤＲ２、および配列番号５７で示されるアミノ酸配列を含むＣＤＲ３を含む軽鎖可変領域を含む、ヒトＣＤ３３に結合するモノクローナル抗体または該抗体の断片。
２．配列番号５２で示されるアミノ酸配列を含むＣＤＲ１、配列番号９６で示されるアミノ酸配列を含むＣＤＲ２、および配列番号５４で示されるアミノ酸配列を含むＣＤＲ３を含む重鎖可変領域、並びに配列番号５５で示されるアミノ酸配列を含むＣＤＲ１、配列番号５６で示されるアミノ酸配列を含むＣＤＲ２、および配列番号５７で示されるアミノ酸配列を含むＣＤＲ３を有する軽鎖可変領域を含む、ヒトＣＤ３３に結合するモノクローナル抗体または該抗体の断片。
３．配列番号８６で示されるアミノ酸配列を含む重鎖可変領域および配列番号８５で示されるアミノ酸配列を含む軽鎖可変領域を含む、前項２に記載のモノクローナル抗体または該抗体の断片。
４．配列番号８７で示されるアミノ酸配列を含む重鎖可変領域および配列番号８５で示されるアミノ酸配列を含む軽鎖可変領域を含む、前項２に記載のモノクローナル抗体または該抗体の断片。
５．（ｉ）配列番号３４で示されるアミノ酸配列を含むＣＤＲ１、配列番号３５で示されるアミノ酸配列を含むＣＤＲ２、および配列番号３６で示されるアミノ酸配列を含むＣＤＲ３を含む重鎖可変領域、並びに配列番号３７で示されるアミノ酸配列を含むＣＤＲ１、配列番号３８で示されるアミノ酸配列を含むＣＤＲ２、および配列番号３９で示されるアミノ酸配列を含むＣＤＲ３を含む軽鎖可変領域を含む、ヒトＣＤ３３に結合するモノクローナル抗体または該抗体の断片、
（ｉｉ）配列番号４０で示されるアミノ酸配列を含むＣＤＲ１、配列番号４１で示されるアミノ酸配列を含むＣＤＲ２、および配列番号４２で示されるアミノ酸配列を含むＣＤＲ３を含む重鎖可変領域、並びに配列番号４３で示されるアミノ酸配列を含むＣＤＲ１、配列番号４４で示されるアミノ酸配列を含むＣＤＲ２、および配列番号４５で示されるアミノ酸配列を含むＣＤＲ３を含む軽鎖可変領域を含む、ヒトＣＤ３３に結合するモノクローナル抗体または該抗体の断片、
（ｉｉｉ）配列番号４６で示されるアミノ酸配列を含むＣＤＲ１、配列番号４７で示されるアミノ酸配列を含むＣＤＲ２、および配列番号４８で示されるアミノ酸配列を含むＣＤＲ３を含む重鎖可変領域、並びに配列番号４９示されるアミノ酸配列を含むＣＤＲ１、配列番号５０で示されるアミノ酸配列を含むＣＤＲ２、および配列番号５１で示されるアミノ酸配列を含むＣＤＲ３を含む軽鎖可変領域を含む、ヒトＣＤ３３に結合するモノクローナル抗体または該抗体の断片、
（ｉｖ）配列番号５２で示されるアミノ酸配列を含むＣＤＲ１、配列番号５３で示されるアミノ酸配列を含むＣＤＲ２、および配列番号５４で示されるアミノ酸配列を含むＣＤＲ３を含む重鎖可変領域、並びに配列番号５５示されるアミノ酸配列を含むＣＤＲ１、配列番号５６で示されるアミノ酸配列を含むＣＤＲ２、および配列番号５７で示されるアミノ酸配列を含むＣＤＲ３を含む軽鎖可変領域を含む、ヒトＣＤ３３に結合するモノクローナル抗体または該抗体の断片、または
（ｖ）配列番号５８で示されるアミノ酸配列を含むＣＤＲ１、配列番号５９で示されるアミノ酸配列を含むＣＤＲ２、および配列番号６０で示されるアミノ酸配列を含むＣＤＲ３を含む重鎖可変領域、並びに配列番号６１示されるアミノ酸配列を含むＣＤＲ１、配列番号６２で示されるアミノ酸配列を含むＣＤＲ２、および配列番号６３で示されるアミノ酸配列を含むＣＤＲ３を含む軽鎖可変領域を含む、ヒトＣＤ３３に結合するモノクローナル抗体または該抗体の断片。
６．（ｉ）配列番号１３２で示されるアミノ酸配列を含む重鎖可変領域および配列番号１３７で示されるアミノ酸配列を含む軽鎖可変領域を含むヒトＣＤ３３に結合するモノクローナル抗体、
（ｉｉ）配列番号１３３で示されるアミノ酸配列を含む重鎖可変領域および配列番号１３８で示されるアミノ酸配列を含む軽鎖可変領域を含むヒトＣＤ３３に結合するモノクローナル抗体、
（ｉｉｉ）配列番号１３４で示されるアミノ酸配列を含む重鎖可変領域および配列番号１３９で示されるアミノ酸配列を含む軽鎖可変領域を含むヒトＣＤ３３に結合するモノクローナル抗体、
（ｉｖ）配列番号１３５で示されるアミノ酸配列を含む重鎖可変領域および配列番号１４０で示されるアミノ酸配列を含む軽鎖可変領域を含むヒトＣＤ３３に結合するモノクローナル抗体、または
（ｖ）配列番号１３６で示されるアミノ酸配列を含む重鎖可変領域および配列番号１４１で示されるアミノ酸配列を含む軽鎖可変領域を含むヒトＣＤ３３に結合するモノクローナル抗体が結合するヒトＣＤ３３上のエピトープに結合する、ヒトＣＤ３３に結合するモノクローナル抗体または該抗体の断片。
７．（ｉ）配列番号１３２で示されるアミノ酸配列を含む重鎖可変領域および配列番号１３７で示されるアミノ酸配列を含む軽鎖可変領域を含むヒトＣＤ３３に結合するモノクローナル抗体、
（ｉｉ）配列番号１３３で示されるアミノ酸配列を含む重鎖可変領域および配列番号１３８で示されるアミノ酸配列を含む軽鎖可変領域を含むヒトＣＤ３３に結合するモノクローナル抗体、
（ｉｉｉ）配列番号１３４で示されるアミノ酸配列を含む重鎖可変領域および配列番号１３９で示されるアミノ酸配列を含む軽鎖可変領域を含むヒトＣＤ３３に結合するモノクローナル抗体、
（ｉｖ）配列番号１３５で示されるアミノ酸配列を含む重鎖可変領域および配列番号１４０で示されるアミノ酸配列を含む軽鎖可変領域を含むヒトＣＤ３３に結合するモノクローナル抗体、または
（ｖ）配列番号１３６で示されるアミノ酸配列を含む重鎖可変領域および配列番号１４１で示されるアミノ酸配列を含む軽鎖可変領域を含むヒトＣＤ３３に結合するモノクローナル抗体と競合してヒトＣＤ３３に結合するモノクローナル抗または該抗体の断片。
８．ヒトＣＤ３３に対する解離定数ＫＤが４．０×１０－９Ｍ以下である、前項１から７のいずれか１に記載のモノクローナル抗体または該抗体の断片。
９．ヒトＣＤ３３に対する解離定数ＫＤが２．０×１０－９Ｍ以下である、前項８に記載のモノクローナル抗体または該抗体の断片。
１０．重鎖定常領域を含む前項１から９のいずれか１に記載のモノクローナル抗体または該抗体の断片であって、重鎖定常領域のクラスがヒトＩｇＧであるモノクローナル抗体または該抗体の断片。
１１．ヒトＩｇＧのサブクラスがヒトＩｇＧ１である、前項１０に記載のモノクローナル抗体または該抗体の断片。
１２．重鎖定常領域を含む前項１から１１のいずれか１に記載のモノクローナル抗体または該抗体の断片であって、重鎖定常領域のＦｃに結合しているＮ結合複合型糖鎖にフコースが結合していないモノクローナル抗体または該抗体の断片。
１３．軽鎖定常領域を含む前項１から１２のいずれか１に記載のモノクローナル抗体または該抗体の断片であって、軽鎖定常領域のクラスがヒトκであるモノクローナル抗体または該抗体の断片。
１４．Ｆａｂ、Ｆ（ａｂ’）２、Ｆａｂ’、ｓｃＦｖ、ｄｉａｂｏｄｙ、ｄｓＦｖまたはＣＤＲを含むペプチドである、前項１から１３のいずれか１に記載の抗体の断片。
１５．前項１から１４のいずれか１に記載のモノクローナル抗体または該抗体の断片をコードするＤＮＡ。
１６．前項１５に記載のＤＮＡを含有する組換えベクター。
１７．（ｉ）配列番号８９で示されるアミノ酸配列を含む軽鎖可変領域をコードするＤＮＡを含む組換えベクター、
（ｉｉ）配列番号９０で示されるアミノ酸配列を含む重鎖可変領域をコードするＤＮＡを含む組換えベクター、
（ｉｉｉ）配列番号９１で示されるアミノ酸配列を含む重鎖可変領域をコードするＤＮＡを含む組換えベクター、
（ｉｖ）配列番号９０で示されるアミノ酸配列を含む重鎖可変領域をコードするＤＮＡおよび配列番号８９で示されるアミノ酸配列を含む軽鎖可変領域をコードするＤＮＡを含む組換えベクター、
または
（ｖ）配列番号９１で示されるアミノ酸配列を含む重鎖可変領域をコードするＤＮＡおよび配列番号８９で示されるアミノ酸配列を含む軽鎖可変領域をコードするＤＮＡを含む組換えベクター。
１８．前項１６および１７に記載の組換えベクターから選ばれる少なくとも１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体。
１９．以下の（ｉ）または（ｉｉ）の組換えベクターを宿主細胞に導入して得られる前項１８に記載の形質転換体。
（ｉ）配列番号８９で示されるアミノ酸配列を含む軽鎖可変領域をコードするＤＮＡを含む組換えベクターおよび配列番号９０で示されるアミノ酸配列を含む重鎖可変領域をコードするＤＮＡを含む組換えベクター
（ｉｉ）配列番号８９で示されるアミノ酸配列を含む軽鎖可変領域をコードするＤＮＡを含む組換えベクターおよび配列番号９１で示されるアミノ酸配列を含む重鎖可変領域をコードするＤＮＡを含む組換えベクター
２０．前項１８または１９に記載の形質転換体を培地に培養し、培養物中に前項１から１４のいずれか１に記載のモノクローナル抗体または該抗体の断片を生成蓄積させ、該培養物から該モノクローナル抗体または該抗体の断片を採取することを特徴とする、前項１から１４のいずれか１に記載のモノクローナル抗体または該抗体の断片の製造方法。
２１．前項１から１４のいずれか１に記載のモノクローナル抗体または該抗体の断片を有効成分として含有する医薬組成物。
２２．前項１から１４のいずれか１に記載のモノクローナル抗体または該抗体の断片を有効成分として含有する、ＣＤ３３陽性細胞が関与する疾患の治療剤。
２３．ＣＤ３３陽性細胞が関与する疾患が、癌、自己免疫疾患またはアレルギー性疾患である、前項２２に記載の治療剤。
２４．癌が、血液癌、乳癌、子宮癌、大腸癌、食道癌、胃癌、卵巣癌、肺癌、腎臓癌、直腸癌、甲状腺癌、子宮頸癌、小腸癌、前立腺癌または膵臓癌である、前項２３に記載の治療剤。
２５．血液癌が、急性骨髄性白血病、未分化大細胞型リンパ腫、急性リンパ性白血病、骨髄異形成症候群、多発性骨髄腫、ホジキンリンパ腫、または非ホジキンリンパ腫である、前項２４に記載の治療剤。
２６．前項１から１４のいずれか１に記載のモノクローナル抗体または該抗体の断片を哺乳動物に投与する工程を含む、ＣＤ３３陽性細胞が関与する疾患を治療する方法。
２７．ＣＤ３３陽性細胞が関与する疾患が、癌、自己免疫疾患またはアレルギー性疾患である、前項２６に記載の方法。
２８．癌が、血液癌、乳癌、子宮癌、大腸癌、食道癌、胃癌、卵巣癌、肺癌、腎臓癌、直腸癌、甲状腺癌、子宮頸癌、小腸癌、前立腺癌または膵臓癌である、前項２７に記載の方法。
２９．血液癌が、急性骨髄性白血病、未分化大細胞型リンパ腫、急性リンパ性白血病、骨髄異形成症候群、多発性骨髄腫、ホジキンリンパ腫、または非ホジキンリンパ腫である、前項２８に記載の方法。
３０．ＣＤ３３陽性細胞が関与する疾患を治療するための、前項１から１４のいずれか１に記載のモノクローナル抗体または該抗体の断片の使用。
３１．ＣＤ３３陽性細胞が関与する疾患が、癌、自己免疫疾患またはアレルギー性疾患である、前項３０に記載の使用。
３２．癌が、血液癌、乳癌、子宮癌、大腸癌、食道癌、胃癌、卵巣癌、肺癌、腎臓癌、直腸癌、甲状腺癌、子宮頸癌、小腸癌、前立腺癌または膵臓癌である、前項３１に記載の使用。
３３．血液癌が、急性骨髄性白血病、未分化大細胞型リンパ腫、急性リンパ性白血病、骨髄異形成症候群、多発性骨髄腫、ホジキンリンパ腫または非ホジキンリンパ腫である、前項３２に記載の使用。 That is, the present invention is as follows.
1. (I) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 52, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 99, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 54, and SEQ ID NO: 55 A monoclonal antibody that binds to human CD33, comprising a light chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 56, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 56, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 57, or A fragment of the antibody,
(Ii) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 52, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 100, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 54, and SEQ ID NO: 55 A monoclonal antibody that binds to human CD33, comprising a light chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 56, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 56, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 57, or A fragment of the antibody,
(Iii) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 52, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 101, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 54, and SEQ ID NO: 55 A monoclonal antibody that binds to human CD33, comprising a light chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 56, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 56, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 57, or A heavy chain variable comprising a fragment of said antibody, or (iv) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 52, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 102, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 54 Region and the amino acid sequence shown in SEQ ID NO: 55. CDR1, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 56 and a light chain variable region comprising a CDR3 comprising an amino acid sequence shown in SEQ ID NO: 57, a fragment of a monoclonal antibody or the antibody that binds human CD33.
2. A heavy chain variable region comprising CDR1, comprising the amino acid sequence represented by SEQ ID NO: 52, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 96, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 54, and represented by SEQ ID NO: 55 A monoclonal antibody that binds to human CD33, comprising a CDR1 comprising an amino acid sequence, a CDR2 comprising an amino acid sequence represented by SEQ ID NO: 56, and a light chain variable region having a CDR3 comprising an amino acid sequence represented by SEQ ID NO: 57 fragment.
3. The monoclonal antibody or the fragment of the antibody according to item 2, comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 86 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 85.
4). The monoclonal antibody or fragment of the antibody according to item 2, comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 87 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 85.
5. (I) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36, and SEQ ID NO: 37 A monoclonal antibody that binds to human CD33, comprising a light chain variable region comprising CDR1, comprising the amino acid sequence represented by SEQ ID NO: 38, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 38, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 39; A fragment of the antibody,
(Ii) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 40, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 41, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 42, and SEQ ID NO: 43 A monoclonal antibody that binds to human CD33, comprising a light chain variable region comprising CDR1, comprising the amino acid sequence represented by SEQ ID NO: 44, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 44, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 45, or A fragment of the antibody,
(Iii) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 46, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 47, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 48, and SEQ ID NO: 49 A monoclonal antibody that binds to human CD33, comprising a CDR1 comprising the amino acid sequence shown, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 50, and a light chain variable region comprising CDR3 comprising the amino acid sequence represented by SEQ ID NO: 51, or Antibody fragments,
(Iv) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 52, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 53, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 54, and SEQ ID NO: 55 A monoclonal antibody that binds to human CD33 comprising a CDR1 comprising the amino acid sequence shown, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 56, and a light chain variable region comprising CDR3 comprising the amino acid sequence represented by SEQ ID NO: 57 or A heavy chain variable region comprising a fragment of an antibody, or (v) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 58, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 59, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 60 As well as a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 61, CDR2 comprising an amino acid sequence represented by sequence number 62, and comprises a light chain variable region comprising a CDR3 comprising an amino acid sequence represented by SEQ ID NO: 63, a fragment of a monoclonal antibody or the antibody that binds human CD33.
6). (I) a monoclonal antibody that binds to human CD33 comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 132 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 137;
(Ii) a monoclonal antibody that binds to human CD33 comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 133 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 138;
(Iii) a monoclonal antibody that binds to human CD33 comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 134 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 139,
(Iv) a monoclonal antibody that binds to human CD33 comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 135 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 140, or (v) in SEQ ID NO: 136 Binds to human CD33, which binds to an epitope on human CD33 to which a monoclonal antibody that binds to human CD33, which comprises a heavy chain variable region comprising the amino acid sequence shown and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 141 A monoclonal antibody or a fragment of said antibody.
7. (I) a monoclonal antibody that binds to human CD33 comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 132 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 137;
(Ii) a monoclonal antibody that binds to human CD33 comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 133 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 138;
(Iii) a monoclonal antibody that binds to human CD33 comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 134 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 139,
(Iv) a monoclonal antibody that binds to human CD33 comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 135 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 140, or (v) in SEQ ID NO: 136 Monoclonal anti or a fragment of said antibody that binds to human CD33 in competition with a monoclonal antibody that binds to human CD33 comprising a heavy chain variable region comprising the amino acid sequence shown and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 141 .
8). Dissociation constant K D for human CD33 is 4.0 × 10 -9 M or less, a monoclonal antibody or fragment of the antibody according to any one of the preceding 1 7.
9. Dissociation constant K D for human CD33 is 2.0 × 10 -9 M or less, a monoclonal antibody or fragment of the antibody according to [8.
10. 10. The monoclonal antibody or the antibody fragment according to any one of 1 to 9 above, which comprises a heavy chain constant region, wherein the heavy chain constant region class is human IgG or a fragment of the antibody.
11. 11. The monoclonal antibody or fragment of the antibody described in 10 above, wherein the subclass of human IgG is human IgG1.
12 12. The monoclonal antibody according to any one of 1 to 11 above or a fragment of the antibody comprising a heavy chain constant region, wherein fucose is bound to an N-linked complex type sugar chain that is bound to Fc of the heavy chain constant region. Not a monoclonal antibody or a fragment of said antibody.
13. 13. The monoclonal antibody or the antibody fragment according to any one of 1 to 12 above, which comprises a light chain constant region, wherein the light chain constant region class is human κ.
14 14. The antibody fragment according to any one of 1 to 13 above, which is a peptide comprising Fab, F (ab ′) 2 , Fab ′, scFv, diabody, dsFv, or CDR.
15. 15. A DNA encoding the monoclonal antibody or the antibody fragment according to any one of 1 to 14 above.
16. 16. A recombinant vector containing the DNA according to item 15 above.
17. (I) a recombinant vector comprising DNA encoding a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 89;
(Ii) a recombinant vector comprising DNA encoding a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 90;
(Iii) a recombinant vector comprising DNA encoding a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 91;
(Iv) a recombinant vector comprising DNA encoding a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 90 and DNA encoding a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 89;
Or (v) a recombinant vector comprising DNA encoding a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 91 and DNA encoding a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 89.
18. 18. A transformant obtained by introducing at least one recombinant vector selected from the recombinant vectors described in 16 and 17 above into a host cell.
19. 19. The transformant according to 18 above, which is obtained by introducing the following recombinant vector (i) or (ii) into a host cell.
(I) a recombinant vector comprising DNA encoding a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 89 and a recombinant vector comprising DNA encoding a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 90 (Ii) a recombinant vector comprising a DNA encoding a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 89 and a recombinant vector comprising a DNA encoding a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 91 20. The transformant according to the item 18 or 19 is cultured in a medium, and the monoclonal antibody or the fragment of the antibody according to any one of the items 1 to 14 is produced and accumulated in the culture, and the monoclonal antibody is produced from the culture. Alternatively, the method for producing a monoclonal antibody or a fragment of the antibody according to any one of items 1 to 14, wherein a fragment of the antibody is collected.
21. 15. A pharmaceutical composition comprising the monoclonal antibody or the antibody fragment according to any one of items 1 to 14 as an active ingredient.
22. 15. A therapeutic agent for a disease involving CD33 positive cells, comprising the monoclonal antibody or the antibody fragment according to any one of items 1 to 14 as an active ingredient.
23. 23. The therapeutic agent according to item 22 above, wherein the disease involving CD33 positive cells is cancer, autoimmune disease or allergic disease.
24. The preceding paragraph 23, wherein the cancer is blood cancer, breast cancer, uterine cancer, colon cancer, esophageal cancer, stomach cancer, ovarian cancer, lung cancer, kidney cancer, rectal cancer, thyroid cancer, cervical cancer, small intestine cancer, prostate cancer or pancreatic cancer. The therapeutic agent as described in.
25. 25. The therapeutic agent according to 24 above, wherein the blood cancer is acute myeloid leukemia, anaplastic large cell lymphoma, acute lymphocytic leukemia, myelodysplastic syndrome, multiple myeloma, Hodgkin lymphoma, or non-Hodgkin lymphoma.
26. 15. A method for treating a disease involving CD33-positive cells, comprising a step of administering the monoclonal antibody or the antibody fragment according to any one of items 1 to 14 to a mammal.
27. 27. The method according to item 26 above, wherein the disease involving CD33 positive cells is cancer, autoimmune disease or allergic disease.
28. The preceding paragraph 27, wherein the cancer is blood cancer, breast cancer, uterine cancer, colon cancer, esophageal cancer, stomach cancer, ovarian cancer, lung cancer, kidney cancer, rectal cancer, thyroid cancer, cervical cancer, small intestine cancer, prostate cancer or pancreatic cancer. The method described in 1.
29. 29. The method according to item 28 above, wherein the blood cancer is acute myeloid leukemia, anaplastic large cell lymphoma, acute lymphocytic leukemia, myelodysplastic syndrome, multiple myeloma, Hodgkin lymphoma, or non-Hodgkin lymphoma.
30. 15. Use of the monoclonal antibody or fragment of the antibody according to any one of 1 to 14 above for treating a disease involving CD33 positive cells.
31. 31. The use according to item 30 above, wherein the disease involving CD33 positive cells is cancer, autoimmune disease or allergic disease.
32. Paragraph 31 above, wherein the cancer is blood cancer, breast cancer, uterine cancer, colon cancer, esophageal cancer, stomach cancer, ovarian cancer, lung cancer, kidney cancer, rectal cancer, thyroid cancer, cervical cancer, small intestine cancer, prostate cancer or pancreatic cancer. Use as described in.
33. 33. The use according to item 32 above, wherein the blood cancer is acute myeloid leukemia, anaplastic large cell lymphoma, acute lymphocytic leukemia, myelodysplastic syndrome, multiple myeloma, Hodgkin lymphoma or non-Hodgkin lymphoma.
　実施例でも示されるように、本発明のモノクローナル抗体は高いアフィニティーでヒトＣＤ３３細胞外領域に結合し、かつ高いＡＤＣＣ活性を発揮する。従って、本発明は、該抗体または該断片、該抗体または該断片をコードするＤＮＡ、該ＤＮＡを含むベクター、該ベクターを導入して得られる形質転換体、該形質転換体を用いる該抗体または該断片の製造方法、並びに該抗体または該断片を含む治療剤および診断剤を提供することができる。 As shown in the Examples, the monoclonal antibody of the present invention binds to the human CD33 extracellular region with high affinity and exhibits high ADCC activity. Therefore, the present invention relates to the antibody or the fragment, the DNA encoding the antibody or the fragment, the vector containing the DNA, the transformant obtained by introducing the vector, the antibody using the transformant or the A method for producing a fragment, and a therapeutic agent and a diagnostic agent containing the antibody or the fragment can be provided.
図１Ａは、ヒトＣＤ３３発現ベクター構築の概略図を示す。FIG. 1A shows a schematic diagram of human CD33 expression vector construction. 図１Ｂは、カニクイザルＣＤ３３発現ベクター構築の概略図を示す。FIG. 1B shows a schematic diagram of cynomolgus monkey CD33 expression vector construction. 図１Ｃは、チンパンジーＣＤ３３発現ベクター構築の概略図を示す。FIG. 1C shows a schematic diagram of chimpanzee CD33 expression vector construction. 図２は、ヒスチジンタグ付加可溶型ヒトＣＤ３３発現ベクター構築の概略図を示す。FIG. 2 shows a schematic diagram of the construction of a histidine-tagged soluble human CD33 expression vector. 図３は、ラットおよびマウス抗ＣＤ３３モノクローナル抗体の評価（ＦＣＭ）を示す。FIG. 3 shows the evaluation (FCM) of rat and mouse anti-CD33 monoclonal antibodies. 図４は、ラットおよびマウス抗ＣＤ３３モノクローナル抗体の評価（ＥＬＩＳＡ）を示す。FIG. 4 shows the evaluation (ELISA) of rat and mouse anti-CD33 monoclonal antibodies. 図５Ａは、抗ＣＤ３３キメラ抗体発現ベクター構築概略図を示す。FIG. 5A shows a schematic diagram of construction of an anti-CD33 chimeric antibody expression vector. 図５Ｂは、抗ＣＤ３３キメラ抗体発現ベクター構築概略図を示す。FIG. 5B shows a schematic diagram of construction of an anti-CD33 chimeric antibody expression vector. 図６は、抗ＣＤ３３キメラ抗体の評価（ＦＣＭ）を示す。FIG. 6 shows the evaluation (FCM) of the anti-CD33 chimeric antibody. 図７は、抗ＣＤ３３キメラ抗体の評価（ＥＬＩＳＡ）を示す。FIG. 7 shows the evaluation (ELISA) of anti-CD33 chimeric antibody. 図８は、抗ＣＤ３３キメラ抗体の評価（ＡＤＣＣ）を示す。FIG. 8 shows the evaluation (ADCC) of anti-CD33 chimeric antibody. 図９は、抗ＣＤ３３キメラ抗体の評価（ＣＤＣ）を示す。FIG. 9 shows the evaluation (CDC) of anti-CD33 chimeric antibody. 図１０は、抗ＣＤ３３キメラ抗体の細胞膜上での抗体の結合量（図１０Ａ）および残存率（図１０Ｂ）を示す。FIG. 10 shows the binding amount (FIG. 10A) and residual rate (FIG. 10B) of the antibody on the cell membrane of the anti-CD33 chimeric antibody. 図１１は、ＨＭ１９５－１、－１１３Ｆ抗体発現ベクター構築の概略図を示す。FIG. 11 shows a schematic diagram of HM195-1, -113F antibody expression vector construction. 図１２は、抗ＣＤ３３ヒト化抗体の評価（ＡＤＣＣ）を示す。FIG. 12 shows the anti-CD33 humanized antibody evaluation (ADCC). 図１３は、抗ＣＤ３３ヒト化抗体の細胞膜上での抗体の結合量（図１３Ａ）および残存率（図１３Ｂ）を示す。FIG. 13 shows the amount of antibody bound on the cell membrane of the anti-CD33 humanized antibody (FIG. 13A) and the residual ratio (FIG. 13B). 図１４は、抗ＣＤ３３ヒト化抗体のヒト末梢血中における細胞除去活性を示す。FIG. 14 shows the cell removal activity of anti-CD33 humanized antibody in human peripheral blood. 図１５は、抗ＣＤ３３ヒト化抗体のヒト、カニクイザルおよびチンパンジーＣＤ３３に対する反応性を示す。FIG. 15 shows the reactivity of anti-CD33 humanized antibodies to human, cynomolgus monkey and chimpanzee CD33.
　本発明は、高いアフィニティーでヒトＣＤ３３細胞外領域に結合し、これに伴い、高いＡＤＣＣ活性を有するモノクローナル抗体またはその断片に関する（以下、単に「ＣＤ３３」と表記した場合には、ヒトＣＤ３３を意味するものとする）。 The present invention relates to a monoclonal antibody or a fragment thereof that binds to the extracellular region of human CD33 with high affinity and has high ADCC activity (hereinafter simply referred to as “CD33” means human CD33). Suppose).
　本発明におけるＣＤ３３としては、配列番号３またはＰ２０１３８（ＳＷＩＳＳＰＲＯＴ）で示されるアミノ酸配列を有するポリペプチドまたは配列番号３またはＥＭＢＬアクセッション番号Ｍ２３１９７で示されるアミノ酸配列において１つ以上のアミノ酸が欠失、置換または付加されたアミノ酸配列からなり、かつＣＤ３３の機能を有するポリペプチド、ならびに配列番号３またはＥＭＢＬアクセッション番号Ｍ２３１９７で示されるアミノ酸配列と６０％以上、好ましくは８０％以上、さらに好ましくは９０％以上の相同性を有するアミノ酸配列を有するポリペプチド、最も好ましくは９５％以上の相同性を有するアミノ酸配列からなり、かつＣＤ３３の機能を有するポリペプチドなどが挙げられる。 As CD33 in the present invention, one or more amino acids in the polypeptide having the amino acid sequence represented by SEQ ID NO: 3 or P20148 (SWISSPROT) or the amino acid sequence represented by SEQ ID NO: 3 or EMBL accession number M23197 are deleted or substituted. Or a polypeptide comprising an added amino acid sequence and having the function of CD33, and 60% or more, preferably 80% or more, more preferably 90% or more, with the amino acid sequence represented by SEQ ID NO: 3 or EMBL accession number M23197 And a polypeptide having an amino acid sequence having the homology of (2), most preferably a polypeptide having an amino acid sequence having a homology of 95% or more and having the function of CD33.
　配列番号３またはＰ２０１３８（ＳＷＩＳＳＰＲＯＴ）で示されるアミノ酸配列において１以上のアミノ酸が欠失、置換、または付加されたアミノ酸配列を有するポリペプチドは、部位特異的変異導入法［Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ，Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ，Ｓｅｃｏｎｄ　Ｅｄｉｔｉｏｎ，Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓ（１９８９）、Ｃｕｒｒｅｎｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ，Ｊｏｈｎ　Ｗｉｌｅｙ＆Ｓｏｎｓ（１９８７－１９９７）、Ｎｕｃｌｅｉｃ　Ａｃｉｄｓ　Ｒｅｓｅａｒｃｈ，１０，６４８７（１９８２）、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，７９，６４０９（１９８２）、Ｇｅｎｅ，３４，３１５（１９８５）、Ｎｕｃｌｅｉｃ　Ａｃｉｄｓ　Ｒｅｓｅａｒｃｈ，１３，４４３１（１９８５）、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，８２，４８８（１９８５）］などを用いて、例えば配列番号３で示されるアミノ酸配列を有するポリペプチドをコードするＤＮＡに部位特異的変異を導入することにより得ることができる。欠失、置換または付加されるアミノ酸の数は特に限定されないが、好ましくは１個～数十個、例えば、１～２０個、より好ましくは１個～数個、例えば、１～５個のアミノ酸である。 A polypeptide having an amino acid sequence in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 3 or P20148 (SWISSPROT) can be obtained by site-directed mutagenesis [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997), Nucleic 64 Proc. Natl. Acad. Sci. USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci. USA, 82, 488 (1985)] and the like, for example, can be obtained by introducing a site-specific mutation into DNA encoding a polypeptide having the amino acid sequence represented by SEQ ID NO: 3. The number of amino acids to be deleted, substituted or added is not particularly limited, but is preferably 1 to several tens, for example 1 to 20, more preferably 1 to several, for example 1 to 5 amino acids. It is.
　ＣＤ３３をコードする遺伝子としては、配列番号４またはＥＭＢＬアクセッション番号Ｍ２３１９７で示される塩基配列が挙げられる。配列番号４またはＥＭＢＬアクセッション番号Ｍ２３１９７で示される塩基配列において、１以上の塩基が欠失、置換または付加された塩基配列からなり、かつＣＤ３３の機能を有するポリペプチドをコードするＤＮＡを含む遺伝子、配列番号４またはＥＭＢＬアクセッション番号Ｍ２３１９７で示される塩基配列と少なくとも６０％以上の相同性を有する塩基配列、好ましくは８０％以上の相同性を有する塩基配列、さらに好ましくは９５％以上の相同性を有する塩基配列からなり、かつＣＤ３３の機能を有するポリペプチドをコードするＤＮＡを含む遺伝子、ならびに配列番号４またはＥＭＢＬアクセッション番号Ｍ２３１９７で示される塩基配列を有するＤＮＡとストリンジェントな条件下でハイブリダイズするＤＮＡからなり、かつＣＤ３３の機能を有するポリペプチドをコードするＤＮＡを含む遺伝子なども本発明のＣＤ３３をコードする遺伝子に包含される。 Examples of the gene encoding CD33 include the nucleotide sequence represented by SEQ ID NO: 4 or EMBL accession number M23197. A gene comprising a DNA encoding a polypeptide consisting of a base sequence in which one or more bases are deleted, substituted or added in the base sequence represented by SEQ ID NO: 4 or EMBL accession number M23197, and having a CD33 function; A nucleotide sequence having at least 60% homology with the nucleotide sequence represented by SEQ ID NO: 4 or EMBL accession number M23197, preferably a nucleotide sequence having 80% or more homology, more preferably 95% or more homology It hybridizes under stringent conditions with a gene comprising a DNA comprising a nucleotide sequence having a CD33 function and a DNA encoding a polypeptide having a CD33 function, and a DNA having the nucleotide sequence represented by SEQ ID NO: 4 or EMBL accession number M23197 Consisting of DNA, and Such as a gene containing DNA encoding a polypeptide having the function of D33 is also included in the gene encoding CD33 of the present invention.
　ストリンジェントな条件下でハイブリダイズするＤＮＡとしては、配列番号４またはＥＭＢＬアクセッション番号Ｍ２３１９７で示される塩基配列を有するＤＮＡをプローブに用いた、コロニー・ハイブリダイゼーション法、プラーク・ハイブリダイゼーション法、サザンブロット・ハイブリダイゼーション法、またはＤＮＡマイクロアレイ法などにより得られるハイブリダイズ可能なＤＮＡを意味する。 As DNA that hybridizes under stringent conditions, DNA having the base sequence represented by SEQ ID NO: 4 or EMBL accession number M23197 was used as a probe, and colony hybridization, plaque hybridization, Southern blot -It means a hybridizable DNA obtained by a hybridization method or a DNA microarray method.
　具体的には、ハイブリダイズしたコロニー若しくはプラーク由来のＤＮＡ、または該配列を有するＰＣＲ産物若しくはオリゴＤＮＡを固定化したフィルターまたはスライドガラスを用いて、０．７～１．０ｍｏｌ／Ｌの塩化ナトリウム存在下、６５℃でハイブリダイゼーション［Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ，Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ，Ｓｅｃｏｎｄ　Ｅｄｉｔｉｏｎ，Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓ（１９８９）、Ｃｕｒｒｅｎｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ，Ｊｏｈｎ　Ｗｉｌｅｙ＆Ｓｏｎｓ（１９８７－１９９７）、ＤＮＡ　Ｃｌｏｎｉｎｇ　１：　Ｃｏｒｅ　Ｔｅｃｈｎｉｑｕｅｓ，Ａ　Ｐｒａｃｔｉｃａｌ　Ａｐｐｒｏａｃｈ，Ｓｅｃｏｎｄ　Ｅｄｉｔｉｏｎ，Ｏｘｆｏｒｄ　Ｕｎｉｖｅｒｓｉｔｙ，（１９９５）］を行った後、０．１～２倍濃度のＳＳＣ溶液（１倍濃度のＳＳＣ溶液の組成は、１５０ｍｍｏｌ／Ｌ塩化ナトリウム、１５ｍｍｏｌ／Ｌクエン酸ナトリウムよりなる）を用い、６５℃条件下でフィルターまたはスライドグラスを洗浄することにより同定できるＤＮＡを挙げることができる。 Specifically, 0.7 to 1.0 mol / L of sodium chloride is present using a DNA or DNA derived from a hybridized colony or plaque, or a filter or slide glass on which a PCR product or oligo DNA having the sequence is immobilized. Hybridization at 65 ° C. [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in BioClinology, BioClinology, BioClinology, BioClinology, BioClinicology App oach, Second Edition, Oxford University, (1995)], followed by 0.1-2 fold concentration of SSC solution (1 fold concentration of SSC solution is 150 mmol / L sodium chloride, 15 mmol / L sodium citrate And a DNA that can be identified by washing a filter or glass slide at 65 ° C.
　ハイブリダイズ可能なＤＮＡとしては、配列番号４またはＥＭＢＬアクセッション番号Ｍ２３１９７で示される塩基配列と少なくとも６０％以上の相同性を有するＤＮＡ、好ましくは８０％以上の相同性を有するＤＮＡ、さらに好ましくは９５％以上の相同性を有するＤＮＡをあげることができる。 The DNA capable of hybridizing is DNA having at least 60% homology with the base sequence represented by SEQ ID NO: 4 or EMBL accession number M23197, preferably DNA having 80% homology or more, more preferably 95 % Of DNA having a homology of at least%.
　真核生物の蛋白質をコードする遺伝子の塩基配列には、しばしば遺伝子の多型が認められる。本発明において用いられる遺伝子に、このような多型によって塩基配列に小規模な変異を生じた遺伝子も、本発明のＣＤ３３をコードする遺伝子に包含される。 In the base sequence of a gene encoding a eukaryotic protein, a polymorphism of the gene is often observed. A gene in which a small-scale variation is caused in the nucleotide sequence due to such polymorphism in the gene used in the present invention is also included in the gene encoding CD33 of the present invention.
　本発明における相同性の数値は、特に明示した場合を除き、当業者に公知の相同性検索プログラムを用いて算出される数値であってよいが、塩基配列については、ＢＬＡＳＴ［Ｊ．Ｍｏｌ．Ｂｉｏｌ．，２１５，４０３（１９９０）］においてデフォルトのパラメータを用いて算出される数値など、アミノ酸配列については、ＢＬＡＳＴ２［Ｎｕｃｌｅｉｃ　Ａｃｉｄｓ　Ｒｅｓ．，２５，３３８９（１９９７）、Ｇｅｎｏｍｅ　Ｒｅｓ．，７，６４９（１９９７）、ｈｔｔｐ：／／ｗｗｗ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ／Ｅｄｕｃａｔｉｏｎ／ＢＬＡＳＴｉｎｆｏ／ｉｎｆｏｒｍａｔｉｏｎ３．ｈｔｍｌ］においてデフォルトのパラメータを用いて算出される数値などが挙げられる。 The numerical value of homology in the present invention may be a numerical value calculated using a homology search program known to those skilled in the art unless otherwise specified, but the base sequence may be BLAST [J. Mol. Biol. , 215, 403 (1990)], for amino acid sequences such as numerical values calculated using default parameters, BLAST2 [Nucleic Acids Res. , 25, 3389 (1997), Genome Res. , 7, 649 (1997), http: // www. ncbi. nlm. nih. gov / Education / BLASTinfo / information3. numerical values calculated using default parameters in [html].
　デフォルトのパラメータとしては、Ｇ（Ｃｏｓｔ　ｔｏ　ｏｐｅｎ　ｇａｐ）が塩基配列の場合は５、アミノ酸配列の場合は１１、－Ｅ（Ｃｏｓｔ　ｔｏ　ｅｘｔｅｎｄ　ｇａｐ）が塩基配列の場合は２、アミノ酸配列の場合は１、－ｑ（Ｐｅｎａｌｔｙ　ｆｏｒ　ｎｕｃｌｅｏｔｉｄｅ　ｍｉｓｍａｔｃｈ）が－３、－ｒ（ｒｅｗａｒｄ　ｆｏｒ　ｎｕｃｌｅｏｔｉｄｅ　ｍａｔｃｈ）が１、－ｅ（ｅｘｐｅｃｔ　ｖａｌｕｅ）が１０、－Ｗ（ｗｏｒｄｓｉｚｅ）が塩基配列の場合は１１残基、アミノ酸配列の場合は３残基、－ｙ［Ｄｒｏｐｏｆｆ（Ｘ）ｆｏｒ　ｂｌａｓｔ　ｅｘｔｅｎｓｉｏｎｓ　ｉｎ　ｂｉｔｓ］がｂｌａｓｔｎ　の場合は２０、ｂｌａｓｔｎ以外のプログラムでは７、－Ｘ（Ｘ　ｄｒｏｐｏｆｆ　ｖａｌｕｅ　ｆｏｒ　ｇａｐｐｅｄ　ａｌｉｇｎｍｅｎｔ　ｉｎ　ｂｉｔｓ）が１５および－Ｚ（ｆｉｎａｌ　Ｘ　ｄｒｏｐｏｆｆ　ｖａｌｕｅ　ｆｏｒ　ｇａｐｐｅｄ　ａｌｉｇｎｍｅｎｔ　ｉｎ　ｂｉｔｓ）がｂｌａｓｔｎ　の場合は５０、ｂｌａｓｔｎ以外のプログラムでは２５である（ｈｔｔｐ：／／ｗｗｗ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ／ｂｌａｓｔ／ｈｔｍｌ／ｂｌａｓｔｃｇｉｈｅｌｐ．ｈｔｍｌ）。 The default parameters are 5 if G (Cost to open gap) is a base sequence, 11 if it is an amino acid sequence, 2 if -E (Cost to extend gap) is a base sequence, and 1 if it is an amino acid sequence. , -Q (Penalty for nucleotide mismatch) is -3, -r (reward for nucleotide match) is 1, -e (expect value) is 10, 11 residues when -W (wordsize) is a base sequence, amino acid sequence 3 residues, -y [Dropoff (X) for blast extensions in bits] is 20 if blastn, 7 for programs other than blastn, -X (X dropoff value f If the r aligned alignment in bits) is 15 and -Z (final X dropoff value for gapd alignment in bits) is blastn, it is 25 for programs other than blastn (http: // www. gov / blast / html / blastcgihelp.html).
　配列番号３またはＰ２０１３８（ＳＷＩＳＳＰＲＯＴ）で示されるアミノ酸配列の部分配列からなるポリペプチドは、当業者に公知の方法によって作製することができる。例えば、配列番号３で示されるアミノ酸配列をコードするＤＮＡの一部を欠失させ、これを含む発現ベクターを導入した形質転換体を培養することにより作製することができる。また、上記の方法で作製されるポリペプチドまたはＤＮＡに基づいて、上記と同様の方法により、配列番号３またはＰ２０１３８（ＳＷＩＳＳＰＲＯＴ）で示されるアミノ酸配列の部分配列において１以上のアミノ酸が欠失、置換または付加されたアミノ酸配列を有するポリペプチドを得ることができる。さらに、配列番号３またはＰ２０１３８（ＳＷＩＳＳＰＲＯＴ）で示されるアミノ酸配列の部分配列からなるポリペプチド、または配列番号３またはＰ２０１３８（ＳＷＩＳＳＰＲＯＴ）で示されるアミノ酸配列の部分配列において１以上のアミノ酸が欠失、置換または付加されたアミノ酸配列を有するポリペプチドは、フルオレニルメチルオキシカルボニル（Ｆｍｏｃ）法、ｔ－ブチルオキシカルボニル（ｔＢｏｃ）法などの化学合成法によって製造することもできる。 A polypeptide comprising a partial sequence of the amino acid sequence represented by SEQ ID NO: 3 or P20148 (SWISSPROT) can be prepared by methods known to those skilled in the art. For example, it can be produced by culturing a transformant in which a part of DNA encoding the amino acid sequence represented by SEQ ID NO: 3 has been deleted and an expression vector containing the DNA is introduced. Further, based on the polypeptide or DNA produced by the above method, one or more amino acids are deleted or substituted in the partial sequence of the amino acid sequence represented by SEQ ID NO: 3 or P20148 (SWISSPROT) by the same method as above. Alternatively, a polypeptide having an added amino acid sequence can be obtained. Further, a polypeptide comprising a partial sequence of the amino acid sequence represented by SEQ ID NO: 3 or P20148 (SWISSPROT), or one or more amino acids in the partial sequence of the amino acid sequence represented by SEQ ID NO: 3 or P20138 (SWISSPROT) is deleted or substituted Alternatively, a polypeptide having an added amino acid sequence can also be produced by a chemical synthesis method such as a fluorenylmethyloxycarbonyl (Fmoc) method or a t-butyloxycarbonyl (tBoc) method.
　本発明におけるＣＤ３３の細胞外領域としては、例えば、配列番号３で示される該ポリペプチドのアミノ酸配列を公知の膜貫通領域予測プログラムＳＯＳＵＩ（ｈｔｔｐ：／／ｂｐ．ｎｕａｐ．ｎａｇｏｙａ－ｕ．ａｃ．ｊｐ／ＳＯＳＵＩ／ＳＯＳＵＩ＿ｓｕｂｍｉｔ．）、ＴＭＨＭＭ　ｖｅｒ．２（ｈｔｔｐ：／／ｗｗｗ．ｃｂｓ．ｄｔｕ．ｄｋ／ｓｅｒｖｉｃｅｓ／ＴＭＨＭＭ－２．０／）またはＥｘＰＡＳｙ　Ｐｒｏｔｅｏｍｉｃｓ　Ｓｅｒｖｅｒ（ｈｔｔｐ：／／Ｃａ．ｅｘｐａｓｙ．ｏｒｇ／）などを用いて予測された領域などが挙げられる。具体的には、ＳＯＳＵＩにおいて予測される細胞外ドメインであるＮ末より２４２番目までが挙げられる。細胞外ドメインは、２つのイムノグロブリンスーパーファミリードメインを有しており、いずれのドメインも含まれる。 As the extracellular region of CD33 in the present invention, for example, the amino acid sequence of the polypeptide represented by SEQ ID NO: 3 is converted into a known transmembrane region prediction program SOSUI (http://bp.nuap.nagoya-u.ac.jp / SOSUI / SOSUI_submit.), TMHMM ver. 2 (http://www.cbs.dtu.dk/services/TMHMM-2.0/) or ExPASy Proteomics Server (http://Ca.expasy.org/) It is done. Specifically, the 242nd from the N-terminal which is an extracellular domain predicted in SOSUI is mentioned. The extracellular domain has two immunoglobulin superfamily domains, and both domains are included.
　本発明におけるＣＤ３３の細胞外領域としては、配列番号３またはＰ２０１３８（ＳＷＩＳＳＰＲＯＴ）で示されるアミノ酸配列を有するＣＤ３３の細胞外領域が天然状態でとりうる構造と同等の構造を有していればいずれの構造でもよい。ＣＤ３３の細胞外領域が天然状態でとりうる構造とは、細胞膜上に発現しているＣＤ３３の天然型の立体構造のことをいう。 The extracellular region of CD33 in the present invention is any as long as the extracellular region of CD33 having the amino acid sequence represented by SEQ ID NO: 3 or P20148 (SWISSPROT) has a structure equivalent to the structure that can be taken in the natural state. It may be a structure. The structure that the extracellular region of CD33 can take in the natural state refers to the natural three-dimensional structure of CD33 expressed on the cell membrane.
　ＣＤ３３の機能について詳細は不明である。本発明におけるＣＤ３３の機能としては、例えば、骨髄系細胞の分化段階において発現し、シアル酸を持った細胞、例えばＣＤ３４陽性幹細胞と接着し、細胞の分化の制御に関与することが挙げられる。また、ＣＤ３３はシアル酸結合たんぱく質である事から、シアル酸分子との結合活性、およびシアル酸分子との結合による細胞内シグナル伝達機能、または細胞の運動能の変化もＣＤ３３の機能として挙げられる。 The details of the function of CD33 are unknown. Examples of the function of CD33 in the present invention include expression in the differentiation stage of myeloid cells, adhesion to sialic acid-containing cells such as CD34 positive stem cells, and involvement in the control of cell differentiation. Further, since CD33 is a sialic acid binding protein, the activity of binding to sialic acid molecules and the intracellular signal transduction function or the motility of cells due to the binding with sialic acid molecules can also be mentioned as functions of CD33.
　本発明の抗体またはその断片が、ＣＤ３３細胞外領域に結合することは、固相サンドイッチ法などを用いたラジオイムノアッセイ、または酵素免疫測定法（ＥＬＩＳＡ）などを用いたＣＤ３３を発現した細胞に対する公知の免疫学的検出法、好ましくは蛍光細胞染色法などの特定の抗原を発現した細胞と特定抗原に対する抗体の結合性を調べることができる方法により確認することができる。 The binding of the antibody of the present invention or a fragment thereof to the extracellular region of CD33 is known for cells expressing CD33 using a radioimmunoassay using a solid phase sandwich method or an enzyme immunoassay (ELISA). It can be confirmed by an immunological detection method, preferably a method capable of examining the binding ability of a cell expressing a specific antigen and an antibody to the specific antigen, such as a fluorescent cell staining method.
　例えば、ＦＭＡＴ８１００ＨＴＳシステム（アプライドバイオシステム社製）などを用いる蛍光抗体染色法［Ｃａｎｃｅｒ　Ｉｍｍｕｎｏｌ．Ｉｍｍｕｎｏｔｈｅｒ．，３６，３７３（１９９３）］、フローサイトメトリーを用いる蛍光細胞染色法、またはＢｉａｃｏｒｅシステム（ジーイーヘルスケア社製）などを用いた表面プラズモン共鳴などの方法が挙げられる。また、公知の免疫学的検出法［Ｍｏｎｏｃｌｏｎａｌ　Ａｎｔｉｂｏｄｉｅｓ－Ｐｒｉｎｃｉｐｌｅｓ　ａｎｄ　ｐｒａｃｔｉｃｅ，Ｔｈｉｒｄ　ｅｄｉｔｉｏｎ，Ａｃａｄｅｍｉｃ　Ｐｒｅｓｓ（１９９６）、Ａｎｔｉｂｏｄｉｅｓ－Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ，Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ（１９８８）、単クローン抗体実験マニュアル，講談社サイエンティフィック（１９８７）］などを組み合わせて確認することもできる。 For example, a fluorescent antibody staining method using a FMAT8100HTS system (Applied Biosystems) or the like [Cancer Immunol. Immunother. , 36, 373 (1993)], fluorescent cell staining using flow cytometry, or surface plasmon resonance using a Biacore system (manufactured by GE Healthcare). In addition, known immunological detection methods [Monoclonal Antibodies-Principles and practices, Third edition, Academic Press (1996), Antibodies-A Laboratory Manual, ColdHordrology, ColdHordrology, ColdHorman, ColdHordrology, ColdHorman, ColdHorman (1987)] etc. can also be confirmed.
　ＣＤ３３を発現した細胞としては、ＣＤ３３を発現していればいずれの細胞でもよく、例えばヒト体内に天然に存在する細胞、ヒト体内に天然に存在する細胞から樹立された細胞株、または遺伝子組換え技術により得られた細胞などが挙げられる。 The cells expressing CD33 may be any cells as long as they express CD33. For example, cells naturally existing in the human body, cell lines established from cells naturally existing in the human body, or genetic recombination Examples include cells obtained by technology.
　ヒト体内に天然に存在する細胞としては、例えば、正常単球、顆粒球または骨髄系細胞のほか、骨髄系白血病、例えば、ＡＭＬ患者体内において該ポリペプチドが発現している細胞が挙げられる。例えば、バイオプシーなどで得られた腫瘍細胞のうちでＣＤ３３が発現している細胞などが挙げられる。 Examples of cells that naturally exist in the human body include normal monocytes, granulocytes, or myeloid cells, as well as myeloid leukemia, for example, cells in which the polypeptide is expressed in AML patients. For example, among the tumor cells obtained by biopsy or the like, cells expressing CD33 can be mentioned.
　ヒト体内に天然に存在する細胞から樹立された細胞株としては、上記の癌患者から得られたＣＤ３３が発現している細胞を株化して得られた細胞株のうち、ＣＤ３３を発現している細胞株が挙げられる。例えば、ヒトから樹立された細胞株であるヒト骨髄系白血病細胞株ＮＢ－４［ＤＳＭＺ番号：ＡＣＣ２０７］またはＨＬ－６０［ＡＴＣＣ番号：ＣＣＬ－２４０］などが挙げられる。 As a cell line established from cells naturally existing in the human body, CD33 is expressed among the cell lines obtained by stocking cells expressing CD33 obtained from the above cancer patients. Cell lines. For example, human myeloid leukemia cell line NB-4 [DSMZ number: ACC207] or HL-60 [ATCC number: CCL-240], which are cell lines established from humans, can be mentioned.
　遺伝子組換え技術により得られた細胞としては、具体的には、例えば、該ＣＤ３３をコードするｃＤＮＡを含む発現ベクターを昆虫細胞または動物細胞などに導入することにより得られる、該ＣＤ３３を発現した細胞などが挙げられる。 Specifically, the cells obtained by gene recombination techniques include, for example, cells expressing CD33 obtained by introducing an expression vector containing cDNA encoding CD33 into insect cells or animal cells. Etc.
　本発明の抗体は、ヒトＣＤ３３の細胞外領域に結合し、かつ高いＡＤＣＣ活性を有するヒトＣＤ３３に対するモノクローナル抗体および該抗体の断片であって、ヒトＣＤ３３に高いアフィニティーを有することが好ましい。ヒトＣＤ３３に高いアフィニティーを有するとは、ヒトＣＤ３３に対する解離定数が低いことをいう。 The antibody of the present invention is a monoclonal antibody against human CD33 that binds to the extracellular region of human CD33 and has high ADCC activity, and a fragment of the antibody, and preferably has high affinity for human CD33. Having high affinity for human CD33 means that the dissociation constant for human CD33 is low.
　本発明の抗体のヒトＣＤ３３に対する解離定数（以下、ＫＤと表記することがある）は、４．０×１０－９Ｍ以下であることが好ましく、より好ましくは２．０×１０－９Ｍ以下である。例えば、０．５×１０－９Ｍ～４．０×１０－９Ｍ、１．０×１０－９Ｍ～４．０×１０－９Ｍ、０．５×１０－９Ｍ～２．０×１０－９Ｍまたは１．０×１０－９Ｍ～２．０×１０－９Ｍである。 Dissociation constant for human CD33 antibodies of the present invention (hereinafter sometimes referred to as K D) is preferably at 4.0 × 10 -9 M or less, more preferably 2.0 × 10 -9 M It is as follows. For example, 0.5 × 10 −9 M to 4.0 × 10 −9 M, 1.0 × 10 −9 M to 4.0 × 10 −9 M, 0.5 × 10 −9 M to 2.0 × 10 −9 M or 1.0 × 10 −9 M to 2.0 × 10 −9 M.
　ヒトＣＤ３３に対する解離定数ＫＤを４．０×１０－９Ｍ以下とすることにより、治療用抗体として十分なアフィニティーでＣＤ３３の細胞外領域に結合することができ、かつ高いＡＤＣ活性および抗腫瘍活性を有することができる。既に臨床試験が実施されているｌｉｎｔｕｚｕｍａｂ（ＨｕＭ１９５）は、解離定数ＫＤが４×１０－９Ｍ程度のアフィニティーであり、治療効果を示していることから、本発明の抗体は、更に高い治療効果を示すことができる。 With a dissociation constant K D for human CD33 4.0 × 10 -9 M or less, can bind to the extracellular region of CD33 with sufficient affinity as a therapeutic antibody, and high ADC activity and antitumor activity Can have. Already lintuzumab clinical trials have been conducted (HuM195) the dissociation constant, K D, is 4 × 10 -9 M about affinity, since the show a therapeutic effect, the antibodies of the invention, higher therapeutic effects Can be shown.
　アフィニティーとは、反応速度論的な解析により測定されるものであり、例えばＢｉａｃｏｒｅ　Ｔ１００（ＧＥヘルスケアバイオサイエンス社製）などを用いて測定することができる。 Affinity is measured by reaction kinetic analysis, and can be measured using, for example, Biacore T100 (manufactured by GE Healthcare Bioscience).
　また、本発明の抗体は、抗原との解離が非常に緩やかである事を特徴とする。解離が緩やかである事、または、解離が緩やかである事および細胞内への取り込みが緩やかである事から、細胞表面上に結合した抗体の量および残存率が高いという利点を有する。 In addition, the antibody of the present invention is characterized by very slow dissociation from the antigen. Since the dissociation is slow, or the dissociation is slow and the incorporation into the cell is slow, there is an advantage that the amount of antibody bound to the cell surface and the residual ratio are high.
　本発明においてモノクローナル抗体としては、ハイブリドーマにより生産される抗体、または抗体遺伝子を含む発現ベクターで形質転換した形質転換体により生産される遺伝子組換え抗体をあげることができる。 In the present invention, examples of the monoclonal antibody include an antibody produced by a hybridoma or a recombinant antibody produced by a transformant transformed with an expression vector containing an antibody gene.
　モノクローナル抗体とは、単一クローンの抗体産生細胞が分泌する抗体であり、ただ一つのエピトープ（抗原決定基ともいう）を認識し、モノクローナル抗体を構成するアミノ酸配列（１次構造）が均一である。 A monoclonal antibody is an antibody that is secreted by an antibody-producing cell of a single clone, recognizes only one epitope (also referred to as an antigenic determinant), and has a uniform amino acid sequence (primary structure) constituting the monoclonal antibody. .
　エピトープとは、モノクローナル抗体が認識し、結合する単一のアミノ酸配列、アミノ酸配列からなる立体構造、糖鎖が結合したアミノ酸配列および糖鎖が結合したアミノ酸配列からなる立体構造などが挙げられる。 Epitopes include a single amino acid sequence that is recognized and bound by a monoclonal antibody, a three-dimensional structure composed of amino acid sequences, an amino acid sequence linked with sugar chains, and a three-dimensional structure composed of amino acid sequences combined with sugar chains.
　本発明のモノクローナル抗体が認識し、結合するエピトープとしては、好ましくはＣＤ３３の細胞外領域、配列番号３で示されるアミノ酸配列の１８～２５９番目のアミノ酸配列に含まれる複数のアミノ酸残基からなるエピトープ、より好ましくはＣＤ３３の細胞外領域の細胞膜から遠いイムノグロブリンドメインに含まれる複数のアミノ酸残基からなるエピトープ、またはＣＤ３３の細胞外領域の細胞膜に近いイムノグロブリンドメインに含まれる複数のアミノ酸残基からなるエピトープなどをあげることができる。 The epitope that is recognized and bound by the monoclonal antibody of the present invention is preferably an epitope consisting of a plurality of amino acid residues contained in the extracellular region of CD33, the 18th to 259th amino acid sequences of the amino acid sequence shown in SEQ ID NO: 3. More preferably, an epitope consisting of a plurality of amino acid residues contained in an immunoglobulin domain far from the cell membrane of the extracellular region of CD33, or a plurality of amino acid residues contained in an immunoglobulin domain close to the cell membrane of the extracellular region of CD33 The epitope etc. which become can be mention | raise | lifted.
　本発明において、解離が遅いとは、Ｂｉａｃｏｒｅ　Ｔ１００において算出される抗体の解離速度定数ｋｄの値が、より小さな値を示すことである。解離速度定数ｋｄは、例えばＢｉａｃｏｒｅ　Ｔ１００（ＧＥヘルスケアバイオサイエンス社製）を用いて測定され、付属ソフトウエアＢｉａｃｏｒｅ　Ｔ１００　ｅｖａｌｕａｔｉｏｎ　ｓｏｆｔｗａｒｅ（Ｂｉａｃｏｒｅ社製）により算出することができる。 In the present invention, slow dissociation means that the value of the antibody dissociation rate constant kd calculated in Biacore T100 shows a smaller value. The dissociation rate constant kd is measured using, for example, Biacore T100 (manufactured by GE Healthcare Bioscience), and can be calculated using attached software Biacore T100 evaluation software (manufactured by Biacore).
　本発明において、ＡＤＣＣ活性を有する抗体とは、ＣＤ３３発現細胞に対して、同時に複数の抗体のＡＤＣＣ活性を、公知の測定方法［Ｃａｎｃｅｒ　Ｉｍｍｕｎｏｌ．Ｉｍｍｕｎｏｔｈｅｒ．，３６，３７３（１９９３）］を用いて測定した場合、検出可能なＡＤＣＣ活性を有する抗体である。 In the present invention, an antibody having ADCC activity refers to a known measurement method [Cancer Immunol. Immunother. , 36, 373 (1993)], which is an antibody having detectable ADCC activity.
　本発明において、ＣＤＣ活性を有する抗体とは、ＣＤ３３発現細胞に対して、同時に複数の抗体のＣＤＣ活性を、ヒト補体を用いて公知のＣＤＣ測定方法［Ｃａｎｃｅｒ　Ｉｍｍｕｎｏｌ．Ｉｍｍｕｎｏｔｈｅｒ．，３６，３７３（１９９３）］を用いて測定した場合、検出可能なＣＤＣ活性を有する抗体である。 In the present invention, an antibody having CDC activity refers to the CDC activity of a plurality of antibodies at the same time against CD33-expressing cells using a known CDC measurement method [Cancer Immunol. Immunother. , 36, 373 (1993)], which is an antibody having detectable CDC activity.
　また、本発明のモノクローナル抗体としては、具体的には、下記（ｉ）～（ｖ）のいずれか１の抗体が結合するヒトＣＤ３３上のエピトープと同じエピトープに結合するモノクローナル抗体、下記（ｉ）～（ｖ）のいずれか１の抗体と競合してヒトＣＤ３３に結合するモノクローナル抗体、および下記（ｉ）～（ｖ）のいずれか１の抗体と同等の反応性を有する抗体等が挙げられる。
（ｉ）抗体のＶＨが配列番号１３２で表されるアミノ酸配列であり、かつ抗体のＶＬが配列番号１３７で表されるアミノ酸配列であるモノクローナル抗体
（ｉｉ）抗体のＶＨが配列番号１３３で表されるアミノ酸配列であり、かつ抗体のＶＬが配列番号１３８で表されるアミノ酸配列であるモノクローナル抗体
（ｉｉｉ）抗体のＶＨが配列番号１３４で表されるアミノ酸配列であり、かつ抗体のＶＬが配列番号１３９で表されるアミノ酸配列であるモノクローナル抗体
（ｉｖ）抗体のＶＨが配列番号１３５で表されるアミノ酸配列であり、かつ抗体のＶＬが配列番号１４０で表されるアミノ酸配列であるモノクローナル抗体
（ｖ）抗体のＶＨが配列番号１３６で表されるアミノ酸配列であり、かつ抗体のＶＬが配列番号１４１で表されるアミノ酸配列であるモノクローナル抗体が結合するヒトＣＤ３３上のエピトープに結合するモノクローナル抗体 The monoclonal antibody of the present invention specifically includes a monoclonal antibody that binds to the same epitope as that on human CD33 to which any one of the following antibodies (i) to (v) binds: Examples thereof include a monoclonal antibody that competes with any one of the antibodies (1) to (v) and binds to human CD33, and an antibody having the same reactivity as any one of the following antibodies (i) to (v).
(I) VH of an antibody is an amino acid sequence represented by SEQ ID NO: 132, and VL of an antibody is an amino acid sequence represented by SEQ ID NO: 137. (ii) VH of an antibody is represented by SEQ ID NO: 133 And the VL of the antibody is the amino acid sequence represented by SEQ ID NO: 138. The VH of the monoclonal antibody (iii) antibody is the amino acid sequence represented by SEQ ID NO: 134, and the VL of the antibody is SEQ ID NO: The monoclonal antibody (v) of the monoclonal antibody (iv) antibody having the amino acid sequence represented by 139 is the amino acid sequence represented by SEQ ID NO: 135, and the monoclonal antibody (v ) An amino acid sequence in which the VH of the antibody is an amino acid sequence represented by SEQ ID NO: 136 and the VL of the antibody is represented by SEQ ID NO: 141 Monoclonal antibody that binds to an epitope on human CD33 monoclonal antibody that is acid sequence binds
　また、本発明のモノクローナル抗体としては、下記（ｉ）～（ｖ）のいずれか１の抗体が結合するヒトＣＤ３３上のエピトープと同じエピトープに結合するモノクローナル抗体、下記（ｉ）～（ｖ）のいずれか１の抗体と競合してヒトＣＤ３３に結合するモノクローナル抗体、および下記（ｉ）～（ｖ）のいずれか１の抗体と同等の反応性を有する抗体等が挙げられる。
（ｉ）抗体のＶＨが配列番号１０３で表されるアミノ酸配列であり、かつ抗体のＶＬが配列番号９５で表されるアミノ酸配列であるモノクローナル抗体
（ｉｉ）抗体のＶＨが配列番号１０４で表されるアミノ酸配列であり、かつ抗体のＶＬが配列番号９５で表されるアミノ酸配列であるモノクローナル抗体
（ｉｉｉ）抗体のＶＨが配列番号１０５で表されるアミノ酸配列であり、かつ抗体のＶＬが配列番号９５で表されるアミノ酸配列であるモノクローナル抗体
（ｉｖ）抗体のＶＨが配列番号９３で表されるアミノ酸配列であり、かつ抗体のＶＬが配列番号９５で表されるアミノ酸配列であるモノクローナル抗体
（ｖ）抗体のＶＨが配列番号１０６で表されるアミノ酸配列であり、かつ抗体のＶＬが配列番号９５で表されるアミノ酸配列であるモノクローナル抗体 In addition, the monoclonal antibody of the present invention includes a monoclonal antibody that binds to the same epitope on human CD33 to which any one of the following antibodies (i) to (v) binds, and the following (i) to (v) Examples thereof include a monoclonal antibody that competes with any one of the antibodies and binds to human CD33, and an antibody having reactivity equivalent to any one of the following antibodies (i) to (v).
(I) VH of an antibody is an amino acid sequence represented by SEQ ID NO: 103, and VL of an antibody is an amino acid sequence represented by SEQ ID NO: 95. (ii) VH of an antibody is represented by SEQ ID NO: 104 And the VL of the antibody is the amino acid sequence represented by SEQ ID NO: 95. The VH of the monoclonal antibody (iii) antibody is the amino acid sequence represented by SEQ ID NO: 105, and the VL of the antibody is SEQ ID NO: The monoclonal antibody (v) of the monoclonal antibody (iv) having the amino acid sequence represented by 95 is the amino acid sequence represented by SEQ ID NO: 93, and the monoclonal antibody (v ) The VH of the antibody is the amino acid sequence represented by SEQ ID NO: 106, and the VL of the antibody is the amino acid sequence represented by SEQ ID NO: 95 Monoclonal antibody
　さらに本発明のモノクローナル抗体としては、以下の（ｉ）～（ｖ）のヒトＣＤ３３に結合するモノクローナル抗体が挙げられる。
（ｉ）配列番号３４で示されるアミノ酸配列を含むＣＤＲ１、配列番号３５で示されるアミノ酸配列を含むＣＤＲ２、および配列番号３６で示されるアミノ酸配列を含むＣＤＲ３を含む重鎖可変領域、並びに配列番号３７で示されるアミノ酸配列を含むＣＤＲ１、配列番号３８で示されるアミノ酸配列を含むＣＤＲ２、および配列番号３９で示されるアミノ酸配列を含むＣＤＲ３を含む軽鎖可変領域を含む、ヒトＣＤ３３に結合するモノクローナル抗体
（ｉｉ）配列番号４０で示されるアミノ酸配列を含むＣＤＲ１、配列番号４１で示されるアミノ酸配列を含むＣＤＲ２、および配列番号４２で示されるアミノ酸配列を含むＣＤＲ３を含む重鎖可変領域、並びに配列番号４３で示されるアミノ酸配列を含むＣＤＲ１、配列番号４４で示されるアミノ酸配列を含むＣＤＲ２、および配列番号４５で示されるアミノ酸配列を含むＣＤＲ３を含む軽鎖可変領域を含む、ヒトＣＤ３３に結合するモノクローナル抗体
（ｉｉｉ）配列番号４６で示されるアミノ酸配列を含むＣＤＲ１、配列番号４７で示されるアミノ酸配列を含むＣＤＲ２、および配列番号４８で示されるアミノ酸配列を含むＣＤＲ３を含む重鎖可変領域、並びに配列番号４９示されるアミノ酸配列を含むＣＤＲ１、配列番号５０で示されるアミノ酸配列を含むＣＤＲ２、および配列番号５１で示されるアミノ酸配列を含むＣＤＲ３を含む軽鎖可変領域を含む、ヒトＣＤ３３に結合するモノクローナル抗体
（ｉｖ）配列番号５２で示されるアミノ酸配列を含むＣＤＲ１、配列番号５３で示されるアミノ酸配列を含むＣＤＲ２、および配列番号５４で示されるアミノ酸配列を含むＣＤＲ３を含む重鎖可変領域、並びに配列番号５５示されるアミノ酸配列を含むＣＤＲ１、配列番号５６で示されるアミノ酸配列を含むＣＤＲ２、および配列番号５７で示されるアミノ酸配列を含むＣＤＲ３を含む軽鎖可変領域を含む、ヒトＣＤ３３に結合するモノクローナル抗体
（ｖ）配列番号５８で示されるアミノ酸配列を含むＣＤＲ１、配列番号５９で示されるアミノ酸配列を含むＣＤＲ２、および配列番号６０で示されるアミノ酸配列を含むＣＤＲ３を含む重鎖可変領域、並びに配列番号６１示されるアミノ酸配列を含むＣＤＲ１、配列番号６２で示されるアミノ酸配列を含むＣＤＲ２、および配列番号６３で示されるアミノ酸配列を含むＣＤＲ３を含む軽鎖可変領域を含む、ヒトＣＤ３３に結合するモノクローナル抗体 Furthermore, the monoclonal antibodies of the present invention include the following monoclonal antibodies that bind to human CD33 (i) to (v).
(I) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36, and SEQ ID NO: 37 A monoclonal antibody that binds to human CD33, comprising a light chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 38, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 38, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 39. ii) a heavy chain variable region comprising CDR1, comprising the amino acid sequence represented by SEQ ID NO: 40, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 41, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 42, and SEQ ID NO: 43 CDR1, comprising the amino acid sequence shown, represented by SEQ ID NO: 44 A monoclonal antibody (iii) that binds human CD33, comprising a light chain variable region comprising CDR2 comprising a amino acid sequence and CDR3 comprising an amino acid sequence represented by SEQ ID NO: 45, CDR1 comprising an amino acid sequence represented by SEQ ID NO: 46, A heavy chain variable region comprising CDR2 comprising the amino acid sequence represented by SEQ ID NO: 47, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 48, and a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 49, the amino acid represented by SEQ ID NO: 50 A monoclonal antibody that binds human CD33 (iv) comprising a CDR2 comprising a sequence and a light chain variable region comprising a CDR3 comprising an amino acid sequence represented by SEQ ID NO: 51, CDR1 comprising an amino acid sequence represented by SEQ ID NO: 52, SEQ ID NO: CDR2, comprising the amino acid sequence represented by 53, And a heavy chain variable region comprising CDR3 comprising the amino acid sequence represented by SEQ ID NO: 54, and CDR1 comprising the amino acid sequence represented by SEQ ID NO: 55, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 56, and SEQ ID NO: 57. A monoclonal antibody that binds to human CD33 comprising a light chain variable region comprising CDR3 comprising an amino acid sequence (v) a CDR1 comprising an amino acid sequence represented by SEQ ID NO: 58, a CDR2 comprising an amino acid sequence represented by SEQ ID NO: 59, and a sequence A heavy chain variable region comprising CDR3 comprising the amino acid sequence represented by number 60, and CDR1 comprising the amino acid sequence represented by SEQ ID NO: 61, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 62, and an amino acid sequence represented by SEQ ID NO: 63 A human CD comprising a light chain variable region comprising CDR3 comprising Monoclonal antibody binding to 33
　さらには、本発明のモノクローナル抗体には、配列番号５２で示されるアミノ酸配列を含むＣＤＲ１、配列番号５３で示されるアミノ酸配列を含むＣＤＲ２、および配列番号５４で示されるアミノ酸配列を含むＣＤＲ３を含む重鎖可変領域、並びに配列番号５５で示されるアミノ酸配列を含むＣＤＲ１、配列番号５６で示されるアミノ酸配列を含むＣＤＲ２、および配列番号５７で示されるアミノ酸配列を含むＣＤＲ３を含む軽鎖可変領域を含む、ヒトＣＤ３３に結合するモノクローナル抗体のＶ領域にＮ結合型糖鎖が結合するコンセンサス配列を有さないように改変した抗体（以下、改変型抗体と表記することがある）も包含される。 Furthermore, the monoclonal antibody of the present invention comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 52, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 53, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 54. A chain variable region, and a light chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 55, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 56, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 57, An antibody modified so as not to have a consensus sequence for binding an N-linked sugar chain to the V region of a monoclonal antibody that binds to human CD33 (hereinafter sometimes referred to as a modified antibody) is also included.
　本発明のモノクローナル抗体としては、具体的には、抗体のＶＨのＣＤＲ１およびＣＤＲ３が、それぞれ配列番号５２および５４で示されるアミノ酸配列を含み、かつ、抗体のＶＬのＣＤＲ１、ＣＤＲ２およびＣＤＲ３が、それぞれ配列番号５５、５６および５７で示されるアミノ酸配列を含むモノクローナル抗体が挙げられる。 Specifically, as the monoclonal antibody of the present invention, CDR1 and CDR3 of VH of the antibody include the amino acid sequences shown by SEQ ID NOs: 52 and 54, respectively, and CDR1, CDR2 and CDR3 of VL of the antibody are respectively And monoclonal antibodies comprising the amino acid sequences shown in SEQ ID NOs: 55, 56 and 57.
　上記の抗体のＶＨのＣＤＲ２としては、改変型抗体のＶＨのＣＤＲ２であって、例えば、配列番号５３で示されるアミノ酸配列の３番目のＡｓｎを他のアミノ酸残基に置換する改変、および５番目のＳｅｒを他のアミノ酸残基に置換する改変のうち、少なくとも１つの改変が導入されたアミノ酸配列を含むＣＤＲ２が挙げられる。 The VH CDR2 of the above antibody is the CDR2 of the modified antibody VH, for example, a modification in which the third Asn in the amino acid sequence represented by SEQ ID NO: 53 is substituted with another amino acid residue, and the fifth CDR2 containing an amino acid sequence in which at least one modification is introduced among the modifications that substitute Ser in the other amino acid residues.
　具体的には、例えば、以下の（１）～（５）のアミノ酸配列などが挙げられる。
（１）配列番号５３で示されるアミノ酸配列の３番目のＡｓｎをＴｙｒに置換した配列番号９９で表されるアミノ酸配列
（２）配列番号５３で示されるアミノ酸配列の３番目のＡｓｎをＳｅｒに置換した配列番号１００で表されるアミノ酸配列
（３）配列番号５３で示されるアミノ酸配列の３番目のＡｓｎをＧｌｎに置換した配列番号１０１で表されるアミノ酸配列
（４）配列番号５３で示されるアミノ酸配列の５番目のＳｅｒをＡｌａに置換した配列番号９６で表されるアミノ酸配列
（５）配列番号５３で示されるアミノ酸配列の５番目のＳｅｒをＧｌｙに置換した配列番号１０２で表されるアミノ酸配列 Specific examples include the following amino acid sequences (1) to (5).
(1) The amino acid sequence represented by SEQ ID NO: 99 in which the third Asn of the amino acid sequence represented by SEQ ID NO: 53 is substituted with Tyr (2) The third Asn of the amino acid sequence represented by SEQ ID NO: 53 is substituted with Ser The amino acid sequence represented by SEQ ID NO: 100 (3) The amino acid sequence represented by SEQ ID NO: 101 in which the third Asn of the amino acid sequence represented by SEQ ID NO: 53 was replaced with Gln (4) The amino acid represented by SEQ ID NO: 53 An amino acid sequence represented by SEQ ID NO: 96 in which the fifth Ser of the sequence is substituted with Ala (5) An amino acid sequence represented by SEQ ID NO: 102 in which the fifth Ser of the amino acid sequence represented by SEQ ID NO: 53 is substituted with Gly
　ハイブリドーマは、例えば上記のＣＤ３３を発現した細胞などを抗原として調製し、該抗原を免疫した動物より抗原特異性を有する抗体生産細胞を誘導し、さらに、該抗体生産細胞と骨髄腫細胞とを融合させることにより、調製することができる。該ハイブリドーマを培養するか、または該ハイブリドーマを動物に投与して該動物を腹水癌化させ、該培養液または腹水から分離、精製することにより抗ＣＤ３３モノクローナル抗体を取得することができる。 The hybridoma is prepared by using, for example, cells expressing the above-mentioned CD33 as an antigen, inducing antibody-producing cells having antigen specificity from an animal immunized with the antigen, and further fusing the antibody-producing cells and myeloma cells. Can be prepared. The anti-CD33 monoclonal antibody can be obtained by culturing the hybridoma or administering the hybridoma to an animal to cause the animal to undergo ascites cancer, and separating and purifying the culture medium or ascites.
　抗原を免疫する動物としてはハイブ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が、好ましくはマウス、ラット、ハムスター、ニワトリ、ラビットおよびウシなどが用いられる。また、このような動物から抗体産生能を有する細胞を取得し、該細胞にｉｎ　ｖｉｔｒｏで免疫を施した後に、骨髄腫細胞と融合して作製したハイブリドーマが生産する抗体なども本発明の抗体に包含される。 Any animal can be used as the animal to immunize with the antigen as long as it can produce a hybridoma, but mice, rats, hamsters, chickens, rabbits, cows and the like are preferably used. An antibody produced by a hybridoma prepared by obtaining a cell capable of producing an antibody from such an animal, immunizing the cell in vitro, and then fusing it with a myeloma cell is also an antibody of the present invention. Is included.
　本発明において遺伝子組換え抗体としては、例えば、ヒト型キメラ抗体、ヒト型ＣＤＲ移植抗体（ヒト化抗体という場合もある）、ヒト抗体または抗体断片など、遺伝子組換えにより製造される抗体が挙げられる。 Examples of the recombinant antibody in the present invention include antibodies produced by genetic recombination, such as human chimeric antibodies, human CDR-grafted antibodies (sometimes referred to as humanized antibodies), human antibodies or antibody fragments. .
　遺伝子組換え抗体において、モノクローナル抗体の特徴を有し、抗原性が低く、血中半減期が延長されたものは、治療剤として好ましい。遺伝子組換え抗体は、例えば、上記本発明のモノクローナル抗体を遺伝子組換え技術を用いて改変したものが挙げられる。 A recombinant antibody having characteristics of a monoclonal antibody, low antigenicity, and extended blood half-life is preferable as a therapeutic agent. Examples of the recombinant antibody include those obtained by modifying the monoclonal antibody of the present invention using a gene recombination technique.
　ヒト型キメラ抗体は、ヒト以外の動物の抗体のＶＨおよびＶＬとヒト抗体の重鎖定常領域（以下、ＣＨと表記することがある）および軽鎖定常領域（以下、ＣＬと表記することがある）とからなる抗体をいう。 The human chimeric antibody is sometimes referred to as VH and VL of a non-human animal antibody, heavy chain constant region of human antibody (hereinafter sometimes referred to as CH) and light chain constant region (hereinafter referred to as CL). ).
　本発明のヒト型キメラ抗体は、ＣＤ３３を特異的に認識し、かつＣＤ３３の細胞外領域に結合するモノクローナル抗体を産生するハイブリドーマより、ＶＨおよびＶＬをコードするｃＤＮＡを取得し、ヒト抗体のＣＨおよびＣＬをコー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、動物細胞へ導入することにより発現させ、製造することができる。 The human chimeric antibody of the present invention obtains cDNAs encoding VH and VL from a hybridoma that specifically recognizes CD33 and produces a monoclonal antibody that binds to the extracellular region of CD33. A human chimeric antibody expression vector can be constructed by inserting into an expression vector for animal cells having a gene encoding CL, and introduced into animal cells for expression and production.
　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、ｈＩｇと表記することがある）に属すればいかなるものでもよいが、好ましくはｈＩｇＧクラスのものが用いられ、さらにｈＩｇＧクラスに属するｈＩｇＧ１、ｈＩｇＧ２、ｈＩｇＧ３または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、ｈＩｇに属すればいずれのものでもよく、κクラスまたはλクラスのものを用いることができる。 The CH of the human chimeric antibody may be any as long as it belongs to human immunoglobulin (hereinafter sometimes referred to as hIg), but preferably the hIgG class is used, and hIgG1, which belongs to the hIgG class, Any of the subclasses such as hIgG2, hIgG3 or hIgG4 can be used. The CL of the human chimeric antibody may be any as long as it belongs to hIg, and those of κ class or λ class can be used.
　更に、本発明のキメラ抗体としては、上述のモノクローナル抗体と競合して、ＣＤ３３の細胞外領域に結合するキメラ抗体および上述のキメラ抗体が結合するＣＤ３３の細胞外領域上に存在するエピトープと、同じエピトープに結合するキメラ抗体を挙げることができる。 Furthermore, the chimeric antibody of the present invention is the same as the chimeric antibody that binds to the extracellular region of CD33 and the epitope present on the extracellular region of CD33 to which the chimeric antibody binds by competing with the monoclonal antibody described above. Mention may be made of chimeric antibodies that bind to an epitope.
　ヒト型ＣＤＲ移植抗体は、ヒト以外の動物の抗体のＶＨおよびＶＬ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いう。本発明のヒト型ＣＤＲ移植抗体は、ＣＤ３３を特異的に認識し、かつ該細胞外領域に結合するヒト以外の動物のモノクローナル抗体を産生するハイブリドーマから産生されるヒト以外の動物の抗体のＶＨおよびＶＬのＣＤＲのアミノ酸配列を任意のヒト抗体のＶＨおよびＶＬのフレームワーク領域（以下、ＦＲと表記することがある）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ＣＬをコー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ＣＤＲ移植抗体発現ベクターを構築し、動物細胞へ導入することにより発現させ、製造することができる。 The human CDR-grafted antibody refers to an antibody obtained by grafting the VH and VL CDR amino acid sequences of non-human animal antibodies to appropriate positions of the human antibody VH and VL. The human CDR-grafted antibody of the present invention specifically recognizes V33 of a non-human animal antibody produced from a hybridoma that specifically recognizes CD33 and produces a monoclonal antibody of a non-human animal that binds to the extracellular region. A cDNA encoding the V region in which the amino acid sequence of the CDR of the VL was grafted to the VH and VL framework regions of the human antibody (hereinafter sometimes referred to as FR) was constructed, and the CH and CL of the human antibody were A human CDR-grafted antibody expression vector can be constructed by inserting it into an expression vector for animal cells having a gene to be encoded, and introduced into animal cells for expression and production.
　ヒト型ＣＤＲ移植抗体のＣＨとしては、ｈＩｇに属すればいかなるものでもよいが、好ましくはｈＩｇＧクラスのものが用いられ、さらにｈＩｇＧクラスに属するｈＩｇＧ１、ｈＩｇＧ２、ｈＩｇＧ３、または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ＣＤＲ移植抗体のＣＬとしては、ｈＩｇに属すればいずれのものでもよく、κクラスまたはλクラスのものを用いることができる。 The CH of the human CDR-grafted antibody may be any as long as it belongs to hIg, but preferably the hIgG class is used, and any subclass such as hIgG1, hIgG2, hIgG3, or hIgG4 belonging to the hIgG class is used. be able to. The CL of the human CDR-grafted antibody may be any CL as long as it belongs to hIg, and those of κ class or λ class can be used.
　本発明のヒト型ＣＤＲ移植抗体としては、具体的には、例えば、抗体のＶＨが、配列番号８４で表されるアミノ酸配列、または配列番号８４で表されるアミノ酸配列中の２５番目のＳｅｒ、２７番目のＧｌｙ、２８番目のＳｅｒ、２９番目のＶａｌ、３０番目のＳｅｒ、４０番目のＧｌｎ、４５番目のＧｌｙ、４６番目のＬｅｕ、４９番目のＩｌｅ、７２番目のＶａｌ、９３番目のＶａｌ、９５番目のＴｙｒ、９７番目のＡｌａおよび１０６番目のＴｈｒから選ばれる少なくとも１つのアミノ酸残基が他のアミノ酸残基に置換されたアミノ酸配列を含み、および／または、抗体のＶＬが、配列番号８５で表されるアミノ酸配列、または配列番号８５で表されるアミノ酸配列中の２番目のＩｌｅ、８番目のＰｒｏ、１１番目のＬｅｕ、１５番目のＰｒｏおよび９０番目のＶａｌから選ばれる少なくとも１つのアミノ酸残基が他のアミノ酸残基に置換されたアミノ酸配列を含む、ヒト化抗体などが挙げられるが、導入される改変の数に制限はない。 As the human CDR-grafted antibody of the present invention, specifically, for example, the VH of the antibody has the amino acid sequence represented by SEQ ID NO: 84 or the 25th Ser in the amino acid sequence represented by SEQ ID NO: 84, 27th Gly, 28th Ser, 29th Val, 30th Ser, 40th Gln, 45th Gly, 46th Leu, 49th Ile, 72nd Val, 93th Val, An amino acid sequence in which at least one amino acid residue selected from the 95th Tyr, the 97th Ala and the 106th Thr is substituted with another amino acid residue, and / or the VL of the antibody is SEQ ID NO: 85; Or the second Ile in the amino acid sequence represented by SEQ ID NO: 85, the 8th Pro, the 11th Leu, the 15th And a humanized antibody including an amino acid sequence in which at least one amino acid residue selected from Pro and 90th Val is substituted with another amino acid residue, but the number of modifications to be introduced is not limited .
　例えば、以下に示すヒト化抗体が挙げられる。
　抗体のＶＨが配列番号８４で表されるアミノ酸配列中の２５番目のＳｅｒ、２７番目のＧｌｙ、２８番目のＳｅｒ、２９番目のＶａｌ、３０番目のＳｅｒ、４０番目のＧｌｎ、４５番目のＧｌｙ、４６番目のＬｅｕ、４９番目のＩｌｅ、７２番目のＶａｌ、９３番目のＶａｌ、９５番目のＴｙｒ、９７番目のＡｌａおよび１０６番目のＴｈｒから選ばれる少なくとも１のアミノ酸残基が、他のアミノ酸残基に置換されたアミノ酸配列を含むヒト化抗体。 For example, the following humanized antibodies can be mentioned.
The VH of the antibody is the 25th Ser, 27th Gly, 28th Ser, 29th Val, 30th Ser, 40th Gln, 45th Gly in the amino acid sequence represented by SEQ ID NO: 84, At least one amino acid residue selected from 46th Leu, 49th Ile, 72nd Val, 93th Val, 95th Tyr, 97th Ala and 106th Thr is another amino acid residue A humanized antibody comprising an amino acid sequence substituted with.
　前記ヒト化抗体の中でも、以下の（１）～（３）が好ましい。
（１）抗体のＶＨが配列番号８４で表されるアミノ酸配列中の２７番目のＧｌｙ、４０番目のＧｌｎ、４５番目のＧｌｙ、４６番目のＬｅｕ、４９番目のＩｌｅ、７２番目のＶａｌ、および９７番目のＡｌａが、他のアミノ酸残基に置換されたアミノ酸配列を含むヒト化抗体
（２）抗体のＶＨが配列番号８４で表されるアミノ酸配列中の２７番目のＧｌｙ、２８番目のＳｅｒ、２９番目のＶａｌ、３０番目のＳｅｒ、７２番目のＶａｌ、および９７番目のＡｌａが、他のアミノ酸残基に置換されたアミノ酸配列を含むヒト化抗体
（３）抗体のＶＨが配列番号８４で表されるアミノ酸配列中の２７番目のＧｌｙ、および７２番目のＶａｌが、他のアミノ酸残基に置換されたアミノ酸配列を含むヒト化抗体 Among the humanized antibodies, the following (1) to (3) are preferable.
(1) 27th Gly, 40th Gln, 45th Gly, 46th Leu, 49th Ile, 72nd Val, and 97 in the amino acid sequence represented by SEQ ID NO: 84 as the VH of the antibody The humanized antibody comprising the amino acid sequence in which the No. Ala is substituted with another amino acid residue (2) 27th Gly, 28th Ser, 29 in the amino acid sequence in which the VH of the antibody is represented by SEQ ID NO: 84 The VH of the humanized antibody (3) antibody comprising the amino acid sequence in which the No. Val, the 30th Ser, the 72nd Val, and the 97th Ala are substituted with other amino acid residues is represented by SEQ ID NO: 84 Humanized antibody comprising an amino acid sequence in which the 27th Gly and the 72nd Val in the amino acid sequence are substituted with other amino acid residues
　上記のアミノ酸改変の結果、得られる抗体のＶＨのアミノ酸配列としては、例えば、配列番号８４で表されるアミノ酸配列中の２５番目のＳｅｒをＴｈｒに、２７番目のＧｌｙをＴｙｒに、２８番目のＳｅｒをＴｈｒに、２９番目のＶａｌをＩｌｅに、３０番目のＳｅｒをＴｈｒに、４０番目のＧｌｎをＬｙｓに、４５番目のＧｌｙをＡｒｇに、４６番目のＬｅｕをＭｅｔに、４９番目のＩｌｅをＭｅｔに、７２番目のＶａｌをＡｒｇに、９３番目のＶａｌをＴｈｒに、９５番目のＴｙｒをＰｈｅに、９７番目のＡｌａをＴｈｒに、および１０６番目のＴｈｒをＶａｌに置換する改変から選ばれる少なくとも１つの改変が導入されたアミノ酸配列が挙げられる。 As a result of the above amino acid modification, the amino acid sequence of the VH of the obtained antibody is, for example, the 25th Ser in the amino acid sequence represented by SEQ ID NO: 84, the 27th Gly in the Tyr, and the 28th in the amino acid sequence. Ser to Thr, 29th Val to Ile, 30th Ser to Thr, 40th Gln to Lys, 45th Gly to Arg, 46th Leu to Met, 49th Ile Met, at least selected from a modification in which the 72nd Val is replaced with Arg, the 93rd Val is replaced with Thr, the 95th Tyr is replaced with Phe, the 97th Ala is replaced with Thr, and the 106th Thr is replaced with Val. An amino acid sequence into which one modification has been introduced is mentioned.
　１４個の改変が導入されたＶＨのアミノ酸配列としては、具体的には、例えば、配列番号８４で表されるアミノ酸配列中の２５番目のＳｅｒをＴｈｒに、２７番目のＧｌｙをＴｙｒに、２８番目のＳｅｒをＴｈｒに、２９番目のＶａｌをＩｌｅに、３０番目のＳｅｒをＴｈｒに、４０番目のＧｌｎをＬｙｓに、４５番目のＧｌｙをＡｒｇに、４６番目のＬｅｕをＭｅｔに、４９番目のＩｌｅをＭｅｔに、７２番目のＶａｌをＡｒｇに、９３番目のＶａｌをＴｈｒに、９５番目のＴｙｒをＰｈｅに、９７番目のＡｌａをＴｈｒに、および１０６番目のＴｈｒをＶａｌに置換したアミノ酸配列が挙げられる。 Specific examples of the amino acid sequence of VH into which 14 modifications have been introduced include, for example, 25th Ser in the amino acid sequence represented by SEQ ID NO: 84, Thr, 27th Gly in Tyr, 28 The th Ser, Thr, the 29th Val to Ile, the 30th Ser to Thr, the 40th Gln to Lys, the 45th Gly to Arg, the 46th Leu to Met, and the 49th An amino acid sequence in which Ile is replaced by Met, 72nd Val is replaced by Arg, 93rd Val is replaced by Thr, 95th Tyr is replaced by Phe, 97th Ala is replaced by Thr, and 106th Thr is replaced by Val. Can be mentioned.
　１３個の改変が導入されたＶＨのアミノ酸配列としては、具体的には、例えば、以下の（１）～（１４）のアミノ酸配列が挙げられる。
（１）配列番号８４で表されるアミノ酸配列中の２７番目のＧｌｙをＴｙｒに、２８番目のＳｅｒをＴｈｒに、２９番目のＶａｌをＩｌｅに、３０番目のＳｅｒをＴｈｒに、４０番目のＧｌｎをＬｙｓに、４５番目のＧｌｙをＡｒｇに、４６番目のＬｅｕをＭｅｔに、４９番目のＩｌｅをＭｅｔに、７２番目のＶａｌをＡｒｇに、９３番目のＶａｌをＴｈｒに、９５番目のＴｙｒをＰｈｅに、９７番目のＡｌａをＴｈｒに、および１０６番目のＴｈｒをＶａｌに置換したアミノ酸配列
（２）配列番号８４で表されるアミノ酸配列中の２５番目のＳｅｒをＴｈｒに、２８番目のＳｅｒをＴｈｒに、２９番目のＶａｌをＩｌｅに、３０番目のＳｅｒをＴｈｒに、４０番目のＧｌｎをＬｙｓに、４５番目のＧｌｙをＡｒｇに、４６番目のＬｅｕをＭｅｔに、４９番目のＩｌｅをＭｅｔに、７２番目のＶａｌをＡｒｇに、９３番目のＶａｌをＴｈｒに、９５番目のＴｙｒをＰｈｅに、９７番目のＡｌａをＴｈｒに、および１０６番目のＴｈｒをＶａｌに置換したアミノ酸配列
（３）配列番号８４で表されるアミノ酸配列中の２５番目のＳｅｒをＴｈｒに、２７番目のＧｌｙをＴｙｒに、２９番目のＶａｌをＩｌｅに、３０番目のＳｅｒをＴｈｒに、４０番目のＧｌｎをＬｙｓに、４５番目のＧｌｙをＡｒｇに、４６番目のＬｅｕをＭｅｔに、４９番目のＩｌｅをＭｅｔに、７２番目のＶａｌをＡｒｇに、９３番目のＶａｌをＴｈｒに、９５番目のＴｙｒをＰｈｅに、９７番目のＡｌａをＴｈｒに、および１０６番目のＴｈｒをＶａｌに置換したアミノ酸配列
（４）配列番号８４で表されるアミノ酸配列中の２５番目のＳｅｒをＴｈｒに、２７番目のＧｌｙをＴｙｒに、２８番目のＳｅｒをＴｈｒに、３０番目のＳｅｒをＴｈｒに、４０番目のＧｌｎをＬｙｓに、４５番目のＧｌｙをＡｒｇに、４６番目のＬｅｕをＭｅｔに、４９番目のＩｌｅをＭｅｔに、７２番目のＶａｌをＡｒｇに、９３番目のＶａｌをＴｈｒに、９５番目のＴｙｒをＰｈｅに、９７番目のＡｌａをＴｈｒに、および１０６番目のＴｈｒをＶａｌに置換したアミノ酸配列
（５）配列番号８４で表されるアミノ酸配列中の２５番目のＳｅｒをＴｈｒに、２７番目のＧｌｙをＴｙｒに、２８番目のＳｅｒをＴｈｒに、２９番目のＶａｌをＩｌｅに、４０番目のＧｌｎをＬｙｓに、４５番目のＧｌｙをＡｒｇに、４６番目のＬｅｕをＭｅｔに、４９番目のＩｌｅをＭｅｔに、７２番目のＶａｌをＡｒｇに、９３番目のＶａｌをＴｈｒに、９５番目のＴｙｒをＰｈｅに、９７番目のＡｌａをＴｈｒに、および１０６番目のＴｈｒをＶａｌに置換したアミノ酸配列
（６）配列番号８４で表されるアミノ酸配列中の２５番目のＳｅｒをＴｈｒに、２７番目のＧｌｙをＴｙｒに、２８番目のＳｅｒをＴｈｒに、２９番目のＶａｌをＩｌｅに、３０番目のＳｅｒをＴｈｒに、４５番目のＧｌｙをＡｒｇに、４６番目のＬｅｕをＭｅｔに、４９番目のＩｌｅをＭｅｔに、７２番目のＶａｌをＡｒｇに、９３番目のＶａｌをＴｈｒに、９５番目のＴｙｒをＰｈｅに、９７番目のＡｌａをＴｈｒに、および１０６番目のＴｈｒをＶａｌに置換したアミノ酸配列
（７）配列番号８４で表されるアミノ酸配列中の２５番目のＳｅｒをＴｈｒに、２７番目のＧｌｙをＴｙｒに、２８番目のＳｅｒをＴｈｒに、２９番目のＶａｌをＩｌｅに、３０番目のＳｅｒをＴｈｒに、４０番目のＧｌｎをＬｙｓに、４６番目のＬｅｕをＭｅｔに、４９番目のＩｌｅをＭｅｔに、７２番目のＶａｌをＡｒｇに、９３番目のＶａｌをＴｈｒに、９５番目のＴｙｒをＰｈｅに、９７番目のＡｌａをＴｈｒに、および１０６番目のＴｈｒをＶａｌに置換したアミノ酸配列
（８）配列番号８４で表されるアミノ酸配列中の２５番目のＳｅｒをＴｈｒに、２７番目のＧｌｙをＴｙｒに、２８番目のＳｅｒをＴｈｒに、２９番目のＶａｌをＩｌｅに、３０番目のＳｅｒをＴｈｒに、４０番目のＧｌｎをＬｙｓに、４５番目のＧｌｙをＡｒｇに、４９番目のＩｌｅをＭｅｔに、７２番目のＶａｌをＡｒｇに、９３番目のＶａｌをＴｈｒに、９５番目のＴｙｒをＰｈｅに、９７番目のＡｌａをＴｈｒに、および１０６番目のＴｈｒをＶａｌに置換したアミノ酸配列
（９）配列番号８４で表されるアミノ酸配列中の２５番目のＳｅｒをＴｈｒに、２７番目のＧｌｙをＴｙｒに、２８番目のＳｅｒをＴｈｒに、２９番目のＶａｌをＩｌｅに、３０番目のＳｅｒをＴｈｒに、４０番目のＧｌｎをＬｙｓに、４５番目のＧｌｙをＡｒｇに、４６番目のＬｅｕをＭｅｔに、７２番目のＶａｌをＡｒｇに、９３番目のＶａｌをＴｈｒに、９５番目のＴｙｒをＰｈｅに、９７番目のＡｌａをＴｈｒに、および１０６番目のＴｈｒをＶａｌに置換したアミノ酸配列
（１０）配列番号８４で表されるアミノ酸配列中の２５番目のＳｅｒをＴｈｒに、２７番目のＧｌｙをＴｙｒに、２８番目のＳｅｒをＴｈｒに、２９番目のＶａｌをＩｌｅに、３０番目のＳｅｒをＴｈｒに、４０番目のＧｌｎをＬｙｓに、４５番目のＧｌｙをＡｒｇに、４６番目のＬｅｕをＭｅｔに、４９番目のＩｌｅをＭｅｔに、９３番目のＶａｌをＴｈｒに、９５番目のＴｙｒをＰｈｅに、９７番目のＡｌａをＴｈｒに、および１０６番目のＴｈｒをＶａｌに置換したアミノ酸配列
（１１）配列番号８４で表されるアミノ酸配列中の２５番目のＳｅｒをＴｈｒに、２７番目のＧｌｙをＴｙｒに、２８番目のＳｅｒをＴｈｒに、２９番目のＶａｌをＩｌｅに、３０番目のＳｅｒをＴｈｒに、４０番目のＧｌｎをＬｙｓに、４５番目のＧｌｙをＡｒｇに、４６番目のＬｅｕをＭｅｔに、４９番目のＩｌｅをＭｅｔに、７２番目のＶａｌをＡｒｇに、９５番目のＴｙｒをＰｈｅに、９７番目のＡｌａをＴｈｒに、および１０６番目のＴｈｒをＶａｌに置換したアミノ酸配列
（１２）配列番号８４で表されるアミノ酸配列中の２５番目のＳｅｒをＴｈｒに、２７番目のＧｌｙをＴｙｒに、２８番目のＳｅｒをＴｈｒに、２９番目のＶａｌをＩｌｅに、３０番目のＳｅｒをＴｈｒに、４０番目のＧｌｎをＬｙｓに、４５番目のＧｌｙをＡｒｇに、４６番目のＬｅｕをＭｅｔに、４９番目のＩｌｅをＭｅｔに、７２番目のＶａｌをＡｒｇに、９３番目のＶａｌをＴｈｒに、９７番目のＡｌａをＴｈｒに、および１０６番目のＴｈｒをＶａｌに置換したアミノ酸配列
（１３）配列番号８４で表されるアミノ酸配列中の２５番目のＳｅｒをＴｈｒに、２７番目のＧｌｙをＴｙｒに、２８番目のＳｅｒをＴｈｒに、２９番目のＶａｌをＩｌｅに、３０番目のＳｅｒをＴｈｒに、４０番目のＧｌｎをＬｙｓに、４５番目のＧｌｙをＡｒｇに、４６番目のＬｅｕをＭｅｔに、４９番目のＩｌｅをＭｅｔに、７２番目のＶａｌをＡｒｇに、９３番目のＶａｌをＴｈｒに、９５番目のＴｙｒをＰｈｅに、および１０６番目のＴｈｒをＶａｌに置換したアミノ酸配列
（１４）配列番号８４で表されるアミノ酸配列中の２５番目のＳｅｒをＴｈｒに、２７番目のＧｌｙをＴｙｒに、２８番目のＳｅｒをＴｈｒに、２９番目のＶａｌをＩｌｅに、３０番目のＳｅｒをＴｈｒに、４０番目のＧｌｎをＬｙｓに、４５番目のＧｌｙをＡｒｇに、４６番目のＬｅｕをＭｅｔに、４９番目のＩｌｅをＭｅｔに、７２番目のＶａｌをＡｒｇに、９３番目のＶａｌをＴｈｒに、９５番目のＴｙｒをＰｈｅに、および９７番目のＡｌａをＴｈｒに置換したアミノ酸配列 Specific examples of the amino acid sequence of VH into which 13 modifications have been introduced include the following amino acid sequences (1) to (14).
(1) The 27th Gly in the amino acid sequence represented by SEQ ID NO: 84 is Tyr, the 28th Ser is Thr, the 29th Val is Ile, the 30th Ser is Thr, and the 40th Gln To Lys, 45th Gly to Arg, 46th Leu to Met, 49th Ile to Met, 72nd Val to Arg, 93rd Val to Thr, 95th Tyr to Phe In the amino acid sequence represented by SEQ ID NO: 84, the 97th Ala is replaced with Thr and the 106th Thr is replaced with Val. The 25th Ser in the amino acid sequence represented by SEQ ID NO: 84 is Thr, and the 28th Ser is Thr. 29th Val to Ile, 30th Ser to Thr, 40th Gln to Lys, 45th Gly to Arg, 46th Leu to M t, 49th Ile to Met, 72nd Val to Arg, 93rd Val to Thr, 95th Tyr to Phe, 97th Ala to Thr, and 106th Thr to Val (3) In the amino acid sequence represented by SEQ ID NO: 84, the 25th Ser in Thr, the 27th Gly in Tyr, the 29th Val in Ile, and the 30th Ser in Thr 40th Gln to Lys, 45th Gly to Arg, 46th Leu to Met, 49th Ile to Met, 72nd Val to Arg, 93th Val to Thr, 95 An amino acid sequence in which the thirr is replaced with Phe, the 97th Ala with Thr, and the 106th thr with Val (4) The 25th Ser in the no acid sequence is Thr, the 27th Gly is Tyr, the 28th Ser is Thr, the 30th Ser is Thr, the 40th Gln is Lys, and the 45th Gly is Arg, 46th Leu to Met, 49th Ile to Met, 72nd Val to Arg, 93rd Val to Thr, 95th Tyr to Phe, 97th Ala to Thr , And the amino acid sequence in which the 106th Thr is substituted with Val (5), the 25th Ser in the amino acid sequence represented by SEQ ID NO: 84, the 27th Gly in the Tyr, and the 28th Ser in the Thr 29th Val to Ile, 40th Gln to Lys, 45th Gly to Arg, 46th Leu to Met, 49th Ile to Met The amino acid sequence (6) in which the 72nd Val is replaced with Arg, the 93rd Val is replaced with Thr, the 95th Tyr is replaced with Phe, the 97th Ala is replaced with Thr, and the 106th Thr is replaced with Val. In the amino acid sequence represented by number 84, the 25th Ser is set to Thr, the 27th Gly to Tyr, the 28th Ser to Thr, the 29th Val to Ile, the 30th Ser to Thr, 45th Gly to Arg, 46th Leu to Met, 49th Ile to Met, 72nd Val to Arg, 93rd Val to Thr, 95th Tyr to Phe, 97th Amino acid sequence in which Ala is replaced with Thr, and Thr at 106th is replaced with Val (7) The 25th Ser in the amino acid sequence represented by SEQ ID NO: 84 is hr, 27th Gly to Tyr, 28th Ser to Thr, 29th Val to Ile, 30th Ser to Thr, 40th Gln to Lys, 46th Leu to Met The 49th Ile is replaced by Met, the 72nd Val is replaced by Arg, the 93rd Val is replaced by Thr, the 95th Tyr is replaced by Phe, the 97th Ala is replaced by Thr, and the 106th Thr is replaced by Val. In the amino acid sequence represented by SEQ ID NO: 84, the 25th Ser in Thr, the 27th Gly in Tyr, the 28th Ser in Thr, the 29th Val in Ile, 30 The 41st Ser to Thr, the 40th Gln to Lys, the 45th Gly to Arg, the 49th Ile to Met, and the 72nd Val to Arg An amino acid sequence in which the 93rd Val is replaced with Thr, the 95th Tyr is replaced with Phe, the 97th Ala is replaced with Thr, and the 106th Thr is replaced with Val (9) in the amino acid sequence represented by SEQ ID NO: 84 25th Ser to Thr, 27th Gly to Tyr, 28th Ser to Thr, 29th Val to Ile, 30th Ser to Thr, 40th Gln to Lys, 45th Gly in Arg, 46th Leu in Met, 72nd Val in Arg, 93rd Val in Thr, 95th Tyr in Phe, 97th Ala in Thr, and 106th Amino acid sequence in which Thr is replaced with Val (10) The 25th Ser in the amino acid sequence represented by SEQ ID NO: 84 is Thr, and the 27th Gly is T yr, 28th Ser to Thr, 29th Val to Ile, 30th Ser to Thr, 40th Gln to Lys, 45th Gly to Arg, 46th Leu to Met Amino acid sequence in which 49th Ile is replaced by Met, 93rd Val is replaced by Thr, 95th Tyr is replaced by Phe, 97th Ala is replaced by Thr, and 106th Thr is replaced by Val (11) In the amino acid sequence represented by 84, the 25th Ser is Thr, the 27th Gly is Tyr, the 28th Ser is Thr, the 29th Val is Ile, the 30th Ser is Thr, 40 The 41st Gln is Lys, the 45th Gly is Arg, the 46th Leu is Met, the 49th Ile is Met, and the 72nd Val is Arg. The amino acid sequence in which the 95th Tyr is substituted with Phe, the 97th Ala with Thr, and the 106th Thr with Val (12), the 25th Ser in the amino acid sequence represented by SEQ ID NO: 84, with Thr, 27th Gly as Tyr, 28th Ser as Thr, 29th Val as Ile, 30th Ser as Thr, 40th Gln as Lys, 45th Gly as Arg, 46th Amino acid sequence in which Leu is replaced with Met, 49th Ile is replaced with Met, 72nd Val is replaced with Arg, 93rd Val is replaced with Thr, 97th Ala is replaced with Thr, and 106th Thr is replaced with Val. (13) The 25th Ser in the amino acid sequence represented by SEQ ID NO: 84 is Thr, the 27th Gly is Tyr, and the 28th Ser Thr, 29th Val to Ile, 30th Ser to Thr, 40th Gln to Lys, 45th Gly to Arg, 46th Leu to Met, 49th Ile to Met An amino acid sequence in which the 72nd Val is replaced with Arg, the 93rd Val is replaced with Thr, the 95th Tyr is replaced with Phe, and the 106th Thr is replaced with Val (14) in the amino acid sequence represented by SEQ ID NO: 84 25th Ser to Thr, 27th Gly to Tyr, 28th Ser to Thr, 29th Val to Ile, 30th Ser to Thr, 40th Gln to Lys, 45 The 41st Gly is Arg, the 46th Leu is Met, the 49th Ile is Met, the 72nd Val is Arg, and the 93rd Val is Thr. Amino acid sequence in which the 95th Tyr is substituted with Phe and the 97th Ala with Thr
　１０個の改変が導入されたＶＨのアミノ酸配列としては、具体的には、例えば、以下の（１）～（３）のアミノ酸配列が挙げられる。
（１）配列番号８４で表されるアミノ酸配列中の２５番目のＳｅｒをＴｈｒに、２７番目のＧｌｙをＴｙｒに、２８番目のＳｅｒをＴｈｒに、２９番目のＶａｌをＩｌｅに、３０番目のＳｅｒをＴｈｒに、７２番目のＶａｌをＡｒｇに、９３番目のＶａｌをＴｈｒに、９５番目のＴｙｒをＰｈｅに、９７番目のＡｌａをＴｈｒに、および１０６番目のＴｈｒをＶａｌに置換したアミノ酸配列
（２）配列番号８４で表されるアミノ酸配列中の２７番目のＧｌｙをＴｙｒに、２８番目のＳｅｒをＴｈｒに、２９番目のＶａｌをＩｌｅに、３０番目のＳｅｒをＴｈｒに、４０番目のＧｌｎをＬｙｓに、４５番目のＧｌｙをＡｒｇに、４６番目のＬｅｕをＭｅｔに、４９番目のＩｌｅをＭｅｔに、７２番目のＶａｌをＡｒｇに、および９７番目のＡｌａをＴｈｒに置換したアミノ酸配列
（３）配列番号８４で表されるアミノ酸配列中の２５番目のＳｅｒをＴｈｒに、２７番目のＧｌｙをＴｙｒに、４０番目のＧｌｎをＬｙｓに、４５番目のＧｌｙをＡｒｇに、４６番目のＬｅｕをＭｅｔに、４９番目のＩｌｅをＭｅｔに、７２番目のＶａｌをＡｒｇに、９３番目のＶａｌをＴｈｒに、９５番目のＴｙｒをＰｈｅに、および９７番目のＡｌａをＴｈｒに置換したアミノ酸配列 Specific examples of the amino acid sequence of VH into which 10 modifications have been introduced include the following amino acid sequences (1) to (3).
(1) The 25th Ser in the amino acid sequence represented by SEQ ID NO: 84 is Thr, the 27th Gly is Tyr, the 28th Ser is Thr, the 29th Val is Ile, and the 30th Ser Is replaced with Thr, 72nd Val with Arg, 93rd Val with Thr, 95th Tyr with Phe, 97th Ala with Thr, and 106th Thr with Val. ) In the amino acid sequence represented by SEQ ID NO: 84, the 27th Gly is Tyr, the 28th Ser is Thr, the 29th Val is Ile, the 30th Ser is Thr, and the 40th Gln is Lys. 45th Gly to Arg, 46th Leu to Met, 49th Ile to Met, 72nd Val to Arg, and 97th Al In the amino acid sequence represented by SEQ ID NO: 84, the 25th Ser in Thr, the 27th Gly in Tyr, the 40th Gln in Lys, and the 45th Gly in Arg, 46th Leu to Met, 49th Ile to Met, 72nd Val to Arg, 93rd Val to Thr, 95th Tyr to Phe, and 97th Ala to Thr Amino acid sequence replaced with
　８個の改変が導入されたＶＨのアミノ酸配列としては、具体的には、例えば、配列番号８４で表されるアミノ酸配列中の２８番目のＳｅｒをＴｈｒに、２９番目のＶａｌをＩｌｅに、３０番目のＳｅｒをＴｈｒに、４０番目のＧｌｎをＬｙｓに、４５番目のＧｌｙをＡｒｇに、４６番目のＬｅｕをＭｅｔに、４９番目のＩｌｅをＭｅｔに、および９７番目のＡｌａをＴｈｒに置換したアミノ酸配列などが挙げられる Specifically, the amino acid sequence of VH into which 8 modifications have been introduced is, for example, 28th Ser in the amino acid sequence represented by SEQ ID NO: 84, Thr, 29th Val in Ile, 30 An amino acid in which the th Ser is replaced with Thr, the 40th Gln with Lys, the 45th Gly with Arg, the 46th Leu with Met, the 49th Ile with Met, and the 97th Ala with Thr Examples include sequences
　７個の改変が導入されたＶＨのアミノ酸配列としては、具体的には、例えば、配列番号８４で表されるアミノ酸配列中の２７番目のＧｌｙをＴｙｒに、４０番目のＧｌｎをＬｙｓに、４５番目のＧｌｙをＡｒｇに、４６番目のＬｅｕをＭｅｔに、４９番目のＩｌｅをＭｅｔに、７２番目のＶａｌをＡｒｇに、および９７番目のＡｌａをＴｈｒに置換したアミノ酸配列などが挙げられる Specific examples of the amino acid sequence of VH into which seven modifications have been introduced include, for example, the 27th Gly in the amino acid sequence represented by SEQ ID NO: 84 as Tyr, the 40th Gln as Lys, and 45 Examples include amino acid sequences in which the first Gly is replaced by Arg, the 46th Leu is replaced by Met, the 49th Ile is replaced by Met, the 72nd Val is replaced by Arg, and the 97th Ala is replaced by Thr.
　６個の改変が導入されたＶＨのアミノ酸配列としては、具体的には、例えば、配列番号８４で表されるアミノ酸配列中の２７番目のＧｌｙをＴｙｒに、２８番目のＳｅｒをＴｈｒに、２９番目のＶａｌをＩｌｅに、３０番目のＳｅｒをＴｈｒに、７２番目のＶａｌをＡｒｇに、および９７番目のＡｌａをＴｈｒに置換したアミノ酸配列などが挙げられる Specifically, the amino acid sequence of VH into which six modifications are introduced is, for example, the 27th Gly in the amino acid sequence represented by SEQ ID NO: 84 as Tyr, the 28th Ser as Thr, 29 Examples include amino acid sequences in which the 1st Val is replaced with Ile, the 30th Ser with Thr, the 72nd Val with Arg, and the 97th Ala with Thr.
　５個の改変が導入されたＶＨのアミノ酸配列としては、具体的には、例えば、配列番号８４で表されるアミノ酸配列中の４０番目のＧｌｎをＬｙｓに、４５番目のＧｌｙをＡｒｇに、４６番目のＬｅｕをＭｅｔに、４９番目のＩｌｅをＭｅｔに、および９７番目のＡｌａをＴｈｒに置換したアミノ酸配列などが挙げられる Specifically, the amino acid sequence of VH into which five modifications are introduced is, for example, that the 40th Gln in the amino acid sequence represented by SEQ ID NO: 84 is Lys, the 45th Gly is Arg, Examples include amino acid sequences in which the first Leu is replaced by Met, the 49th Ile is replaced by Met, and the 97th Ala is replaced by Thr.
　４個の改変が導入されたＶＨのアミノ酸配列としては、具体的には、例えば、以下の（１）～（６）のアミノ酸配列が挙げられる。
（１）配列番号８４で表されるアミノ酸配列中の４０番目のＧｌｎをＬｙｓに、４５番目のＧｌｙをＡｒｇに、４６番目のＬｅｕをＭｅｔに、および４９番目のＩｌｅをＭｅｔに置換したアミノ酸配列
（２）配列番号８４で表されるアミノ酸配列中の２７番目のＧｌｙをＴｙｒに、２８番目のＳｅｒをＴｈｒに、２９番目のＶａｌをＩｌｅに、および３０番目のＳｅｒをＴｈｒに置換したアミノ酸配列
（３）配列番号８４で表されるアミノ酸配列中の４０番目のＧｌｎをＬｙｓに、４５番目のＧｌｙをＡｒｇに、４６番目のＬｅｕをＭｅｔに、および９７番目のＡｌａをＴｈｒに置換したアミノ酸配列
（４）配列番号８４で表されるアミノ酸配列中の４０番目のＧｌｎをＬｙｓに、４５番目のＧｌｙをＡｒｇに、４９番目のＩｌｅをＭｅｔに、および９７番目のＡｌａをＴｈｒに置換したアミノ酸配列
（５）配列番号８４で表されるアミノ酸配列中の４０番目のＧｌｎをＬｙｓに、４６番目のＬｅｕをＭｅｔに、４９番目のＩｌｅをＭｅｔに、および９７番目のＡｌａをＴｈｒに置換したアミノ酸配列
（６）配列番号８４で表されるアミノ酸配列中の４５番目のＧｌｙをＡｒｇに、４６番目のＬｅｕをＭｅｔに、４９番目のＩｌｅをＭｅｔに、および９７番目のＡｌａをＴｈｒに置換したアミノ酸配列 Specific examples of the amino acid sequence of VH into which four modifications have been introduced include the following amino acid sequences (1) to (6).
(1) An amino acid sequence in which the 40th Gln in the amino acid sequence represented by SEQ ID NO: 84 is substituted with Lys, the 45th Gly with Arg, the 46th Leu with Met, and the 49th Ile with Met (2) An amino acid sequence in which the 27th Gly in the amino acid sequence represented by SEQ ID NO: 84 is substituted with Tyr, the 28th Ser with Thr, the 29th Val with Ile, and the 30th Ser with Thr. (3) An amino acid sequence in which the 40th Gln in the amino acid sequence represented by SEQ ID NO: 84 is substituted with Lys, the 45th Gly with Arg, the 46th Leu with Met, and the 97th Ala with Thr (4) The 40th Gln in the amino acid sequence represented by SEQ ID NO: 84 is Lys, the 45th Gly is Arg, and the 49th Ile is Met. And amino acid sequence in which 97th Ala is replaced with Thr (5) In the amino acid sequence represented by SEQ ID NO: 84, 40th Gln is Lys, 46th Leu is Met, 49th Ile is Met, In the amino acid sequence represented by SEQ ID NO: 84, the 45th Gly is Arg, the 46th Leu is Met, the 49th Ile is Met, and the 97th Ala is replaced with Thr. And amino acid sequence in which 97th Ala is replaced with Thr
　２個の改変が導入されたＶＨのアミノ酸配列としては、具体的には、例えば、以下の（１）～（１２）のアミノ酸配列が挙げられる。
（１）配列番号８４で表されるアミノ酸配列中の２７番目のＧｌｙをＴｙｒに、および７２番目のＶａｌをＡｒｇに置換したアミノ酸配列
（２）配列番号８４で表されるアミノ酸配列中の２７番目のＧｌｙをＴｙｒに、および９７番目のＡｌａをＴｈｒに置換したアミノ酸配列
（３）配列番号８４で表されるアミノ酸配列中の４０番目のＧｌｎをＬｙｓに、および４５番目のＧｌｙをＡｒｇに置換したアミノ酸配列
（４）配列番号８４で表されるアミノ酸配列中の４０番目のＧｌｎをＬｙｓに、および４６番目のＬｅｕをＭｅｔに置換したアミノ酸配列
（５）配列番号８４で表されるアミノ酸配列中の４０番目のＧｌｎをＬｙｓに、および４９番目のＩｌｅをＭｅｔに置換したアミノ酸配列
（６）配列番号８４で表されるアミノ酸配列中の４０番目のＧｌｎをＬｙｓに、および９７番目のＡｌａをＴｈｒに置換したアミノ酸配列
（７）配列番号８４で表されるアミノ酸配列中の４５番目のＧｌｙをＡｒｇに、および４６番目のＬｅｕをＭｅｔに置換したアミノ酸配列
（８）配列番号８４で表されるアミノ酸配列中の４５番目のＧｌｙをＡｒｇに、および４９番目のＩｌｅをＭｅｔに置換したアミノ酸配列
（９）配列番号８４で表されるアミノ酸配列中の４５番目のＧｌｙをＡｒｇに、および９７番目のＡｌａをＴｈｒに置換したアミノ酸配列
（１０）配列番号８４で表されるアミノ酸配列中の４６番目のＬｅｕをＭｅｔに、および４９番目のＩｌｅをＭｅｔに置換したアミノ酸配列
（１１）配列番号８４で表されるアミノ酸配列中の４６番目のＬｅｕをＭｅｔに、および９７番目のＡｌａをＴｈｒに置換したアミノ酸配列
（１２）配列番号８４で表されるアミノ酸配列中の４９番目のＩｌｅをＭｅｔに、および９７番目のＡｌａをＴｈｒに置換したアミノ酸配列 Specific examples of the amino acid sequence of VH into which two modifications have been introduced include the following amino acid sequences (1) to (12).
(1) An amino acid sequence in which the 27th Gly in the amino acid sequence represented by SEQ ID NO: 84 is substituted with Tyr and the 72nd Val is substituted with Arg (2) The 27th amino acid in the amino acid sequence represented by SEQ ID NO: 84 In the amino acid sequence represented by SEQ ID NO: 84, the 40th Gln in the amino acid sequence represented by SEQ ID NO: 84 was replaced with Lys, and the 45th Gly was replaced with Arg. Amino acid sequence (4) In the amino acid sequence represented by SEQ ID NO: 84, the 40th Gln in the amino acid sequence was replaced with Lys, and the 46th Leu was replaced with Met (5) in the amino acid sequence represented by SEQ ID NO: 84 An amino acid sequence in which the 40th Gln is replaced with Lys and the 49th Ile is replaced with Met (6) 4 in the amino acid sequence represented by SEQ ID NO: 84 The amino acid sequence in which the Ginth Gln is replaced with Lys and the 97th Ala is replaced with Thr (7) The 45th Gly in the amino acid sequence represented by SEQ ID NO: 84 is replaced with Arg, and the 46th Leu is replaced with Met Amino acid sequence (8) In the amino acid sequence represented by SEQ ID NO: 84, the 45th Gly in the amino acid sequence represented by Arg and the 49th Ile substituted with Met (9) In the amino acid sequence represented by SEQ ID NO: 84 In the amino acid sequence represented by SEQ ID NO: 84, the 45th Gly in Arg was replaced with Arg, and the 97th Ala was replaced with Thr. The 46th Leu in the amino acid sequence represented by SEQ ID NO: 84 was designated as Met, and the 49th Ile was designated as Met. The amino acid sequence substituted with (11) the 46th Leu in the amino acid sequence represented by SEQ ID NO: 84, and the 97th la amino acid sequence is substituted with Thr (12) with Met 49th Ile in the amino acid sequence represented by SEQ ID NO: 84, and 97 amino acid sequence was replaced with Thr to Ala
　１個の改変が導入されたＶＨのアミノ酸配列としては、具体的には、例えば、配列番号８４で表されるアミノ酸配列中の２５番目のＳｅｒをＴｈｒに置換したアミノ酸配列、２７番目のＧｌｙをＴｙｒに置換したアミノ酸配列、２８番目のＳｅｒをＴｈｒに置換したアミノ酸配列、２９番目のＶａｌをＩｌｅに置換したアミノ酸配列、３０番目のＳｅｒをＴｈｒに置換したアミノ酸配列、４０番目のＧｌｎをＬｙｓに置換したアミノ酸配列、４５番目のＧｌｙをＡｒｇに置換したアミノ酸配列、４６番目のＬｅｕをＭｅｔに置換したアミノ酸配列、４９番目のＩｌｅをＭｅｔに置換したアミノ酸配列、７２番目のＶａｌをＡｒｇに置換したアミノ酸配列、９３番目のＶａｌをＴｈｒに置換したアミノ酸配列、９５番目のＴｙｒをＰｈｅに置換したアミノ酸配列、９７番目のＡｌａをＴｈｒに置換したアミノ酸配列、または、１０６番目のＴｈｒをＶａｌに置換したアミノ酸配列、が挙げられる。 Specifically, the amino acid sequence of VH into which one modification is introduced is, for example, an amino acid sequence in which the 25th Ser in the amino acid sequence represented by SEQ ID NO: 84 is replaced with Thr, and the 27th Gly is Amino acid sequence substituted with Tyr, amino acid sequence substituted with 28th Ser with Thr, amino acid sequence substituted with 29th Val with Ile, amino acid sequence substituted with 30th Ser with Thr, 40th Gln with Lys Substituted amino acid sequence, amino acid sequence in which the 45th Gly was replaced with Arg, amino acid sequence in which the 46th Leu was replaced with Met, amino acid sequence in which the 49th Ile was replaced with Met, and the 72nd Val was replaced with Arg Amino acid sequence, amino acid sequence in which 93rd Val is replaced with Thr, 95th Tyr is placed in Phe Amino acid sequence, the 97th Ala amino acid sequence is substituted with Thr, or the amino acid sequence obtained by replacing 106 th Thr in Val, and the like.
　抗体のＶＬについては、例えば、配列番号８５のアミノ酸配列中の２番目のＩｌｅ、８番目のＰｒｏ、１１番目のＬｅｕ、１５番目のＰｒｏ、および９０番目のＶａｌから選ばれる少なくとも１のアミノ酸残基が、他のアミノ酸残基に置換されたアミノ酸配列を含むヒト化抗体が挙げられる。　 For antibody VL, for example, at least one amino acid residue selected from the second Ile, the eighth Pro, the eleventh Leu, the fifteenth Pro, and the 90th Val in the amino acid sequence of SEQ ID NO: 85 Are humanized antibodies comprising amino acid sequences substituted with other amino acid residues. *
　前記ヒト化抗体の中でも、以下の（１）～（４）が好ましい。
（１）配列番号８５のアミノ酸配列中の２番目のＩｌｅ、１１番目のＬｅｕ、１５番目のＰｒｏ、および９０番目のＶａｌが、他のアミノ酸残基に置換されたアミノ酸配列を含むヒト化抗体
（２）配列番号８５のアミノ酸配列中の２番目のＩｌｅ、１５番目のＰｒｏ、および９０番目のＶａｌが、他のアミノ酸残基に置換されたアミノ酸配列を含むヒト化抗体。
（３）配列番号８５のアミノ酸配列中の２番目のＩｌｅ、１１番目のＬｅｕ、および１５番目のＰｒｏが、他のアミノ酸残基に置換されたアミノ酸配列を含むヒト化抗体。
（４）配列番号８５のアミノ酸配列中の２番目のＩｌｅ、および１５番目のＰｒｏが、他のアミノ酸残基に置換されたアミノ酸配列を含むヒト化抗体 Among the humanized antibodies, the following (1) to (4) are preferable.
(1) A humanized antibody comprising an amino acid sequence in which the second Ile, the 11th Leu, the 15th Pro, and the 90th Val in the amino acid sequence of SEQ ID NO: 85 are substituted with other amino acid residues ( 2) A humanized antibody comprising an amino acid sequence in which the second Ile, 15th Pro, and 90th Val in the amino acid sequence of SEQ ID NO: 85 are substituted with other amino acid residues.
(3) A humanized antibody comprising an amino acid sequence in which the second Ile, 11th Leu, and 15th Pro in the amino acid sequence of SEQ ID NO: 85 are substituted with other amino acid residues.
(4) A humanized antibody comprising an amino acid sequence in which the second Ile and 15th Pro in the amino acid sequence of SEQ ID NO: 85 are substituted with other amino acid residues
　上記のアミノ酸改変の結果、得られる抗体ＶＬのアミノ酸配列としては、配列番号８５のアミノ酸配列中の２番目のＩｌｅをＶａｌに、８番目のＰｒｏをＬｅｕに、１１番目のＬｅｕをＧｌｎに、１５番目のＰｒｏをＬｅｕに、および９０番目のＶａｌがＬｅｕに置換する改変から選ばれる少なくとも１つの改変が導入されたアミノ酸配列が挙げられる。 The amino acid sequence of the antibody VL obtained as a result of the above amino acid modification is as follows. The second Ile in the amino acid sequence of SEQ ID NO: 85 is Val, the 8th Pro is Leu, the 11th Leu is Gln, 15 An amino acid sequence in which at least one modification selected from the modification in which the 1st Pro is replaced with Leu and the 90th Val is replaced with Leu is introduced.
　５個の改変が導入されたＶＬのアミノ酸配列としては、具体的には、例えば、配列番号８５のアミノ酸配列中の２番目のＩｌｅをＶａｌに、８番目のＰｒｏをＬｅｕに、１１番目のＬｅｕをＧｌｎに、１５番目のＰｒｏをＬｅｕに、および９０番目のＶａｌがＬｅｕに置換したアミノ酸配列が挙げられる。 Specifically, as the amino acid sequence of VL into which five modifications are introduced, for example, the second Ile in the amino acid sequence of SEQ ID NO: 85 is Val, the 8th Pro is Leu, and the 11th Leu Amino acid sequence in which is replaced with Gln, 15th Pro is replaced with Leu, and 90th Val is replaced with Leu.
　４個の改変が導入されたＶＬのアミノ酸配列としては、具体的には、例えば、以下の（１）～（５）のアミノ酸配列が挙げられる。
（１）配列番号８５のアミノ酸配列中の８番目のＰｒｏをＬｅｕに、１１番目のＬｅｕをＧｌｎに、１５番目のＰｒｏをＬｅｕに、および９０番目のＶａｌがＬｅｕに置換したアミノ酸配列
（２）配列番号８５のアミノ酸配列中の２番目のＩｌｅをＶａｌに、１１番目のＬｅｕをＧｌｎに、１５番目のＰｒｏをＬｅｕに、および９０番目のＶａｌがＬｅｕに置換したアミノ酸配列
（３）配列番号８５のアミノ酸配列中の２番目のＩｌｅをＶａｌに、８番目のＰｒｏをＬｅｕに、１５番目のＰｒｏをＬｅｕに、および９０番目のＶａｌがＬｅｕに置換したアミノ酸配列
（４）配列番号８５のアミノ酸配列中の２番目のＩｌｅをＶａｌに、８番目のＰｒｏをＬｅｕに、１１番目のＬｅｕをＧｌｎに、および９０番目のＶａｌがＬｅｕに置換したアミノ酸配列
（５）配列番号８５のアミノ酸配列中の２番目のＩｌｅをＶａｌに、８番目のＰｒｏをＬｅｕに、１１番目のＬｅｕをＧｌｎに、および１５番目のＰｒｏをＬｅｕに置換したアミノ酸配列 Specific examples of the amino acid sequence of VL into which four modifications have been introduced include the following amino acid sequences (1) to (5).
(1) Amino acid sequence in which the 8th Pro in the amino acid sequence of SEQ ID NO: 85 is replaced with Leu, the 11th Leu is replaced with Gln, the 15th Pro is replaced with Leu, and the 90th Val is replaced with Leu (2) The amino acid sequence in which the second Ile in the amino acid sequence of SEQ ID NO: 85 is replaced with Val, the 11th Leu is replaced with Gln, the 15th Pro is replaced with Leu, and the 90th Val is replaced with Leu (3) SEQ ID NO: 85 The amino acid sequence of SEQ ID NO: 85 in which the second Ile is replaced with Val, the 8th Pro is replaced with Leu, the 15th Pro is replaced with Leu, and the 90th Val is replaced with Leu. The second Ile is replaced with Val, the 8th Pro is replaced with Leu, the 11th Leu is replaced with Gln, and the 90th Val is replaced with Leu. Acid sequence of (5) the second Ile in the amino acid sequence of SEQ ID NO: 85 to Val, a Leu 8th Pro, 11 th Leu to Gln, and the amino acid sequence obtained by replacing the 15th Pro to Leu
　３個の改変が導入されたＶＬのアミノ酸配列としては、具体的には、例えば、以下の（１）～（３）のアミノ酸配列が挙げられる。
（１）配列番号８５のアミノ酸配列中の２番目のＩｌｅをＶａｌに、８番目のＰｒｏをＬｅｕに、および１５番目のＰｒｏをＬｅｕに置換したアミノ酸配列
（２）配列番号８５のアミノ酸配列中の２番目のＩｌｅをＶａｌに、１１番目のＬｅｕをＧｌｎに、および１５番目のＰｒｏをＬｅｕに置換したアミノ酸配列
（３）配列番号８５のアミノ酸配列中の２番目のＩｌｅをＶａｌに、１５番目のＰｒｏをＬｅｕに、および９０番目のＶａｌがＬｅｕに置換したアミノ酸配列 Specific examples of the amino acid sequence of VL into which three modifications have been introduced include the following amino acid sequences (1) to (3).
(1) Amino acid sequence in which the second Ile in the amino acid sequence of SEQ ID NO: 85 is replaced with Val, the 8th Pro is replaced with Leu, and the 15th Pro is replaced with Leu (2) in the amino acid sequence of SEQ ID NO: 85 The amino acid sequence in which the second Ile is replaced with Val, the 11th Leu is replaced with Gln, and the 15th Pro is replaced with Leu. (3) The second Ile in the amino acid sequence of SEQ ID NO: 85 is Val, and the 15th Amino acid sequence in which Pro is replaced with Leu and 90th Val is replaced with Leu
　２個の改変が導入されたＶＬのアミノ酸配列としては、具体的には、例えば、以下の（１）～（７）のアミノ酸配列が挙げられる。
（１）配列番号８５のアミノ酸配列中の２番目のＩｌｅをＶａｌに、および８番目のＰｒｏをＬｅｕに置換したアミノ酸配列
（２）配列番号８５のアミノ酸配列中の２番目のＩｌｅをＶａｌに、および１１番目のＬｅｕをＧｌｎに置換したアミノ酸配列
（３）配列番号８５のアミノ酸配列中の２番目のＩｌｅをＶａｌに、および１５番目のＰｒｏをＬｅｕに置換したアミノ酸配列
（４）配列番号８５のアミノ酸配列中の２番目のＩｌｅをＶａｌに、および９０番目のＶａｌがＬｅｕに置換したアミノ酸配列
（５）配列番号８５のアミノ酸配列中の８番目のＰｒｏをＬｅｕに、および１５番目のＰｒｏをＬｅｕに置換したアミノ酸配列
（６）配列番号８５のアミノ酸配列中の１１番目のＬｅｕをＧｌｎに、および１５番目のＰｒｏをＬｅｕに置換したアミノ酸配列
（７）配列番号８５のアミノ酸配列中の１５番目のＰｒｏをＬｅｕに、および９０番目のＶａｌがＬｅｕに置換したアミノ酸配列 Specific examples of the amino acid sequence of VL into which two modifications are introduced include the following amino acid sequences (1) to (7).
(1) An amino acid sequence in which the second Ile in the amino acid sequence of SEQ ID NO: 85 is substituted with Val, and the 8th Pro is substituted with Leu. (2) The second Ile in the amino acid sequence of SEQ ID NO: 85 is Val. And the amino acid sequence (4) of SEQ ID NO: 85 in which the second Ile in the amino acid sequence of SEQ ID NO: 85 is replaced with Val, and the 15th Pro is replaced with Leu. The amino acid sequence in which the second Ile in the amino acid sequence is substituted with Val and the 90th Val is substituted with Leu (5) The 8th Pro in the amino acid sequence of SEQ ID NO: 85 is Leu, and the 15th Pro is Leu In the amino acid sequence of SEQ ID NO: 85, the 11th Leu in Gln and the 15th Pro in Leu Was the 15 th Pro amino acid sequence (7) in the amino acid sequence of SEQ ID NO: 85 to Leu, and 90 amino acid sequence Val has been substituted with Leu
　１個の改変が導入されたＶＬのアミノ酸配列としては、具体的には、例えば、配列番号８５のアミノ酸配列中の２番目のＩｌｅをＶａｌに置換したアミノ酸配列、８番目のＰｒｏをＬｅｕに置換したアミノ酸配列、１１番目のＬｅｕをＧｌｎに置換したアミノ酸配列、１５番目のＰｒｏをＬｅｕに置換したアミノ酸配列、または、９０番目のＶａｌがＬｅｕに置換したアミノ酸配列、が挙げられる。 Specifically, the amino acid sequence of VL into which one modification is introduced is, for example, an amino acid sequence in which the second Ile in the amino acid sequence of SEQ ID NO: 85 is substituted with Val, and the eighth Pro is substituted with Leu. An amino acid sequence in which the 11th Leu is replaced with Gln, an amino acid sequence in which the 15th Pro is replaced with Leu, or an amino acid sequence in which the 90th Val is replaced with Leu.
　ヒト抗体は、元来、ヒト体内に天然に存在する抗体をいうが、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ーおよびヒト抗体産生トランスジェニック動物から得られる抗体なども含まれる。 A human antibody originally refers to an antibody that naturally exists in the human body, but a human antibody phage library and a human antibody-producing transgene prepared by recent advances in genetic engineering, cell engineering, and developmental engineering techniques. Also included are antibodies obtained from transgenic animals.
　ヒト体内に天然に存在する抗体は、例えば、ヒト末梢血リンパ球を単離し、ＥＢウイルスなどを感染させ不死化し、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、培養上清中より該抗体を精製することができる。 The antibody naturally present in the human body can be cultured by, for example, isolating human peripheral blood lymphocytes, infecting and immortalizing EB virus, etc., and cloning the lymphocytes that produce the antibody. The antibody can be further 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またはｓｃＦｖなどの抗体断片をファージ表面に発現させたライブラリーである。該ライブラリーより、抗原を固定化した基質に対する結合活性を指標として所望の抗原結合活性を有する抗体断片を表面に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、さらに、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 or scFv are expressed on the phage surface by inserting antibody genes prepared from human B cells into the phage genes. From the library, phages expressing an antibody fragment having a desired antigen-binding activity on the surface can be collected using the binding activity to the substrate on which the antigen is immobilized as an index. The antibody fragment can be further converted into a human antibody molecule comprising two complete heavy chains and two complete light chains by genetic engineering techniques.
　ヒト抗体産生トランスジェニック動物は、ヒト抗体遺伝子が細胞内に組込まれた動物を意味する。具体的には、例えば、マウスＥＳ細胞へヒト抗体遺伝子を導入し、該ＥＳ細胞を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ェニックマウスを作製することができる。ヒト抗体産生トランスジェニック動物からのヒト抗体は、通常のヒト以外の動物で行われているハイブリドーマ作製方法を用い、ヒト抗体産生ハイブリドーマを取得し、培養することで培養上清中にヒト抗体を産生蓄積させることにより作製できる。 A human antibody-producing transgenic animal means an animal in which a human antibody gene is incorporated into cells. Specifically, for example, a human antibody-producing transgenic mouse can be produced by introducing a human antibody gene into a mouse ES cell, transplanting the ES cell into an early mouse embryo, and generating it. For human antibodies from human antibody-producing transgenic animals, human antibody-producing hybridomas are obtained and cultured using normal hybridoma production methods performed in non-human animals to produce human antibodies in the culture supernatant. It can be produced by accumulating.
　上述の抗体または抗体断片を構成するアミノ酸配列において、１つ以上のアミノ酸が欠失、付加、置換または挿入され、かつ上述の抗体またはその断片と同様な活性を有するモノクローナル抗体またはその断片も、本発明のモノクローナル抗体またはその断片に包含される。 A monoclonal antibody or fragment thereof in which one or more amino acids are deleted, added, substituted or inserted in the amino acid sequence constituting the above-described antibody or antibody fragment and has the same activity as the above-described antibody or fragment thereof, It is included in the monoclonal antibody of the invention or a fragment thereof.
　欠失、置換、挿入および／または付加されるアミノ酸の数は１個以上でありその数は特に限定されないが、部位特異的変異導入法［Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ　２ｎｄ　Ｅｄｉｔｉｏｎ，Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓ（１９８９）、Ｃｕｒｒｅｎｔ　ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ，Ｊｏｈｎ　Ｗｉｌｅｙ＆Ｓｏｎｓ（１９８７－１９９７）、Ｎｕｃｌｅｉｃ　Ａｃｉｄｓ　Ｒｅｓｅａｒｃｈ，１０，６４８７（１９８２）、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．，ＵＳＡ，７９，６４０９（１９８２）、Ｇｅｎｅ，３４，３１５（１９８５）、Ｎｕｃｌｅｉｃ　Ａｃｉｄｓ　Ｒｅｓｅａｒｃｈ，１３，４４３１（１９８５）、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ　ＵＳＡ，８２，４８８（１９８５）］などの周知の技術により、欠失、置換もしくは付加できる程度の数である。例えば、１～数十個、好ましくは１～２０個、より好ましくは１～１０個、さらに好ましくは１～５個である。 The number of amino acids to be deleted, substituted, inserted and / or added is one or more, and the number is not particularly limited, but site-specific mutagenesis [Molecular Cloning, 2nd Edition, Cold Spring Harbor Laboratory Press (1989), Current protocols in Molecular Biology, John Wiley & Sons (1987-1997), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci. USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci USA, 82, 488 (1985)], etc., and the number that can be deleted, substituted, or added. For example, the number is 1 to several tens, preferably 1 to 20, more preferably 1 to 10, and still more preferably 1 to 5.
　上記の抗体のアミノ酸配列において１つ以上のアミノ酸残基が欠失、置換、挿入または付加されたとは、次のことを示す。即ち、同一配列中の任意、かつ１または複数のアミノ酸配列中において、１または複数のアミノ酸残基の欠失、置換、挿入または付加があることを意味する。また、欠失、置換、挿入または付加が同時に生じる場合もあり、置換、挿入または付加されるアミノ酸残基は天然型と非天然型いずれの場合もある。 The deletion, substitution, insertion or addition of one or more amino acid residues in the amino acid sequence of the above antibody indicates the following. That is, it means that there is a deletion, substitution, insertion or addition of one or a plurality of amino acid residues in any one and a plurality of amino acid sequences in the same sequence. In addition, deletion, substitution, insertion or addition may occur simultaneously, and the amino acid residue to be substituted, inserted or added may be either a natural type or a non-natural type.
　天然型アミノ酸残基としては、例えば、Ｌ－アラニン、Ｌ－アスパラギン、Ｌ－アスパラギン酸、Ｌ－グルタミン、Ｌ－グルタミン酸、グリシン、Ｌ－ヒスチジン、Ｌ－イソロイシン、Ｌ－ロイシン、Ｌ－リジン、Ｌ－メチオニン、Ｌ－フェニルアラニン、Ｌ－プロリン、Ｌ－セリン、Ｌ－スレオニン、Ｌ－トリプトファン、Ｌ－チロシン、Ｌ－バリン、またはＬ－システインなどが挙げられる。 Examples of natural amino acid residues include L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L -Methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, or L-cysteine.
　以下に、相互に置換可能なアミノ酸残基の好ましい例を示す。同一群に含まれるアミノ酸残基は相互に置換可能である。
　Ａ群：ロイシン、イソロイシン、ノルロイシン、バリン、ノルバリン、アラニン、２－アミノブタン酸、メチオニン、Ｏ－メチルセリン、ｔ－ブチルグリシン、ｔ－ブチルアラニン、シクロヘキシルアラニン
　Ｂ群：アスパラギン酸、グルタミン酸、イソアスパラギン酸、イソグルタミン酸、２－アミノアジピン酸、２－アミノスベリン酸
　Ｃ群：アスパラギン、グルタミン
　Ｄ群：リジン、アルギニン、オルニチン、２，４－ジアミノブタン酸、２，３－ジアミノプロピオン酸
　Ｅ群：プロリン、３－ヒドロキシプロリン、４－ヒドロキシプロリン
　Ｆ群：セリン、スレオニン、ホモセリン
　Ｇ群：フェニルアラニン、チロシン The preferred examples of amino acid residues that can be substituted with each other are shown below. Amino acid residues contained in the same group can be substituted for each other.
Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, O-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine Group B: aspartic acid, glutamic acid, isoaspartic acid, Isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid Group C: asparagine, glutamine Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid Group E: proline, 3 -Hydroxyproline, 4-hydroxyproline Group F: serine, threonine, homoserine Group G: phenylalanine, tyrosine
　本発明において、抗体断片としては、例えば、Ｆａｂ、Ｆ（ａｂ’）２、Ｆａｂ’、ｓｃＦｖ、ｄｉａｂｏｄｙ、ｄｓＦｖおよびＣＤＲを含むペプチドなどが挙げられる。 In the present invention, antibody fragments include, for example, peptides including Fab, F (ab ′) 2 , Fab ′, scFv, diabody, dsFv and CDR.
　Ｆａｂは、ＩｇＧを蛋白質分解酵素であるパパインで処理して得られる断片のうち（Ｈ鎖の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５万の抗原結合活性を有する抗体断片である。 Fab is a fragment obtained by treating IgG with papain, a proteolytic enzyme (cleaved at the 224th amino acid residue of the H chain), about half of the N chain side of the H chain and the entire L chain are disulfides. It is an antibody fragment having an antigen binding activity with a molecular weight of about 50,000 bound by binding.
　本発明のＦａｂは、ＣＤ３３を特異的に認識し、かつ該細胞外領域に結合するモノクローナル抗体をパパインで処理して得ることができる。また、該抗体のＦａｂをコードするＤＮＡを原核生物用発現ベクターまたは真核生物用発現ベクターに挿入し、該ベクターを原核生物または真核生物へ導入することにより発現させ、Ｆａｂを製造することもできる。 The Fab of the present invention can be obtained by treating a monoclonal antibody that specifically recognizes CD33 and binds to the extracellular region with papain. Alternatively, a Fab may be produced by inserting a DNA encoding the Fab of the antibody into a prokaryotic expression vector or a eukaryotic expression vector, and expressing the vector by introducing the vector into a prokaryotic or eukaryotic organism. it can.
　Ｆ（ａｂ’）２は、ＩｇＧのヒンジ領域の２個のジスルフィド結合の下部を蛋白質分解酵素であるペプシンで分解して得られた、２つのＦａｂ領域がヒンジ部分で結合して構成された、分子量約１０万の抗原結合活性を有する断片である。本発明のＦ（ａｂ’）２は、ＣＤ３３を特異的に認識し、かつその細胞外領域に結合するモノクローナル抗体をペプシンで処理して得ることができる。また、下記のＦａｂ’をチオエーテル結合またはジスルフィド結合させ、作製することもできる。 F (ab ′) 2 was obtained by decomposing the lower part of two disulfide bonds in the hinge region of IgG with pepsin, a proteolytic enzyme, and was constructed by binding two Fab regions at the hinge portion. This fragment has an antigen binding activity with a molecular weight of about 100,000. The F (ab ′) 2 of the present invention can be obtained by treating a monoclonal antibody specifically recognizing CD33 and binding to its extracellular region with pepsin. Alternatively, Fab ′ described below can be produced by thioether bond or disulfide bond.
　Ｆａｂ’は、上記Ｆ（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５万の抗原結合活性を有する抗体断片である。本発明のＦａｂ’は、本発明のＣＤ３３を特異的に認識し、かつ該細胞外領域に結合するＦ（ａｂ’）２をジチオスレイトールなどの還元剤で処理して得ることができる。また、Ｆａｂ’断片をコードするＤＮＡを原核生物用発現ベクターまたは真核生物用発現ベクターに挿入し、該ベクターを原核生物または真核生物へ導入することにより発現させ、Ｆａｂ’を製造することもできる。 Fab ′ is an antibody fragment having an antigen-binding activity having a molecular weight of about 50,000, which is obtained by cleaving the disulfide bond in the hinge region of F (ab ′) 2 . Fab ′ of the present invention can be obtained by treating F (ab ′) 2 that specifically recognizes CD33 of the present invention and binds to the extracellular region with a reducing agent such as dithiothreitol. Alternatively, Fab ′ may be produced by inserting a DNA encoding a Fab ′ fragment into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryotic or eukaryotic organism to express the Fab ′ fragment. it can.
　ｓｃＦｖは、１本のＶＨと１本のＶＬとを適当なペプチドリンカー（以下、Ｐと表記する）を用いて連結した、ＶＨ－Ｐ－ＶＬまたはＶＬ－Ｐ－ＶＨポリペプチドで、抗原結合活性を有する抗体断片である。 scFv is 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), and has antigen-binding activity. It is an antibody fragment having
　本発明のｓｃＦｖは、本発明のＣＤ３３を特異的に認識し、かつ該細胞外領域に結合するモノクローナル抗体のＶＨおよびＶＬをコードするｃＤＮＡを取得し、ｓｃＦｖをコードするＤＮＡを構築し、該ＤＮＡを原核生物用発現ベクターまたは真核生物用発現ベクターに挿入し、該発現ベクターを原核生物または真核生物へ導入することにより発現させ、製造することができる。 The scFv of the present invention specifically recognizes the CD33 of the present invention and obtains cDNA encoding the VH and VL of a monoclonal antibody that binds to the extracellular region, constructs a DNA encoding scFv, Can be expressed and produced by inserting the expression vector into a prokaryotic expression vector or eukaryotic expression vector and introducing the expression vector into a prokaryotic or eukaryotic organism.
　ｄｉａｂｏｄｙは、ｓｃＦｖが二量体化した抗体断片で、二価の抗原結合活性を有する抗体断片である。二価の抗原結合活性は、同一であることもできるし、一方を異なる抗原結合活性とすることもできる。 Diabody is an antibody fragment obtained by dimerizing scFv and is an antibody fragment having a bivalent antigen-binding activity. The bivalent antigen binding activity can be the same, or one can be a different antigen binding activity.
　本発明のｄｉａｂｏｄｙは、本発明のＣＤ３３を特異的に認識し、かつ該細胞外領域に結合するモノクローナル抗体のＶＨおよびＶＬをコードするｃＤＮＡを取得し、ｓｃＦｖをコードするＤＮＡをペプチドリンカーのアミノ酸配列の長さが８残基以下となるように構築し、該ＤＮＡを原核生物用発現ベクターまたは真核生物用発現ベクターに挿入し、該発現ベクターを原核生物または真核生物へ導入することにより発現させ、製造することができる。 The diabody of the present invention obtains cDNA encoding VH and VL of a monoclonal antibody that specifically recognizes CD33 of the present invention and binds to the extracellular region, and converts the DNA encoding scFv into the amino acid sequence of the peptide linker. Is constructed so that its length is 8 residues or less, and the DNA is inserted into a prokaryotic expression vector or a eukaryotic expression vector, and the expression vector is introduced into a prokaryotic or eukaryotic expression. And can be manufactured.
　ｄｓＦｖは、ＶＨおよびＶＬ中のそれぞれ１アミノ酸残基をシステイン残基に置換したポリペプチドを該システイ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。システイン残基に置換するアミノ酸残基は既知の方法［Ｐｒｏｔｅｉｎ　Ｅｎｇｉｎｅｅｒｉｎｇ，７，６９７（１９９４）］に従って、抗体の立体構造予測に基づいて選択することができる。 DsFv refers to a polypeptide in which one amino acid residue in each of VH and VL is substituted with a cysteine residue and bonded via a disulfide bond between the cysteine residues. The amino acid residue to be substituted for the cysteine residue can be selected based on the three-dimensional structure prediction of the antibody according to a known method [Protein Engineering, 7, 697 (1994)].
　本発明のｄｓＦｖは、本発明のＣＤ３３を特異的に認識し、かつ該細胞外領域に結合するモノクローナル抗体の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を原核生物用発現ベクターまたは真核生物用発現ベクターに挿入し、該発現ベクターを原核生物または真核生物へ導入することにより発現させ、製造することができる。 The dsFv of the present invention specifically recognizes the CD33 of the present invention and obtains cDNA encoding the VH and VL of the monoclonal antibody that binds to the extracellular region, constructs a DNA encoding dsFv, Can be expressed and produced by inserting the expression vector into a prokaryotic expression vector or eukaryotic expression vector and introducing the expression vector into a prokaryotic or eukaryotic organism.
　ＣＤＲを含むペプチドは、ＶＨまたはＶＬの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を含むペプチドは、直接または適当なペプチドリンカーを介して結合させることができる。本発明のＣＤＲを含むペプチドは、本発明のＣＤ３３を特異的に認識し、かつ該細胞外領域に結合するモノクローナル抗体のＶＨおよびＶＬのＣＤＲをコードするＤＮＡを構築し、該ＤＮＡを原核生物用発現ベクターまたは真核生物用発現ベクターに挿入し、該発現ベクターを原核生物またいは真核生物へ導入することにより発現させ、製造することができる。また、ＣＤＲを含むペプチドは、Ｆｍｏｃ法、またはｔＢｏｃ法などの化学合成法によって製造することもできる。 The peptide containing CDR is configured to contain at least one region of CDR of VH or VL. Peptides containing multiple CDRs can be linked directly or via a suitable peptide linker. The peptide comprising the CDR of the present invention constructs DNA encoding the CDRs of VH and VL of the monoclonal antibody that specifically recognizes CD33 of the present invention and binds to the extracellular region, and uses the DNA for prokaryotes It can be expressed and produced by inserting into an expression vector or eukaryotic expression vector and introducing the expression vector into a prokaryotic or eukaryotic organism. Moreover, the peptide containing CDR can also be manufactured by chemical synthesis methods, such as Fmoc method or tBoc method.
　本発明のモノクローナル抗体は、本発明のＣＤ３３の細胞外領域を特異的に認識し、かつ該細胞外領域に結合するモノクローナル抗体またはその断片に放射性同位元素、低分子の薬剤、高分子の薬剤、蛋白質、または抗体医薬などを化学的若しくは遺伝子工学的に結合させた抗体の誘導体を包含する。 The monoclonal antibody of the present invention specifically recognizes the extracellular region of CD33 of the present invention and binds to the extracellular region with a radioisotope, a low molecular drug, a high molecular drug, It includes a derivative of an antibody in which a protein or an antibody drug is chemically or genetically bound.
　本発明における、抗体の誘導体は、本発明のＣＤ３３の細胞外領域を特異的に認識し、かつ該細胞外領域に結合するモノクローナル抗体またはその断片のＨ鎖若しくはＬ鎖のＮ末端側あるいはＣ末端側、抗体またはその断片中の適当な置換基若しくは側鎖、さらにはモノクローナル抗体またはその断片中の糖鎖などに、放射性同位元素、低分子の薬剤、高分子の薬剤、免疫賦活剤、蛋白質または抗体医薬などを化学的手法［抗体工学入門，地人書館（１９９４）］により結合させることにより製造することができる。 The derivative of the antibody in the present invention specifically recognizes the extracellular region of CD33 of the present invention and binds to the extracellular region, or the N-terminal side or C-terminal of the H chain or L chain thereof. On the side, an appropriate substituent or side chain in the antibody or fragment thereof, or a sugar chain in the monoclonal antibody or fragment thereof, etc., radioisotope, low molecular weight drug, high molecular weight drug, immunostimulant, protein or It can be produced by combining antibody drugs and the like by chemical methods [Introduction to Antibody Engineering, Jinshokan (1994)].
　また、本発明のＣＤ３３の細胞外領域を特異的に認識し、かつ該細胞外領域に結合するモノクローナル抗体またはその断片をコードするＤＮＡと、結合させたい蛋白質または抗体医薬をコードするＤＮＡとを連結させて発現ベクターに挿入し、該発現ベクターを適当な宿主細胞へ導入し、発現させる遺伝子工学的手法より製造することができる。 Further, a DNA encoding a monoclonal antibody or a fragment thereof that specifically recognizes the extracellular region of CD33 of the present invention and binds to the extracellular region is linked to a DNA encoding a protein or antibody drug to be bound. And then inserted into an expression vector, introduced into an appropriate host cell, and expressed by a genetic engineering technique.
　放射性同位元素としては、例えば、１３１Ｉ、１２５Ｉ、９０Ｙ、６４Ｃｕ、９９Ｔｃ、７７Ｌｕ、または２１１Ａｔなどが挙げられる。放射性同位元素は、クロラミンＴ法などによって抗体に直接結合させることができる。また、放射性同位元素をキレートする物質を抗体に結合させてもよい。キレート剤としては、例えば、１－イソチオシアネートベンジル－３－メチルジエチレントリアミンペンタ酢酸（ＭＸ－ＤＴＰＡ）などが挙げられる。 Examples of the radioisotope include 131 I, 125 I, 90 Y, 64 Cu, 99 Tc, 77 Lu, and 211 At. The radioisotope can be directly bound to the antibody by the chloramine T method or the like. Further, a substance that chelates a radioisotope may be bound to the antibody. Examples of the chelating agent include 1-isothiocyanate benzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA).
　低分子の薬剤としては、例えば、アルキル化剤、ニトロソウレア剤、代謝拮抗剤、抗生物質、植物アルカロイド、トポイソメラーゼ阻害剤、ホルモン療法剤、ホルモン拮抗剤、アロマターゼ阻害剤、Ｐ糖蛋白阻害剤、白金錯体誘導体、Ｍ期阻害剤若しくはキナーゼ阻害剤などの抗癌剤［臨床腫瘍学，癌と化学療法社（１９９６）］、ハイドロコーチゾン若しくはプレドニゾンなどのステロイド剤、アスピリン若しくはインドメタシンなどの非ステロイド剤、金チオマレート若しくはペニシラミンなどの免疫調節剤、サイクロフォスファミド若しくはアザチオプリンなどの免疫抑制剤、またはマレイン酸クロルフェニラミン若しくはクレマシチンのような抗ヒスタミン剤などの抗炎症剤［炎症と抗炎症療法，医歯薬出版株式会社（１９８２）］などが挙げられる。 Examples of low molecular weight drugs include alkylating agents, nitrosourea agents, antimetabolites, antibiotics, plant alkaloids, topoisomerase inhibitors, hormone therapy agents, hormone antagonists, aromatase inhibitors, P glycoprotein inhibitors, platinum Anticancer agents such as complex derivatives, M phase inhibitors or kinase inhibitors [Clinical Oncology, Cancer and Chemotherapy (1996)], steroidal agents such as hydrocortisone or prednisone, non-steroidal agents such as aspirin or indomethacin, gold thiomalate or Anti-inflammatory agents such as penicillamine, immunosuppressive agents such as cyclophosphamide or azathioprine, or antihistamines such as chlorpheniramine maleate or clemacitine [Inflammation and anti-inflammatory therapy, Ishiyaku Publishing Co., Ltd. ( (1982) And the like.
　抗癌剤としては、例えば、アミフォスチン（エチオール）、シスプラチン、ダカルバジン（ＤＴＩＣ）、ダクチノマイシン、メクロレタミン（ナイトロジェンマスタード）、ストレプトゾシン、シクロフォスファミド、イホスファミド、カルムスチン（ＢＣＮＵ）、ロムスチン（ＣＣＮＵ）、ドキソルビシン（アドリアマイシン）、エピルビシン、ゲムシタビン（ゲムザール）、ダウノルビシン、プロカルバジン、マイトマイシン、シタラビン、エトポシド、メトトレキセート、５－フルオロウラシル、フルオロウラシル、ビンブラスチン、ビンクリスチン、ブレオマイシン、ダウノマイシン、ペプロマイシン、エストラムスチン、パクリタキセル（タキソール）、ドセタキセル（タキソテア）、アルデスロイキン、アスパラギナーゼ、ブスルファン、カルボプラチン、オキサリプラチン、ネダプラチン、クラドリビン、カンプトテシン、１０－ヒドロキシ－７－エチル－カンプトテシン（ＳＮ３８）、フロクスウリジン、フルダラビン、ヒドロキシウレア、イホスファミド、イダルビシン、メスナ、イリノテカン（ＣＰＴ－１１）、ノギテカン、ミトキサントロン、トポテカン、ロイプロリド、メゲストロール、メルファラン、メルカプトプリン、ヒドロキシカルバミド、プリカマイシン、ミトタン、ペガスパラガーゼ、ペントスタチン、ピポブロマン、ストレプトゾシン、タモキシフェン、ゴセレリン、リュープロレニン、フルタミド、テニポシド、テストラクトン、チオグアニン、チオテパ、ウラシルマスタード、ビノレルビン、クロラムブシル、ハイドロコーチゾン、プレドニゾロン、メチルプレドニゾロン、ビンデシン、ニムスチン、セムスチン、カペシタビン、トムデックス、アザシチジン、ＵＦＴ、オキザロプラチン、ゲフィチニブ（イレッサ）、イマチニブ（ＳＴＩ５７１）、エルロチニブ、ＦＭＳ－ｌｉｋｅ　ｔｙｒｏｓｉｎｅ　ｋｉｎａｓｅ　３（Ｆｌｔ３）阻害剤、ｖａｓｃｕｌａｒ　ｅｎｄｏｔｈｅｌｉａｌ　ｇｒｏｗｔｈ　ｆａｃｏｔｒ　ｒｅｃｅｐｔｏｒ（ＶＥＧＦＲ）阻害剤、ｆｉｂｒｏｂｌａｓｔ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ　ｒｅｃｅｐｔｏｒ（ＦＧＦＲ）阻害剤、イレッサ、タルセバなどのｅｐｉｄｅｒｍａｌ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ　ｒｅｃｅｐｔｏｒ（ＥＧＦＲ）阻害剤、ラディシコール、１７－アリルアミノ－１７－デメトキシゲルダナマイシン、ラパマイシン、アムサクリン、オール－トランスレチノイン酸、サリドマイド、アナストロゾール、ファドロゾール、レトロゾール、エキセメスタン、金チオマレート、Ｄ－ペニシラミン、ブシラミン、アザチオプリン、ミゾリビン、シクロスポリン、ラパマイシン、ヒドロコルチゾン、ベキサロテン（ターグレチン）、タモキシフェン、デキサメタゾン、プロゲスチン類、エストロゲン類、アナストロゾール（アリミデックス）、ロイプリン、アスピリン、インドメタシン、セレコキシブ、アザチオプリン、ペニシラミン、金チオマレート、マレイン酸クロルフェニラミン、クロロフェニラミン、クレマシチン、トレチノイン、ベキサロテン、砒素、ボルテゾミブ、アロプリノール、カリケアマイシン、イブリツモマブチウキセタン、タルグレチン、オゾガミン、クラリスロマシン、ロイコボリン、イファスファミド、ケトコナゾール、アミノグルテチミド、スラミン、メトトレキセート、マイタンシノイド、またはその誘導体、などが挙げられる。 Examples of anticancer agents include amifostine (ethiol), cisplatin, dacarbazine (DTIC), dactinomycin, mechlorethamine (nitrogen mustard), streptozocin, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), doxorubicin (Adriamycin), epirubicin, gemcitabine (gemzar), daunorubicin, procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil, fluorouracil, vinblastine, vincristine, bleomycin, daunomycin, pepromycin, estramustine, paclitaxel, paclitaxel, paclitaxel Taxotea), aldesleukin, asparaginase, buoy Ruphan, carboplatin, oxaliplatin, nedaplatin, cladribine, camptothecin, 10-hydroxy-7-ethyl-camptothecin (SN38), floxuridine, fludarabine, hydroxyurea, ifosfamide, idarubicin, mesna, irinotecan (CPT-11), nogitane, Mitoxantrone, topotecan, leuprolide, megestrol, melphalan, mercaptopurine, hydroxycarbamide, pricamycin, mitotan, pegsparagase, pentostatin, pipobroman, streptozocin, tamoxifen, goserelin, leuprorenin, flutamide, teniposide, test Lactone, thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil, hydrocortisone, Donisolone, methylprednisolone, vindesine, nimustine, semstin, capecitabine, tomdex, azacitidine, UFT, oxaloplatin, gefitinib (Iressa), imatinib (STI571), erlotinib, FMS-like tyrosinek inhibitor facotr receptor (VEGFR) inhibitor, fibroblast growth factor receptor (FGFR) inhibitor, epidermal growth factor receptor (EGFR) inhibitors such as Iressa, Tarceva, methoxyamino 17 A Musacrine, all-trans retinoic acid, thalidomide, anastrozole, fadrozole, letrozole, exemestane, gold thiomalate, D-penicillamine, bucillamine, azathioprine, mizoribine, cyclosporine, rapamycin, hydrocortisone, bexarotene (targretin), tamoxifen, dexamethasone , Estrogens, anastrozole (Arimidex), leupurine, aspirin, indomethacin, celecoxib, azathioprine, penicillamine, gold thiomalate, chlorpheniramine maleate, chloropheniramine, clemacitine, tretinoin, bexarotene, arsenic, bortezomib, allopurinol, cali Keamycin, Ibritumomab tiuxetan, Targretin, Ozoga Emissions, clarithromycin machine, leucovorin, Ifasufamido, ketoconazole, aminoglutethimide, suramin, methotrexate, maytansinoid, or derivatives, and the like.
　低分子の薬剤と抗体とを結合させる方法としては、例えば、グルタールアルデヒドを介して薬剤と抗体のアミノ基間を結合させる方法、または水溶性カルボジイミドを介して薬剤のアミノ基と抗体のカルボキシル基を結合させる方法などが挙げられる。 Examples of a method for binding a low molecular weight drug and an antibody include, for example, a method of binding a drug and an amino group of an antibody via glutaraldehyde, or a drug amino group and an antibody carboxyl group via a water-soluble carbodiimide. And the like.
　高分子の薬剤としては、例えば、ポリエチレングリコール（以下、ＰＥＧと表記することがある）、アルブミン、デキストラン、ポリオキシエチレン、スチレンマレイン酸コポリマー、ポリビニルピロリドン、ピランコポリマー、またはヒドロキシプロピルメタクリルアミドなどが挙げられる。これらの高分子化合物を抗体またはその断片に結合させることにより、（１）化学的、物理的あるいは生物的な種々の因子に対する安定性の向上、（２）血中半減期の顕著な延長、（３）免疫原性の消失または抗体産生の抑制、などの効果が期待される［バイオコンジュゲート医薬品，廣川書店（１９９３）］。例えば、ＰＥＧと抗体を結合させる方法としては、ＰＥＧ化修飾試薬と反応させる方法などが挙げられる［バイオコンジュゲート医薬品，廣川書店（１９９３）］。 Examples of the polymer drug include polyethylene glycol (hereinafter sometimes referred to as PEG), albumin, dextran, polyoxyethylene, styrene maleic acid copolymer, polyvinyl pyrrolidone, pyran copolymer, or hydroxypropyl methacrylamide. It is done. By binding these macromolecular compounds to antibodies or fragments thereof, (1) improved stability against various chemical, physical or biological factors, (2) significant increase in blood half-life, ( 3) Effects such as loss of immunogenicity or suppression of antibody production are expected [Bioconjugate Pharmaceuticals, Yodogawa Shoten (1993)]. For example, as a method of linking PEG and an antibody, a method of reacting with a PEGylation modifying reagent and the like can be mentioned [Bioconjugate Pharmaceutical, Yodogawa Shoten (1993)].
　ＰＥＧ化修飾試薬としては、例えば、リジンのε－アミノ基への修飾剤（日本国特開昭６１－１７８９２６号公報）、アスパラギン酸およびグルタミン酸のカルボキシル基への修飾剤（日本国特開昭５６－２３５８７号公報）、またはアルギニンのグアニジノ基への修飾剤（日本国特開平２－１１７９２０号公報）などが挙げられる。 Examples of the PEGylation modifying reagent include a modifying agent for ε-amino group of lysine (Japanese Unexamined Patent Publication No. 61-178926), a modifying agent for carboxyl group of aspartic acid and glutamic acid (Japanese Unexamined Patent Publication No. Sho 56). No. -23587), or a modifier for guanidino group of arginine (Japanese Patent Laid-Open No. 2-117920).
　免疫賦活剤としては、イムノアジュバントとして知られている天然物でもよく、具体例としては、免疫を亢進する薬剤が、β（１→３）グルカン（レンチナン、シゾフィラン）、またはαガラクトシルセラミドなどが挙げられる。 The immunostimulant may be a natural product known as an immunoadjuvant, and specific examples thereof include β (1 → 3) glucan (lentinan, schizophyllan), α galactosylceramide and the like. It is done.
　蛋白質としては、例えば、ＮＫ細胞、マクロファージ、または好中球などの免疫担当細胞を活性化するサイトカイン若しくは増殖因子、または毒素蛋白質などが挙げられる。 Examples of proteins include cytokines or growth factors that activate immunocompetent cells such as NK cells, macrophages, or neutrophils, or toxin proteins.
　サイトカインまたは増殖因子としては、例えば、インターフェロン（以下、ＩＮＦと記す）－α、ＩＮＦ－β、ＩＮＦ－γ、インターロイキン（以下、ＩＬと記す）－２、ＩＬ－１２、ＩＬ－１５、ＩＬ－１８、ＩＬ－２１、ＩＬ－２３、顆粒球コロニー刺激因子（Ｇ－ＣＳＦ）、顆粒球／マクロファージコロニー刺激因子（ＧＭ－ＣＳＦ）、またはマクロファージコロニー刺激因子（Ｍ－ＣＳＦ）などが挙げられる。 Examples of cytokines or growth factors include interferon (hereinafter referred to as INF) -α, INF-β, INF-γ, interleukin (hereinafter referred to as IL) -2, IL-12, IL-15, IL- 18, IL-21, IL-23, granulocyte colony stimulating factor (G-CSF), granulocyte / macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF) and the like.
　毒素蛋白質としては、例えば、リシン、ジフテリアトキシンなどが挙げられ、毒性を調節するためにタンパク質に変異を導入したタンパク毒素も含まれる。 Examples of the toxin protein include ricin and diphtheria toxin, and also include protein toxins in which mutations are introduced into the protein in order to regulate toxicity.
　抗体医薬としては、例えば、抗体の結合によりアポトーシスが誘導される抗原、腫瘍の病態形成に関わる抗原または免疫機能を調節する抗原、病変部位の血管新生に関与する抗原に対する抗体が挙げられる。 Examples of the antibody drug include an antigen against which apoptosis is induced by antibody binding, an antigen associated with tumor pathogenesis or an antigen that regulates immune function, and an antibody against an antigen involved in angiogenesis of a lesion site.
　抗体の結合によりアポトーシスが誘導される抗原としては、例えば、ＣＤ４、ＣＤ１９、ＣＤ２０、ＣＤ２１、ＣＤ２２、ＣＤ２３、ＣＤ２４、ＣＤ３７、ＣＤ５３、ＣＤ７２、ＣＤ７３、ＣＤ７４、ＣＤｗ７５、ＣＤｗ７６、ＣＤ７７、ＣＤｗ７８、ＣＤ７９ａ、ＣＤ７９ｂ、ＣＤ８０（Ｂ７．１）、ＣＤ８１、ＣＤ８２、ＣＤ８３、ＣＤｗ８４、ＣＤ８５、ＣＤ８６（Ｂ７．２）、ｈｕｍａｎ　ｌｅｕｋｏｃｙｔｅ　ａｎｔｉｇｅｎ（ＨＬＡ）－Ｃｌａｓｓ　ＩＩ、またはＥｐｉｄｅｒｍａｌ　Ｇｒｏｗｔｈ　Ｆａｃｔｏｒ　Ｒｅｃｅｐｔｏｒ（ＥＧＦＲ）などが挙げられる。 Antigens that induce apoptosis by antibody binding include, for example, CD4, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b , CD80 (B7.1), CD81, CD82, CD83, CDw84, CD85, CD86 (B7.2), human leukocyte antigen (HLA) -Class II, or Epidermal Growth Factor Receptor (EGFR).
　腫瘍の病態形成に関わる抗原または免疫機能を調節する抗体の抗原としては、例えば、ＣＤ４、ＣＤ４０、ＣＤ４０リガンド、Ｂ７ファミリー分子（ＣＤ８０、ＣＤ８６、ＣＤ２７４、Ｂ７－ＤＣ、Ｂ７－Ｈ２、Ｂ７－Ｈ３、またはＢ７－Ｈ４）、Ｂ７ファミリー分子のリガンド（ＣＤ２８、ＣＴＬＡ－４、ＩＣＯＳ、ＰＤ－１、またはＢＴＬＡ）、ＯＸ－４０、ＯＸ－４０リガンド、ＣＤ１３７、ｔｕｍｏｒ　ｎｅｃｒｏｓｉｓ　ｆａｃｔｏｒ（ＴＮＦ）受容体ファミリー分子（ＤＲ４、ＤＲ５、ＴＮＦＲ１、またはＴＮＦＲ２）、ＴＮＦ－ｒｅｌａｔｅｄ　ａｐｏｐｔｏｓｉｓ－ｉｎｄｕｃｉｎｇ　ｌｉｇａｎｄ　ｒｅｃｅｐｔｏｒ（ＴＲＡＩＬ）ファミリー分子、ＴＲＡＩＬファミリー分子の受容体ファミリー（ＴＲＡＩＬ－Ｒ１、ＴＲＡＩＬ－Ｒ２、ＴＲＡＩＬ－Ｒ３、またはＴＲＡＩＬ－Ｒ４）、ｒｅｃｅｐｔｏｒ　ａｃｔｉｖａｔｏｒ　ｏｆ　ｎｕｃｌｅａｒ　ｆａｃｔｏｒ　ｋａｐｐａ　Ｂ　ｌｉｇａｎｄ（ＲＡＮＫ）、ＲＡＮＫリガンド、ＣＤ２５、葉酸受容体４、サイトカイン［ＩＬ－１α、ＩＬ－１β、ＩＬ－４、ＩＬ－５、ＩＬ－６、ＩＬ－１０、ＩＬ－１３、ｔｒａｎｓｆｏｒｍｉｎｇ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ（ＴＧＦ）β、またはＴＮＦαなど］、これらのサイトカインの受容体、ケモカイン（ＳＬＣ、ＥＬＣ、Ｉ－３０９、ＴＡＲＣ、ＭＤＣ、またはＣＴＡＣＫなど）、またはこれらのケモカインの受容体が挙げられる。 Examples of antigens involved in tumor pathogenesis or antibodies that regulate immune functions include CD4, CD40, CD40 ligand, B7 family molecules (CD80, CD86, CD274, B7-DC, B7-H2, B7-H3, Or B7-H4), a ligand of a B7 family molecule (CD28, CTLA-4, ICOS, PD-1, or BTLA), OX-40, OX-40 ligand, CD137, a tumor necrosis factor (TNF) receptor family molecule ( DR4, DR5, TNFR1, or TNFR2), TNF-related apoptosis-inducing ligand receptor (TRAIL) family molecule, TRAIL family molecule receptor family (TRAIL-R1, RAIL-R2, TRAIL-R3, or TRAIL-R4), receptor activator of nuclear factor kappa B ligand (RANK), RANK ligand, CD25, folate receptor 4, cytokine [IL-1α, IL-1β, IL-4, IL-5, IL-6, IL-10, IL-13, transforming growth factor (TGF) β, or TNFα, etc.], receptors for these cytokines, chemokines (SLC, ELC, I-309, TARC, MDC, Or CTACK), or receptors for these chemokines.
　病変部位の血管新生を阻害する抗体の抗原としては、例えば、ｖａｓｃｕｌａｒ　ｅｎｄｏｔｈｅｌｉａｌ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ（ＶＥＧＦ）、Ａｎｇｉｏｐｏｉｅｔｉｎ、ｆｉｂｒｏｂｌａｓｔ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ（ＦＧＦ）、ＥＧＦ、ｐｌａｔｅｌｅｔ－ｄｅｒｉｖｅｄ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ（ＰＤＧＦ）、ｉｎｓｕｌｉｎ－ｌｉｋｅ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ（ＩＧＦ）、ｅｒｙｔｈｒｏｐｏｉｅｔｉｎ（ＥＰＯ）、ＴＧＦβ、ＩＬ－８、Ｅｐｈｉｌｉｎ、ＳＤＦ－１、またはこれらの受容体などが挙げられる。 Examples of antigens for antibodies that inhibit angiogenesis of a lesion site include, for example, vascular electrical growth factor (VEGF), anangiopoietin, fibroblast growth factor (FGF), EGF, and platelet-derived GF. IGF), erythropoietin (EPO), TGFβ, IL-8, Ephilin, SDF-1, or a receptor thereof.
　ＣＤ３３に結合するモノクローナル抗体またはその断片と、蛋白質または抗体医薬との融合抗体は、該モノクローナル抗体または該断片をコードするｃＤＮＡに、蛋白質または該抗体医薬をコードするｃＤＮＡを連結させ、融合抗体をコードするＤＮＡを構築し、該ＤＮＡを原核生物または真核生物用発現ベクターに挿入し、該発現ベクターを原核生物または真核生物へ導入することにより発現させ、該融合抗体を製造することができる。 A fusion antibody of a monoclonal antibody or a fragment thereof that binds to CD33 and a protein or antibody drug is linked to a cDNA encoding the monoclonal antibody or the fragment, and a cDNA encoding the protein or the antibody drug is linked to encode the fusion antibody. The fusion antibody can be produced by constructing a DNA to be expressed, inserting the DNA into a prokaryotic or eukaryotic expression vector, and introducing the expression vector into a prokaryotic or eukaryotic organism.
　上記抗体の誘導体を検出方法、定量方法、検出用試薬、定量用試薬または診断剤として使用する場合に、本発明のＣＤ３３の細胞外領域を特異的に認識し、かつ該細胞外領域に結合するモノクローナル抗体またはそのその断片に結合する薬剤としては、通常の免疫学的検出または測定法で用いられる標識体が挙げられる。 When the derivative of the antibody is used as a detection method, a quantification method, a detection reagent, a quantification reagent or a diagnostic agent, the extracellular region of CD33 of the present invention is specifically recognized and binds to the extracellular region. Examples of the drug that binds to the monoclonal antibody or a fragment thereof include a label used in a usual immunological detection or measurement method.
　標識体としては、例えば、アルカリフォスファターゼ、ペルオキシダーゼ若しくはルシフェラーゼなどの酵素、アクリジニウムエステル若しくはロフィンなどの発光物質、またはフルオレセインイソチオシアネート（ＦＩＴＣ）若しくはテトラメチルローダミンイソチオシアネート（ＲＩＴＣ）などの蛍光物質などが挙げられる。 Examples of the label include an enzyme such as alkaline phosphatase, peroxidase or luciferase, a luminescent substance such as acridinium ester or lophine, or a fluorescent substance such as fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (RITC). Can be mentioned.
　また、本発明は、ＣＤ３３の細胞外領域を特異的に認識し、かつ該細胞外領域に結合するモノクローナル抗体またはその断片を有効成分として含有するＣＤ３３陽性細胞が関与する疾患の治療剤に関する。 The present invention also relates to a therapeutic agent for a disease involving CD33-positive cells that specifically recognize the extracellular region of CD33 and contain a monoclonal antibody or fragment thereof that binds to the extracellular region as an active ingredient.
　ＣＤ３３陽性細胞が関与する疾患としては、ＣＤ３３が発現している細胞が関与する疾患であればいかなるものでもよく、例えば、癌、自己免疫疾患、またはアレルギー性疾患が挙げられる。 The disease involving CD33-positive cells may be any disease involving cells expressing CD33, and examples thereof include cancer, autoimmune diseases, and allergic diseases.
　ＣＤ３３陽性細胞としては、例えば、ＣＤ３３陽性細胞である単球、マクロファージ、顆粒球などが挙げられる。ＣＤ３３が発現する単球・好中球は微生物などの異物除去に関与する細胞である事から、異物貪食が関与する疾患が挙げられる。 Examples of CD33 positive cells include monocytes, macrophages, granulocytes and the like which are CD33 positive cells. Since monocytes and neutrophils expressing CD33 are cells involved in the removal of foreign substances such as microorganisms, diseases involving foreign body phagocytosis can be mentioned.
　癌としては、例えば、血液癌、乳癌、子宮癌、大腸癌、食道癌、胃癌、卵巣癌、肺癌、腎臓癌、直腸癌、甲状腺癌、子宮頸癌、小腸癌、前立腺癌または膵臓癌などが挙げられ、好ましくは血液癌、食道癌、胃癌、大腸癌、肝癌または前立腺癌が挙げられる。 Examples of cancer include blood cancer, breast cancer, uterine cancer, colon cancer, esophageal cancer, stomach cancer, ovarian cancer, lung cancer, kidney cancer, rectal cancer, thyroid cancer, cervical cancer, small intestine cancer, prostate cancer or pancreatic cancer. Preferably, hematological cancer, esophageal cancer, stomach cancer, colon cancer, liver cancer or prostate cancer can be mentioned.
　血液癌としては、例えば、ＡＭＬ、未分化大細胞型リンパ腫（Ａｎａｐｌａｓｔｉｃ　ｌａｒｇｅ　ｃｅｌｌ　ｌｙｍｐｈｏｍａ，ＡＬＣＬ）、急性リンパ性白血病（Ａｃｕｔｅ　ｌｙｍｐａｔｉｃ　ｌｅｕｋｅｍｉａ，ＡＬＬ）、骨髄異形成症候群（ｍｙｅｌｏｄｙｓｐｌａｓｔｉｃ　ｓｙｎｄｒｏｍｅ、ＭＤＳ）その他リンパ性白血病、多発性骨髄腫、ホジキンリンパ腫、または非ホジキンリンパ腫などが挙げられる。 Examples of hematological cancers include AML, anaplastic large cell lymphoma (ALCL), acute lymphocytic leukemia (ALL), myelodysplastic syndrome, MD, and other myelodysplastic syndromes. , Multiple myeloma, Hodgkin lymphoma, or non-Hodgkin lymphoma.
　自己免疫疾患としては、例えば、関節リウマチ、乾癬、クローン病、強直性脊椎炎、多発性硬化症、Ｉ型糖尿病、肝炎、心筋炎、シェーグレン症候群、または移植後拒絶反応などが挙げられる。 Examples of the autoimmune disease include rheumatoid arthritis, psoriasis, Crohn's disease, ankylosing spondylitis, multiple sclerosis, type I diabetes, hepatitis, myocarditis, Sjogren's syndrome, or rejection after transplantation.
　アレルギー性疾患としては、例えば、急性若しくは慢性気道過敏症、気管支喘息、アトピー性皮膚炎、またはアレルギー性鼻炎などが挙げられる。 Examples of allergic diseases include acute or chronic airway hypersensitivity, bronchial asthma, atopic dermatitis, or allergic rhinitis.
　本発明の治療剤としては、上述した本発明のモノクローナル抗体またはその断片を有効成分として含有する。 The therapeutic agent of the present invention contains the above-described monoclonal antibody of the present invention or a fragment thereof as an active ingredient.
　本発明の抗体またはその断片を含有する治療剤は、有効成分としての該抗体もしくは該断片のみを含むものであってもよいが、通常は薬理学的に許容される１以上の担体と一緒に混合し、製剤学の技術分野において公知の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。 The therapeutic agent containing the antibody of the present invention or a fragment thereof may contain only the antibody or the fragment as an active ingredient, but usually together with one or more pharmacologically acceptable carriers. It is desirable to mix and provide as a pharmaceutical formulation produced by any method known in the art of pharmaceutical sciences.
　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好ましく、例えば、経口投与、口腔内、気道内、直腸内、皮下、筋肉内または静脈内などの非経口投与が挙げられ、好ましくは静脈内投与が挙げられる。 It is preferable to use the administration route that is most effective for treatment, and examples include oral administration, oral administration, intratracheal, rectal, subcutaneous, intramuscular or intravenous administration, preferably Intravenous administration is mentioned.
　投与形態としては、例えば、噴霧剤、カプセル剤、錠剤、散剤、顆粒剤、シロップ剤、乳剤、座剤、注射剤、軟膏、またはテープ剤などが挙げられる。 Examples of administration forms include sprays, capsules, tablets, powders, granules, syrups, emulsions, suppositories, injections, ointments, or tapes.
　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、および体重などにより異なるが、通常成人１日当たり１０μｇ／ｋｇ～２０ｍｇ／ｋｇである。 The dose or frequency of administration varies depending on the intended therapeutic effect, administration method, treatment period, age, weight, etc., but is usually 10 μg / kg to 20 mg / kg per day for an adult.
　さらに、本発明は、ＣＤ３３の細胞外領域を特異的に認識し、かつ該細胞外領域に結合するモノクローナル抗体またはその断片を有効成分として含有する、ＣＤ３３の免疫学的検出または測定方法、ＣＤ３３の免疫学的検出用または測定用試薬、ＣＤ３３が発現する細胞の免疫学的検出または測定方法、およびＣＤ３３陽性細胞が関与する疾患の診断剤に関する。 Furthermore, the present invention relates to a method for immunological detection or measurement of CD33, which contains as an active ingredient a monoclonal antibody or a fragment thereof that specifically recognizes the extracellular region of CD33 and binds to the extracellular region. The present invention relates to a reagent for immunological detection or measurement, an immunological detection or measurement method for cells expressing CD33, and a diagnostic agent for a disease involving CD33-positive cells.
　本発明においてＣＤ３３の量を検出または測定する方法としては、任意の公知の方法が挙げられる。例えば、免疫学的検出または測定方法などが挙げられる。 In the present invention, any known method may be used as a method for detecting or measuring the amount of CD33. Examples include immunological detection or measurement methods.
　免疫学的検出または測定方法とは、標識を施した抗原または抗体を用いて、抗体量または抗原量を検出または測定する方法である。免疫学的検出または測定方法としては、例えば、放射性物質標識免疫抗体法（ＲＩＡ）、酵素免疫測定法（ＥＩＡまたはＥＬＩＳＡ）、蛍光免疫測定法（ＦＩＡ）、発光免疫測定法（ｌｕｍｉｎｅｓｃｅｎｔ　ｉｍｍｕｎｏａｓｓａｙ）、ウェスタンブロット法または物理化学的手法などが挙げられる。 The immunological detection or measurement method is a method of detecting or measuring the amount of antibody or the amount of antigen using a labeled antigen or antibody. Examples of immunological detection or measurement methods include radiolabeled immunoassay (RIA), enzyme immunoassay (EIA or ELISA), fluorescence immunoassay (FIA), luminescence immunoassay (Western immunoassay), Western Examples include blotting or physicochemical techniques.
　本発明のモノクローナル抗体またはその断片を用いてＣＤ３３が発現した細胞を検出または測定することにより、ＣＤ３３が関連する疾患を診断することができる。 By detecting or measuring cells expressing CD33 using the monoclonal antibody of the present invention or a fragment thereof, a disease associated with CD33 can be diagnosed.
　該ポリペプチドが発現している細胞の検出には、公知の免疫学的検出法を用いることができるが、免疫沈降法、蛍光細胞染色法、免疫組織染色法、または免疫組織染色法などが、好ましく用いられる。また、ＦＭＡＴ８１００ＨＴＳシステム（アプライドバイオシステム社製）などの蛍光抗体染色法なども用いることができる。 Known immunological detection methods can be used for detection of cells expressing the polypeptide, but immunoprecipitation, fluorescent cell staining, immunohistochemical staining, immunohistological staining, etc. Preferably used. Moreover, fluorescent antibody staining methods such as FMAT8100HTS system (Applied Biosystems) can also be used.
　本発明においてＣＤ３３を検出または測定する対象となる生体試料としては、例えば、組織細胞、骨髄細胞、骨髄液、血液、血漿、血清、膵液、尿、糞便、組織液、または培養液など、ＣＤ３３を発現した細胞を含む可能性のあるものであれば特に限定されない。 Examples of biological samples to be detected or measured for CD33 in the present invention include CD33 expression such as tissue cells, bone marrow cells, bone marrow fluid, blood, plasma, serum, pancreatic juice, urine, stool, tissue fluid, or culture fluid. The cell is not particularly limited as long as it may contain the cells.
　本発明のモノクローナル抗体またはその断片を含有する診断剤は、目的の診断法に応じて、抗原抗体反応を行なうための試薬、該反応の検出用試薬を含んでもよい。抗原抗体反応を行なうための試薬としては、例えば、緩衝剤または塩などが挙げられる。検出用試薬としては、例えば、該モノクローナル抗体若しくはその断片を認識する標識された二次抗体、または標識に対応した基質などの通常の免疫学的検出若しくは測定法に用いられる試薬が挙げられる。 The diagnostic agent containing the monoclonal antibody or fragment thereof of the present invention may contain a reagent for conducting an antigen-antibody reaction and a reagent for detecting the reaction, depending on the target diagnostic method. Examples of the reagent for performing the antigen-antibody reaction include a buffer or a salt. Examples of the reagent for detection include a labeled secondary antibody that recognizes the monoclonal antibody or a fragment thereof, or a reagent used for usual immunological detection or measurement methods such as a substrate corresponding to the label.
　以下に、本発明の抗体の製造方法、疾患の治療方法、および疾患の診断方法について、具体的に説明する。 The antibody production method, disease treatment method, and disease diagnosis method of the present invention will be specifically described below.
　１．モノクローナル抗体の製造方法
（１）抗原の調製
　抗原となるＣＤ３３またはＣＤ３３を発現させた細胞は、ＣＤ３３全長またはその部分長をコードするｃＤＮＡを含む発現ベクターを、大腸菌、酵母、昆虫細胞、または動物細胞などに導入することにより、得ることができる。また、ＣＤ３３を多量に発現している各種ヒト腫瘍培養細胞、ヒト組織などからＣＤ３３を精製し、得ることが出来る。また、該腫瘍培養細胞、または該組織などをそのまま抗原として用いることもできる。さらに、Ｆｍｏｃ法、またはｔＢｏｃ法などの化学合成法によりＣＤ３３の部分配列を有する合成ペプチドを調製し、抗原に用いることもできる。 1. Method for Producing Monoclonal Antibody (1) Preparation of antigen Cells expressing CD33 or CD33 as an antigen are expressed in E. coli, yeast, insect cells, or animal cells using an expression vector containing cDNA encoding CD33 full length or a partial length thereof. It can be obtained by introducing into the above. In addition, CD33 can be purified and obtained from cultured human tumor cells, human tissues, etc. that express CD33 in large amounts. Further, the cultured tumor cells or the tissue can be used as an antigen as it is. Furthermore, a synthetic peptide having a partial sequence of CD33 can be prepared by a chemical synthesis method such as the Fmoc method or the tBoc method and used as an antigen.
　本発明で用いられるＣＤ３３は、Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ，Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ，Ｓｅｃｏｎｄ　Ｅｄｉｔｉｏｎ，Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓ（１９８９）またはＣｕｒｒｅｎｔ　Ｐｒｏｔｏｃｏｌｓ　Ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ，Ｊｏｈｎ　Ｗｉｌｅｙ＆Ｓｏｎｓ（１９８７－１９９７）などに記載された方法などを用い、例えば、以下の方法により、該ＣＤ３３をコードするＤＮＡを宿主細胞中で発現させて、製造することができる。 The CD33 used in the present invention is described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) or Current Protocols in MolecularJoule7, 198 For example, the DNA encoding CD33 can be expressed in a host cell and produced by the following method.
　まず、ＣＤ３３をコードする部分を含む完全長ｃＤＮＡを適当な発現ベクターのプロモーターの下流に挿入することにより、組換えベクターを作製する。上記完全長ｃＤＮＡの代わりに、完全長ｃＤＮＡをもとにして調製された、ポリペプチドをコードする部分を含む適当な長さのＤＮＡ断片を用いてもよい。次に、得られた該組換えベクターを、該組換えベクターに適合した宿主細胞に導入することにより、ポリペプチドを生産する形質転換体を得ることができる。 First, a recombinant vector is prepared by inserting a full-length cDNA containing a portion encoding CD33 downstream of a promoter of an appropriate expression vector. Instead of the full-length cDNA, a DNA fragment of an appropriate length containing a polypeptide-encoding portion prepared based on the full-length cDNA may be used. Next, a transformant producing a polypeptide can be obtained by introducing the obtained recombinant vector into a host cell suitable for the recombinant vector.
　発現ベクターとしては、使用する宿主細胞における自律複製または染色体中への組込みが可能で、ポリペプチドをコードするＤＮＡを転写できる位置に、適当なプロモーターを含有しているものであればいずれも用いることができる。 Any expression vector can be used as long as it can autonomously replicate in the host cell to be used or can be integrated into the chromosome, and contains an appropriate promoter at a position where DNA encoding the polypeptide can be transcribed. Can do.
　宿主細胞としては、大腸菌などのエシェリヒア属などに属する微生物、酵母、昆虫細胞、または動物細胞など、目的とする遺伝子を発現できるものであればいずれも用いることができる。 As the host cell, any microorganism that belongs to the genus Escherichia such as Escherichia coli, yeast, insect cells, or animal cells can be used so long as it can express the target gene.
　大腸菌などの原核生物を宿主細胞として用いる場合、組換えベクターは、原核生物中で自律複製が可能であると同時に、プロモーター、リボソーム結合配列、ＣＤ３３をコードする部分を含むＤＮＡ、および転写終結配列を含むベクターであることが好ましい。また、該組換えベクターには、転写終結配列は必ずしも必要ではないが、構造遺伝子の直下に転写終結配列を配置することが好ましい。さらに、該組換えベクターには、プロモーターを制御する遺伝子を含んでいてもよい。 When a prokaryote such as E. coli is used as a host cell, the recombinant vector is capable of autonomous replication in a prokaryote and at the same time contains a promoter, a ribosome binding sequence, DNA containing a CD33-encoding portion, and a transcription termination sequence. It is preferable that it is a vector containing. The recombinant vector does not necessarily require a transcription termination sequence, but it is preferable to place the transcription termination sequence immediately below the structural gene. Furthermore, the recombinant vector may contain a gene that controls the promoter.
　該組換えベクターとしては、リボソーム結合配列であるシャイン・ダルガルノ配列（ＳＤ配列ともいう）と開始コドンとの間を適当な距離（例えば６～１８塩基）に調節したプラスミドを用いることが好ましい。 As the recombinant vector, it is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence (also referred to as SD sequence), which is a ribosome binding sequence, and the start codon is adjusted to an appropriate distance (eg, 6 to 18 bases).
　また、該ＣＤ３３をコードするＤＮＡの塩基配列としては、宿主内での発現に最適なコドンとなるように塩基を置換することができ、これにより目的とするＣＤ３３の生産率を向上させることができる。 In addition, the base sequence of the DNA encoding CD33 can be substituted so that the codon is optimal for expression in the host, thereby improving the production rate of the target CD33. .
　発現ベクターとしては、使用する宿主細胞中で機能を発揮できるものであればいずれも用いることができる。例えば、ｐＢＴｒｐ２、ｐＢＴａｃ１、ｐＢＴａｃ２（以上、ロシュ・ダイアグノスティックス社製）、ｐＫＫ２３３―２（ファルマシア社製）、ｐＳＥ２８０（インビトロジェン社製）、ｐＧＥＭＥＸ－１（プロメガ社製）、ｐＱＥ－８（キアゲン社製）、ｐＫＹＰ１０（日本国特開昭５８－１１０６００号公報）、ｐＫＹＰ２００［Ａｇｒｉｃｕｌｔｕｒａｌ　Ｂｉｏｌｏｇｉｃａｌ　Ｃｈｅｍｉｓｔｒｙ，４８，６６９（１９８４）］、ｐＬＳＡ１［Ａｇｒｉｃ．Ｂｉｏｌ．Ｃｈｅｍ．，５３，２７７（１９８９）］、ｐＧＥＬ１［Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，８２，４３０６（１９８５）］、ｐＢｌｕｅｓｃｒｉｐｔ　ＩＩ　ＳＫ（－）（ストラタジーン社製）、ｐＴｒｓ３０［大腸菌ＪＭ１０９／ｐＴｒＳ３０（ＦＥＲＭ　ＢＰ－５４０７）より調製］、ｐＴｒｓ３２［大腸菌ＪＭ１０９／ｐＴｒＳ３２（ＦＥＲＭ　ＢＰ－５４０８）より調製］、ｐＧＨＡ２［大腸菌ＩＧＨＡ２（ＦＥＲＭ　ＢＰ－４００）より調製、日本国特開昭６０－２２１０９１号公報］、ｐＧＫＡ２［大腸菌ＩＧＫＡ２（ＦＥＲＭ　ＢＰ－６７９８）より調製、日本国特開昭６０－２２１０９１号公報］、ｐＴｅｒｍ２（米国特許第４６８６１９１号明細書、米国特許第４９３９０９４号明細書、米国特許第５１６０７３５号明細書）、ｐＳｕｐｅｘ、ｐＵＢ１１０、ｐＴＰ５、ｐＣ１９４、ｐＥＧ４００［Ｊ．Ｂａｃｔｅｒｉｏｌ．，１７２，２３９２（１９９０）］、ｐＧＥＸ（ファルマシア社製）、ｐＥＴシステム（ノバジェン社製）、またはｐＭＥ１８ＳＦＬ３などが挙げられる。 Any expression vector can be used as long as it can function in the host cell to be used. For example, pBTrp2, pBTac1, pBTac2 (above, Roche Diagnostics), pKK233-2 (Pharmacia), pSE280 (Invitrogen), pGEMEX-1 (Promega), pQE-8 (Qiagen) PKYP10 (Japanese Unexamined Patent Publication No. 58-110600), pKYP200 [Agricultural Biological Chemistry, 48, 669 (1984)], pLSA1 [Agric. Biol. Chem. , 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], pBluescript II SK (-) (manufactured by Stratagene), pTrs30 [prepared from E. coli JM109 / pTrS30 (FERM BP-5407)], pTrs32 [E. coli JM109 / pTrS32 (FERM BP-5408). ) Prepared], pGHA2 [prepared from E. coli IGHA2 (FERM BP-400), Japanese Unexamined Patent Publication No. 60-221091], pGKA2 [prepared from E. coli IGKA2 (FERM BP-6798), Japanese Unexamined Patent Publication 60- 221091], pTerm2 (US Pat. No. 4,686,191, US Pat. No. 4,939,094, US Pat. No. 5,160,735), pSupex, pUB110, pTP5, pC194, pEG400 [J. Bacteriol. 172, 2392 (1990)], pGEX (Pharmacia), pET system (Novagen), or pME18SFL3.
　プロモーターとしては、使用する宿主細胞中で機能を発揮できるものであればいかなるものでもよい。例えば、ｔｒｐプロモーター（Ｐｔｒｐ）、ｌａｃプロモーター、ＰＬプロモーター、ＰＲプロモーター、またはＴ７プロモーターなどの、大腸菌やファージなどに由来するプロモーターを挙げることができる。また、例えば、Ｐｔｒｐを２つ直列させたタンデムプロモーター、ｔａｃプロモーター、ｌａｃＴ７プロモーター、またはｌｅｔ　Ｉプロモーターなどの人為的に設計改変されたプロモーターなども用いることができる。 As the promoter, any promoter can be used as long as it can function in the host cell to be used. For example, promoters derived from Escherichia coli or phage, such as trp promoter (Ptrp), lac promoter, PL promoter, PR promoter, or T7 promoter can be mentioned. In addition, artificially designed and modified promoters such as a tandem promoter, tac promoter, lacT7 promoter, or let I promoter in which two Ptrps are connected in series can also be used.
　宿主細胞としては、例えば、大腸菌ＸＬ１－Ｂｌｕｅ、大腸菌ＸＬ２－Ｂｌｕｅ、大腸菌ＤＨ１、大腸菌ＭＣ１０００、大腸菌ＫＹ３２７６、大腸菌Ｗ１４８５、大腸菌ＪＭ１０９、大腸菌ＨＢ１０１、大腸菌Ｎｏ．４９、大腸菌Ｗ３１１０、大腸菌ＮＹ４９、または大腸菌ＤＨ５αなどが挙げられる。 Examples of host cells include E. coli XL1-Blue, E. coli XL2-Blue, E. coli DH1, E. coli MC1000, E. coli KY3276, E. coli W1485, E. coli JM109, E. coli HB101, E. coli No. 49, E. coli W3110, E. coli NY49, or E. coli DH5α.
　宿主細胞への組換えベクターの導入方法としては、使用する宿主細胞へＤＮＡを導入する方法であればいずれも用いることができ、例えば、カルシウムイオンを用いる方法［Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，６９，２１１０（１９７２）、Ｇｅｎｅ，１７，１０７（１９８２）、Ｍｏｌｅｃｕｌａｒ＆Ｇｅｎｅｒａｌ　Ｇｅｎｅｔｉｃｓ，１６８，１１１（１９７９）］が挙げられる。 Any method can be used for introducing a recombinant vector into a host cell as long as it is a method for introducing DNA into the host cell to be used. For example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972), Gene, 17, 107 (1982), Molecular & General Genetics, 168, 111 (1979)].
　動物細胞を宿主として用いる場合、発現ベクターとしては、動物細胞中で機能を発揮できるものであればいずれも用いることができる。例えば、ｐｃＤＮＡＩ、ｐｃＤＭ８（フナコシ社製）、ｐＡＧＥ１０７［日本国特開平３－２２９７９号公報；Ｃｙｔｏｔｅｃｈｎｏｌｏｇｙ，３，１３３（１９９０）］、ｐＡＳ３－３（日本国特開平２－２２７０７５号公報）、ｐＣＤＭ８［Ｎａｔｕｒｅ，３２９，８４０（１９８７）］、ｐｃＤＮＡＩ／Ａｍｐ（インビトロジェン社製）、ｐｃＤＮＡ３．１（インビトロジェン社製）、ｐＲＥＰ４（インビトロジェン社製）、ｐＡＧＥ１０３［Ｊ．Ｂｉｏｃｈｅｍｉｓｔｒｙ，１０１，１３０７（１９８７）］、ｐＡＧＥ２１０、ｐＭＥ１８ＳＦＬ３、またはｐＫＡＮＴＥＸ９３（国際公開第９７／１０３５４号公報）などが挙げられる。 When animal cells are used as a host, any expression vector can be used as long as it can function in animal cells. For example, pcDNAI, pcDM8 (manufactured by Funakoshi), pAGE107 [Japanese Unexamined Patent Publication No. 3-22979; Cytotechnology, 3, 133 (1990)], pAS3-3 (Japanese Unexamined Patent Publication No. 2227705), pCDM8 [ Nature, 329, 840 (1987)], pcDNAI / Amp (Invitrogen), pcDNA3.1 (Invitrogen), pREP4 (Invitrogen), pAGE103 [J. Biochemistry, 101, 1307 (1987)], pAGE210, pME18SFL3, or pKANTEX93 (International Publication No. 97/10354).
　プロモーターとしては、動物細胞中で機能を発揮できるものであればいずれも用いることができる。例えば、サイトメガロウイルス（ＣＭＶ）のｉｍｍｅｄｉａｔｅ　ｅａｒｌｙ（ＩＥ）遺伝子のプロモーター、ＳＶ４０の初期プロモーター、レトロウイルスのプロモーター、メタロチオネインプロモーター、ヒートショックプロモーター、ＳＲαプロモーター、またはモロニーマウス白血病ウイルスのプロモーターまたはエンハンサーが挙げられる。また、ヒトＣＭＶのＩＥ遺伝子のエンハンサーをプロモーターと共に用いてもよい。 Any promoter can be used as long as it can function in animal cells. Examples include cytomegalovirus (CMV) immediate early (IE) gene promoter, SV40 early promoter, retroviral promoter, metallothionein promoter, heat shock promoter, SRα promoter, or Moloney murine leukemia virus promoter or enhancer. . In addition, an enhancer of human CMV IE gene may be used together with a promoter.
　宿主細胞としては、例えば、ヒト白血病細胞Ｎａｍａｌｗａ細胞、サル細胞ＣＯＳ細胞、チャイニーズ・ハムスター卵巣細胞ＣＨＯ細胞（Ｊｏｕｒｎａｌ　ｏｆ　Ｅｘｐｅｒｉｍｅｎｔａｌ　Ｍｅｄｉｃｉｎｅ，１０８，９４５（１９５８）；　Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，６０，１２７５（１９６８）；　Ｇｅｎｅｔｉｃｓ，５５，５１３（１９６８）；　Ｃｈｒｏｍｏｓｏｍａ，４１，１２９（１９７３）；　Ｍｅｔｈｏｄｓ　ｉｎ　Ｃｅｌｌ　Ｓｃｉｅｎｃｅ，１８，１１５（１９９６）；　Ｒａｄｉａｔｉｏｎ　Ｒｅｓｅａｒｃｈ，１４８，２６０（１９９７）；　Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，７７，４２１６（１９８０）；　Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．，６０，１２７５（１９６８）；　Ｃｅｌｌ，６，１２１（１９７５）；　Ｍｏｌｅｃｕｌａｒ　Ｃｅｌｌ　Ｇｅｎｅｔｉｃｓ，Ａｐｐｅｎｄｉｘ　Ｉ，ＩＩ（ｐｐ．８８３－９００）；）、ＣＨＯ／ＤＧ４４、ＣＨＯ－Ｋ１（ＡＴＣＣ　ＣＣＬ－６１）、ＤＵｋＸＢ１１（ＡＴＣＣ　ＣＣＬ－９０９６）、Ｐｒｏ－５（ＡＴＣＣ　ＣＣＬ－１７８１）、ＣＨＯ－Ｓ（Ｌｉｆｅ　Ｔｅｃｈｎｏｌｏｇｉｅｓ，Ｃａｔ　＃　１１６１９）、Ｐｒｏ－３、ラットミエローマ細胞ＹＢ２／３ＨＬ．Ｐ２．Ｇ１１．１６Ａｇ．２０（またはＹＢ２／０ともいう）、マウスミエローマ細胞ＮＳＯ、マウスミエローマ細胞ＳＰ２／０－Ａｇ１４、シリアンハムスター細胞ＢＨＫまたはＨＢＴ５６３７（日本国特開昭６３－０００２９９号公報）、などが挙げられる。 Examples of host cells include human leukemia cells Namalwa cells, monkey cells COS cells, Chinese hamster ovary cells CHO cells (Journal of Experimental Medicine, 108, 945 (1958); Proc. Natl. Acad. Sci. USA, 60, 1275 (1968); Genetics, 55, 513 (1968); Chromoma, 41, 129 (1973); Methods in Cell Science, 18, 115 (1996); Radiation Research, 148, 260 (1997); Proc. Natl. Sci.USA, 77, 4216 (1980); Proc.Natl.Acad.Sci., 60,127 (1968); Cell, 6, 121 (1975); Molecular Cell Genetics, Appendix I, II (pp. 883-900);), CHO / DG44, CHO-K1 (ATCC CCL-61), DUkXB11 (ATCC CCL- 9096), Pro-5 (ATCC CCL-1781), CHO-S (Life Technologies, Cat # 11619), Pro-3, rat myeloma cells YB2 / 3HL. P2. G11.16 Ag. 20 (also referred to as YB2 / 0), mouse myeloma cell NSO, mouse myeloma cell SP2 / 0-Ag14, Syrian hamster cell BHK or HBT5637 (Japanese Unexamined Patent Publication No. 63-000299), and the like.
　宿主細胞への組換えベクターの導入方法としては、動物細胞にＤＮＡを導入する方法であればいずれも用いることができ、例えば、エレクトロポレーション法［Ｃｙｔｏｔｅｃｈｎｏｌｏｇｙ，３，１３３（１９９０）］、リン酸カルシウム法（日本国特開平２－２２７０７５号公報）、またはリポフェクション法［Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，８４，７４１３（１９８７）］などが挙げられる。 As a method for introducing a recombinant vector into a host cell, any method can be used as long as it introduces DNA into animal cells. For example, electroporation method [Cytotechnology, 3, 133 (1990)], calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), or lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)].
　以上のようにして得られるＣＤ３３をコードするＤＮＡを組み込んだ組換えベクターを保有する微生物、または動物細胞などに由来する形質転換体を培地に培養し、培養物中に該ＣＤ３３を生成蓄積させ、該培養物から採取することにより、ＣＤ３３を製造することができる。該形質転換体を培地に培養する方法は、宿主の培養に用いられる通常の方法に従って行うことができる。 Culturing a transformant derived from a microorganism or animal cell having a recombinant vector into which DNA encoding CD33 obtained as described above is incorporated in a medium, and producing and accumulating the CD33 in the culture, By collecting from the culture, CD33 can be produced. The method of culturing the transformant in a medium can be performed according to a usual method used for culturing a host.
　真核生物由来の細胞で発現させた場合には、糖または糖鎖が付加されたＣＤ３３を得ることができる。 When expressed in cells derived from eukaryotes, CD33 with an added sugar or sugar chain can be obtained.
　誘導性のプロモーターを用いた組換えベクターで形質転換した微生物を培養するときには、必要に応じてインデューサーを培地に添加してもよい。例えば、ｌａｃプロモーターを用いた組換えベクターで形質転換した微生物を培養する場合にはイソプロピル－β－Ｄ－チオガラクトピラノシドなどを、ｔｒｐプロモーターを用いた組換えベクターで形質転換した微生物を培養する場合にはインドールアクリル酸などを培地に添加してもよい。 When culturing a microorganism transformed with a recombinant vector using an inducible promoter, an inducer may be added to the medium as necessary. For example, when cultivating a microorganism transformed with a recombinant vector using the lac promoter, cultivate a microorganism transformed with isopropyl-β-D-thiogalactopyranoside or the like using a recombinant vector using the trp promoter. In this case, indole acrylic acid or the like may be added to the medium.
　動物細胞を宿主として得られた形質転換体を培養する培地としては、例えば、一般に使用されているＲＰＭＩ１６４０培地［Ｔｈｅ　Ｊｏｕｒｎａｌ　ｏｆ　ｔｈｅ　Ａｍｅｒｉｃａｎ　Ｍｅｄｉｃａｌ　Ａｓｓｏｃｉａｔｉｏｎ，１９９，５１９（１９６７）］、ＥａｇｌｅのＭＥＭ培地［Ｓｃｉｅｎｃｅ，１２２，５０１（１９５２）］、ダルベッコ改変ＭＥＭ培地［Ｖｉｒｏｌｏｇｙ，８，３９６（１９５９）］、１９９培地［Ｐｒｏｃ．Ｓｏｃ．Ｅｘｐ．Ｂｉｏｌ．Ｍｅｄ．，７３，１（１９５０）］、Ｉｓｃｏｖｅ’ｓ　Ｍｏｄｉｆｉｅｄ　Ｄｕｌｂｅｃｃｏ’ｓ　Ｍｅｄｉｕｍ（ＩＭＤＭ）培地、またはこれら培地に牛胎児血清（ＦＢＳ）などを添加した培地などが挙げられる。培養は、通常ｐＨ６～８、３０～４０℃、５％ＣＯ２存在下などの条件下で１～７日間行う。また、培養中必要に応じて、カナマイシン、ペニシリンなどの抗生物質を培地に添加してもよい。 Examples of a medium for culturing a transformant obtained using an animal cell as a host include, for example, a commonly used RPMI 1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], Eagle's MEM medium [Science]. , 122, 501 (1952)], Dulbecco's modified MEM medium [Virology, 8, 396 (1959)], 199 medium [Proc. Soc. Exp. Biol. Med. 73, 1 (1950)], Iscove's Modified Dulbecco's Medium (IMDM) medium, or a medium obtained by adding fetal bovine serum (FBS) or the like to these mediums. The culture is usually carried out for 1 to 7 days under conditions such as pH 6 to 8, 30 to 40 ° C., and 5% CO 2 . Moreover, you may add antibiotics, such as kanamycin and penicillin, to a culture medium as needed during culture | cultivation.
　ＣＤ３３をコードする遺伝子の発現方法としては、例えば、直接発現以外に、分泌生産または融合蛋白質発現などの方法［Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ，Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ，Ｓｅｃｏｎｄ　Ｅｄｉｔｉｏｎ，Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓ（１９８９）］が挙げられる。 As an expression method of the gene encoding CD33, for example, methods such as secretory production or fusion protein expression other than direct expression [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)] can be mentioned. .
　ＣＤ３３の生産方法としては、例えば、宿主細胞内に生産させる方法、宿主細胞外に分泌させる方法、または宿主細胞外膜上に生産させる方法が挙げられ、使用する宿主細胞や、生産させるＣＤ３３の構造を変えることにより、適切な方法を選択することができる。 Examples of the production method of CD33 include a method of producing in a host cell, a method of secreting it outside the host cell, and a method of producing it on the host cell outer membrane. The host cell to be used and the structure of CD33 to be produced are exemplified. By changing, an appropriate method can be selected.
　ＣＤ３３が宿主細胞内又は宿主細胞外膜上に生産される場合、ポールソンらの方法［Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．，２６４，１７６１９（１９８９）］、ロウらの方法［Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．，ＵＳＡ，８６，８２２７（１９８９）、Ｇｅｎｅｓ　Ｄｅｖｅｌｏｐ．，４，１２８８（１９９０）］、日本国特開平０５－３３６９６３号公報、または国際公開第９４／２３０２１号公報などに記載の方法を用いることにより、ＣＤ３３を宿主細胞外に積極的に分泌させることができる。 When CD33 is produced in the host cell or on the host cell outer membrane, the method of Paulson et al. [J. Biol. Chem. , 264, 17619 (1989)], the method of Lowe et al. [Proc. Natl. Acad. Sci. USA, 86, 8227 (1989), Genes Develop. , 4, 1288 (1990)], and actively secreting CD33 outside the host cell by using the method described in Japanese Patent Application Laid-Open No. 05-336963 or International Publication No. 94/23021. Can do.
　また、ジヒドロ葉酸還元酵素遺伝子などを用いた遺伝子増幅系（日本国特開平２－２２７０７５号公報）を利用してＣＤ３３の生産量を上昇させることもできる。 In addition, the production amount of CD33 can be increased by using a gene amplification system using a dihydrofolate reductase gene or the like (Japanese Patent Laid-Open No. 2-227075).
　得られたＣＤ３３は、例えば、以下のようにして単離、精製することができる。 The obtained CD33 can be isolated and purified as follows, for example.
　ＣＤ３３が細胞内に溶解状態で発現した場合には、培養終了後に細胞を遠心分離により回収し、水系緩衝液に懸濁後、超音波破砕機、フレンチプレス、マントンガウリンホモゲナイザー、またはダイノミルなどを用いて細胞を破砕し、無細胞抽出液を得る。該無細胞抽出液を遠心分離することにより得られる上清から、通常の蛋白質の単離精製法、即ち、溶媒抽出法、硫安などによる塩析法、脱塩法、有機溶媒による沈殿法、ジエチルアミノエチル（ＤＥＡＥ）－セファロース、ＤＩＡＩＯＮ　ＨＰＡ－７５（三菱化学社製）などのレジンを用いた陰イオン交換クロマトグラフィー法、Ｓ－Ｓｅｐｈａｒｏｓｅ　ＦＦ（ファルマシア社製）などのレジンを用いた陽イオン交換クロマトグラフィー法、ブチルセファロース、フェニルセファロースなどのレジンを用いた疎水性クロマトグラフィー法、分子篩を用いたゲルろ過法、アフィニティークロマトグラフィー法、クロマトフォーカシング法、または等電点電気泳動などの電気泳動法などの手法を単独または組み合わせて用い、精製標品を得ることができる。 When CD33 is expressed in a dissolved state in the cells, the cells are collected by centrifugation after culturing, suspended in an aqueous buffer solution, and then used with an ultrasonic crusher, a French press, a Manton Gaurin homogenizer, or a dynomill. The cells are disrupted to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, an ordinary protein isolation and purification method, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, diethylamino Anion exchange chromatography using a resin such as ethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Chemical), cation exchange chromatography using a resin such as S-Sepharose FF (manufactured by Pharmacia) , Methods such as hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, or electrophoresis such as isoelectric focusing Can be used alone or in combination to obtain purified preparations. Can.
　ＣＤ３３が細胞内に不溶体を形成して発現した場合は、上記と同様に細胞を回収後破砕し、遠心分離を行うことにより、沈殿画分として該ＣＤ３３の不溶体を回収する。回収した該ＣＤ３３の不溶体を蛋白質変性剤で可溶化する。該可溶化液を希釈または透析することにより、該ＣＤ３３を正常な立体構造に戻した後、上記と同様の単離精製法によりポリペプチドの精製標品を得ることができる。 When CD33 is expressed by forming an insoluble substance in the cell, the cell is recovered and crushed in the same manner as described above, and centrifuged to collect the insoluble substance of CD33 as a precipitate fraction. The recovered insoluble matter of CD33 is solubilized with a protein denaturant. The solubilized solution is diluted or dialyzed to return the CD33 to a normal three-dimensional structure, and then a purified polypeptide preparation can be obtained by the same isolation and purification method as described above.
　ＣＤ３３またはその糖修飾体などの誘導体が細胞外に分泌された場合には、培養上清において該ＣＤ３３またはその糖修飾体などの誘導体を回収することができる。該培養物を上記と同様に遠心分離などの手法により処理することにより可溶性画分を取得し、該可溶性画分から、上記と同様の単離精製法を用いることにより、精製標品を得ることができる。 When a derivative such as CD33 or a modified sugar thereof is secreted extracellularly, the derivative such as CD33 or a modified sugar thereof can be recovered from the culture supernatant. A soluble fraction can be obtained by treating the culture by a method such as centrifugation as described above, and a purified preparation can be obtained from the soluble fraction by using the same isolation and purification method as described above. it can.
　ＣＤ３３に適当なペプチドを付加する事により容易に精製が可能となる。例えば、イムノグロブリンのＦｃ領域をＣＤ３３に付加する事により、細胞外に分泌されたＣＤ３３－Ｆｃ融合タンパク質をプロテインＡまたはプロテインＧなどを用いることにより、精製標品を得ることが出来る。 Purification can be easily performed by adding an appropriate peptide to CD33. For example, by adding the Fc region of immunoglobulin to CD33 and using CD33-Fc fusion protein secreted extracellularly using protein A or protein G, a purified preparation can be obtained.
　また、本発明において用いられるＣＤ３３は、Ｆｍｏｃ法、またはｔＢｏｃ法などの化学合成法によっても製造することができる。また、アドバンストケムテック社製、パーキン・エルマー社製、ファルマシア社製、プロテインテクノロジインストルメント社製、シンセセル－ベガ社製、パーセプチブ社製、または島津製作所社製などのペプチド合成機を利用して化学合成することもできる。 Further, CD33 used in the present invention can also be produced by a chemical synthesis method such as the Fmoc method or the tBoc method. In addition, chemical synthesis using peptide synthesizers such as Advanced Chemtech, Perkin Elmer, Pharmacia, Protein Technology Instrument, Synthecel-Vega, Perceptive, or Shimadzu It can also be synthesized.
（２）動物の免疫と融合用抗体産生細胞の調製
　３～２０週令のマウス、ラットまたはハムスターなどの動物に、（１）で得られる抗原を免疫して、その動物の脾、リンパ節、末梢血中の抗体産生細胞を採取する。また、免疫原性が低く上記の動物で充分な抗体価の上昇が認められない場合には、ＣＤ３３ノックアウトマウスを被免疫動物として用いることもできる。 (2) Immunization of animals and preparation of antibody-producing cells for fusion 3 to 20-week-old animals such as mice, rats or hamsters are immunized with the antigen obtained in (1), and the spleen, lymph nodes, Collect antibody-producing cells in peripheral blood. In addition, CD33 knockout mice can be used as immunized animals when the immunogenicity is low and the above-mentioned animals do not have a sufficient increase in antibody titer.
　免疫は、動物の皮下、静脈内または腹腔内に、例えば、フロインドの完全アジュバント、または水酸化アルミニウムゲルと百日咳菌ワクチンなどの適当なアジュバントとともに抗原を投与することにより行う。抗原が部分ペプチドである場合には、ＢＳＡ（ウシ血清アルブミン）、またはＫＬＨ（Ｋｅｙｈｏｌｅ　Ｌｉｍｐｅｔ　ｈｅｍｏｃｙａｎｉｎ）などのキャリア蛋白質とコンジュゲートを作製し、これを免疫原として用いる。 Immunization is performed by administering the antigen subcutaneously, intravenously or intraperitoneally to the animal together with an appropriate adjuvant such as Freund's complete adjuvant or aluminum hydroxide gel and pertussis vaccine. When the antigen is a partial peptide, a conjugate with a carrier protein such as BSA (bovine serum albumin) or KLH (Keyhole limpet hemocyanin) is prepared and used as an immunogen.
　抗原の投与は、１回目の投与の後、１～２週間おきに５～１０回行う。各投与後３～７日目に眼底静脈叢より採血し、その血清の抗体価を酵素免疫測定法［Ａｎｔｉｂｏｄｉｅｓ－Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ，Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ（１９８８）］などを用いて測定する。免疫に用いた抗原に対し、その血清が十分な抗体価を示した動物を融合用抗体産生細胞の供給源とする。 The antigen is administered 5 to 10 times every 1 to 2 weeks after the first administration. Three to seven days after each administration, blood is collected from the fundus venous plexus, and the antibody titer of the serum is measured using an enzyme immunoassay [Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988)]. An animal whose serum shows a sufficient antibody titer against the antigen used for immunization is used as a source of antibody producing cells for fusion.
　抗原の最終投与後３～７日目に、免疫した動物より脾臓などの抗体産生細胞を含む組織を摘出し、抗体産生細胞を採取する。脾臓細胞を用いる場合には、脾臓を細断、ほぐした後、遠心分離し、さらに赤血球を除去して融合用抗体産生細胞を取得する。 3 to 7 days after the final administration of the antigen, tissues containing antibody-producing cells such as the spleen are removed from the immunized animal, and antibody-producing cells are collected. When spleen cells are used, the spleen is shredded and loosened, and then centrifuged, and the erythrocytes are removed to obtain antibody producing cells for fusion.
（３）骨髄腫細胞の調製
　骨髄腫細胞としては、マウスから得られた株化細胞を用いる。例えば、８－アザグアニン耐性マウス（ＢＡＬＢ／ｃ由来）骨髄腫細胞株Ｐ３－Ｘ６３Ａｇ８－Ｕ１（Ｐ３－Ｕ１）［Ｃｕｒｒｅｎｔ　Ｔｏｐｉｃｓ　ｉｎ　Ｍｉｃｒｏｂｉｏｌｏｇｙ　ａｎｄ　Ｉｍｍｕｎｏｌｏｇｙ，１８，１（１９７８）］、Ｐ３－ＮＳ１／１－Ａｇ４１（ＮＳ－１）［Ｅｕｒｏｐｅａｎ　Ｊ．Ｉｍｍｕｎｏｌｏｇｙ，６，５１１（１９７６）］、ＳＰ２／０－Ａｇ１４（ＳＰ－２）［Ｎａｔｕｒｅ，２７６，２６９（１９７８）］、Ｐ３－Ｘ６３－Ａｇ８６５３（６５３）［Ｊ．Ｉｍｍｕｎｏｌｏｇｙ，１２３，１５４８（１９７９）］、またはＰ３－Ｘ６３－Ａｇ８（Ｘ６３）［Ｎａｔｕｒｅ，２５６，４９５（１９７５）］などが挙げられる。 (3) Preparation of myeloma cells As myeloma cells, cell lines obtained from mice are used. For example, 8-azaguanine resistant mouse (BALB / c-derived) myeloma cell line P3-X63Ag8-U1 (P3-U1) [Current Topics in Microbiology and Immunology, 18, 1 (1978)], P3-NS1 / 1-Ag41 (NS-1) [European J. et al. Immunology, 6, 511 (1976)], SP2 / 0-Ag14 (SP-2) [Nature, 276, 269 (1978)], P3-X63-Ag8653 (653) [J. Immunology, 123, 1548 (1979)], or P3-X63-Ag8 (X63) [Nature, 256, 495 (1975)].
　該骨髄腫細胞は、正常培地［グルタミン、２－メルカプトエタノール、ジェンタマイシン、ＦＢＳ、および８－アザグアニンを加えたＲＰＭＩ１６４０培地］で継代し、細胞融合の３～４日前に正常培地に継代し、融合当日２×１０７個以上の細胞数を確保する。 The myeloma cells are passaged in normal medium [RPMI 1640 medium supplemented with glutamine, 2-mercaptoethanol, gentamicin, FBS, and 8-azaguanine] and passaged to normal medium 3-4 days prior to cell fusion. On the day of fusion, secure a cell count of 2 × 10 7 or more.
（４）細胞融合とモノクローナル抗体産生ハイブリドーマの調製
　（２）で得られる融合用抗体産生細胞と（３）で得られる骨髄腫細胞をＭｉｎｉｍｕｍ　Ｅｓｓｅｎｔｉａｌ　Ｍｅｄｉｕｍ（ＭＥＭ）培地またはＰＢＳ（リン酸二ナトリウム１．８３ｇ、リン酸一カリウム０．２１ｇ、食塩７．６５ｇ、蒸留水１リットル、ｐＨ７．２）でよく洗浄し、細胞数が、融合用抗体産生細胞：骨髄腫細胞＝５～１０：１になるよう混合し、遠心分離した後、上清を除く。 (4) Cell fusion and preparation of monoclonal antibody-producing hybridoma The antibody-producing cells for fusion obtained in (2) and the myeloma cells obtained in (3) were treated with Minimum Essential Medium (MEM) medium or PBS (disodium phosphate 1. 83 g, monopotassium phosphate 0.21 g, sodium chloride 7.65 g, distilled water 1 liter, pH 7.2), and the number of cells becomes fusion antibody-producing cells: myeloma cells = 5 to 10: 1 After mixing and centrifuging, remove the supernatant.
　沈澱した細胞群をよくほぐした後、ポリエチレングリコール－１０００（ＰＥＧ－１０００）、ＭＥＭ培地およびジメチルスルホキシドの混液を３７℃で、攪拌しながら加える。さらに１～２分間毎にＭＥＭ培地１～２ｍＬを数回加えた後、ＭＥＭ培地を加えて全量が５０ｍＬになるようにする。遠心分離後、上清を除く。沈澱した細胞群をゆるやかにほぐした後、融合用抗体産生細胞をＨＡＴ培地［ヒポキサンチン、チミジン、およびアミノプテリンを加えた正常培地］中にゆるやかに懸濁する。この懸濁液を５％ＣＯ２インキュベーター中、３７℃で７～１４日間培養する。 After loosening the precipitated cells, a mixture of polyethylene glycol-1000 (PEG-1000), MEM medium and dimethyl sulfoxide is added at 37 ° C. with stirring. Add 1-2 mL of MEM medium several times every 1-2 minutes, and then add MEM medium to a total volume of 50 mL. After centrifugation, remove the supernatant. After loosening the precipitated cell group, the antibody-producing cells for fusion are gently suspended in a HAT medium [normal medium supplemented with hypoxanthine, thymidine, and aminopterin]. This suspension is cultured for 7-14 days at 37 ° C. in a 5% CO 2 incubator.
　培養後、培養上清の一部を抜き取り、後述のバインディングアッセイなどのハイブリドーマの選択方法により、ＣＤ３３を含む抗原に反応し、ＣＤ３３を含まない抗原に反応しない細胞群を選択する。次に、限界希釈法によりクローニングを２回繰り返し［１回目はＨＴ培地（ＨＡＴ培地からアミノプテリンを除いた培地）、２回目は正常培地を使用する］、安定して強い抗体価の認められたものをモノクローナル抗体産生ハイブリドーマとして選択する。 After culturing, a part of the culture supernatant is extracted, and a cell group that reacts with an antigen containing CD33 and does not react with an antigen not containing CD33 is selected by a hybridoma selection method such as a binding assay described later. Next, cloning was repeated twice by the limiting dilution method (first time was HT medium (medium obtained by removing aminopterin from HAT medium), second time normal medium was used), and a stable and strong antibody titer was observed. One is selected as a monoclonal antibody-producing hybridoma.
（５）精製モノクローナル抗体の調製
　プリスタン処理［２，６，１０，１４－テトラメチルペンタデカン（Ｐｒｉｓｔａｎｅ）０．５ｍｌを腹腔内投与し、２週間飼育する］した８～１０週令のマウスまたはヌードマウスに、（４）で得られるモノクローナル抗体産生ハイブリドーマを腹腔内に注射する。１０～２１日でハイブリドーマは腹水癌化する。このマウスから腹水を採取し、遠心分離して固形分を除去後、４０～５０％硫酸アンモニウムで塩析し、カプリル酸沈殿法、ＤＥＡＥ－セファロースカラム、プロテインＡ－カラムまたはゲル濾過カラムによる精製を行ない、ＩｇＧまたはＩｇＭ画分を集め、精製モノクローナル抗体とする。 (5) Preparation of Purified Monoclonal Antibody 8- to 10-week-old mice or nude mice treated with pristane [2,6,10,14-tetramethylpentadecane (Pristane) 0.5 ml intraperitoneally and bred for 2 weeks] Then, the monoclonal antibody-producing hybridoma obtained in (4) is injected intraperitoneally. In 10 to 21 days, the hybridoma becomes ascites tumor. Ascites was collected from this mouse, centrifuged to remove solids, salted out with 40-50% ammonium sulfate, and purified by caprylic acid precipitation, DEAE-Sepharose column, Protein A-column or gel filtration column. The IgG or IgM fractions are collected and used as a purified monoclonal antibody.
　また、（４）で得られるモノクローナル抗体産生ハイブリドーマを、１０％ＦＢＳ添加を添加したＲＰＭＩ１６４０培地などで培養した後、遠心分離により上清を除き、Ｈｙｂｒｉｄｏｍａ　ＳＦＭ培地に懸濁し、３～７日間培養する。得られた細胞懸濁液を遠心分離し、得られた上清よりプロテインＡ－カラムまたはプロテインＧ－カラムによる精製を行ない、ＩｇＧ画分を集め、精製モノクローナル抗体を得ることもできる。なお、Ｈｙｂｒｉｄｏｍａ　ＳＦＭ培地には５％ダイゴＧＦ２１を添加することもできる。 In addition, the monoclonal antibody-producing hybridoma obtained in (4) is cultured in an RPMI1640 medium supplemented with 10% FBS, the supernatant is removed by centrifugation, and the suspension is suspended in a Hybridoma SFM medium and cultured for 3 to 7 days. . The obtained cell suspension is centrifuged, and purified using a protein A-column or protein G-column from the resulting supernatant, and the IgG fraction is collected to obtain a purified monoclonal antibody. In addition, 5% Digo GF21 can also be added to the Hybridoma SFM medium.
　抗体のサブクラスの決定は、サブクラスタイピングキットを用いて酵素免疫測定法により行う。蛋白量の定量は、ローリー法または２８０ｎｍでの吸光度より算出する。 The antibody subclass is determined by enzyme immunoassay using a sub-clustering kit. The amount of protein is calculated from the Raleigh method or absorbance at 280 nm.
（６）モノクローナル抗体の選択
　モノクローナル抗体の選択は以下に示す酵素免疫測定法によるバインディングアッセイ、およびＢｉａｃｏｒｅによるｋｉｎｅｔｉｃｓ解析により行う。 (6) Selection of monoclonal antibody The monoclonal antibody is selected by a binding assay by the enzyme immunoassay shown below and a kinetic analysis by Biacore.
（６－ａ）バインディングアッセイ
　抗原としては、例えば、（１）で得られるＣＤ３３をコードするｃＤＮＡを含む発現ベクターを大腸菌、酵母、昆虫細胞若しくは動物細胞などに導入して得られた遺伝子導入細胞、リコンビナント蛋白質、またはヒト組織から得た精製ポリペプチド若しくは部分ペプチドなどが挙げられる。抗原が部分ペプチドである場合には、ＢＳＡまたはＫＬＨなどのキャリア蛋白質とコンジュゲートを作製して、これを用いる。 (6-a) Binding assay As an antigen, for example, a gene-transferred cell obtained by introducing an expression vector containing cDNA encoding CD33 obtained in (1) into Escherichia coli, yeast, insect cells or animal cells, Recombinant protein, or purified polypeptide or partial peptide obtained from human tissue. When the antigen is a partial peptide, a conjugate with a carrier protein such as BSA or KLH is prepared and used.
　抗原を９６ウェルプレートなどのプレートに分注し、固相化した後、第１抗体として血清、ハイブリドーマの培養上清または精製モノクローナル抗体などの被験物質を分注し、反応させる。ＰＢＳまたはＰＢＳ－Ｔｗｅｅｎなどで、よく洗浄した後、第２抗体としてビオチン、酵素、化学発光物質または放射線化合物などで標識した抗イムノグロブリン抗体を分注して反応させる。ＰＢＳ－Ｔｗｅｅｎでよく洗浄した後、第２抗体の標識物質に応じた反応を行ない、免疫原に対し特異的に反応するモノクローナル抗体を選択する。 After dispensing the antigen to a plate such as a 96-well plate and immobilizing the antigen, a test substance such as serum, hybridoma culture supernatant or purified monoclonal antibody is dispensed as the first antibody and allowed to react. After thoroughly washing with PBS or PBS-Tween or the like, an anti-immunoglobulin antibody labeled with biotin, an enzyme, a chemiluminescent substance, a radiation compound or the like is dispensed and reacted as a second antibody. After thoroughly washing with PBS-Tween, a reaction is performed according to the labeling substance of the second antibody, and a monoclonal antibody that specifically reacts with the immunogen is selected.
　また、本発明の抗ＣＤ３３モノクローナル抗体と競合してＣＤ３３に結合するモノクローナル抗体は、上述のバインティングアッセイ系に、被検抗体を添加して反応させることで取得できる。すなわち、被検抗体を加えた時にモノクローナル抗体の結合が阻害される抗体をスクリーニングすることにより、ＣＤ３３細胞外領域への結合について、取得したモノクローナル抗体と競合するモノクローナル抗体を取得することができる。 In addition, a monoclonal antibody that competes with the anti-CD33 monoclonal antibody of the present invention and binds to CD33 can be obtained by adding a test antibody to the above-described binding assay system for reaction. That is, by screening for an antibody that inhibits the binding of the monoclonal antibody when the test antibody is added, a monoclonal antibody that competes with the acquired monoclonal antibody for binding to the CD33 extracellular region can be obtained.
　更に、本発明のＣＤ３３の細胞外領域に結合するモノクローナル抗体が認識して、結合するエピトープと、同じエピトープに結合する抗体は、上述のバインティングアッセイ系で取得された抗体のエピトープを同定し、同定したエピトープの、部分的な合成ペプチド、またはエピトープの立体構造に擬態させた合成ペプチド等を作製し、免疫することで、取得することができる。 Furthermore, the monoclonal antibody that binds to the extracellular region of CD33 of the present invention recognizes and binds to the same epitope as the epitope that identifies the epitope of the antibody obtained by the above-described binding assay system, A partial synthetic peptide of the identified epitope, or a synthetic peptide mimicking the three-dimensional structure of the epitope can be prepared and immunized.
（６－ｂ）Ｂｉａｃｏｒｅによるｋｉｎｅｔｉｃｓ解析
　Ｂｉａｃｏｒｅ　Ｔ１００を用い、抗原と被験物の間の結合におけるｋｉｎｅｔｉｃｓを測定し、その結果を機器付属の解析ソフトウエアで解析をする。抗マウスＩｇＧ抗体をセンサーチップＣＭ５にアミンカップリング法により固定した後、ハイブリドーマ培養上清または精製モノクローナル抗体などの被験物質を流し、適当量結合させ、更に濃度既知の複数濃度の抗原を流し、結合、解離を測定する。得られたデータを機器付属のソフトウエアを用い、１：１バインディングモデルによりｋｉｎｅｔｉｃｓ解析を行い、各種パラメータを取得する。 (6-b) Kinetics analysis by Biacore Using Biacore T100, kinetics in the binding between an antigen and a test substance is measured, and the result is analyzed by analysis software attached to the instrument. After immobilizing the anti-mouse IgG antibody on the sensor chip CM5 by the amine coupling method, a test substance such as a hybridoma culture supernatant or a purified monoclonal antibody is allowed to flow, and an appropriate amount is bound. Measure dissociation. Using the software attached to the device, the obtained data is subjected to kinetic analysis using a 1: 1 binding model, and various parameters are acquired.
　または、ヒトＣＤ３３をセンサーチップ上に、例えばアミンカップリング法により固定した後、濃度既知の複数濃度の精製モノクローナル抗体を流し、結合、解離を測定する。得られたデータを機器付属のソフトウエアを用い、バイバレントバインディングモデルによりｋｉｎｅｔｉｃｓ解析を行い、各種パラメータを取得する。 Alternatively, human CD33 is immobilized on a sensor chip by, for example, an amine coupling method, and then purified monoclonal antibodies having a plurality of known concentrations are flowed to measure binding and dissociation. The obtained data is subjected to kinetic analysis using a software that is included with the device, using a binding binding model, and various parameters are acquired.
２．遺伝子組換え抗体の作製
　遺伝子組換え抗体の作製例として、以下にヒト型キメラ抗体およびヒト型ＣＤＲ移植抗体の作製方法を示す。
（１）遺伝子組換え抗体発現用ベクターの構築
　遺伝子組換え抗体発現用ベクターは、ヒト抗体のＣＨおよびＣＬをコードするＤＮＡが組み込まれた動物細胞用発現ベクターであり、動物細胞用発現ベクターにヒト抗体のＣＨおよびＣＬをコードするＤＮＡ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。 2. Production of Recombinant Antibody As an example of producing a recombinant antibody, a method for producing a human chimeric antibody and a human CDR-grafted antibody is shown below.
(1) Construction of Recombinant Antibody Expression Vector A recombinant antibody expression vector is an animal cell expression vector in which DNAs encoding human antibodies CH and CL are incorporated, and the animal cell expression vector is human. It can be constructed by cloning DNAs encoding antibody CH and CL, respectively.
　ヒト抗体のＣ領域は任意のヒト抗体のＣＨおよびＣＬを用いることができる。例えば、ヒト抗体のγ１サブクラスのＣＨおよびκクラスのＣＬなどを用いる。ヒト抗体のＣＨおよびＣＬをコードするＤＮＡには、ｃＤＮＡを用いるが、エキソンとイントロンからなる染色体ＤＮＡを用いることもできる。動物細胞用発現ベクターには、ヒト抗体のＣ領域をコードする遺伝子を組込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。 Any human antibody CH and CL can be used for the C region of a human antibody. For example, γ1 subclass CH and κ class CL of human antibodies are used. Although cDNA is used for DNA encoding CH and CL of human antibodies, chromosomal DNA consisting of exons and introns can also be used. Any animal cell expression vector can be used as long as it can incorporate and express a gene encoding the C region of a human antibody.
　例えば、ｐＡＧＥ１０７［Ｃｙｔｏｔｅｃｈｎｏｌ．，３，１３３（１９９０）］、ｐＡＧＥ１０３［Ｊ．Ｂｉｏｃｈｅｍ．，１０１，１３０７（１９８７）〕、ｐＨＳＧ２７４［Ｇｅｎｅ，２７，２２３（１９８４）］、ｐＫＣＲ［Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，７８，１５２７（１９８１）］、ｐＳＧ１ｂｄ２－４［Ｃｙｔｏｔｅｃｈｎｏｌ．，４，１７３（１９９０）］、またはｐＳＥ１ＵＫ１Ｓｅｄ１－３［Ｃｙｔｏｔｅｃｈｎｏｌ．，１３，７９（１９９３）］などが挙げられる。 For example, pAGE107 [Cytotechnol. , 3, 133 (1990)], pAGE103 [J. Biochem. , 101, 1307 (1987)], pHSG274 [Gene, 27, 223 (1984)], pKCR [Proc. Natl. Acad. Sci. USA, 78, 1527 (1981)], pSG1bd2-4 [Cytotechnol. , 4, 173 (1990)], or pSE1UK1Sed1-3 [Cytotechnol. , 13, 79 (1993)].
　動物細胞用発現ベクターのうちプロモーターとエンハンサーとしては、例えば、ＳＶ４０の初期プロモーター［Ｊ．Ｂｉｏｃｈｅｍ．，１０１，１３０７（１９８７）］、モロニーマウス白血病ウイルスＬＴＲ［Ｂｉｏｃｈｅｍ．Ｂｉｏｐｈｙｓ．Ｒｅｓ．Ｃｏｍｍｕｎ．，１４９，９６０（１９８７）〕、または免疫グロブリンＨ鎖のプロモーター［Ｃｅｌｌ，４１，４７９（１９８５）］とエンハンサー［Ｃｅｌｌ，３３，７１７（１９８３）］などが挙げられる。 Among the expression vectors for animal cells, examples of promoters and enhancers include SV40 early promoter [J. Biochem. , 101, 1307 (1987)], Moloney murine leukemia virus LTR [Biochem. Biophys. Res. Commun. , 149, 960 (1987)], or an immunoglobulin heavy chain promoter [Cell, 41, 479 (1985)] and an enhancer [Cell, 33, 717 (1983)].
　遺伝子組換え抗体発現用ベクターには、遺伝子組換え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Ｌ鎖の発現量のバランスが均衡するなどの点から、抗体Ｈ鎖およびＬ鎖が同一のベクター上に存在するタイプ（タンデム型）の遺伝子組換え抗体発現用ベクター［Ｊ．Ｉｍｍｕｎｏｌ．Ｍｅｔｈｏｄｓ，１６７，２７１（１９９４）］を用いるが、抗体Ｈ鎖およびＬ鎖が別々のベクター上に存在するタイプを用いることもできる。タンデム型の遺伝子組換え抗体発現用ベクターには、ｐＫＡＮＴＥＸ９３（国際公開第９７／１０３５４号）、ｐＥＥ１８［Ｈｙｂｒｉｄｏｍａ，１７，５５９（１９９８）］などを用いる。 Recombinant antibody expression vectors balance the ease of construction of recombinant antibody expression vectors, the ease of introduction into animal cells, and the balance of expression levels of antibody H and L chains in animal cells. In view of the above, a vector for expressing a recombinant antibody of a type (tandem type) in which the antibody H chain and L chain are present on the same vector [J. Immunol. Methods, 167, 271 (1994)], but it is also possible to use a type in which the antibody H chain and L chain are present on separate vectors. As a tandem recombinant antibody expression vector, pKANTEX93 (International Publication No. 97/10354), pEE18 [Hybridoma, 17, 559 (1998)] or the like is used.
（２）ヒト以外の動物由来の抗体のＶ領域をコードするｃＤＮＡの取得およびアミノ酸配列の解析
　非ヒト抗体のＶＨ及びＶＬをコードするｃＤＮＡの取得およびアミノ酸配列の解析は以下のようにして行うことができる。 (2) Obtaining cDNA encoding the V region of an antibody derived from a non-human animal and analyzing the amino acid sequence Obtaining cDNA encoding the VH and VL of the non-human antibody and analyzing the amino acid sequence should be performed as follows. Can do.
　非ヒト抗体を産生するハイブリドーマ細胞よりｍＲＮＡを抽出し、ｃＤＮＡを合成する。合成したｃＤＮＡをファージまたはプラスミドなどのベクターにクローニングしてｃＤＮＡライブラリーを作製する。該ライブラリーより、マウス抗体のＣ領域部分またはＶ領域部分をコードするＤＮＡをプローブとして用い、ＶＨまたはＶＬをコードするｃＤＮＡを有する組換えファージまたは組換えプラスミドをそれぞれ単離する。組換えファージまたは組換えプラスミド上の目的とするマウス抗体のＶＨまたはＶＬの全塩基配列をそれぞれ決定し、塩基配列よりＶＨまたはＶＬの全アミノ酸配列をそれぞれ推定する。 MRNA is extracted from hybridoma cells producing non-human 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. Recombinant phages or recombinant plasmids having cDNA encoding VH or VL are isolated from the library using DNA encoding the C region portion or V region portion of the mouse antibody as a probe. The entire base sequence of VH or VL of the target mouse antibody on the recombinant phage or recombinant plasmid is determined, respectively, and the total amino acid sequence of VH or VL is estimated from the base sequence.
　非ヒト抗体を産生するハイブリドーマ細胞を作製するヒト以外の動物には、マウス、ラット、ハムスター、またはラビットなどを用いるが、ハイブリドーマ細胞を作製することが可能であれば、いかなる動物も用いることができる。 As a non-human animal for producing a hybridoma cell producing a non-human antibody, a mouse, rat, hamster, rabbit or the like is used, but any animal can be used as long as it can produce a hybridoma cell. .
　ハイブリドーマ細胞からの全ＲＮＡの調製には、チオシアン酸グアニジン－トリフルオロ酢酸セシウム法［Ｍｅｔｈｏｄｓ　ｉｎ　Ｅｎｚｙｍｏｌ．，１５４，３（１９８７）］、またはＲＮＡ　ｅａｓｙ　ｋｉｔ（キアゲン社製）などのキットなどを用いる。 For preparation of total RNA from hybridoma cells, the guanidine thiocyanate-cesium trifluoroacetate method [Methods in Enzymol. , 154, 3 (1987)], or a kit such as RNA easy kit (manufactured by Qiagen).
　全ＲＮＡからのｍＲＮＡの調製には、オリゴ（ｄＴ）固定化セルロースカラム法［Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ，Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ，Ｓｅｃｏｎｄ　Ｅｄｉｔｉｏｎ，Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓ（１９８９）］、またはＯｌｉｇｏ－ｄＴ３０＜Ｓｕｐｅｒ＞　ｍＲＮＡ　Ｐｕｒｉｆｉｃａｔｉｏｎ　Ｋｉｔ（タカラバイオ社製）などのキットなどを用いる。また、Ｆａｓｔ　Ｔｒａｃｋ　ｍＲＮＡ　Ｉｓｏｌａｔｉｏｎ　Ｋｉｔ（インビトロジェン社製）、またはＱｕｉｃｋＰｒｅｐ　ｍＲＮＡ　Ｐｕｒｉｆｉｃａｔｉｏｎ　Ｋｉｔ（ファルマシア社製）などのキットを用いてハイブリドーマ細胞からｍＲＮＡを調製することもできる。 For preparation of mRNA from total RNA, oligo (dT) -immobilized cellulose column method [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Press (1989)] or Oligo-dT> SurPiSpR> <SpiK> Use a kit such as (Takara Bio). Alternatively, mRNA can be prepared from hybridoma cells using a kit such as Fast Track mRNA Isolation Kit (manufactured by Invitrogen) or QuickPrep mRNA Purification Kit (manufactured by Pharmacia).
　ｃＤＮＡの合成およびｃＤＮＡライブラリーの作製には、公知の方法［Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ，Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ，Ｓｅｃｏｎｄ　Ｅｄｉｔｉｏｎ，Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓ（１９８９）、Ｃｕｒｒｅｎｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ，Ｓｕｐｐｌｅｍｅｎｔ　１，Ｊｏｈｎ　Ｗｉｌｅｙ＆Ｓｏｎｓ（１９８７－１９９７）］、またはＳｕｐｅｒＳｃｒｉｐｔ　Ｐｌａｓｍｉｄ　Ｓｙｓｔｅｍ　ｆｏｒ　ｃＤＮＡ　Ｓｙｎｔｈｅｓｉｓ　ａｎｄ　Ｐｌａｓｍｉｄ　Ｃｌｏｎｉｎｇ（インビトロジェン社製）、またはＺＡＰ－ｃＤＮＡ　Ｓｙｎｔｈｅｓｉｓ　Ｋｉｔ（ストラタジーン社製）などのキットなどを用いる。 For the synthesis of cDNA and preparation of cDNA library, known methods [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in BioProsthetics, BioProsthetics, BioProsthetics, BioProsthetics, BioProsthetics, BioProsthetics. )], Or a kit such as SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning (manufactured by Invitrogen), or ZAP-cDNA Synthesis Kit (manufactured by Stratagene) Used.
　ｃＤＮＡライブラリーの作製の際、ハイブリドーマ細胞から抽出したｍＲＮＡを鋳型として合成したｃＤＮＡを組み込むベクターには、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［Ｓｔｒａｔｅｇｉｅｓ，５，５８（１９９２）］、ｐＢｌｕｅｓｃｒｉｐｔ　ＩＩ　ＳＫ（＋）［Ｎｕｃｌｅｉｃ　Ａｃｉｄｓ　Ｒｅｓｅａｒｃｈ，１７，９４９４（１９８９）］、λＺＡＰＩＩ（Ｓｔｒａｔａｇｅｎｅ社製）、λｇｔ１０、λｇｔ１１［ＤＮＡ　Ｃｌｏｎｉｎｇ：Ａ　Ｐｒａｃｔｉｃａｌ　Ａｐｐｒｏａｃｈ，Ｉ，４９（１９８５）］、Ｌａｍｂｄａ　ＢｌｕｅＭｉｄ（クローンテック社製）、λＥｘＣｅｌｌ、ｐＴ７Ｔ３－１８Ｕ（ファルマシア社製）、ｐｃＤ２［Ｍｏｌ．Ｃｅｌｌ．Ｂｉｏｌ．，３，２８０（１９８３）］、またはｐＵＣ１８［Ｇｅｎｅ，３３，１０３（１９８５）］などが挙げられる。 When preparing a cDNA library, any vector can be used as a vector into which cDNA synthesized using mRNA extracted from a hybridoma cell as a template is incorporated. For example, ZAP Express [Stratesies, 5, 58 (1992)], pBluescript II SK (+) [Nucleic Acids Research, 17, 9494 (1989)], λZAPIII (manufactured by Stratagene), λgt10, Clgt11A: Clgt11 DNA Approach, I, 49 (1985)], Lambda BlueMid (Clontech), λExCell, pT7T3-18U (Pharmacia), pcD2 [Mol. Cell. Biol. 3, 280 (1983)], or pUC18 [Gene, 33, 103 (1985)].
　ファージまた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には、該ｃＤＮＡライブラリーを導入、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、ＸＬ１－Ｂｌｕｅ　ＭＲＦ’［Ｓｔｒａｔｅｇｉｅｓ，５，８１（１９９２）］、Ｃ６００［Ｇｅｎｅｔｉｃｓ，３９，４４０（１９５４）］、Ｙ１０８８、Ｙ１０９０［Ｓｃｉｅｎｃｅ，２２２，７７８（１９８３）］、ＮＭ５２２［Ｊ．Ｍｏｌ．Ｂｉｏｌ．，１６６，１（１９８３）］、Ｋ８０２［Ｊ．Ｍｏｌ．Ｂｉｏｌ．，１６，１１８（１９６６）］、またはＪＭ１０５［Ｇｅｎｅ，３８，２７５（１９８５）］などが挙げられる。 Any Escherichia coli into which a cDNA library constructed by a phage or plasmid vector is introduced can be used as long as the cDNA library can be introduced, expressed and maintained. For example, XL1-Blue MRF '[Stratesies, 5, 81 (1992)], C600 [Genetics, 39, 440 (1954)], Y1088, Y1090 [Science, 222, 778 (1983)], NM522 [J. Mol. Biol. , 166, 1 (1983)], K802 [J. Mol. Biol. 16, 118 (1966)] or JM105 [Gene, 38, 275 (1985)].
　ｃＤＮＡライブラリーからの非ヒト抗体のＶＨまたはＶＬをコードするｃＤＮＡクローンを選択する方法としては、例えば、アイソトープ若しくは蛍光標識したプローブを用いたコロニー・ハイブリダイゼーション法、またはプラーク・ハイブリダイゼーション法［Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ，Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ，Ｓｅｃｏｎｄ　Ｅｄｉｔｉｏｎ，Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓ（１９８９）］などが挙げられる。 As a method for selecting a cDNA clone encoding VH or VL of a non-human antibody from a cDNA library, for example, a colony hybridization method using an isotope or fluorescently labeled probe, or a plaque hybridization method [Molecular Cloning] , A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)].
　また、プライマーを調製し、ｍＲＮＡから合成したｃＤＮＡまたはｃＤＮＡライブラリーを鋳型として、Ｐｏｌｙｍｅｒａｓｅ　Ｃｈａｉｎ　Ｒｅａｃｔｉｏｎ法［以下、ＰＣＲ法と表記する、Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ，Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ，Ｓｅｃｏｎｄ　Ｅｄｉｔｉｏｎ，Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓ（１９８９）、Ｃｕｒｒｅｎｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ，Ｓｕｐｐｌｅｍｅｎｔ　１，Ｊｏｈｎ　Ｗｉｌｅｙ＆Ｓｏｎｓ（１９８７－１９９７）］を行うことよりＶＨまたはＶＬをコードするｃＤＮＡを調製することもできる。 In addition, using primers prepared and cDNA synthesized from mRNA or cDNA library as a template, Polymerase Chain Reaction method (hereinafter referred to as PCR method, Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Labor 198) ), Current Protocols in Molecular Biology, Supplement 1, John Wiley & Sons (1987-1997)], cDNA encoding VH or VL can also be prepared.
　選択されたｃＤＮＡを、適当な制限酵素などで切断後、ｐＢｌｕｅｓｃｒｉｐｔ　ＳＫ（－）（ストラタジーン社製）など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などにより該ｃＤＮＡの塩基配列を決定する。塩基配列解析方法には、例えば、ジデオキシ法［Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，７４，５４６３（１９７７）］などの反応を行った後、ＡＢＩ　ＰＲＩＳＭ３７００（ＰＥバイオシステムズ社製）またはＡ．Ｌ．Ｆ．ＤＮＡシークエンサー（ファルマシア社製）などの塩基配列自動分析装置などを用いる。 The selected cDNA is cleaved with an appropriate restriction enzyme and then cloned into a plasmid such as pBluescript SK (-) (Stratagene), and the nucleotide sequence of the cDNA is determined by a commonly used nucleotide sequence analysis method. . Examples of the base sequence analysis method include the dideoxy method [Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)], etc., followed by ABI PRISM 3700 (manufactured by PE Biosystems) or A.P. L. F. An automatic base sequence analyzer such as a DNA sequencer (Pharmacia) is used.
　決定した塩基配列からＶＨおよびＶＬの全アミノ酸配列をそれぞれ推定し、既知の抗体のＶＨおよびＶＬの全アミノ酸配列［Ｓｅｑｕｅｎｃｅｓ　ｏｆ　Ｐｒｏｔｅｉｎｓ　ｏｆ　Ｉｍｍｕｎｏｌｏｇｉｃａｌ　Ｉｎｔｅｒｅｓｔ，ＵＳ　Ｄｅｐｔ．Ｈｅａｌｔｈ　ａｎｄ　Ｈｕｍａｎ　Ｓｅｒｖｉｃｅｓ（１９９１）］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の完全なアミノ酸配列をコードしているかをそれぞれ確認する。 From the determined base sequence, all amino acid sequences of VH and VL are estimated, respectively, and all amino acid sequences of known antibodies VH and VL [Sequences of Proteins of Immunological Interest, US Dept. Comparison with Health and Human Services (1991)] confirms whether the obtained cDNA encodes the complete amino acid sequence of VH and VL of the antibody including the secretory signal sequence.
　分泌シグナル配列を含む抗体のＶＨおよびＶＬの完全なア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［Ｓｅｑｕｅｎｃｅｓ　ｏｆ　Ｐｒｏｔｅｉｎｓ　ｏｆ　Ｉｍｍｕｎｏｌｏｇｉｃａｌ　Ｉｎｔｅｒｅｓｔ，ＵＳ　Ｄｅｐｔ．Ｈｅａｌｔｈ　ａｎｄ　Ｈｕｍａｎ　Ｓｅｒｖｉｃｅｓ（１９９１）］と比較することにより、分泌シグナル配列の長さおよびＮ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。 Regarding the complete amino acid sequence of VH and VL of an antibody including a secretory signal sequence, all amino acid sequences of known antibodies VH and VL [Sequences of Proteins of Immunological Interest, US Dept. By comparing with Health and Human Services (1991)], the length of the secretory signal sequence and the N-terminal amino acid sequence can be estimated, and further, the subgroup to which they belong can be known.
　また、ＶＨおよびＶＬ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ＶＬのアミノ酸配列［Ｓｅｑｕｅｎｃｅｓ　ｏｆ　Ｐｒｏｔｅｉｎｓ　ｏｆ　Ｉｍｍｕｎｏｌｏｇｉｃａｌ　Ｉｎｔｅｒｅｓｔ，ＵＳ　Ｄｅｐｔ．Ｈｅａｌｔｈ　ａｎｄ　Ｈｕｍａｎ　Ｓｅｒｖｉｃｅｓ（１９９１）］と比較することによって見出すことができる。 Also, regarding the amino acid sequences of CDRs of VH and VL, the VH and VL amino acid sequences of known antibodies [Sequences of Proteins of Immunological Interest, US Dept. It can be found by comparing with Health and Human Services (1991)].
　また、得られたＶＨおよびＶＬの完全なアミノ酸配列を用いて、例えば、ＳＷＩＳＳ－ＰＲＯＴまたはＰＩＲ－Ｐｒｏｔｅｉｎなどの任意のデータベースに対してＢＬＡＳＴ法［Ｊ．Ｍｏｌ．Ｂｉｏｌ．，２１５，４０３（１９９０）］などの相同性検索を行い、ＶＨおよびＶＬの完全なアミノ酸配列が新規なものかを確認できる。 Also, using the obtained VH and VL complete amino acid sequences, for example, BLAST method [J. Mol. Biol. , 215, 403 (1990)], and the like, it can be confirmed whether the complete amino acid sequences of VH and VL are novel.
（３）Ｖ領域にＮ結合型糖鎖が結合するコンセンサス配列を有さない遺伝子組換え抗体の作製
　Ｖ領域に、Ｎ結合型糖鎖が結合するコンセンサス配列（Ａｓｎ－Ｘａａ－Ｓｅｒ／Ｔｈｒ）を有さない抗体（改変型抗体）のＶ領域は、Ｎ結合型糖鎖が結合するコンセンサス配列のＡｓｎ残基および／またはＳｅｒ残基／Ｔｈｒ残基を他のアミノ酸残基に置換することにより、作製することができる。 (3) Production of recombinant antibody having no consensus sequence for binding N-linked sugar chain to V region Consensus sequence (Asn-Xaa-Ser / Thr) for binding N-linked sugar chain to V region The V region of an antibody that does not have (modified antibody) can be obtained by substituting other amino acid residues for the Asn residue and / or the Ser residue / Thr residue of the consensus sequence to which the N-linked sugar chain binds. Can be produced.
　抗体のＶ領域、特にＣＤＲは、抗体の抗原への結合性を規定する重要な領域である。従って、抗体のＶ領域、特にＣＤＲにおける、任意のアミノ酸残基への置換は、抗体の抗原への結合性が変化してしまう可能性がある。従って、上記コンセンサス配列を有さないＶ領域を作製する際には、抗体の抗原への結合性が変化しないアミノ酸配列に改変する必要がある。その具体的な方法を以下に示す。 Antibody V region, in particular CDR, is an important region that regulates antibody binding to antigen. Therefore, substitution to any amino acid residue in the V region of an antibody, particularly in a CDR, may change the binding property of the antibody to an antigen. Therefore, when producing a V region that does not have the consensus sequence, it is necessary to modify the amino acid sequence so that the binding property of the antibody to the antigen does not change. The specific method is shown below.
　Ｖ領域のＮ結合型糖鎖が結合するコンセンサス配列中のＡｓｎ残基、並びにＳｅｒ残基またはＴｈｒ残基を抗体の抗原への結合性が変化しないアミノ酸配列に改変するためには、抗原への結合性に影響を与える可能性の少ないアミノ酸残基の予測が重要である。そのため、Ｘ線結晶構造解析［Ｊ．Ｍｏｌ．Ｂｉｏｌ．，１１２，５３５（１９７７）］またはコンピューターモデリング［Ｐｒｏｔｅｉｎ　Ｅｎｇｉｎｅｅｒｉｎｇ，７，１５０１（１９９４）］等による抗体の立体構造の構築および解析を行う。 To change the Asn residue in the consensus sequence to which the N-linked sugar chain of the V region binds, as well as the Ser residue or Thr residue, to an amino acid sequence that does not change the binding property of the antibody to the antigen, It is important to predict amino acid residues that are less likely to affect binding. Therefore, X-ray crystal structure analysis [J. Mol. Biol. 112, 535 (1977)] or computer modeling [Protein Engineering, 7, 1501 (1994)], etc., to construct and analyze the three-dimensional structure of the antibody.
　しかしながら、これら抗体の立体構造の情報に基づいても、抗体のＶ領域、特にＣＤＲへの変異の導入は、抗体の抗原への結合性を変化させる可能性がある。そのため、変異の導入に際しては、数種の改変体を作製し、アミノ酸の改変との抗原結合活性との相関を検討する等の、試行錯誤が必要である。 However, even based on the information on the three-dimensional structure of these antibodies, introduction of mutations into the V region of the antibody, particularly the CDR, may change the binding property of the antibody to the antigen. Therefore, trials and errors, such as preparing several types of modified products and examining the correlation between the amino acid modification and the antigen binding activity, are necessary for introducing the mutation.
　このようにして、抗体の抗原への結合性に影響を与える可能性の少ないアミノ酸残基の部位特異的変異を推定した後、変異が導入された抗体Ｖ領域のア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なる数本の合成ＤＮＡを合成し、それらを用いてＰＣＲ反応を行う。この場合、ＰＣＲ反応での反応効率及び合成可能なＤＮＡの長さから、好ましくはＨ鎖、Ｌ鎖とも６本の合成ＤＮＡを設計する。 In this way, after estimating site-specific mutations of amino acid residues that are less likely to affect the binding of an antibody to an antigen, a DNA sequence encoding the amino acid sequence of the antibody V region into which the mutation has been introduced is obtained. design. Based on the designed DNA sequence, several synthetic DNAs having a length of about 100 bases are synthesized, and PCR reaction is performed using them. In this case, preferably 6 synthetic DNAs are designed for both the H chain and the L chain from the reaction efficiency in the PCR reaction and the length of the synthesizable DNA.
　または、変異を導入する基となった抗体のＶ領域をコードするｃＤＮＡにクンケル法などの公知の部位特異的変異導入法［Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ，Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ，Ｓｅｃｏｎｄ　Ｅｄｉｔｉｏｎ，Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓ（１９８９）］を用いたり、変異を導入する基となった抗体のＶ領域をコードするｃＤＮＡ中に存在する制限酵素認識部位を利用して、Ｎ結合型糖鎖が結合するコンセンサス配列を含む部分のアミノ酸配列をコードするＤＮＡに、ＰＣＲ等で変異を導入し、変異を導入する基となった抗体のＶ領域をコードするｃＤＮＡと置換して、設計したＤＮＡ配列を有するｃＤＮＡを含むプラスミドを作製することもできる。 Alternatively, a known site-directed mutagenesis method such as Kunkel method [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) The amino acid sequence of the portion containing a consensus sequence to which an N-linked sugar chain binds using a restriction enzyme recognition site present in the cDNA encoding the V region of the antibody from which the mutation was introduced A plasmid containing a cDNA having a designed DNA sequence may be prepared by introducing a mutation into DNA encoding DNA by PCR or the like and substituting it with the cDNA encoding the V region of the antibody from which the mutation was introduced. it can.
　Ｖ領域のアミノ酸残基は、上記のようにして得られた遺伝子組換え抗体のＶＨまたはＶＬのアミノ酸配列をコードするＤＮＡ配列を有するｃＤＮＡを含むプラスミドの塩基配列を上記（２）に記載の方法により決定し、目的の改変が施されたことを確認する。 The method according to (2) above, wherein the amino acid residue of the V region is a base sequence of a plasmid containing cDNA having a DNA sequence encoding the VH or VL amino acid sequence of the recombinant antibody obtained as described above. Confirm that the target modification has been made.
（４）ヒト型キメラ抗体発現ベクターの構築
　（１）で得られる遺伝子組換え抗体発現用ベクターのヒト抗体のＣＨまたはＣＬをコードするそれぞれの遺伝子の上流に、非ヒト抗体のＶＨまたはＶＬをコードするｃＤＮＡをそれぞれクローニングすることで、ヒト型キメラ抗体発現ベクターを構築することができる。 (4) Construction of human chimeric antibody expression vector The non-human antibody VH or VL is encoded upstream of each gene encoding the human antibody CH or CL of the recombinant antibody expression vector obtained in (1). A human chimeric antibody expression vector can be constructed by cloning each of the cDNAs.
　非ヒト抗体のＶＨまたはＶＬをコードするｃＤＮＡの３’末端側と、ヒト抗体のＣＨまたはＣＬの５’末端側とを連結するために、連結部分の塩基配列が適切なアミノ酸をコードし、かつ適当な制限酵素認識配列になるように設計したＶＨおよびＶＬのｃＤＮＡを作製する。作製されたＶＨおよびＶＬのｃＤＮＡを、（１）で得られるヒト型ＣＤＲ移植抗体発現用ベクターのヒト抗体のＣＨまたはＣＬをコードするそれぞれの遺伝子の上流にそれらが適切な形で発現する様にそれぞれクローニングし、ヒト型キメラ抗体発現ベクターを構築する。 In order to link the 3 ′ end of cDNA encoding VH or VL of a non-human antibody and the 5 ′ end of CH or CL of a human antibody, the base sequence of the linking portion encodes an appropriate amino acid, and VH and VL cDNAs designed to be appropriate restriction enzyme recognition sequences are prepared. The prepared VH and VL cDNAs are expressed in an appropriate form upstream of each gene encoding the human antibody CH or CL of the human CDR-grafted antibody expression vector obtained in (1). Each is cloned to construct a human chimeric antibody expression vector.
　また、非ヒト抗体ＶＨまたはＶＬをコードするｃＤＮＡを、適当な制限酵素の認識配列を両端に有する合成ＤＮＡを用いてＰＣＲ法によりそれぞれ増幅し、（１）で得られる遺伝子組換え抗体発現用ベクターにクローニングすることもできる。 Further, a cDNA encoding the non-human antibody VH or VL is amplified by a PCR method using a synthetic DNA having a recognition sequence of an appropriate restriction enzyme at both ends, and the recombinant antibody expression vector obtained in (1) Can also be cloned.
（５）ヒト型ＣＤＲ移植抗体のＶ領域をコードするｃＤＮＡの構築
　ヒト型ＣＤＲ移植抗体のＶＨまたはＶＬをコードするｃＤＮＡは、以下のようにして構築することができる。 (5) Construction of cDNA encoding V region of human CDR-grafted antibody A cDNA encoding VH or VL of human CDR-grafted antibody can be constructed as follows.
　非ヒト抗体のＶＨまたはＶＬのＣＤＲのアミノ酸配列を移植するヒト抗体のＶＨまたはＶＬのＦＲのアミノ酸配列をそれぞれ選択する。選択するＦＲのアミノ酸配列には、ヒト抗体由来のものであれば、いずれのものでも用いることができる。例えば、Ｐｒｏｔｅｉｎ　Ｄａｔａ　Ｂａｎｋなどのデータベースに登録されているヒト抗体のＦＲのアミノ酸配列、またはヒト抗体のＦＲの各サブグループの共通アミノ酸配列［Ｓｅｑｕｅｎｃｅｓ　ｏｆ　Ｐｒｏｔｅｉｎｓ　ｏｆ　Ｉｍｍｕｎｏｌｏｇｉｃａｌ　Ｉｎｔｅｒｅｓｔ，ＵＳ　Ｄｅｐｔ．Ｈｅａｌｔｈ　ａｎｄ　Ｈｕｍａｎ　Ｓｅｒｖｉｃｅｓ（１９９１）］などを用いる。抗体の結合活性の低下を抑えるため、元の抗体のＶＨまたはＶＬ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）のＦＲのアミノ酸配列を選択する。 The amino acid sequence of the VH or VL FR of the human antibody to be grafted with the VH or VL CDR amino acid sequence of the non-human antibody is selected. Any amino acid sequence can be used as long as it is derived from a human antibody. For example, the FR amino acid sequence of human antibodies registered in databases such as Protein Data Bank, or the common amino acid sequence of each subgroup of FRs of human antibodies [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)] are used. In order to suppress a decrease in the binding activity of the antibody, an FR amino acid sequence having the highest homology (at least 60% or more) with the FR amino acid sequence of the VH or VL of the original antibody is selected.
　次に、選択したヒト抗体のＶＨまたはＶＬのＦＲのアミノ酸配列に、もとの抗体のＣＤＲのアミノ酸配列をそれぞれ移植し、ヒト型ＣＤＲ移植抗体のＶＨまたはＶＬのアミノ酸配列をそれぞれ設計する。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［Ｓｅｑｕｅｎｃｅｓ　ｏｆ　Ｐｒｏｔｅｉｎｓ　ｏｆ　Ｉｍｍｕｎｏｌｏｇｉｃａｌ　Ｉｎｔｅｒｅｓｔ，ＵＳ　Ｄｅｐｔ．Ｈｅａｌｔｈ　ａｎｄ　Ｈｕｍａｎ　Ｓｅｒｖｉｃｅｓ（１９９１）］を考慮してＤＮＡ配列に変換し、ヒト型ＣＤＲ移植抗体のＶＨまたはＶＬのアミノ酸配列をコードするＤＮＡ配列をそれぞれ設計する。 Next, the amino acid sequence of the CDR of the original antibody is transplanted to the amino acid sequence of VH or VL of the selected human antibody, respectively, and the amino acid sequence of VH or VL of the human CDR-grafted antibody is designed. Frequency of codon usage of the designed amino acid sequence in the nucleotide sequence of the antibody gene [Sequences of Proteins of Immunological Interest, US Dept. Considering Health and Human Services (1991)], the DNA sequence is converted into a DNA sequence, and the DNA sequence encoding the VH or VL amino acid sequence of the human CDR-grafted antibody is designed.
　設計したＤＮＡ配列に基づき、１００塩基前後の長さからなる数本の合成ＤＮＡを合成し、それらを用いてＰＣＲ反応を行う。この場合、ＰＣＲ反応での反応効率及び合成可能なＤＮＡの長さから、好ましくはＨ鎖、Ｌ鎖とも６本の合成ＤＮＡを設計する。 Based on the designed DNA sequence, several synthetic DNAs having a length of about 100 bases are synthesized, and PCR reaction is performed using them. In this case, preferably 6 synthetic DNAs are designed for both the H chain and the L chain from the reaction efficiency in the PCR reaction and the length of the synthesizable DNA.
　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、（１）で得られるヒト型ＣＤＲ移植抗体発現用ベクターに容易にヒト型ＣＤＲ移植抗体のＶＨまたはＶＬをコードするｃＤＮＡをクローニングすることができる。 In addition, by introducing an appropriate restriction enzyme recognition sequence into the 5 ′ end of the synthetic DNA located at both ends, the human CDR-grafted antibody VH can be easily added to the human CDR-grafted antibody expression vector obtained in (1). Alternatively, cDNA encoding VL can be cloned.
　または、設計したＤＮＡ配列に基づき、１本のＤＮＡとして合成された各Ｈ鎖、Ｌ鎖全長合成ＤＮＡを用いることで実施できる。 Alternatively, it can be carried out by using each H chain and L chain full-length synthetic DNA synthesized as one DNA based on the designed DNA sequence.
　ＰＣＲ反応後、増幅産物をｐＢｌｕｅｓｃｒｉｐｔ　ＳＫ（－）（ストラタジーン社製）などのプラスミドにそれぞれクローニングし、（２）に記載の方法と同様の方法により、塩基配列を決定し、所望のヒト型ＣＤＲ移植抗体のＶＨまたはＶＬのアミノ酸配列をコードするＤＮＡ配列を有するプラスミドを取得する。 After the PCR reaction, the amplified product is cloned into a plasmid such as pBluescript SK (-) (manufactured by Stratagene), the base sequence is determined by the same method as described in (2), and the desired human CDR is obtained. A plasmid having a DNA sequence encoding the amino acid sequence of the VH or VL of the transplanted antibody is obtained.
（６）ヒト型ＣＤＲ移植抗体のＶ領域のアミノ酸配列の改変
　ヒト型ＣＤＲ移植抗体は、非ヒト抗体の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、その抗原結合活性は元の非ヒト抗体に比べて低下する［ＢＩＯ／ＴＥＣＨＮＯＬＯＧＹ，９，２６６（１９９１）］。ヒト型ＣＤＲ移植抗体では、ヒト抗体のＶＨおよびＶＬのＦＲのアミノ酸配列の中で、直接抗原との結合に関与しているアミノ酸残基、ＣＤＲのアミノ酸残基と相互作用するアミノ酸残基、および抗体の立体構造を維持し、間接的に抗原との結合に関与しているアミノ酸残基を同定し、それらのアミノ酸残基を元の非ヒト抗体のアミノ酸残基に置換することにより、低下した抗原結合活性を上昇させることができる。 (6) Modification of amino acid sequence of V region of human CDR-grafted antibody A human CDR-grafted antibody can be obtained by transplanting only the non-human antibody VH and VL CDRs into the human antibody VH and VL FRs. The binding activity is reduced compared to the original non-human antibody [BIO / TECHNOLOGY, 9, 266 (1991)]. In the human CDR-grafted antibody, among the amino acid sequences of human antibody VH and VL FRs, amino acid residues that are directly involved in antigen binding, amino acid residues that interact with CDR amino acid residues, and Reduced by maintaining the conformation of the antibody, identifying amino acid residues that are indirectly involved in antigen binding, and substituting those amino acid residues with the amino acid residues of the original non-human antibody Antigen binding activity can be increased.
　抗原結合活性に関わるＦＲのアミノ酸残基を同定するために、Ｘ線結晶解析［Ｊ．Ｍｏｌ．Ｂｉｏｌ．，１１２，５３５（１９７７）］またはコンピューターモデリング［Ｐｒｏｔｅｉｎ　Ｅｎｇｉｎｅｅｒｉｎｇ，７，１５０１（１９９４）］などを用いることにより、抗体の立体構造の構築および解析を行うことができる。また、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ことを繰り返し、試行錯誤することで必要な抗原結合活性を有する改変型ヒト型ＣＤＲ移植抗体を取得できる。 In order to identify FR amino acid residues involved in antigen binding activity, X-ray crystallography [J. Mol. Biol. , 112, 535 (1977)] or computer modeling [Protein Engineering, 7, 1501 (1994)] or the like, the three-dimensional structure of the antibody can be constructed and analyzed. In addition, several types of variants are prepared for each antibody, and the correlation with each antigen binding activity is repeatedly studied. By trial and error, a modified human CDR-grafted antibody having the necessary antigen binding activity is obtained. You can get it.
　ヒト抗体のＶＨ及びＶＬのＦＲのアミノ酸残基は、改変用合成ＤＮＡを用いて（４）に記載のＰＣＲ反応を行うことにより、改変させることができる。ＰＣＲ反応後の増幅産物について（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 amino acid residues of FR of human antibody VH and VL can be modified by performing the PCR reaction described in (4) using the synthetic DNA for modification. For the amplified product after the PCR reaction, the base sequence is determined by the method described in (2) and it is confirmed that the target modification has been performed.
（７）ヒト型ＣＤＲ移植抗体発現ベクターの構築
　（１）で得られる遺伝子組換え抗体発現用ベクターのヒト抗体のＣＨまたはＣＬをコードするそれぞれの遺伝子の上流に、構築した遺伝子組換え抗体のＶＨまたはＶＬをコードするｃＤＮＡをそれぞれクローニングし、ヒト型ＣＤＲ移植抗体発現ベクターを構築することができる。 (7) Construction of human CDR-grafted antibody expression vector VH of the constructed recombinant antibody upstream of each gene encoding the human antibody CH or CL of the recombinant antibody expression vector obtained in (1) Alternatively, cDNA encoding VL can be cloned, and a human CDR-grafted antibody expression vector can be constructed.
　例えば、（４）および（５）で得られるヒト型ＣＤＲ移植抗体のＶＨまたはＶＬを構築する際に用いる合成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）で得られるヒト型ＣＤＲ移植抗体発現用ベクターのヒト抗体のＣＨまたはＣＬをコードするそれぞれの遺伝子の上流にそれらが適切な形で発現するようにそれぞれクローニングする。 For example, among the synthetic DNAs used in constructing the VH or VL of the human CDR-grafted antibody obtained in (4) and (5), a recognition sequence for an appropriate restriction enzyme at the 5 ′ end of the synthetic DNA located at both ends Are introduced into the human CDR-grafted antibody expression vector obtained in (1), and cloned so as to be expressed in an appropriate form upstream of each gene encoding CH or CL of the human antibody.
（８）遺伝子組換え抗体の一過性発現
　（３）および（６）で得られる遺伝子組換え抗体発現ベクター、またはそれらを改変した発現ベクターを用いて遺伝子組換え抗体の一過性発現を行い、作製した多種類のヒト型ＣＤＲ移植抗体の抗原結合活性を効率的に評価することができる。 (8) Transient expression of recombinant antibodies Transient expression of recombinant antibodies using the recombinant antibody expression vectors obtained in (3) and (6) or modified expression vectors Thus, the antigen-binding activity of the prepared various types of human CDR-grafted antibodies can be efficiently evaluated.
　発現ベクターを導入する宿主細胞としては、遺伝子組換え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、例えば、ＣＯＳ－７細胞［Ａｍｅｒｉｃａｎ　Ｔｙｐｅ　Ｃｕｌｔｕｒｅ　Ｃｏｌｌｅｃｔｉｏｎ（ＡＴＣＣ）番号：ＣＲＬ１６５１］が挙げられる［Ｍｅｔｈｏｄｓ　ｉｎ　Ｎｕｃｌｅｉｃ　Ａｃｉｄｓ　Ｒｅｓ．，ＣＲＣ　ｐｒｅｓｓ，２８３（１９９１）］。 As the host cell into which the expression vector is introduced, any cell can be used as long as it can express the recombinant antibody. For example, COS-7 cells [American Type Culture Collection (ATCC) number: CRL1651] [Methods in Nucleic Acids Res. , CRC press, 283 (1991)].
　ＣＯＳ－７細胞への発現ベクターの導入には、ＤＥＡＥ－デキストラン法［Ｍｅｔｈｏｄｓ　ｉｎ　Ｎｕｃｌｅｉｃ　Ａｃｉｄｓ　Ｒｅｓ．，ＣＲＣ　ｐｒｅｓｓ（１９９１）］、またはリポフェクション法［Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，８４，７４１３（１９８７）］などを用いる。 For the introduction of expression vectors into COS-7 cells, the DEAE-dextran method [Methods in Nucleic Acids Res. , CRC press (1991)], or lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)].
　発現ベクターの導入後、培養上清中の遺伝子組換え抗体の発現量および抗原結合活性は酵素免疫抗体法［Ｍｏｎｏｃｌｏｎａｌ　Ａｎｔｉｂｏｄｉｅｓ－Ｐｒｉｎｃｉｐｌｅｓ　ａｎｄ　ｐｒａｃｔｉｃｅ，Ｔｈｉｒｄ　ｅｄｉｔｉｏｎ，Ａｃａｄｅｍｉｃ　Ｐｒｅｓｓ（１９９６）、Ａｎｔｉｂｏｄｉｅｓ－Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ，Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ（１９８８）、単クローン抗体実験マニュアル，講談社サイエンティフィック（１９８７）］などを用いて測定する。 After the introduction of the expression vector, the expression amount and antigen binding activity of the recombinant antibody in the culture supernatant are measured by enzyme immunoantibody method [Monoclonal Antibodies-Principles and Practice, Third edition, Academic Press (1996), Antibodies-ALaboratory. Cold Spring Harbor Laboratory (1988), monoclonal antibody experiment manual, Kodansha Scientific (1987)] and the like.
（９）遺伝子組換え抗体を安定に発現する形質転換株の取得と遺伝子組換え抗体の調製
　（３）および（６）で得られた遺伝子組換え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遺伝子組換え抗体を安定に発現する形質転換株を得ることができる。 (9) Obtaining a transformant that stably expresses the recombinant antibody and preparation of the recombinant antibody. Introducing the recombinant antibody expression vector obtained in (3) and (6) into an appropriate host cell. Thus, a transformed strain that stably expresses the recombinant antibody can be obtained.
　宿主細胞への発現ベクターの導入には、エレクトロポレーション法［日本国特開平２－２５７８９１号公報、Ｃｙｔｏｔｅｃｈｎｏｌｏｇｙ，３，１３３（１９９０）］などを用いる。 For introduction of an expression vector into a host cell, an electroporation method [Japanese Patent Laid-Open No. 2-257891, Cytotechnology, 3, 133 (1990)] or the like is used.
　遺伝子組換え抗体発現ベクターを導入する宿主細胞には、遺伝子組換え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、ＣＨＯ－Ｋ１（ＡＴＣＣ　ＣＣＬ－６１）、ＤＵｋＸＢ１１（ＡＴＣＣ　ＣＣＬ－９０９６）、Ｐｒｏ－５（ＡＴＣＣ　ＣＣＬ－１７８１）、ＣＨＯ－Ｓ（Ｌｉｆｅ　Ｔｅｃｈｎｏｌｏｇｉｅｓ，Ｃａｔ　＃　１１６１９）、ラットミエローマ細胞ＹＢ２／３ＨＬ．Ｐ２．Ｇ１１．１６Ａｇ．２０（またはＹＢ２／０ともいう）、マウスミエローマ細胞ＮＳＯ、マウスミエローマ細胞ＳＰ２／０－Ａｇ１４（ＡＴＣＣ番号：ＣＲＬ１５８１）、マウスＰ３Ｘ６３－Ａｇ８．６５３細胞（ＡＴＣＣ番号：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ＣＨＯ細胞［Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，７７，４２１６（１９８０）］、レクチン耐性を獲得したＬｅｃ１３［Ｓｏｍａｔｉｃ　Ｃｅｌｌ　ａｎｄ　Ｍｏｌｅｃｕｌａｒ　ｇｅｎｅｔｉｃｓ，１２，５５（１９８６）］、α１，６－フコース転移酵素遺伝子が欠損したＣＨＯ細胞（国際公開第２００５／０３５５８６号、国際公開第０２／３１１４０号）、ラットＹＢ２／３ＨＬ．Ｐ２．Ｇ１１．１６Ａｇ．２０細胞（ＡＴＣＣ番号：ＣＲＬ１６６２）などが挙げられる。 Any host cell capable of expressing a recombinant antibody can be used as a host cell into which the recombinant antibody expression vector is introduced. For example, CHO-K1 (ATCC CCL-61), DUkXB11 (ATCC CCL-9096), Pro-5 (ATCC CCL-1781), CHO-S (Life Technologies, Cat # 11619), rat myeloma cell YB2 / 3HL. P2. G11.16 Ag. 20 (also referred to as YB2 / 0), mouse myeloma cell NSO, mouse myeloma cell SP2 / 0-Ag14 (ATCC number: CRL1581), mouse P3X63-Ag8.653 cell (ATCC number: CRL1580), dihydrofolate reductase gene ( (Hereinafter referred to as dhfr) deficient CHO cells [Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)], Lec13 [Somatic Cell and Molecular genetics, 12, 55 (1986)] that has acquired lectin resistance, CHO cells deficient in the α1,6-fucose transferase gene (International Publication No. 2005 / 035586, WO 02/31140), rat YB2 / 3HL. P2. G11.16 Ag. 20 cells (ATCC number: CRL1662) and the like.
　また、例えば、細胞内糖ヌクレオチドＧＤＰ－フコースの合成に関与する酵素などの蛋白質、Ｎ－グリコシド結合複合型糖鎖の還元末端のＮ－アセチルグルコサミンの６位にフコースの１位がα結合する糖鎖修飾に関与する酵素などの蛋白質、または細胞内糖ヌクレオチドＧＤＰ－フコースのゴルジ体への輸送に関与する蛋白質などの活性が低下または欠失した宿主細胞、例えばα１，６－フコース転移酵素遺伝子が欠損したＣＨＯ細胞（国際公開第２００５／０３５５８６号、国際公開第０２／３１１４０号）などが挙げられる。 In addition, for example, a protein such as an enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose, a sugar in which the 1-position of fucose is α-bonded to the 6-position of N-acetylglucosamine at the reducing end of the N-glycoside-linked complex sugar chain Host cells in which the activity of a protein such as an enzyme involved in chain modification or a protein involved in the transport of intracellular sugar nucleotide GDP-fucose to the Golgi apparatus is reduced or deleted, such as α1,6-fucose transferase gene Deficient CHO cells (WO 2005/035586, WO 02/31140) and the like can be mentioned.
　発現ベクターの導入後、遺伝子組換え抗体を安定に発現する形質転換株は、Ｇ４１８硫酸塩（以下、Ｇ４１８と表記する）など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する（日本国特開平２－２５７８９１号公報）。 After the introduction of the expression vector, a transformant that stably expresses the recombinant antibody is selected by culturing in an animal cell culture medium containing a drug such as G418 sulfate (hereinafter referred to as G418) (Japan). (Japanese Patent Laid-Open No. 2-257891).
　動物細胞培養用培地としては、例えば、ＲＰＭＩ１６４０培地（インビトロジェン社製）、ＧＩＴ培地（日本製薬社製）、ＥＸ－ＣＥＬＬ３０１培地（ジェイアールエイチ社製）、ＩＭＤＭ培地（インビトロジェン社製）、Ｈｙｂｒｉｄｏｍａ－ＳＦＭ培地（インビトロジェン社製）、またはこれら培地にＦＢＳなどの各種添加物を添加した培地などが挙げられる。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遺伝子組換え抗体を発現蓄積させる。培養上清中の遺伝子組換え抗体の発現量および抗原結合活性はＥＬＩＳＡ法などにより測定できる。また、形質転換株は、ＤＨＦＲ増幅系（日本国特開平２－２５７８９１号公報）などを利用して遺伝子組換え抗体の発現量を上昇させることができる。 Examples of the animal cell culture medium include RPMI 1640 medium (manufactured by Invitrogen), GIT medium (manufactured by Nippon Pharmaceutical Co., Ltd.), EX-CELL301 medium (manufactured by JRH), IMDM medium (manufactured by Invitrogen), Hybridoma-SFM. Examples thereof include a medium (manufactured by Invitrogen) or a medium obtained by adding various additives such as FBS to these mediums. By culturing the obtained transformant in a medium, the recombinant antibody is expressed and accumulated in the culture supernatant. The expression level and antigen binding activity of the recombinant antibody in the culture supernatant can be measured by ELISA method or the like. The transformed strain can increase the expression level of the recombinant antibody using a DHFR amplification system (Japanese Patent Laid-Open No. 2-257891).
　遺伝子組換え抗体は、形質転換株の培養上清よりプロテインＡ－カラムを用いて精製する［Ｍｏｎｏｃｌｏｎａｌ　Ａｎｔｉｂｏｄｉｅｓ－Ｐｒｉｎｃｉｐｌｅｓ　ａｎｄ　ｐｒａｃｔｉｃｅ，Ｔｈｉｒｄ　ｅｄｉｔｉｏｎ，Ａｃａｄｅｍｉｃ　Ｐｒｅｓｓ（１９９６）、Ａｎｔｉｂｏｄｉｅｓ－Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ，Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ（１９８８）］。また、ゲル濾過、イオン交換クロマトグラフィーおよび限外濾過などの蛋白質の精製で用いられる方法を組み合わせることもできる。 The recombinant antibody is purified from the culture supernatant of the transformant using a protein A-column [Monoclonal Antibodies-Principles and Practice, Third edition, Academic Press (1996), Antibodies-A LaboratoryLaboratoryLaboratoryLaboratoryLaboratory. (1988)]. In addition, methods used in protein purification such as gel filtration, ion exchange chromatography, and ultrafiltration can be combined.
　精製した遺伝子組換え抗体の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法［Ｎａｔｕｒｅ，２２７，６８０（１９７０）］、またはウェスタンブロッティング法［Ｍｏｎｏｃｌｏｎａｌ　Ａｎｔｉｂｏｄｉｅｓ－Ｐｒｉｎｃｉｐｌｅｓ　ａｎｄ　ｐｒａｃｔｉｃｅ，Ｔｈｉｒｄ　ｅｄｉｔｉｏｎ，Ａｃａｄｅｍｉｃ　Ｐｒｅｓｓ（１９９６）、Ａｎｔｉｂｏｄｉｅｓ－Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ，Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ（１９８８）］など用いて測定することができる。 The molecular weight of the purified recombinant antibody H chain, L chain, or whole antibody molecule is determined by polyacrylamide gel electrophoresis [Nature, 227, 680 (1970)] or Western blotting [Monoclonal Antibodies-Principles and practicies, Third]. edition, Academic Press (1996), Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988)].
３．精製モノクローナル抗体またはその断片の活性評価
　精製した本発明のモノクローナル抗体またはその断片の活性は、以下のように評価することができる。 3. Evaluation of activity of purified monoclonal antibody or fragment thereof The activity of the purified monoclonal antibody or fragment thereof of the present invention can be evaluated as follows.
　ＣＤ３３発現細胞株に対する結合活性は、前述の１－（６ａ）記載のバインディングアッセイおよび（６ｂ）記載のＢｉａｃｏｒｅシステムなどを用いた表面プラズモン共鳴法を用いて測定する。また、蛍光抗体法［Ｃａｎｃｅｒ　Ｉｍｍｕｎｏｌ．Ｉｍｍｕｎｏｔｈｅｒ．，３６，３７３（１９９３）］などを用いて測定できる。 The binding activity to the CD33-expressing cell line is measured using the above-described binding assay described in 1- (6a) and the surface plasmon resonance method using the Biacore system described in (6b). In addition, the fluorescent antibody method [Cancer Immunol. Immunother. , 36, 373 (1993)].
　抗原陽性培養細胞株に対するＣＤＣ活性、またはＡＤＣＣ活性は公知の測定方法［Ｃａｎｃｅｒ　Ｉｍｍｕｎｏｌ．Ｉｍｍｕｎｏｔｈｅｒ．，３６，３７３（１９９３）］により測定する。 CDC activity or ADCC activity against an antigen-positive cultured cell line is measured by a known measurement method [Cancer Immunol. Immunother. , 36, 373 (1993)].
４．抗体のエフェクター活性を制御する方法
　本発明の抗ＣＤ３３モノクローナル抗体のエフェクター活性を制御する方法としては、抗体のＦｃ領域の２９７番目のアスパラギン（Ａｓｎ）に結合するＮ結合複合型糖鎖の還元末端に存在するＮ－アセチルグルコサミン（ＧｌｃＮＡｃ）にα－１，６結合するフコース（コアフコースともいう）の量を制御する方法（国際公開第２００５／０３５５８６号、国際公開第２００２／３１１４０号、国際公開第００／６１７３９号）や、抗体のＦｃ領域のアミノ酸残基を改変することで制御する方法などが知られている。本発明の抗ＣＤ３３モノクローナル抗体にはいずれの方法を用いても、エフェクター活性を制御することができる。 4). Method for Controlling Effector Activity of Antibody As a method for controlling the effector activity of the anti-CD33 monoclonal antibody of the present invention, an N-linked complex type sugar chain that binds to the 297th asparagine (Asn) of the Fc region of the antibody Methods for controlling the amount of fucose (also referred to as core fucose) that binds α-1,6 to existing N-acetylglucosamine (GlcNAc) (WO 2005/035586, WO 2002/31140, WO 00) No. / 61739) and a method of controlling by modifying amino acid residues in the Fc region of an antibody are known. The effector activity can be controlled using any method for the anti-CD33 monoclonal antibody of the present invention.
　エフェクター活性とは、抗体のＦｃ領域を介して引き起こされる抗体依存性の活性をいい、ＡＤＣＣ活性、ＣＤＣ活性、またはマクロファージ若しくは樹状細胞などの食細胞による抗体依存性ファゴサイトーシス（Ａｎｔｉｂｏｄｙ－ｄｅｐｅｎｄｅｎｔ　ｐｈａｇｏｃｙｔｏｓｉｓ，ＡＤＰ活性）などが知られている。 The effector activity refers to antibody-dependent activity caused through the Fc region of an antibody, and includes ADCC activity, CDC activity, or antibody-dependent phagocytosis by phagocytic cells such as macrophages or dendritic cells (Antibody-dependent phagocytosis). , ADP activity) and the like.
　抗体のＦｃのＮ結合複合型糖鎖のコアフコースの含量を制御することで、抗体のエフェクター活性を増加または低下させることができる。抗体のＦｃに結合しているＮ結合複合型糖鎖に結合するフコースの含量を低下させる方法としては、α１，６－フコース転移酵素遺伝子が欠損したＣＨＯ細胞を用いて抗体を発現することで、フコースが結合していない抗体を取得することができる。 By controlling the content of core fucose in the N-linked complex sugar chain of the Fc of the antibody, the effector activity of the antibody can be increased or decreased. As a method for reducing the content of fucose bound to the N-linked complex type sugar chain bound to the Fc of the antibody, expressing the antibody using CHO cells deficient in the α1,6-fucose transferase gene, An antibody to which fucose is not bound can be obtained.
　フコースが結合していない抗体は高いＡＤＣＣ活性を有する。一方、抗体のＦｃに結合しているＮ結合複合型糖鎖に結合するフコースの含量を増加させる方法としては、α１，６－フコース転移酵素遺伝子を導入した宿主細胞を用いて抗体を発現させることで、フコースが結合している抗体を取得できる。フコースが結合している抗体は、フコースが結合していない抗体よりも低いＡＤＣＣ活性を有する。 An antibody to which fucose is not bound has high ADCC activity. On the other hand, as a method for increasing the content of fucose bound to the N-linked complex type sugar chain bound to the Fc of the antibody, the antibody is expressed using a host cell into which an α1,6-fucose transferase gene has been introduced. Thus, an antibody to which fucose is bound can be obtained. An antibody to which fucose is bound has a lower ADCC activity than an antibody to which fucose is not bound.
　また、抗体のＦｃ領域のアミノ酸残基を改変することでＡＤＣＣ活性またはＣＤＣ活性を増加または低下させることができる。例えば、米国特許出願公開第２００７／０１４８１６５号明細書に記載のＦｃ領域のアミノ酸配列を用いることで、抗体のＣＤＣ活性を増加させることができる。 Also, ADCC activity or CDC activity can be increased or decreased by modifying amino acid residues in the Fc region of the antibody. For example, the CDC activity of an antibody can be increased by using the amino acid sequence of the Fc region described in US Patent Application Publication No. 2007/0148165.
　また、米国特許第６，７３７，０５６号明細書、米国特許第７，２９７，７７５号明細書または米国特許第７，３１７，０９１号明細書に記載のアミノ酸改変を行うことで、ＡＤＣＣ活性またはＣＤＣ活性を、増加させることも低下させることもできる。 Further, by performing amino acid modification described in US Pat. No. 6,737,056, US Pat. No. 7,297,775 or US Pat. No. 7,317,091, ADCC activity or CDC activity can be increased or decreased.
　更に、上述の方法を組み合わせて、一つの抗体に使用することにより、抗体のエフェクター活性が制御された抗体を取得することができる。 Furthermore, an antibody with controlled effector activity of an antibody can be obtained by using the above method in combination with one antibody.
５．本発明の抗ＣＤ３３モノクローナル抗体またはその断片を用いた疾患の治療方法
　本発明のモノクローナル抗体またはその断片は、ＣＤ３３陽性細胞が関与する疾患の治療に用いることができる。 5. Method for Treating Disease Using Anti-CD33 Monoclonal Antibody or Fragment thereof of the Present Invention The monoclonal antibody or fragment thereof of the present invention can be used for the treatment of diseases involving CD33 positive cells.
　本発明のモノクローナル抗体またはその断片を含有する治療剤は、有効成分としての該抗体もしくは該断片のみを含むものであってもよいが、通常は薬理学的に許容される１以上の担体と一緒に混合し、製剤学の技術分野において公知の方法により製造した医薬製剤として提供される。 The therapeutic agent containing the monoclonal antibody or fragment thereof of the present invention may contain only the antibody or fragment as an active ingredient, but usually together with one or more pharmacologically acceptable carriers. And prepared as a pharmaceutical preparation produced by a method known in the technical field of pharmaceutics.
　投与経路としては、例えば、経口投与、または口腔内、気道内、直腸内、皮下、筋肉内または静脈内などの非経口投与が挙げられる。投与形態としては、例えば、噴霧剤、カプセル剤、錠剤、散剤、顆粒剤、シロップ剤、乳剤、座剤、注射剤、軟膏、またはテープ剤などが挙げられる。 Examples of the administration route include oral administration and parenteral administration such as intraoral, intratracheal, rectal, subcutaneous, intramuscular or intravenous. Examples of the dosage form include sprays, capsules, tablets, powders, granules, syrups, emulsions, suppositories, injections, ointments, or tapes.
　経口投与に適当な製剤は、乳剤、シロップ剤、カプセル剤、錠剤、散剤、または顆粒剤などである。 Suitable formulations for oral administration are emulsions, syrups, capsules, tablets, powders, or granules.
　乳剤またはシロップ剤のような液体調製物は、水、ショ糖、ソルビトール若しくは果糖などの糖類、ポリエチレングリコール若しくはプロピレングリコールなどのグリコール類、ごま油、オリーブ油若しくは大豆油などの油類、ｐ－ヒドロキシ安息香酸エステル類などの防腐剤、またはストロベリーフレーバー若しくはペパーミントなどのフレーバー類などを添加剤として用いて製造する。 Liquid preparations such as emulsions or syrups include saccharides such as water, sucrose, sorbitol or fructose, glycols such as polyethylene glycol or propylene glycol, oils such as sesame oil, olive oil or soybean oil, p-hydroxybenzoic acid Manufactured using preservatives such as esters, or flavors such as strawberry flavor or peppermint as additives.
　カプセル剤、錠剤、散剤または顆粒剤などは、乳糖、ブドウ糖、ショ糖若しくはマンニトールなどの賦形剤、デンプン若しくはアルギン酸ナトリウムなどの崩壊剤、ステアリン酸マグネシウム若しくはタルクなどの滑沢剤、ポリビニルアルコール、ヒドロキシプロピルセルロース若しくはゼラチンなどの結合剤、脂肪酸エステルなどの界面活性剤またはグリセリンなどの可塑剤などを添加剤として用いて製造する。 Capsules, tablets, powders or granules include excipients such as lactose, glucose, sucrose or mannitol, disintegrants such as starch or sodium alginate, lubricants such as magnesium stearate or talc, polyvinyl alcohol, hydroxy A binder such as propylcellulose or gelatin, a surfactant such as fatty acid ester, or a plasticizer such as glycerin is used as an additive.
　非経口投与に適当な製剤としては、例えば、注射剤、座剤または噴霧剤などが挙げられる。 Examples of preparations suitable for parenteral administration include injections, suppositories, and sprays.
　注射剤は、塩溶液、ブドウ糖溶液、またはその両者の混合物からなる担体などを用いて製造する。 Injection is manufactured using a carrier made of 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などの担体を用いて製造する。 Suppositories are produced using a carrier such as cacao butter, hydrogenated fat or carboxylic acid.
　噴霧剤は受容者の口腔および気道粘膜を刺激せず、かつ本発明のモノクローナル抗体またはその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などを用いて製造する。担体としては、例えば乳糖またはグリセリンなどを用いる。また、エアロゾルまたはドライパウダーとして製造することもできる。 The propellant is produced using a carrier that does not irritate the recipient's oral cavity and airway mucosa, and in which the monoclonal antibody of the present invention or a fragment thereof is dispersed as fine particles to facilitate absorption. As the carrier, for example, lactose or glycerin is used. It can also be produced as an aerosol or dry powder.
　さらに、上記非経口剤においても、経口投与に適当な製剤で添加剤として例示した成分を添加することもできる。 Furthermore, in the above parenteral preparations, the components exemplified as additives in preparations suitable for oral administration can also be added.
６．本発明の抗ＣＤ３３モノクローナル抗体またはその断片を用いた疾患の診断方法
　本発明のモノクローナル抗体またはその断片を用いて、ＣＤ３３またはＣＤ３３が発現した細胞を検出または測定することにより、ＣＤ３３が関連する疾患を診断することができる。 6). Method of diagnosing disease using anti-CD33 monoclonal antibody or fragment thereof of the present invention By detecting or measuring cells expressing CD33 or CD33 using the monoclonal antibody or fragment thereof of the present invention, a disease associated with CD33 can be detected. Can be diagnosed.
　ＣＤ３３が関連する疾患の一つである癌の診断は、例えば、以下のようにＣＤ３３を検出または測定して行うことができる。 The diagnosis of cancer, which is one of the diseases associated with CD33, can be performed by detecting or measuring CD33 as follows, for example.
　患者体内の癌細胞に発現しているＣＤ３３をフローサイトメーターなどの免疫学的手法を用いて検出することにより診断を行うことができる。 Diagnosis can be made by detecting CD33 expressed in cancer cells in a patient using an immunological technique such as a flow cytometer.
　免疫学的手法とは、標識を施した抗原または抗体を用いて、抗体量または抗原量を検出または測定する方法である。例えば、放射性物質標識免疫抗体法、酵素免疫測定法、蛍光免疫測定法、発光免疫測定法、ウェスタンブロット法または物理化学的手法などが挙げられる。 An immunological technique is a method for detecting or measuring the amount of antibody or the amount of antigen using a labeled antigen or antibody. For example, a radioactive substance-labeled immunoantibody method, an enzyme immunoassay method, a fluorescence immunoassay method, a luminescence immunoassay method, a Western blot method, a physicochemical method, or the like can be given.
　放射性物質標識免疫抗体法は、例えば、抗原または抗原を発現した細胞などに、本発明の抗体またはその断片を反応させ、さらに放射線標識を施した抗イムノグロブリン抗体またはその結合を反応させた後、シンチレーションカウンターなどで測定する。 The radioactive substance-labeled immunoantibody method is, for example, reacting an antigen or a cell expressing the antigen with the antibody of the present invention or a fragment thereof, and further reacting with a radiolabeled anti-immunoglobulin antibody or a binding thereof, Measure with a scintillation counter.
　酵素免疫測定法は、例えば、抗原または抗原を発現した細胞などに、本発明の抗体またはその断片を反応させ、さらに標識を施した抗イムノグロブリン抗体またはその結合断片を反応させた後、発色色素を吸光光度計で測定する。例えば、サンドイッチＥＬＩＳＡ法などを用いる。 In the enzyme immunoassay, for example, an antigen or a cell expressing the antigen is reacted with the antibody of the present invention or a fragment thereof, and further, a labeled anti-immunoglobulin antibody or a binding fragment thereof is reacted, and then a coloring dye. Is measured with an absorptiometer. For example, a sandwich ELISA method is used.
　酵素免疫測定法で用いる標識体としては、公知［酵素免疫測定法，医学書院（１９８７）］の酵素標識を用いることができる。例えば、アルカリフォスファターゼ標識、ペルオキシダーゼ標識、ルシフェラーゼ標識、またはビオチン標識などを用いる。 As the label used in the enzyme immunoassay, a known enzyme label [enzyme immunoassay, Medical School (1987)] can be used. For example, alkaline phosphatase label, peroxidase label, luciferase label, biotin label or the like is used.
　サンドイッチＥＬＩＳＡ法は、固相に抗体を結合させた後、検出または測定対象である抗原をトラップさせ、トラップされた抗原に第２の抗体を反応させる方法である。該ＥＬＩＳＡ法では、検出または測定したい抗原を認識する抗体またはその断片であって、抗原認識部位の異なる２種類の抗体を準備し、そのうち、第１の抗体またはその断片を予めプレート（例えば、９６ウェルプレート）に吸着させ、次に第２の抗体またはその断片をＦＩＴＣなどの蛍光物質、ペルオキシダーゼなどの酵素、またはビオチンなどで標識しておく。 Sandwich ELISA is a method in which an antibody to be bound to a solid phase, an antigen to be detected or measured is trapped, and a second antibody is reacted with the trapped antigen. In the ELISA method, two types of antibodies that recognize an antigen to be detected or measured or fragments thereof and having different antigen recognition sites are prepared, and the first antibody or fragment thereof is preliminarily placed on a plate (for example, 96 Next, the second antibody or a fragment thereof is labeled with a fluorescent substance such as FITC, an enzyme such as peroxidase, or biotin.
　前記の抗体が吸着したプレートに、生体内から分離された、細胞またはその破砕液、組織またはその破砕液、細胞培養上清、血清、胸水、腹水、または眼液などを反応させた後、標識したモノクローナル抗体またはその断片を反応させ、標識物質に応じた検出反応を行う。濃度既知の抗原を段階的に希釈して作製した検量線より、被験サンプル中の抗原濃度を算出する。 After the cells adsorbed with the antibody are reacted with cells or a lysate thereof, tissue or a lysate thereof, cell culture supernatant, serum, pleural effusion, ascites or ocular fluid, the label is obtained. The detected monoclonal antibody or a fragment thereof is reacted, and a detection reaction corresponding to the labeling substance is performed. The antigen concentration in the test sample is calculated from a calibration curve prepared by diluting antigens with known concentrations stepwise.
　サンドイッチＥＬＩＳＡ法に用いる抗体としては、ポリクローナル抗体またはモノクローナル抗体のいずれを用いてもよく、Ｆａｂ、Ｆａｂ’、またはＦ（ａｂ）２などの抗体フラグメントを用いてもよい。サンドイッチＥＬＩＳＡ法で用いる２種類の抗体の組み合わせとしては、異なるエピトープを認識するモノクローナル抗体またはその断片の組み合わせでもよいし、ポリクローナル抗体とモノクローナル抗体またはその断片との組み合わせでもよい。 As an antibody used in the sandwich ELISA method, either a polyclonal antibody or a monoclonal antibody may be used, and an antibody fragment such as Fab, Fab ′, or F (ab) 2 may be used. The combination of the two types of antibodies used in the sandwich ELISA method may be a combination of a monoclonal antibody that recognizes different epitopes or a fragment thereof, or a combination of a polyclonal antibody and a monoclonal antibody or a fragment thereof.
　蛍光免疫測定法は、文献［Ｍｏｎｏｃｌｏｎａｌ　Ａｎｔｉｂｏｄｉｅｓ－Ｐｒｉｎｃｉｐｌｅｓ　ａｎｄ　ｐｒａｃｔｉｃｅ，Ｔｈｉｒｄ　ｅｄｉｔｉｏｎ，Ａｃａｄｅｍｉｃ　Ｐｒｅｓｓ（１９９６）、単クローン抗体実験マニュアル，講談社サイエンティフィック（１９８７）］などに記載された方法で測定する。蛍光免疫測定法で用いる標識体としては、公知［蛍光抗体法，ソフトサイエンス社（１９８３）］の蛍光標識を用いることができる。例えば、ＦＩＴＣ、またはＲＩＴＣなどを用いる。 The fluorescence immunoassay is measured by the method described in the literature [Monoclonal Antibodies-Principles and practices, Third edition, Academic Press (1996), Monoclonal Antibody Experiment Manual, Kodansha Scientific (1987)]. As a label used in the fluorescence immunoassay, a fluorescent label known in the art [fluorescent antibody method, Soft Science (1983)] can be used. For example, FITC or RITC is used.
　発光免疫測定法は文献［生物発光と化学発光　臨床検査４２，廣川書店（１９９８）］などに記載された方法で測定する。発光免疫測定法で用いる標識体としては、公知の発光体標識が挙げられ、例えば、アクリジニウムエステルまたはロフィンなどが挙げられる。 The luminescent immunoassay is measured by the method described in the literature [Bioluminescence and chemiluminescence, clinical examination 42, Yodogawa Shoten (1998)]. Examples of the label used in the luminescent immunoassay include known luminescent labels, such as acridinium ester or lophine.
　ウェスタンブロット法は、抗原または抗原を発現した細胞などをＳＤＳ（ドデシル硫酸ナトリウム）－ＰＡＧＥ［Ａｎｔｉｂｏｄｉｅｓ－Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ　Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ（１９８８）］で分画した後、該ゲルをポリフッ化ビニリデン（ＰＶＤＦ）膜またはニトロセルロース膜にブロッティングし、該膜に抗原を認識する抗体またはその断片を反応させ、さらにＦＩＴＣなどの蛍光物質、ペルオキシダーゼなどの酵素標識、またはビオチン標識などを施した抗マウスＩｇＧ抗体またはその結合断片を反応させた後、該標識を可視化することによって測定する。一例を以下に示す。 In Western blotting, an antigen or cells expressing the antigen are fractionated with SDS (sodium dodecyl sulfate) -PAGE [Antibodies-A Laboratory Manual Spring Spring Laboratory (1988)], and the gel is then polyvinylidene fluoride (PVDF). B) blotting on a membrane or a nitrocellulose membrane, reacting an antibody that recognizes the antigen or a fragment thereof on the membrane, and further applying an anti-mouse IgG antibody or a fluorescent substance such as FITC, an enzyme label such as peroxidase, or a biotin label After the bound fragment is reacted, the label is measured by visualization. An example is shown below.
　配列番号３で示されるアミノ酸配列を含むポリペプチドを発現している細胞または組織を溶解し、還元条件下でレーンあたりのタンパク量として０．１～３０μｇをＳＤＳ－ＰＡＧＥ法により泳動する。泳動されたタンパク質をＰＶＤＦ膜にトランスファーし１～１０％ＢＳＡを含むＰＢＳ（以下、ＢＳＡ－ＰＢＳと表記することがある）に室温で３０分間反応させブロッキング操作を行う。ここで本発明のモノクローナル抗体を反応させ、０．０５～０．１％のＴｗｅｅｎ－２０を含むＰＢＳ（以下、Ｔｗｅｅｎ－ＰＢＳと表記することがある）で洗浄し、ペルオキシダーゼ標識したヤギ抗マウスＩｇＧを室温で２時間反応させる。 A cell or tissue expressing a polypeptide containing the amino acid sequence represented by SEQ ID NO: 3 is lysed, and 0.1 to 30 μg of protein per lane is electrophoresed by SDS-PAGE under reducing conditions. The migrated protein is transferred to a PVDF membrane and reacted with PBS containing 1 to 10% BSA (hereinafter sometimes referred to as BSA-PBS) at room temperature for 30 minutes to perform a blocking operation. Here, the monoclonal antibody of the present invention was reacted, washed with PBS containing 0.05 to 0.1% Tween-20 (hereinafter sometimes referred to as Tween-PBS), and peroxidase-labeled goat anti-mouse IgG. For 2 hours at room temperature.
　Ｔｗｅｅｎ－ＰＢＳで洗浄し、ＥＣＬ　Ｗｅｓｔｅｒｎ　Ｂｌｏｔｔｉｎｇ　Ｄｅｔｅｃｔｉｏｎ　Ｒｅａｇｅｎｔｓ（アマシャム社製）などを用いてモノクローナル抗体が結合したバンドを検出することにより、配列番号３で示されるアミノ酸配列を含むポリペプチドを検出する。ウェスタンブロッティングでの検出に用いられる抗体としては、天然型の立体構造を保持していないポリペプチドに結合できる抗体が用いられる。 By washing with Tween-PBS and detecting the band to which the monoclonal antibody is bound using ECL Western Blotting Detection Reagents (manufactured by Amersham), a polypeptide containing the amino acid sequence represented by SEQ ID NO: 3 is detected. As an antibody used for detection by Western blotting, an antibody capable of binding to a polypeptide that does not have a natural three-dimensional structure is used.
　物理化学的手法は、例えば、抗原であるＣＤ３３と本発明のモノクローナル抗体またはその断片とを結合させることにより凝集体を形成させて、この凝集体を検出することにより行う。この他に物理化学的手法としては、例えば、毛細管法、一次元免疫拡散法、免疫比濁法またはラテックス免疫比濁法［臨床検査法提要，金原出版（１９９８）］などが挙げられる。 The physicochemical method is performed by, for example, forming an aggregate by binding the antigen CD33 and the monoclonal antibody of the present invention or a fragment thereof, and detecting the aggregate. In addition, examples of the physicochemical method include a capillary method, a one-dimensional immunodiffusion method, an immunoturbidimetric method, or a latex immunoturbidimetric method [Proposal for Clinical Laboratory Methods, Kanbara Publishing (1998)].
　ラテックス免疫比濁法は、抗体または抗原を感作させた粒径０．１～１μｍ程度のポリスチレンラテックスなどの担体を用い、対応する抗原または抗体により抗原抗体反応を起こさせると、反応液中の散乱光は増加し、透過光は減少する。この変化を吸光度または積分球濁度として検出することにより被験サンプル中の抗原濃度などを測定する。 Latex immunoturbidimetry is a method in which an antibody or antigen-sensitized carrier such as polystyrene latex having a particle size of about 0.1 to 1 μm is used to cause an antigen-antibody reaction with the corresponding antigen or antibody. Scattered light increases and transmitted light decreases. By detecting this change as absorbance or integrating sphere turbidity, the antigen concentration or the like in the test sample is measured.
　一方、ＣＤ３３が発現している細胞の検出または測定は、公知の免疫学的検出法を用いることができるが、好ましくは免疫沈降法、免疫細胞染色法、免疫組織染色法または蛍光抗体染色法などを用いる。 On the other hand, for the detection or measurement of cells expressing CD33, a known immunological detection method can be used. Preferably, immunoprecipitation method, immune cell staining method, immunohistochemical staining method, fluorescent antibody staining method, etc. Is used.
　免疫沈降法は、ＣＤ３３を発現した細胞などを本発明のモノクローナル抗体またはその断片と反応させた後、プロテインＧ－セファロースなどのイムノグロブリンに特異的な結合能を有する担体を加えて抗原抗体複合体を沈降させる。または以下のような方法によっても行なうことができる。 In the immunoprecipitation method, a cell expressing CD33 or the like is reacted with the monoclonal antibody of the present invention or a fragment thereof, and then a carrier having a specific binding ability to immunoglobulin such as protein G-sepharose is added to the antigen-antibody complex. To settle. Alternatively, the following method can be used.
　ＥＬＩＳＡ用９６ウェルプレートに上述した本発明のモノクローナル抗体またはその断片を固相化した後、ＢＳＡ－ＰＢＳによりブロッキングする。抗体が、例えばハイブリドーマ培養上清などの精製されていない状態である場合には、抗マウスイムノグロブリン、抗ラットイムノグロブリン、プロテイン－Ａまたはプロテイン－ＧなどをあらかじめＥＬＩＳＡ用９６ウェルプレートに固相化し、ＢＳＡ－ＰＢＳでブロッキングした後、ハイブリドーマ培養上清を分注して結合させる。次に、ＢＳＡ－ＰＢＳを捨てＰＢＳでよく洗浄した後、ＣＤ３３を発現している細胞や組織の溶解液を反応させる。よく洗浄した後のプレートより免疫沈降物をＳＤＳ－ＰＡＧＥ用サンプルバッファーで抽出し、上記のウェスタンブロッティングにより検出する。 The above-described monoclonal antibody of the present invention or a fragment thereof is immobilized on a 96-well plate for ELISA, and then blocked with BSA-PBS. When the antibody is in an unpurified state, such as a hybridoma culture supernatant, anti-mouse immunoglobulin, anti-rat immunoglobulin, protein-A or protein-G is immobilized on a 96-well plate for ELISA in advance. After blocking with BSA-PBS, the hybridoma culture supernatant is dispensed and bound. Next, after discarding BSA-PBS and washing well with PBS, a lysate of cells and tissues expressing CD33 is reacted. Immunoprecipitates are extracted from the well-washed plate with SDS-PAGE sample buffer and detected by Western blotting as described above.
　免疫細胞染色法または免疫組織染色法は、抗原を発現した細胞または組織などを、場合によっては抗体の通過性を良くするため界面活性剤またはメタノールなどで処理した後、本発明のモノクローナル抗体と反応させ、さらにＦＩＴＣなどの蛍光標識、ペルオキシダーゼなどの酵素標識またはビオチン標識などを施した抗イムノグロブリン抗体またはその結合断片と反応させた後、該標識を可視化し、顕微鏡にて顕鏡する方法である。 The immune cell staining method or the immunohistochemical staining method is a method in which cells or tissues expressing an antigen are treated with a surfactant or methanol in order to improve antibody passage, and then reacted with the monoclonal antibody of the present invention. And then reacting with a fluorescent label such as FITC, an enzyme label such as peroxidase or a biotin label, or an anti-immunoglobulin antibody or a binding fragment thereof, then the label is visualized and microscopically observed .
　また、蛍光標識の抗体と細胞を反応させ、フロ－サイトメーターにて解析する蛍光抗体染色法［Ｍｏｎｏｃｌｏｎａｌ　Ａｎｔｉｂｏｄｉｅｓ－Ｐｒｉｎｃｉｐｌｅｓ　ａｎｄ　ｐｒａｃｔｉｃｅ，Ｔｈｉｒｄ　ｅｄｉｔｉｏｎ，Ａｃａｄｅｍｉｃ　Ｐｒｅｓｓ（１９９６）、単クローン抗体実験マニュアル，講談社サイエンティフィック（１９８７）］により検出を行うことができる。特に、ＣＤ３３の細胞外領域に結合する、本発明のモノクローナル抗体またはその断片は、蛍光抗体染色法により天然型の立体構造を保持して発現している細胞の検出ができる。 In addition, a fluorescent antibody staining method in which a fluorescently labeled antibody is reacted with a cell and analyzed with a flow cytometer [Monoclonal Antibodies-Principles and Practice, Academic Press (1996), Monoclonal Antibody Experiment Manual, Kodansha Scientific Fick (1987)]. In particular, the monoclonal antibody of the present invention or a fragment thereof that binds to the extracellular region of CD33 can detect cells expressing the natural three-dimensional structure by fluorescent antibody staining.
　また、蛍光抗体染色法のうち、ＦＭＡＴ８１００ＨＴＳシステム（アプライドバイオシステム社製）などを用いた場合には、形成された抗体－抗原複合体と、抗体－抗原複合体の形成に関与していない遊離の抗体または抗原とを分離することなく、抗原量または抗体量を測定できる。 In addition, among the fluorescent antibody staining methods, when the FMAT8100HTS system (Applied Biosystems) or the like is used, the formed antibody-antigen complex and the free that is not involved in the formation of the antibody-antigen complex The amount of antigen or antibody can be measured without separating the antibody or antigen.
　以下、本発明を実施例により具体的に説明するが、本発明は下記実施例に限定されるものではない。 Hereinafter, the present invention will be specifically described by way of examples. However, the present invention is not limited to the following examples.
［実施例１］
　ＣＤ３３トランスフェクタントの作製      
（１）ＣＤ３３発現ベクターの作製
　ベクターｐＣＭＶ／ＳＰＯＲＴ６にヒトＣＤ３３遺伝子を組み込んだベクター（Ｏｐｅｎ　Ｂｉｏｓｙｓｔｅｍｓ社製、Ｃｌｏｎｅ　Ｎｏ．５２１７１８２、以下ｐＣＭＶ／ｈＣＤ３３と表記することがある）から以下のようにしてヒトＣＤ３３遺伝子配列が組み込まれたベクターを作製した。 [Example 1]
Production of CD33 transfectant
(1) Preparation of CD33 expression vector From a vector in which the human CD33 gene is incorporated into the vector pCMV / SPORT6 (Open Biosystems, Clone No. 5217182, hereinafter sometimes referred to as pCMV / hCD33), human CD33 is prepared as follows. A vector incorporating the gene sequence was prepared.
　ｐＣＭＶ／ｈＣＤ３３を鋳型とし、配列番号１、２で表される塩基配列を有したプライマーを用いて通常のＰＣＲ法によりヒトＣＤ３３遺伝子を増幅した。ＰＣＲ産物をＰＣＲ　Ｐｕｒｉｆｉｃａｔｉｏｎ　Ｋｉｔ（ＱＩＡＧＥＮ社製）により精製し、ＳａｃＩおよびＨｉｎｄＩＩＩで制限酵素処理した。ベクターｐＢｌｕｅｓｃｒｉｐｔＩＩ　ｓｋ（－）（ストラタジーン社製）（以下、ｐＢＳと表記することがある）も同様にＳａｃＩおよびＨｉｎｄＩＩＩで制限酵素処理を行い、これら２種類の断片をＬｉｇａｔｉｏｎ　ｈｉｇｈ（ＴＯＹＯＢＯ社製）を用いて添付の説明書に従って連結した。 The human CD33 gene was amplified by a normal PCR method using pCMV / hCD33 as a template and primers having the nucleotide sequences represented by SEQ ID NOs: 1 and 2. The PCR product was purified by PCR Purification Kit (manufactured by QIAGEN) and treated with restriction enzymes with SacI and HindIII. The vector pBluescript II sk (-) (Stratagene) (hereinafter sometimes referred to as pBS) is similarly treated with SacI and HindIII, and these two fragments are ligated high (TOYOBO). Used and ligated according to the attached instructions.
　得られた組換えプラスミドＤＮＡ溶液を用いて大腸菌ＤＨ５α株（ＴＯＹＯＢＯ社製）を形質転換した。形質転換株のクローンよりプラスミドＤＮＡを調製して制限酵素処理により切り出された断片の大きさをアガロースゲル電気泳動にて比較することにより確認し、ヒトＣＤ３３遺伝子が挿入されたプラスミドｐＢＳ／ｈＣＤ３３を取得した。 The obtained recombinant plasmid DNA solution was used to transform E. coli DH5α strain (manufactured by TOYOBO). A plasmid pBS / hCD33 into which the human CD33 gene has been inserted is obtained by preparing plasmid DNA from a clone of the transformant and confirming the size of the fragment excised by restriction enzyme treatment by agarose gel electrophoresis. did.
　こうして得られたヒトＣＤ３３遺伝子が組み込まれたｐＢＳ／ｈＣＤ３３、および動物細胞発現ベクターｐＫＡＮＴＥＸ９３（国際公開第９７／１０３５４号）をＢｓｉＷＩおよびＳｐｅＩで制限酵素処理した。その反応液をアガロースゲル電気泳動により分画し、ｈＣＤ３３のＢｓｉＷＩ－ＳｐｅＩ断片およびｐＫＡＮＴＥＸ９３のＢｓｉＷＩ－ＳｐｅＩ断片をそれぞれ回収した。 The pBS / hCD33 into which the human CD33 gene thus obtained was integrated and the animal cell expression vector pKANTEX93 (International Publication No. 97/10354) were subjected to restriction enzyme treatment with BsiWI and SpeI. The reaction mixture was fractionated by agarose gel electrophoresis, and the BsiWI-SpeI fragment of hCD33 and the BsiWI-SpeI fragment of pKANTEX93 were recovered.
　これら２種類の断片をＬｉｇａｔｉｏｎ　ｈｉｇｈを用いて添付の説明書に従って連結した。得られた組換えプラスミドＤＮＡ溶液を用いて大腸菌ＤＨ５α株を形質転換した。形質転換株のクローンよりプラスミドＤＮＡを調製して制限酵素処理により切り出された断片の大きさをアガロースゲル電気泳動にて比較することにより確認した。 These two types of fragments were ligated using Ligation high according to the attached instructions. The resulting recombinant plasmid DNA solution was used to transform E. coli DH5α strain. Plasmid DNA was prepared from the clone of the transformant and confirmed by comparing the size of the fragment excised by the restriction enzyme treatment by agarose gel electrophoresis.
　さらに該プラスミドに関して、ＢｉｇＤｙｅ　Ｔｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　ＦＳ　Ｒｅａｄｙ　Ｒｅａｃｔｉｏｎ　Ｋｉｔ（アプライドバイオシステムズ社製）を用いて、添付の説明書に従って反応後、同社のシーケンサーＡＢＩ　ＰＲＩＳＭ３７００により塩基配列を解析した。 Further, the base sequence of the plasmid was analyzed using the BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (Applied Biosystems) according to the attached instructions, and then analyzed by the sequencer ABI PRISM 3700 of the company.
　その結果、ヒトＣＤ３３遺伝子をコードするｃＤＮＡがクローニングされたベクターｐＫＡＮＴＥＸ／ｈＣＤ３３を取得した。ベクター構築の概略図を図１Ａに示した。ヒトＣＤ３３のアミノ酸配列を配列番号３、ＣＤ３３の塩基配列を配列番号４に示す。 As a result, a vector pKANTEX / hCD33 in which cDNA encoding the human CD33 gene was cloned was obtained. A schematic diagram of vector construction is shown in FIG. 1A. The amino acid sequence of human CD33 is shown in SEQ ID NO: 3, and the nucleotide sequence of CD33 is shown in SEQ ID NO: 4.
　カニクイザルＣＤ３３遺伝子を組み込んだ発現ベクターｐＫＡＮＴＥＸ／ｃｙｎｏＣＤ３３は、カニクイザル末梢血単核球（Ｐｅｒｉｐｈｅｒａｌ　ｂｌｏｏｄ　ｍｏｎｏｎｕｃｌｅａｒ　ｃｅｌｌ：以下、ＰＢＭＣと表記することがある）から抽出したｍＲＮＡから配列番号１１５、１１６で表される塩基配列を有したプライマーを用いて通常のＰＣＲ法によりヒトＣＤ３３遺伝子を増幅したものから、上記と同様の方法により作製した。ベクター構築の概略図を図１Ｂに示す。上記の解析で確認した塩基配列を配列番号１１８に、該塩基配列から予想されるカニクイザルＣＤ３３のアミノ酸配列を配列番号１１７に示す。 An expression vector pKANTEX / cynoCD33 incorporating a cynomolgus monkey CD33 gene is a base represented by SEQ ID NOs: 115 and 116 from mRNA extracted from a cynomolgus monkey peripheral blood mononuclear cell (Peripheral blood mononuclear cell: hereinafter sometimes referred to as PBMC). A human CD33 gene was amplified by a normal PCR method using a primer having a sequence, and prepared by the same method as described above. A schematic diagram of vector construction is shown in FIG. 1B. The base sequence confirmed by the above analysis is shown in SEQ ID NO: 118, and the amino acid sequence of cynomolgus CD33 predicted from the base sequence is shown in SEQ ID NO: 117.
　チンパンジーＣＤ３３遺伝子は、ＮＣＢＩデータベースにＸＭ＿５１２８５０．２で登録されたチンパンジーＣＤ３３遺伝子の塩基配列を元に、配列番号１１９で表される塩基配列を設計し、該塩基配列がクローニングベクターｐＵＣ５３に組み込まれたプラスミドをＧｅｎＳｃｒｉｐｔ社の遺伝子合成受託サービスを利用して購入した。 The chimpanzee CD33 gene is a plasmid in which the base sequence represented by SEQ ID NO: 119 is designed based on the base sequence of the chimpanzee CD33 gene registered in the NCBI database as XM — 5128850.2, and the base sequence is incorporated into the cloning vector pUC53. Was purchased using a gene synthesis contract service of GenScript.
　前記プラスミドを用いて、チンパンジーＣＤ３３遺伝子組み込んだ発現ベクターｐＫＡＮＴＥＸ／ｃｈｉｍｐＣＤ３３を、上記と同様の方法により作製した。ベクター構築の概略図を図１Ｃに示す。チンパンジーＣＤ３３のアミノ酸配列を配列番号１２０、チンパンジーＣＤ３３の塩基配列を配列番号１２１に示す。 Using the plasmid, an expression vector pKANTEX / chimpCD33 incorporating the chimpanzee CD33 gene was prepared by the same method as described above. A schematic diagram of vector construction is shown in FIG. 1C. The amino acid sequence of chimpanzee CD33 is shown in SEQ ID NO: 120, and the base sequence of chimpanzee CD33 is shown in SEQ ID NO: 121.
（２）ＣＤ３３のＣＨＯ／ＤＧ４４細胞での発現
　上記（１）で作製したＣＤ３３発現ベクターｐＫＡＮＴＥＸ／ｈＣＤ３３、ｐＫＡＮＴＥＸ／ｃｙｎｏＣＤ３３、ｐＫＡＮＴＥＸ／ｃｈｉｍｐＣＤ３３を、エレクトロポレーション法［Ｃｙｔｏｔｅｃｈｎｏｌｏｇｙ，３，１３３（１９９０）］により、以下のようにしてＣＨＯ／ＤＧ４４細胞［Ｓｏｍａｔｉｃ　Ｃｅｌｌ　ａｎｄ　Ｍｏｌｅｃｕｌａｒ　Ｇｅｎｅｔｉｃｓ，１２，５５５（１９８６）］へ導入した。 (2) Expression of CD33 in CHO / DG44 cells The CD33 expression vectors pKANTEX / hCD33, pKANTEX / cynoCD33, and pKANTEX / chimpCD33 prepared in (1) above were prepared by electroporation [Cytotechnology, 3, 133 (1990)]. Then, it was introduced into CHO / DG44 cells [Somatic Cell and Molecular Genetics, 12, 555 (1986)] as follows.
　細胞は、１０％ウシ胎児血清（ライフテクノロジーズ社製）およびＧｅｎｔａｍｉｃｉｎ（ナカライテスク社製、５０μｇ／ｍＬ）を添加したＩＭＤＭ培地（インビトロジェン社製）（以下、Ａ３倍地と表記することがある）に、１×ＨＴ　ｓｕｐｐｌｅｍｅｎｔ（インビトロジェン社製）を添加した培地で継代したものを用いた。 Cells were added to IMDM medium (Invitrogen) (hereinafter sometimes referred to as A3 medium) supplemented with 10% fetal calf serum (Life Technologies) and Gentamin (Nacalai Tesque, 50 μg / mL). 1 × HT supplement (manufactured by Invitrogen) was used in a subculture medium.
　ＣＨＯ／ＤＧ４４細胞を１３７ｎｍｏｌ／Ｌ塩化カリウム、２．７ｎｍｏｌ／Ｌ塩化ナトリウム、８．１ｍｍｏｌ／Ｌリン酸一水素二ナトリウム、１．５ｎｍｏｌ／Ｌリン酸二水素一ナトリウムおよび４ｍｍｏｌ／Ｌ塩化マグネシウムを含む緩衝液（以下、Ｋ－ＰＢＳと表記することがある）に懸濁して８×１０６細胞／ｍＬとし、得られた細胞懸濁液２００μＬ（１．６×１０６個の細胞）を各発現ベクター（８μｇ）と混和した。 CHO / DG44 cells contain 137 nmol / L potassium chloride, 2.7 nmol / L sodium chloride, 8.1 mmol / L disodium monohydrogen phosphate, 1.5 nmol / L monosodium dihydrogen phosphate and 4 mmol / L magnesium chloride Suspend in a buffer solution (hereinafter sometimes referred to as K-PBS) to 8 × 10 6 cells / mL, and express 200 μL (1.6 × 10 6 cells) of the resulting cell suspension for each expression. Mixed with vector (8 μg).
　この混和液をキュベット（電極間距離２ｍｍ）に移し、ＧｅｎｅＰｕｌｓｅｒＩＩ（バイオラッド社製）を用いてパルス電圧０．３５ｋＶ、電気容量２５０μＦの条件で遺伝子導入を行った。キュベットを氷上で静置後、キュベット中の細胞懸濁液を、Ａ３培地を含む細胞培養用容器に懸濁し、３７℃、５％炭酸ガス培養器中で培養した。 The mixture was transferred to a cuvette (distance between electrodes: 2 mm), and gene introduction was performed using GenePulser II (Bio-Rad) under conditions of a pulse voltage of 0.35 kV and an electric capacity of 250 μF. After leaving the cuvette on ice, the cell suspension in the cuvette was suspended in a cell culture vessel containing A3 medium, and cultured in a 37 ° C., 5% carbon dioxide incubator.
　その後、Ｇ４１８（インビトロジェン社製、０．５ｍｇ／ｍＬ）を添加した培地で培養し、Ｇ４１８に耐性を有する形質転換細胞株を取得した。更にメソトレキセートを培地に添加し、段階的に濃度を上昇させることにより、メソトレキセート耐性を有するクローンを選抜した。 Thereafter, the cells were cultured in a medium supplemented with G418 (manufactured by Invitrogen, 0.5 mg / mL) to obtain a transformed cell line resistant to G418. Further, methotrexate-resistant clones were selected by adding methotrexate to the medium and gradually increasing the concentration.
　さらに実施例１（３）の方法に従い、これらの細胞を蛍光免疫染色し、フローサイトメーター（ベックマン・コールター社製）により蛍光強度を測定することで、ＣＤ３３を高発現するクローンを選択し、ヒトＣＤ３３発現細胞株、カニクイザルＣＤ３３発現細胞株およびチンパンジーＣＤ３３発現細胞株を取得した。 Furthermore, according to the method of Example 1 (3), these cells were fluorescently immunostained, and the fluorescence intensity was measured with a flow cytometer (manufactured by Beckman Coulter) to select a clone that highly expresses CD33. A CD33-expressing cell line, a cynomolgus monkey CD33-expressing cell line, and a chimpanzee CD33-expressing cell line were obtained.
（３）フローサイトメトリー（ＦＣＭ）による細胞上でのＣＤ３３発現量の比較
　１～５×１０５細胞のＣＤ３３発現細胞に、ヒトＩｇＧ（シグマ社製）を１ｍｇ／ｍＬ加えブロッキングをし、マウス抗ＣＤ３３抗体Ｐ６７．６（Ｓａｎｔａ　Ｃｒｕｚｅ　Ｂｉｏｓｙｓｔｅｍｓ社製）またはＷＭ－５３（Ａｎｃｅｌｌ社製）を０．０２％ＥＤＴＡおよび０．０５％アジ化ナトリウムを含む１％牛血清アルブミン（ＢＳＡ）－ＰＢＳ（以下、ＦＣＭバッファーと表記することがある）で希釈し、全量を１００μＬとした。 (3) Comparison of CD33 expression level on cells by flow cytometry (FCM) 1 to 5 × 10 5 CD33-expressing cells were blocked by adding 1 mg / mL human IgG (manufactured by Sigma), and anti-mouse CD33 antibody P67.6 (Santa Cruze Biosystems) or WM-53 (Ancell) 1% bovine serum albumin (BSA) -PBS (hereinafter referred to as 0.02% EDTA and 0.05% sodium azide) The total volume was adjusted to 100 μL.
　これらの細胞懸濁液を氷上で６０分間反応させた後、ダルベッコリン酸バッファー（Ｐｈｏｓｐｈａｔｅ　ｂｕｆｆｅｒｅｄ　ｓａｌｉｎｅ：以下、ＰＢＳと表記することがある）で２回洗浄を行った。該細胞に、ＦＣＭバッファーで希釈調製したＡｌｅｘａ－４８８標識ヤギ抗マウスＩｇＧ（Ｈ＋Ｌ）（インビトロジェン社製）を３０μＬ添加し、氷上で４０分間反応させた。ＰＢＳで３回洗浄を行った後、ＦＣＭバッファーに懸濁し、フローサイトメーターにより蛍光強度を測定した。 These cell suspensions were reacted for 60 minutes on ice, and then washed twice with Dulbecco's phosphate buffer (hereinafter sometimes referred to as PBS). 30 μL of Alexa-488-labeled goat anti-mouse IgG (H + L) (manufactured by Invitrogen) diluted with FCM buffer was added to the cells and reacted on ice for 40 minutes. After washing 3 times with PBS, it was suspended in FCM buffer and the fluorescence intensity was measured with a flow cytometer.
［実施例２］
ヒスチジンタグ付加可溶型ヒトＣＤ３３（ｈＣＤ３３－Ｈｉｓ）の作製
（１）ｈＣＤ３３－Ｈｉｓ発現ベクターの作製
　実施例１（１）にて取得したヒトＣＤ３３遺伝子が挿入されたプラスミドｐＢＳ／ｈＣＤ３３を鋳型とし、配列番号１、５で表される塩基配列を有したプライマーを用いて通常のＰＣＲ法により６×ヒスチジン配列（以下、Ｈｉｓ配列と表記することがある）をコードする遺伝子配列（以下、ｈｉｓ配列と表記することがある）を３’末端側に付加したｈＣＤ３３遺伝子（以下、ｈＣＤ３３－ｈｉｓと表記することがある）を増幅した。 [Example 2]
Preparation of histidine-tagged soluble human CD33 (hCD33-His) (1) Preparation of hCD33-His expression vector Using plasmid pBS / hCD33 inserted with human CD33 gene obtained in Example 1 (1) as a template, A gene sequence (hereinafter referred to as a his sequence) encoding a 6 × histidine sequence (hereinafter sometimes referred to as a His sequence) by a normal PCR method using a primer having a base sequence represented by SEQ ID NOs: 1 and 5 The hCD33 gene (hereinafter sometimes referred to as hCD33-his) added to the 3 ′ end side was amplified.
　ＰＣＲ産物をＰＣＲ　Ｐｕｒｉｆｉｃａｔｉｏｎ　Ｋｉｔにより精製し、ＢｓｉＷＩおよびＳｐｅＩで制限酵素処理した。ベクターｐＫＡＮＴＥＸ９３を同様にＢｓｉＷＩおよびＳｐｅＩで制限酵素処理した。その反応液をアガロースゲル電気泳動により分画し、ｈＣＤ３３－ｈｉｓのＢｓｉＷＩ－ＳｐｅＩ断片およびｐＫＡＮＴＥＸ９３のＢｓｉＷＩ－ＳｐｅＩ断片をそれぞれ回収した。これら２種類の断片をＬｉｇａｔｉｏｎ　ｈｉｇｈを用いて添付の説明書に従って連結した。 The PCR product was purified by PCR Purification Kit and subjected to restriction enzyme treatment with BsiWI and SpeI. The vector pKANTEX93 was similarly digested with BsiWI and SpeI. The reaction solution was fractionated by agarose gel electrophoresis, and the BsiWI-SpeI fragment of hCD33-his and the BsiWI-SpeI fragment of pKANTEX93 were recovered. These two types of fragments were ligated using Ligation high according to the attached instructions.
　得られた組換えプラスミドＤＮＡ溶液を用いて大腸菌ＤＨ５α株を形質転換した。形質転換株のクローンよりプラスミドＤＮＡを調製して制限酵素処理により確認した。さらに該プラスミドを、ＢｉｇＤｙｅ　Ｔｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　ＦＳ　Ｒｅａｄｙ　Ｒｅａｃｔｉｏｎ　Ｋｉｔを用いて、添付の説明書に従って反応後、同社のシーケンサーＡＢＩ　ＰＲＩＳＭ３７００により塩基配列を解析した。 The obtained recombinant plasmid DNA solution was used to transform E. coli DH5α strain. Plasmid DNA was prepared from the clone of the transformant and confirmed by restriction enzyme treatment. Further, the plasmid was reacted using the BigDye Terminator Cycle Sequencing FS Ready Reaction Kit according to the attached instruction, and the base sequence was analyzed by the sequencer ABI PRISM 3700.
　その結果、ｈＣＤ３３－ｈｉｓをコードするｃＤＮＡがクローニングされたベクターｐＫＡＮＴＥＸ／ｈＣＤ３３－ｈｉｓを取得した。ベクター構築の概略図を図２に示す。ｈＣＤ３３－Ｈｉｓのアミノ酸配列を配列番号６、ｈＣＤ３３－ｈｉｓの塩基配列を配列番号７に示す。 As a result, a vector pKANTEX / hCD33-his in which cDNA encoding hCD33-his was cloned was obtained. A schematic diagram of vector construction is shown in FIG. The amino acid sequence of hCD33-His is shown in SEQ ID NO: 6, and the nucleotide sequence of hCD33-his is shown in SEQ ID NO: 7.
（２）ｈＣＤ３３－ＨｉｓのＣＨＯ／ＤＧ４４細胞での産生
　下記の方法により、ヒトＣＤ３３細胞外領域のＣ末端に連続した６つのヒスチジン配列であるＨｉｓ配列を付加したｈＣＤ３３－Ｈｉｓを取得した。上記（１）で作製したｈＣＤ３３－Ｈｉｓ発現ベクターｐＫＡＮＴＥＸ／ｈＣＤ３３－ｈｉｓを、エレクトロポレーション法により、以下のようにしてＣＨＯ／ＤＧ４４細胞へ導入した。 (2) Production of hCD33-His in CHO / DG44 cells By the following method, hCD33-His to which a His sequence, which is a sequence of six histidines at the C-terminus of the human CD33 extracellular region, was added. The hCD33-His expression vector pKANTEX / hCD33-his prepared in (1) above was introduced into CHO / DG44 cells by electroporation as follows.
　細胞は、Ａ３倍地に、１×ＨＴ　ｓｕｐｐｌｅｍｅｎｔを添加した培地で継代したものを用いた。ＣＨＯ／ＤＧ４４細胞をＫ－ＰＢＳに懸濁して８×１０６細胞／ｍＬとし、得られた細胞懸濁液２００μＬ（１．６×１０６個の細胞）を発現ベクターｐＫＡＮＴＥＸ／ｈＣＤ３３－ｈｉｓ（８μｇ）と混和した。 The cells used were subcultured with a medium supplemented with 1 × HT supplement to A3 medium. CHO / DG44 cells were suspended in K-PBS to 8 × 10 6 cells / mL, and 200 μL (1.6 × 10 6 cells) of the obtained cell suspension was used as an expression vector pKANTEX / hCD33-his (8 μg). ).
　この混和液をキュベット（電極間距離２ｍｍ）に移し、ＧｅｎｅＰｕｌｓｅｒＩＩを用いてパルス電圧０．３５ｋＶ、電気容量２５０μＦの条件で遺伝子導入を行った。キュベットを氷上で静置後、キュベット中の細胞懸濁液を、Ａ３培地を含む細胞培養用容器に懸濁し、３７℃、５％炭酸ガス培養器中で培養した。 The mixture was transferred to a cuvette (distance between electrodes: 2 mm), and gene introduction was performed using GenePulser II under conditions of a pulse voltage of 0.35 kV and an electric capacity of 250 μF. After leaving the cuvette on ice, the cell suspension in the cuvette was suspended in a cell culture vessel containing A3 medium, and cultured in a 37 ° C., 5% carbon dioxide incubator.
　その後、Ｇ４１８を添加した培地で培養し、Ｇ４１８に耐性を有する形質転換細胞株を取得した。更にメソトレキセートを培地に添加し、段階的に濃度を上昇させることにより、メソトレキセート耐性を有するクローンを選抜した。さらに下記（３）の方法に従い、これらの細胞の培養上清中ｈＣＤ３３－Ｈｉｓの濃度を比較することによりｈＣＤ３３－Ｈｉｓを高産生するクローンを選択し、ｈＣＤ３３－Ｈｉｓ産生細胞株を取得した。 Thereafter, the cells were cultured in a medium supplemented with G418 to obtain a transformed cell line having resistance to G418. Further, methotrexate-resistant clones were selected by adding methotrexate to the medium and gradually increasing the concentration. Further, according to the method (3) below, by comparing the concentration of hCD33-His in the culture supernatant of these cells, a clone producing high hCD33-His was selected, and an hCD33-His-producing cell line was obtained.
（３）ＥＬＩＳＡを用いた培養上清中ｈＣＤ３３－Ｈｉｓ濃度の比較
　アッセイには９６ウェルのＥＬＩＳＡ用プレート（グライナー社）を用いた。ＰＢＳにて２マイクログラム／ｍＬに希釈したＰ６７．６を５０μＬ／ウェルで分注し、４℃で一晩静置して吸着させたものを用いた。該プレートをＰＢＳで洗浄後、１％ＢＳＡ－ＰＢＳを１００μＬ／ウェル加え、室温で１時間静置して残っている活性基をブロックした。 (3) Comparison of hCD33-His concentration in culture supernatant using ELISA A 96-well ELISA plate (Greiner) was used for the assay. P67.6 diluted to 2 microgram / mL with PBS was dispensed at 50 μL / well, and allowed to stand overnight at 4 ° C. for adsorption. The plate was washed with PBS, 1% BSA-PBS was added at 100 μL / well, and left at room temperature for 1 hour to block remaining active groups.
　その後、１％　ＢＳＡ－ＰＢＳを捨て、該プレートにｈＣＤ３３－Ｈｉｓを含む細胞培養上清を適宜希釈して５０μＬ／ウェル分注し、１時間静置した。該プレートを０．０５％ポリオキシエチレン（２０）ソルビタンモノラウレート（ＩＣＩ社商標Ｔｗｅｅｎ　２０相当品：和光純薬工業社製）／ＰＢＳ（以下、Ｔｗｅｅｎ／ＰＢＳと表記することがある）で洗浄後、２次抗体としてペルオキシダーゼ標識マウス抗Ｐｅｎｔａ－Ｈｉｓ抗体（ＱＩＡＧＥＮ社製）を５０μＬ／ウェル加えて１時間室温で静置した。 Thereafter, 1% BSA-PBS was discarded, and the cell culture supernatant containing hCD33-His was appropriately diluted on the plate, dispensed at 50 μL / well, and allowed to stand for 1 hour. The plate was washed with 0.05% polyoxyethylene (20) sorbitan monolaurate (ICI trademark Tween 20 equivalent: manufactured by Wako Pure Chemical Industries, Ltd.) / PBS (hereinafter sometimes referred to as Tween / PBS). Thereafter, a peroxidase-labeled mouse anti-Penta-His antibody (manufactured by QIAGEN) was added as a secondary antibody at 50 μL / well and allowed to stand at room temperature for 1 hour.
　該プレートをＴｗｅｅｎ／ＰＢＳで洗浄し、ＡＢＴＳ〔２．２―アジノビス（３―エチルベンゾチアゾール－６－スルホン酸）アンモニウム〕基質液〔１ｍｍｏＬ／Ｌ　ＡＢＴＳ／０．１ｍｏＬ／Ｌ　クエン酸バッファー（ｐＨ４．２）、０．１％Ｈ２Ｏ２〕を５０μＬ／ウェル添加して発色させ、５％ＳＤＳ（Ｓｏｄｉｕｍ　Ｌａｕｒｙｌ　Ｓｕｌｆａｔｅ）溶液を５０μＬ／ウェル添加して反応を止め、サンプル波長４１５ｎｍ、リファレンス波長４９０ｎｍにおける吸光度（ＯＤ４１５－ＯＤ４９０）をプレートリーダー（Ｅｍａｘ　ｍｉｃｒｏｐｌａｔｅ　ｒｅａｄｅｒ、Ｍｏｌｅｃｕｌａｒ　Ｄｅｖｉｃｅｓ社）を用いて測定した。 The plate was washed with Tween / PBS, ABTS [2.2-azinobis (3-ethylbenzothiazole-6-sulfonic acid) ammonium] substrate solution [1 mmoL / L ABTS / 0.1 moL / L citrate buffer (pH 4. 2), 0.1% H 2 O 2 ] was added at 50 μL / well for color development, and 5% SDS (Sodium Lauryl Sulfate) solution was added at 50 μL / well to stop the reaction, and the sample wavelength was 415 nm and the reference wavelength was 490 nm. Absorbance (OD415-OD490) was measured using a plate reader (Emax microplate reader, Molecular Devices).
［実施例３］
抗ＣＤ３３モノクローナル抗体の作製
（１）免疫原の調製
　Ｒ＆Ｄシステム社製リコンビナントヒトＳｉｇｌｅｃ－３（ＣＤ３３）／Ｆｃ　Ｃｈｉｍｅｒａ（カタログ番号　１１３７－ＳＬ）（以下、リコンビナントヒトＣＤ３３－Ｆｃと表記することがある）凍結乾燥品、実施例１に記載した方法で取得したヒトＣＤ３３発現細胞株またはＣＤ３３陽性であるヒト急性単球性白血病細胞株ＴＨＰ－１をＰＢＳにて溶解し、免疫原として用いた。 [Example 3]
Preparation of anti-CD33 monoclonal antibody (1) Preparation of immunogen Recombinant human Siglec-3 (CD33) / Fc Chimera (catalog number 1137-SL) manufactured by R & D Systems (hereinafter sometimes referred to as recombinant human CD33-Fc) The freeze-dried product, human CD33-expressing cell line obtained by the method described in Example 1, or human acute monocytic leukemia cell line THP-1 positive for CD33 was dissolved in PBS and used as an immunogen.
（２）動物の免疫と抗体産生細胞の調製
　上記（１）で調製したリコンビナントヒトＣＤ３３－Ｆｃ　２０μｇ、水酸化アルミニウムアジュバント〔Ａｎｔｉｂｏｄｉｅｓ－Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ，Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ，ｐ９９、１９８８〕２ｍｇを百日咳ワクチン（千葉県血清研究所製）１×１０９細胞とともにＳＤラット（日本ＳＬＣ社）に投与した。投与２週間後より、該ＣＤ３３　２０μｇと百日咳ワクチン１×１０９細胞のみを１週間に１回、計３回投与した。 (2) Immunization of animals and preparation of antibody-producing cells Recombinant human CD33-Fc 20 μg prepared in (1) above, aluminum hydroxide adjuvant [Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory, p99, 1988] 2 mg pertussis vaccine It was administered to SD rats (Japan SLC) together with 1 × 10 9 cells (manufactured by Serological Research Institute, Chiba Prefecture). From 2 weeks after administration, 20 μg of CD33 and pertussis vaccine 1 × 10 9 cells alone were administered once a week for a total of 3 times.
　または、上記（１）で調製したヒトＣＤ３３発現細胞株　１×１０７細胞を百日咳ワクチン１×１０９細胞とともに１週間に１回、計４回ＳＤラット（日本ＳＬＣ社）に投与した。または、上記（１）で調製したＴＨＰ－１　１×１０７細胞を百日咳ワクチン１×１０９細胞とともに１週間に１回、計４回Ｂａｌｂ／ｃマウス（日本ＳＬＣ社）、ＢＸＳＢマウス（日本ＳＬＣ社）に投与した。 Alternatively, the human CD33-expressing cell line 1 × 10 7 cells prepared in (1) above was administered to SD rats (Japan SLC) four times in total, once per week, with pertussis vaccine 1 × 10 9 cells. Alternatively, THP-1 1 × 10 7 cells prepared in (1) above and pertussis vaccine 1 × 10 9 cells once a week for a total of 4 times, Balb / c mice (Japan SLC), BXSB mice (Japan SLC) ).
　該ラットまたは、マウスの眼底静脈より部分採血し、その血清抗体価を以下に示す酵素免疫測定法で確認し、十分な抗体価を示したラットまたはマウスから最終免疫３日後に脾臓を摘出した。脾臓をＭＥＭ（Ｍｉｎｉｍｕｍ　Ｅｓｓｅｎｔｉａｌ　Ｍｅｄｉｕｍ）培地（日水製薬社製）中で細断し、すりつぶして遠心分離（１２００　ｒｐｍ、５分間）した。 Partial blood was collected from the fundus vein of the rat or mouse, the serum antibody titer was confirmed by the enzyme immunoassay shown below, and the spleen was removed 3 days after the final immunization from the rat or mouse showing a sufficient antibody titer. The spleen was shredded in MEM (Minimum Essential Medium) medium (manufactured by Nissui Pharmaceutical), ground and centrifuged (1200 rpm, 5 minutes).
　得られた沈殿画分にトリス－塩化アンモニウム緩衝液（ｐＨ７．６）を添加し、３７℃で１分間処理することにより赤血球を除去した。得られた沈殿画分（細胞画分）をＭＥＭ培地で３回洗浄し、細胞融合に用いた。 To the resulting precipitate fraction, tris-ammonium chloride buffer (pH 7.6) was added and treated at 37 ° C. for 1 minute to remove erythrocytes. The obtained precipitate fraction (cell fraction) was washed 3 times with MEM medium and used for cell fusion.
（３）酵素免疫測定法（バインディングＥＬＩＳＡ）
　アッセイには９６ウェルのＥＬＩＳＡ用プレート（グライナー社）を用いた。リコンビナントヒトＣＤ３３－Ｆｃ、または抗ラットイムノグロブリン抗体（ＤＡＫＯ社）、または抗マウスイムノグロブリン抗体（ＤＡＫＯ社）を５０μＬ／ウェルで分注し、４℃で一晩静置して吸着させたものを用いた。該プレートをＰＢＳで洗浄後、１％牛血清アルブミン（ＢＳＡ）－ＰＢＳを１００μＬ／ウェル加え、室温で１時間静置して残っている活性基をブロックした。 (3) Enzyme immunoassay (binding ELISA)
A 96-well ELISA plate (Greiner) was used for the assay. Recombinant human CD33-Fc, anti-rat immunoglobulin antibody (DAKO) or anti-mouse immunoglobulin antibody (DAKO) was dispensed at 50 μL / well and allowed to stand overnight at 4 ° C. for adsorption. Using. The plate was washed with PBS, 1% bovine serum albumin (BSA) -PBS was added at 100 μL / well, and the plate was allowed to stand at room temperature for 1 hour to block remaining active groups.
　その後、１％　ＢＳＡ－ＰＢＳを捨て、該プレートに一次抗体として被免疫ラットまたはマウス血清を適宜希釈して５０μＬ／ウェル分注し、１時間静置した。該プレートをＴｗｅｅｎ‐ＰＢＳで洗浄後、２次抗体としてペルオキシダーゼ標識ヤギ抗ラットＩｇＧ（Ｈ＋Ｌ鎖）抗体（ＺＹＭＥＤ社製）または、ペルオキシダーゼ標識ヤギ抗マウスＩｇＧ（γ）抗体（ＫＰＬ社製）を５０μＬ／ウェル加えて１時間室温で静置した。 Thereafter, 1% BSA-PBS was discarded, and immunized rat or mouse serum was appropriately diluted as a primary antibody on the plate, dispensed at 50 μL / well, and allowed to stand for 1 hour. After washing the plate with Tween-PBS, 50 μL / peroxidase-labeled goat anti-rat IgG (H + L chain) antibody (ZYMED) or peroxidase-labeled goat anti-mouse IgG (γ) antibody (manufactured by KPL) was used as the secondary antibody. The wells were added and left at room temperature for 1 hour.
　該プレートをＴｗｅｅｎ－ＰＢＳで洗浄し、ＡＢＴＳ〔２．２－アジノビス（３‐エチルベンゾチアゾール－６－スルホン酸）アンモニウム〕基質液〔１ｍｍｏＬ／Ｌ　ＡＢＴＳ／０．１ｍｏＬ／Ｌ　クエン酸バッファー（ｐＨ４．２）、０．１％Ｈ２Ｏ２〕を５０μＬ／ウェル添加して発色させ、５％ＳＤＳ（Ｓｏｄｉｕｍ　Ｌａｕｒｙｌ　Ｓｕｌｆａｔｅ）溶液を５０μＬ／ウェル添加して反応を止め、サンプル波長４１５ｎｍ、リファレンス波長４９０ｎｍにおける吸光度（ＯＤ４１５－ＯＤ４９０）をプレートリーダー（Ｅｍａｘ　ｍｉｃｒｏｐｌａｔｅ　ｒｅａｄｅｒ、Ｍｏｌｅｃｕｌａｒ　Ｄｅｖｉｃｅｓ社製）を用いて測定した。 The plate was washed with Tween-PBS, ABTS [2.2-azinobis (3-ethylbenzothiazole-6-sulfonic acid) ammonium] substrate solution [1 mmoL / L ABTS / 0.1 moL / L citrate buffer (pH 4. 2), 0.1% H 2 O 2 ] was added at 50 μL / well for color development, and 5% SDS (Sodium Lauryl Sulfate) solution was added at 50 μL / well to stop the reaction, and the sample wavelength was 415 nm and the reference wavelength was 490 nm. Absorbance (OD415-OD490) was measured using a plate reader (Emax microplate reader, manufactured by Molecular Devices).
（４）マウス骨髄腫細胞の調製
　８－アザグアニン耐性マウス骨髄腫細胞株Ｐ３Ｘ６３Ａｇ８Ｕ．１（Ｐ３－Ｕ１：ＡＴＣＣより購入）を１０％ウシ胎児血清添加ＲＰＭＩ１６４０（インビトロジェン社製）で培養し、細胞融合時に２×１０７個以上の細胞を確保し、細胞融合の親株として供した。 (4) Preparation of mouse myeloma cells 8-Azaguanine resistant mouse myeloma cell line P3X63Ag8U. 1 (P3-U1: purchased from ATCC) was cultured in RPMI 1640 supplemented with 10% fetal bovine serum (manufactured by Invitrogen), 2 × 10 7 cells or more were secured at the time of cell fusion, and used as a parent strain for cell fusion.
（５）ハイブリドーマの作製
　上記（２）で得られたマウス脾細胞と上記（４）で得られた骨髄腫細胞とを１０：１になるよう混合し、遠心分離（１２００ｒｐｍ、５分間）した。得られた沈澱画分の細胞群をよくほぐした後、攪拌しながら、３７℃で、ポリエチレングリコール－１０００（ＰＥＧ－１０００）１ｇ、ＭＥＭ培地１ｍＬ、およびジメチルスルホキシド（ＤＭＳＯ）０．３５ｍＬの混液を１×１０８個のマウス脾細胞あたり０．５ｍＬ加え、該懸濁液に１分間毎にＭＥＭ培地１ｍＬを数回加えた後、ＭＥＭ培地を加えて全量が５０ｍＬになるようにした。 (5) Production of hybridoma The mouse spleen cells obtained in (2) above and the myeloma cells obtained in (4) above were mixed at 10: 1 and centrifuged (1200 rpm, 5 minutes). After thoroughly disaggregating the cells of the obtained precipitate fraction, a mixed solution of 1 g of polyethylene glycol-1000 (PEG-1000), 1 mL of MEM medium, and 0.35 mL of dimethyl sulfoxide (DMSO) is stirred at 37 ° C. 0.5 mL per 1 × 10 8 mouse spleen cells was added, and 1 mL of MEM medium was added several times to the suspension every minute, and then the MEM medium was added to make the total volume 50 mL.
　該懸濁液を遠心分離（９００ｒｐｍ、５分間）し、得られた沈澱画分の細胞をゆるやかにほぐした後、ＨＡＴ培地〔１０％ウシ胎児血清添加ＲＰＭＩ１６４０培地にＨＡＴ　Ｍｅｄｉａ　Ｓｕｐｐｌｅｍｅｎｔ（インビトロジェン社製）を加えた培地〕１００ｍＬ中にゆるやかに懸濁した。該懸濁液を９６ウェル培養用プレートに２００μＬ／ウェルずつ分注し、５％ＣＯ２インキュベーター中、３７℃で１０～１４日間培養した。 The suspension was centrifuged (900 rpm, 5 minutes), and the cells of the obtained precipitate fraction were loosely loosened. Medium gently added to 100 mL. The suspension was dispensed into a 96-well culture plate at 200 μL / well and cultured at 37 ° C. for 10-14 days in a 5% CO 2 incubator.
　培養後、培養上清を下記（６）に記載のＦＭＡＴ法で測定し、ヒトＣＤ３３発現細胞株には反応し、ＣＨＯ／ＤＧ４４細胞には反応しないウェルを選択した。そこに含まれる細胞から限界希釈法によるクローニングを２回繰り返し、抗ＣＤ３３モノクローナル抗体産生ハイブリドーマ株ＫＭ４０７１、ＫＭ４０７２、ＫＭ４０７３、ＫＭ４０７４、およびＫＭ４０７５を確立した（表１）。なお、例えば、「ＫＭ４０７１」と表記した場合、ハイブリドーマまたは当該ハイブリドーマが産生するモノクローナル抗体を指すものとする（他のハイブリドーマについても同様である。 After culture, the culture supernatant was measured by the FMAT method described in (6) below, and a well that reacted with the human CD33-expressing cell line but did not react with CHO / DG44 cells was selected. Cloning by limiting dilution was repeated twice from cells contained therein to establish anti-CD33 monoclonal antibody-producing hybridoma strains KM4071, KM4072, KM4073, KM4074, and KM4075 (Table 1). For example, the expression “KM4071” refers to a hybridoma or a monoclonal antibody produced by the hybridoma (the same applies to other hybridomas).
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
（６）ＦＭＡＴ法による培養上清の測定
　実施例１に記載した方法で作製したヒトＣＤ３３発現細胞株を、９６ウェルＦＭＡＴ用プレート（アプライドバイオシステム社製）に１×１０４細胞／５０μＬ／ウェルまき、３７℃の５％ＣＯ２インキュベーター内で一晩培養した。該プレートにハイブリドーマの培養上清を１０μＬ／ウェル添加し、すぐに０．０５％アジ化ナトリウム、１％ＢＳＡ－ＰＢＳで希釈したＡｌｅｘａ－６４７標識ヤギ抗ラットＩｇＧ（Ｈ＋Ｌ）抗体（インビトロジェン社製）を５０μＬ／ウェル加え、遮光して室温で３時間静置した。その後、レーザー光　６３３ｎｍ　Ｈｅ／Ｎｅで励起される６５０ｎｍ～６８５ｎｍの波長を８２００　Ｃｅｌｌｕｌａｒ　Ｄｅｔｅｃｔｉｏｎ　Ｓｙｓｔｅｍ（アプライドバイオシステム社製）で測定した。以下、この測定法をＦＭＡＴ法とする。 (6) Measurement of culture supernatant by FMAT method The human CD33-expressing cell line prepared by the method described in Example 1 was placed in a 96-well FMAT plate (Applied Biosystems) at 1 × 10 4 cells / 50 μL / well. Then, the cells were cultured overnight in a 5% CO 2 incubator at 37 ° C. 10 μL / well of hybridoma culture supernatant was added to the plate, and immediately Alexa-647-labeled goat anti-rat IgG (H + L) antibody (manufactured by Invitrogen) diluted with 0.05% sodium azide and 1% BSA-PBS. Was added at 50 μL / well and allowed to stand at room temperature for 3 hours in the dark. Thereafter, the wavelength of 650 nm to 685 nm excited by laser light 633 nm He / Ne was measured with 8200 Cellular Detection System (Applied Biosystems). Hereinafter, this measurement method is referred to as FMAT method.
［実施例４］
ヒトＣＤ３３に対するラットまたはマウス抗ＣＤ３３モノクローナル抗体の活性評価
（１）ＦＣＭにおけるヒトＣＤ３３陽性セルラインとの反応性
　１～５×１０５細胞のＴＨＰ－１に、ヒトＩｇＧ（シグマ社製）を１ｍｇ／ｍＬ加えブロッキングをし、ＫＭ４０７１～ＫＭ４０７５およびＰ６７．６［Ｂｉｏｃｏｍｊｕｇａｔｅ　Ｃｈｅｍ，１３，４７（２００２）］をＦＣＭバッファーで適宜希釈し、全量を１００μＬとした。これらの細胞懸濁液を氷上で６０分間反応させた後、ＰＢＳで２回洗浄を行った。 [Example 4]
Activity evaluation of rat or mouse anti-CD33 monoclonal antibody against human CD33 (1) Reactivity with human CD33-positive cell line in FCM 1-5 × 10 5 cells of THP-1 with human IgG (Sigma) KM4071 to KM4075 and P67.6 [Biocomjugate Chem, 13, 47 (2002)] were appropriately diluted with FCM buffer to make a total volume of 100 μL. These cell suspensions were reacted for 60 minutes on ice and then washed twice with PBS.
　前記細胞に、ＦＣＭバッファーで希釈調製したＡｌｌｅｘａ－４８８標識ヤギ抗ラットＩｇＧ（Ｈ＋Ｌ）抗体（インビトロジェン社製）またはＡｌｌｅｘａ－４８８標識ヤギ抗マウスＩｇＧ（Ｈ＋Ｌ）を３０μＬ添加し、氷上で４０分間反応させた。ＰＢＳで３回洗浄を行った後、ＦＣＭバッファーに懸濁してフローサイトメーター（ベックマン・コールター社製）用いて、蛍光強度を測定した。 30 μL of Allexa-488-labeled goat anti-rat IgG (H + L) antibody (manufactured by Invitrogen) or Allexa-488-labeled goat anti-mouse IgG (H + L) diluted with FCM buffer was added to the cells and allowed to react on ice for 40 minutes. It was. After washing 3 times with PBS, it was suspended in FCM buffer and the fluorescence intensity was measured using a flow cytometer (Beckman Coulter).
　図３に、実施例３で得られた抗ＣＤ３３モノクローナル抗体を３μｇ／ｍＬから４倍希釈で段階的に希釈したものを反応させた場合の平均蛍光強度（以下、ＭＦＩと表記することがある）を示す。図３に示すように、ＫＭ４０７１～ＫＭ４０７５およびｐ６７．６はいずれも濃度依存的にＣＤ３３陽性細胞株へ結合することが確認された。 FIG. 3 shows the average fluorescence intensity when the anti-CD33 monoclonal antibody obtained in Example 3 was reacted stepwise diluted from 3 μg / mL at a 4-fold dilution (hereinafter sometimes referred to as MFI). Indicates. As shown in FIG. 3, it was confirmed that KM4071 to KM4075 and p67.6 all bind to the CD33 positive cell line in a concentration-dependent manner.
（２）ＥＬＩＳＡにおけるリコンビナントヒトＣＤ３３－Ｆｃとの結合活性
　実施例３（３）に記載の方法に従って行った。図４に、実施例３で得られたＫＭ４０７１～ＫＭ４０７５、およびＰ６７．６を、２μｇ／ｍＬから５倍希釈で段階的に希釈したものを１次抗体に用いた結果を示す。 (2) Binding activity with recombinant human CD33-Fc in ELISA This was performed according to the method described in Example 3 (3). FIG. 4 shows the results of using KM4071 to KM4075 and P67.6 obtained in Example 3 stepwise diluted from 2 μg / mL at a 5-fold dilution as the primary antibody.
（３）ＢｉａｃｏｒｅにおけるリコンビナントヒトＣＤ３３－Ｆｃとの結合活性
　ＫＭ４０７１～ＫＭ４０７５およびＰ６７．６のリコンビナントヒトＣＤ３３－Ｆｃに対する結合活性を反応速度論的に解析するため、表面プラズモン共鳴法（ＳＰＲ法）を用いて結合活性測定を行った。以下の操作は全てＢｉａｃｏｒｅ　Ｔ－１００（ＧＥヘルスケアバイオサイエンス社製）を用いて行った。 (3) Binding activity to recombinant human CD33-Fc in Biacore In order to analyze the binding activity of KM4071 to KM4075 and P67.6 to recombinant human CD33-Fc, the surface plasmon resonance method (SPR method) was used. The binding activity was measured. The following operations were all performed using Biacore T-100 (manufactured by GE Healthcare Bioscience).
　Ｍｏｕｓｅ　Ａｎｔｉｂｏｄｙ　Ｃａｐｔｕｒｅ　Ｋｉｔ（ＧＥヘルスケアバイオサイエンス社製）を用い、添付のプロトコールに従い、抗マウスＩｇＧ抗体をアミンカップリング法によりＣＭ５センサーチップ（ＧＥヘルスケアバイオサイエンス社製）に固層化した。抗マウスＩｇＧ抗体を固層化したチップに測定サンプル（ＫＭ４０７１～ＫＭ４０７５およびＰ６７．６を添加し、約２００ＲＵ（レゾナンス　ユニット）になるようにキャプチャーさせた。 Using Mouse Antibody Capture Kit (manufactured by GE Healthcare Bioscience), the anti-mouse IgG antibody was solidified on a CM5 sensor chip (manufactured by GE Healthcare Bioscience) by the amine coupling method according to the attached protocol. Measurement samples (KM4071 to KM4075 and P67.6 were added to a chip on which an anti-mouse IgG antibody was solidified, and the sample was captured so as to be about 200 RU (resonance unit).
　その後１０μｇ／ｍＬから２倍希釈で５段階に希釈したリコンビナントヒトＣＤ３３－Ｆｃを３０μＬ／ｍｉｎの速度でチップ上に流し、各濃度におけるセンサーグラムを取得した。装置添付の解析ソフトを用い、１：１バインディングモデルを用いて解析し、各抗体のヒトＣＤ３３に対する結合速度定数ｋａ及び解離速度定数ｋｄを算出した。 Thereafter, recombinant human CD33-Fc diluted in 10 stages from 10 μg / mL to 2 stages was flowed on the chip at a rate of 30 μL / min, and sensorgrams at each concentration were obtained. Using the analysis software attached to the apparatus, analysis was performed using a 1: 1 binding model, and the binding rate constant ka and the dissociation rate constant kd of each antibody for human CD33 were calculated.
　その結果得られた各抗体のｋａ、ｋｄおよび解離定数ＫＤを表２に示す。 The resulting ka of each antibody, indicating a kd and dissociation constant, K D, in Table 2.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　表２に示すように、ＫＭ４０７１、４０７３、４０７４および４０７５はＰ６７．６よりもＣＤ３３－Ｆｃに高いアフィニティーを示した。 As shown in Table 2, KM4071, 4073, 4074, and 4075 showed higher affinity for CD33-Fc than P67.6.
［実施例５］
抗ＣＤ３３モノクローナル抗体の可変領域をコードするｃＤＮＡの単離、解析
（１）抗ＣＤ３３モノクローナル抗体産生ハイブリドーマ細胞からのｍＲＮＡの調製
　実施例３に記載のＫＭ４０７１～ＫＭ４０７５より、ＲＮＡｅａｓｙ　Ｍｉｎｉ　ｋｉｔ（ＱＩＡＧＥＮ社製）およびＯｌｉｇｏｔｅｘＴＭ－ｄＴ３０＜Ｓｕｐｅｒ＞ｍＲＮＡ　Ｐｕｒｉｆｉｃａｔｉｏｎ　Ｋｉｔ（ＴａＫａＲａ社製）を用いて、添付の使用説明書に従い、ハイブリドーマ５×１０７～１×１０８細胞よりｍＲＮＡを調製した。 [Example 5]
Isolation and Analysis of cDNA Encoding Variable Region of Anti-CD33 Monoclonal Antibody (1) Preparation of mRNA from Anti-CD33 Monoclonal Antibody-Producing Hybridoma Cells From KM4071 to KM4075 described in Example 3, RNAeasy Mini kit (QIAGEN) Using Oligotex ™ -dT30 <Super> mRNA Purification Kit (TaKaRa), mRNA was prepared from hybridoma 5 × 10 7 to 1 × 10 8 cells according to the attached instruction manual.
（２）抗ＣＤ３３モノクローナル抗体のＨ鎖およびＬ鎖可変領域の遺伝子クローニング
　上記（１）で取得したモノクローナル抗体のｍＲＮＡ　０．５μｇから、ＳＭＡＲＴ　ＲＡＣＥ　ｃＤＮＡ　Ａｍｐｌｉｆｉｃａｔｉｏｎ　Ｋｉｔ（Ｃｌｏｎｔｅｃｈ社製）を用いて、添付の使用説明書に従ってｃＤＮＡを取得した。 (2) Gene cloning of H chain and L chain variable regions of anti-CD33 monoclonal antibody From 0.5 μg of the monoclonal antibody mRNA obtained in (1) above, using SMART RACE cDNA Amplification Kit (Clontech), the attached CDNA was obtained according to the instructions for use.
　そのｃＤＮＡを鋳型として、キット添付のユニバーサルプライマーＡ　ｍｉｘと、ラットＩｇＧ１に特異的なプライマー（配列番号８）、またはラットＩｇＧ２ａに特異的なプライマー（配列番号９）、またはマウスＩｇＧ１に特異的なプライマー（配列番号１０）、またはマウスＩｇＧ２ａに特異的なプライマー（配列番号１１）を用いてＰＣＲ反応を行い、各抗体の重鎖可変部（以下、ＶＨと表記することがある）のｃＤＮＡ断片を増幅した。 Using the cDNA as a template, a universal primer A mix attached to the kit, a primer specific to rat IgG1 (SEQ ID NO: 8), a primer specific to rat IgG2a (SEQ ID NO: 9), or a primer specific to mouse IgG1 (SEQ ID NO: 10) or PCR reaction using a primer specific to mouse IgG2a (SEQ ID NO: 11) to amplify the cDNA fragment of each antibody heavy chain variable region (hereinafter sometimes referred to as VH) did.
　また抗体の各サブクラス特異的プライマーの代わりにラットＩｇ（κ）特異的プライマー（配列番号１２）または、マウスＩｇ（κ）特異的なプライマー（配列番号１３）を用いてＰＣＲを行い、各抗体の軽鎖可変部（以下、ＶＬと表記することがある）のｃＤＮＡ断片を増幅した。ＰＣＲは、９４℃で５分間加熱後、９４℃で１５秒間、７２℃で３分間からなる反応サイクルを５回、９４℃で１５秒間、７０℃で３０秒間、７２℃で３分間からなる反応サイクルを５回、９４℃で１５秒間、６８℃で３０秒間、７２℃で３分間からなる反応サイクルを３０回それぞれ行った後、７２℃で１０分間反応させた。ＰＣＲはＰＴＣ－２００　ＤＮＡ　Ｅｎｇｉｎｅ（ＢｉｏＲａｄ社製）を用いて行った。 PCR was performed using a rat Ig (κ) -specific primer (SEQ ID NO: 12) or a mouse Ig (κ) -specific primer (SEQ ID NO: 13) instead of each antibody subclass-specific primer. A cDNA fragment of the light chain variable region (hereinafter sometimes referred to as VL) was amplified. PCR is a reaction consisting of 5 reaction cycles consisting of 94 ° C for 15 seconds and 72 ° C for 3 minutes after heating at 94 ° C for 5 minutes, 94 ° C for 15 seconds, 70 ° C for 30 seconds, and 72 ° C for 3 minutes. The reaction was repeated 30 times, consisting of 5 cycles, 94 ° C for 15 seconds, 68 ° C for 30 seconds, 72 ° C for 3 minutes, and then reacted at 72 ° C for 10 minutes. PCR was performed using PTC-200 DNA Engine (manufactured by BioRad).
　得られたＰＣＲ産物は、クローニングして塩基配列を決定するため、アガロースゲル電気泳動で分離し、Ｈ鎖、Ｌ鎖をＧｅｌ　Ｅｘｔｒａｃｔｉｏｎ　Ｋｉｔ（ＱＩＡＧＥＮ社製）を用いて抽出した。得られた抽出断片にＴａｒｇｅｔ　Ｃｌｏｎｅ　Ｐｌｕｓ（ＴＯＹＯＢＯ社製）を用い、添付の説明書に従ってｄＡ付加反応後、ｐＴＡ２ベクター（ＴＯＹＯＢＯ社製）に各抗体のＶＨまたはＶＬを組み込んだ。 The obtained PCR products were separated by agarose gel electrophoresis in order to clone and determine the base sequence, and the H chain and L chain were extracted using Gel Extraction Kit (manufactured by QIAGEN). Target Clone Plus (manufactured by TOYOBO) was used for the extracted fragment, and after the dA addition reaction according to the attached instruction, VH or VL of each antibody was incorporated into the pTA2 vector (manufactured by TOYOBO).
　こうして得られたベクターを用いて、Ｃｏｍｐｅｔｅｎｔ　Ｑｕｉｃｋ（ＴＯＹＯＢＯ社製）の付属の取扱説明書に従って大腸菌ＤＨ５α株を形質転換した。得られた形質転換体より自動プラスミド抽出機（クラボウ社製）を用いてプラスミドを抽出し、ＢｉｇＤｙｅ　Ｔｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　ＦＳ　Ｒｅａｄｙ　Ｒｅａｃｔｉｏｎ　Ｋｉｔ（ＰＥバイオシステムズ社製）を用い添付の説明書に従って反応後、同社のシーケンサーＡＢＩ　ＰＲＩＳＭ３７００によりクローニングしたＰＣＲ産物の塩基配列を解析した。 Using the vector thus obtained, Escherichia coli DH5α strain was transformed according to the instruction manual attached to Competent Quick (manufactured by TOYOBO). A plasmid was extracted from the obtained transformant using an automatic plasmid extractor (manufactured by Kurabo Industries), and reacted according to the attached instructions using BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (manufactured by PE Biosystems). The base sequence of the PCR product cloned by the sequencer ABI PRISM 3700 was analyzed.
　その結果、ｃＤＮＡの５’末端に開始コドンと推定されるＡＴＧ配列が存在する、完全長のＨ鎖ｃＤＮＡを含むプラスミドおよびＬ鎖ｃＤＮＡを含むプラスミドが取得された。 As a result, a plasmid containing a full-length H chain cDNA and a plasmid containing an L chain cDNA in which an ATG sequence presumed to be the initiation codon was present at the 5 'end of the cDNA were obtained.
（３）抗ＣＤ３３モノクローナル抗体Ｖ領域の遺伝子配列の解析
　上記（２）で得られたプラスミドに含まれていた、ハイブリドーマＫＭ４０７１～ＫＭ４０７５のＶＨの全塩基配列を配列番号１４～１８に、該配列から推定された、シグナル配列を含んだＶＨの全アミノ酸配列を配列番号１９～２３に、ＶＬの全塩基配列を配列番号２４～２８におよび該配列から推定された、シグナル配列を含んだＶＬの全アミノ酸配列を配列番号２９～３３にそれぞれ示す。 (3) Analysis of gene sequence of V region of anti-CD33 monoclonal antibody The entire nucleotide sequence of VH of hybridomas KM4071 to KM4075 contained in the plasmid obtained in (2) above is represented by SEQ ID NOs: 14 to 18, The estimated entire amino acid sequence of VH including the signal sequence is represented by SEQ ID NOs: 19 to 23, the entire nucleotide sequence of VL is represented by SEQ ID NOs: 24-28, and the entire VL including the signal sequence is estimated from the sequences. Amino acid sequences are shown in SEQ ID NOs: 29 to 33, respectively.
　また、配列番号１４～１８に示される塩基配列からシグナル配列を除いた塩基配列を配列番号１２２～１２６に、配列番号２４～２８に示される塩基配列からシグナル配列を除いた塩基配列を配列番号１２７～１３１に、配列番号１９～２３に示されるアミノ酸配列からシグナル配列を除いたアミノ酸配列を配列番号１３２～１３６に、配列番号２９～３３に示されるアミノ酸配列からシグナル配列を除いたアミノ酸配列を配列番号１３７～１４１にそれぞれ示す。 Further, the base sequence obtained by removing the signal sequence from the base sequence shown in SEQ ID NOs: 14 to 18 is shown in SEQ ID NO: 122-126, and the base sequence obtained by removing the signal sequence from the base sequences shown in SEQ ID NOs: 24-28 is shown in SEQ ID NO: 127 -131, the amino acid sequence obtained by removing the signal sequence from the amino acid sequence shown in SEQ ID NOs: 19-23, and the amino acid sequence obtained by removing the signal sequence from the amino acid sequences shown in SEQ ID NOs: 29-33, The numbers 137 to 141 are shown respectively.
　既知のラットおよびマウス抗体の配列データ［ＳＥＱＵＥＮＣＥＳ　ｏｆ　Ｐｒｏｔｅｉｎｓ　ｏｆ　Ｉｍｍｕｎｏｌｏｇｉｃａｌ　Ｉｎｔｅｒｅｓｔ、ＵＳ　Ｄｅｐｔ．Ｈｅａｌｔｈ　ａｎｄ　Ｈｕｍａｎ　Ｓｅｒｖｉｃｅｓ（１９９１）］との比較から、単離した各々のｃＤＮＡは分泌シグナル配列を含むＫＭ４０７１～ＫＭ４０７５をコードする完全長ｃＤＮＡであり、ＫＭ４０７１のＨ鎖については配列番号１９に示されるアミノ酸配列の１から１９番目のアミノ酸配列が、ＫＭ４０７１のＬ鎖については配列番号２９に示されるアミノ酸配列の１から１９番目のアミノ酸配列が分泌シグナル配列であり、ＫＭ４０７２のＨ鎖については配列番号２０に示されるアミノ酸配列の１から１８番目のアミノ酸配列が、ＫＭ４０７２のＬ鎖については配列番号３０に示されるアミノ酸配列の１から２２番目のアミノ酸配列が分泌シグナル配列であり、ＫＭ４０７３のＨ鎖については配列番号２１に示されるアミノ酸配列の１から１８番目のアミノ酸配列が、ＫＭ４０７３のＬ鎖については配列番号３１に示されるアミノ酸配列の１から２０番目のアミノ酸配列が分泌シグナル配列であり、ＫＭ４０７４のＨ鎖については配列番号２２に示されるアミノ酸配列の１から２０番目のアミノ酸配列が、ＫＭ４０７４のＬ鎖については配列番号３２に示されるアミノ酸配列の１から１９番目のアミノ酸配列が分泌シグナル配列であり、ＫＭ４０７５のＨ鎖については配列番号２３に示されるアミノ酸配列の１から２０番目のアミノ酸配列が、ＫＭ４０７５のＬ鎖については配列番号３３に示されるアミノ酸配列の１から１９番目のアミノ酸配列が分泌シグナル配列であることが明らかとなった。 Sequence data of known rat and mouse antibodies [SEQUENCES of Proteins of Immunological Interest, US Dept. According to comparison with Health and Human Services (1991)], each isolated cDNA is a full-length cDNA encoding KM4071 to KM4075 including a secretory signal sequence, and the KM4071 heavy chain is represented by SEQ ID NO: 19. The 1st to 19th amino acid sequence of the sequence is the secretion signal sequence for the KM4071 L chain, the 1st to 19th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 29, and the KM4072 H chain is represented by SEQ ID NO: 20 The amino acid sequence from 1 to 18 of the amino acid sequence shown is the secretory signal sequence for the L chain of KM4072 and the amino acid sequence of 1 to 22 of the amino acid sequence shown in SEQ ID NO: 30 is the sequence for the H chain of KM4073 Ami shown in number 21 The 1st to 18th amino acid sequence of the acid sequence is the secretory signal sequence for the L chain of KM4073, the 1st to 20th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 31, and the SEQ ID NO: 22 for the H chain of KM4074 The amino acid sequence from 1 to 20 of the amino acid sequence shown in Fig. 4 is the secretory signal sequence for the KM4074 L chain, the amino acid sequence from 1 to 19 of the amino acid sequence shown in SEQ ID NO: 32, and the KM4075 H chain is It is clear that the 1st to 20th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 23 is the secretory signal sequence, and the 1st to 19th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 33 for the KM4075 L chain is the secretory signal sequence. became.
　各モノクローナル抗体のＶＨおよびＶＬのＣＤＲを、既知の抗体のアミノ酸配列と比較することにより同定した。 The CDRs of VH and VL of each monoclonal antibody were identified by comparing with the amino acid sequences of known antibodies.
　ＫＭ４０７１のＶＨのＣＤＲ１、ＣＤＲ２およびＣＤＲ３のアミノ酸配列を配列番号３４、３５および３６に、ＶＬのＣＤＲ１、ＣＤＲ２およびＣＤＲ３のアミノ酸配列を配列番号３７、３８および３９にそれぞれ示す。 The amino acid sequences of CDR1, CDR2, and CDR3 of VH of KM4071 are shown in SEQ ID NOs: 34, 35, and 36, and the amino acid sequences of CDR1, CDR2, and CDR3 of VL are shown in SEQ ID NOs: 37, 38, and 39, respectively.
　ＫＭ４０７２のＶＨのＣＤＲ１、ＣＤＲ２およびＣＤＲ３のアミノ酸配列を配列番号４０、４１および４２に、ＶＬのＣＤＲ１、ＣＤＲ２およびＣＤＲ３のアミノ酸配列を配列番号４３、４４および４５にそれぞれ示す。 The amino acid sequences of CDR1, CDR2 and CDR3 of VH of KM4072 are shown in SEQ ID NOs: 40, 41 and 42, and the amino acid sequences of CDR1, CDR2 and CDR3 of VL are shown in SEQ ID NOs: 43, 44 and 45, respectively.
　ＫＭ４０７３のＶＨのＣＤＲ１、ＣＤＲ２およびＣＤＲ３のアミノ酸配列を配列番号４６、４７および４８に、ＶＬのＣＤＲ１、ＣＤＲ２およびＣＤＲ３のアミノ酸配列を配列番号４９、５０および５１にそれぞれ示す。 The amino acid sequences of CDR1, CDR2 and CDR3 of VH of KM4073 are shown in SEQ ID NOs: 46, 47 and 48, and the amino acid sequences of CDR1, CDR2 and CDR3 of VL are shown in SEQ ID NOs: 49, 50 and 51, respectively.
　ＫＭ４０７４のＶＨのＣＤＲ１、ＣＤＲ２およびＣＤＲ３のアミノ酸配列を配列番号５２、５３および５４に、ＶＬのＣＤＲ１、ＣＤＲ２およびＣＤＲ３のアミノ酸配列を配列番号５５、５６および５７にそれぞれ示す。 The amino acid sequences of CDR1, CDR2, and CDR3 of VH of KM4074 are shown in SEQ ID NOs: 52, 53, and 54, and the amino acid sequences of CDR1, CDR2, and CDR3 of VL are shown in SEQ ID NOs: 55, 56, and 57, respectively.
　ＫＭ４０７５のＶＨのＣＤＲ１、ＣＤＲ２およびＣＤＲ３のアミノ酸配列を配列番号５８、５９および６０に、ＶＬのＣＤＲ１、ＣＤＲ２およびＣＤＲ３のアミノ酸配列を配列番号６１、６２および６３にそれぞれ示す。 The amino acid sequences of CDR1, CDR2 and CDR3 of VH of KM4075 are shown in SEQ ID NOs: 58, 59 and 60, and the amino acid sequences of CDR1, CDR2 and CDR3 of VL are shown in SEQ ID NOs: 61, 62 and 63, respectively.
［実施例６］
抗ＣＤ３３キメラ抗体の作製
　ＥＬＩＳＡ、ＦＣＭなどの免疫染色を用いた評価方法において、由来する種の異なる抗体、または同じ種であっても抗体のサブクラスが異なる抗体間の活性は検出抗体の反応性が異なるために比較することが出来ない。そこで、実施例３において確立したＫＭ４０７１～ＫＭ４０７５の定常領域をヒト定常領域配列に置換したキメラ抗体を以下の方法に従い作製した。このように、定常領域をヒト由来の配列に揃えることにより、定常領域に依存する各種活性を測定、比較することも可能となる。 [Example 6]
Preparation of anti-CD33 chimeric antibody In an evaluation method using immunostaining such as ELISA, FCM, etc., the activity between antibodies of different species, or antibodies of different subclasses of the same species, is the reactivity of the detection antibody. It cannot be compared because it is different. Therefore, a chimeric antibody in which the constant regions KM4071 to KM4075 established in Example 3 were replaced with human constant region sequences was prepared according to the following method. Thus, by aligning the constant region with human-derived sequences, various activities depending on the constant region can be measured and compared.
（１）抗ＣＤ３３キメラ抗体発現ベクターの構築
　今回作製するキメラ抗体とは、ヒトＩｇＧ１とヒトＩｇＧ３の混合配列からなる強い補体依存性細胞傷害活性（以下、ＣＤＣ活性と表記することがある）を含む定常領域（以下、当該定常領域を含む抗体を１１３Ｆ型抗体と表記することがある）と、実施例５（２）で得られたモノクローナル抗体の可変領域を含むキメラ抗体である。 (1) Construction of anti-CD33 chimeric antibody expression vector The chimeric antibody produced this time is a strong complement-dependent cytotoxic activity consisting of a mixed sequence of human IgG1 and human IgG3 (hereinafter sometimes referred to as CDC activity). A chimeric antibody comprising a constant region containing (hereinafter, an antibody containing the constant region may be referred to as a 113F antibody) and the variable region of the monoclonal antibody obtained in Example 5 (2).
　方法としては、抗ＣＤ２０抗体の可変部が入った高ＣＤＣ型キメラ抗体発現ベクターＰＫＴＸ９３／１１３Ｆ（米国特許出願公開第２００７／０１４８１６５号明細書に記載）と、実施例５（２）で得られた各モノクローナル抗体のＶＨまたはＶＬをｐＴＡ２ベクターに組み込んだプラスミドを用いて、抗ＣＤ３３キメラ抗体発現ベクターを以下のようにして構築した。 As a method, high CDC type chimeric antibody expression vector PKTX93 / 113F (described in US Patent Application Publication No. 2007/0148165) containing the variable part of anti-CD20 antibody and obtained in Example 5 (2) were used. Using a plasmid in which the VH or VL of each monoclonal antibody was incorporated into the pTA2 vector, an anti-CD33 chimeric antibody expression vector was constructed as follows.
　各モノクローナル抗体のＶＨまたはＶＬを実施例５（２）において作成したＶＨまたはＶＨを組み込んだｐＴＡ２ベクターを鋳型として１００ｎｇ使用し、１０×ＫＯＤ　Ｐｌｕｓ緩衝液５μＬ、２ｍｍｏｌ／Ｌ　ｄＮＴＰ　５μＬ、２５ｍｍｏｌ／Ｌの塩化マグネシウムを２μＬ、ＤＭＳＯを２．５μＬ、ＫＯＤ　ｐｌｕｓ　ｐｏｌｙｍｅｒａｓｅ（ＴＯＹＯＢＯ社製）を１μＬ、各モノクローナル抗体のＶＨおよびＶＬに特異的な１０μｍｏｌ／Ｌのプライマーそれぞれ１μＬを含む総量５０μＬからなる溶液を、９４℃で２分間加熱後、９４℃で１５秒間、６０．６℃で３０秒間、６８℃で１分間の反応を３０サイクル行った。 Using 100 ng of pTA2 vector incorporating VH or VH prepared in Example 5 (2) as a template for VH or VL of each monoclonal antibody, 5 × L of 10 × KOD Plus buffer, 2 μL of dNTP 5 μL, 25 mmol / L A solution consisting of 2 μL of magnesium chloride, 2.5 μL of DMSO, 1 μL of KOD plus polymerase (manufactured by TOYOBO), and a total volume of 50 μL containing 1 μL of each 10 μmol / L primer specific for VH and VL of each monoclonal antibody, 94 After heating at ° C for 2 minutes, the reaction was carried out for 30 cycles of 94 ° C for 15 seconds, 60.6 ° C for 30 seconds, and 68 ° C for 1 minute.
　ＫＭ４０７１のＶＨのプライマーの塩基配列は配列番号６４および６５、ＶＬのプライマーの塩基配列は配列番号６６および６７、ＫＭ４０７２のＶＨのプライマーの塩基配列は配列番号６８および６９、ＶＬのプライマーの塩基配列は配列番号７０および７１、ＫＭ４０７３のＶＨのプライマーの塩基配列は配列番号７２および７３、ＶＬのプライマーの塩基配列は配列番号７４および７５、ＫＭ４０７４のＶＨのプライマーの塩基配列は配列番号７６および７７、ＶＬのプライマーの塩基配列は配列番号７８および７９、ＫＭ４０７５のＶＨのプライマーの塩基配列は配列番号８０および８１、ＶＬのプライマーの塩基配列は配列番号８２および８３に示す。 The base sequence of the KM4071 VH primer is SEQ ID NOs: 64 and 65, the base sequence of the VL primer is SEQ ID NOs: 66 and 67, the base sequence of the KM4072 VH primer is SEQ ID NOs: 68 and 69, and the base sequence of the VL primer is SEQ ID NO: 70 and 71, KM4073 VH primer base sequence is SEQ ID NO: 72 and 73, VL primer base sequence SEQ ID NO: 74 and 75, KM4074 VH primer base sequence is SEQ ID NO: 76 and 77, VL SEQ ID NOs: 78 and 79 are shown in SEQ ID NOs: 78 and 79, SEQ ID NOs: 80 and 81 are shown in the sequence of VH primers in KM 4075, and SEQ ID NOS: 82 and 83 are used in the base sequences of VL primers.
　この反応により、ベクターｐＫＡＮＴＥＸ９３（国際公開第９７／１０３５４号に記載）に挿入するための制限酵素認識配列が付加された各抗体のＶＨまたはＶＬをコードする遺伝子配列を合成した。それぞれのＰＣＲ反応産物をＰＣＲ　Ｐｕｒｉｆｉｃａｔｉｏｎ　Ｋｉｔ（ＱＩＡＧＥＮ社製）を用いて精製した。 By this reaction, a gene sequence encoding VH or VL of each antibody to which a restriction enzyme recognition sequence for insertion into the vector pKANTEX93 (described in WO 97/10354) was added was synthesized. Each PCR reaction product was purified using PCR Purification Kit (manufactured by QIAGEN).
　こうして得られた各抗体のＶＨにＡｐａＩ（Ｎｅｗ　Ｅｎｇｌａｎｄ　Ｂｉｏｌａｂｓ社製）およびＮｏｔＩ（Ｎｅｗ　Ｅｎｇｌａｎｄ　Ｂｉｏｌａｂｓ社製）で制限酵素処理を行い、ＶＨのＮｏｔＩ－ＡｐａＩ断片を取得した。 The VH of each antibody thus obtained was subjected to restriction enzyme treatment with ApaI (manufactured by New England Biolabs) and NotI (manufactured by New England Biolabs) to obtain a NotI-ApaI fragment of VH.
　また、各抗体のＶＬにＢｓｉＷＩ（Ｎｅｗ　Ｅｎｇｌａｎｄ　Ｂｉｏｌａｂｓ社製）およびＥｃｏＲＩ（Ｎｅｗ　Ｅｎｇｌａｎｄ　Ｂｉｏｌａｂｓ社製）で制限酵素処理を行い、ＶＬのＥｃｏＲＩ－ＢｓｉＷＩ断片を取得した。ベクターＰＫＴＸ９３／１１３Ｆも、同様にＮｏｔＩおよびＡｐａＩ、またはＥｃｏＲＩおよびＢｓｉＷＩで制限酵素処理をした。 Further, the VL of each antibody was subjected to restriction enzyme treatment with BsiWI (manufactured by New England Biolabs) and EcoRI (manufactured by New England Biolabs) to obtain an EcoRI-BsiWI fragment of VL. Similarly, the vector PKTX93 / 113F was subjected to restriction enzyme treatment with NotI and ApaI, or EcoRI and BsiWI.
　得られた各モノクローナル抗体のＶＨまたはＶＬ断片を、同様に制限酵素処理したベクターとＬｉｇａｔｉｏｎ　ｈｉｇｈ（ＴＯＹＯＢＯ社製）を用いて添付の説明書に従って連結し、得られた組換えプラスミドＤＮＡ溶液を用いて大腸菌ＤＨ５α株（ＴＯＹＯＢＯ社製）を形質転換した。形質転換株のクローンより各プラスミドＤＮＡを調製して制限酵素処理により確認し、目的のＶＨのＡｐａＩ－ＮｏｔＩ断片またはＶＬのＥｃｏＲＩ－ＢＳＩＷＩ断片が挿入されたプラスミドを取得した。 The obtained VH or VL fragment of each monoclonal antibody was ligated with a restriction enzyme-treated vector and Ligation high (manufactured by TOYOBO) according to the attached instructions, and the resulting recombinant plasmid DNA solution was used. E. coli DH5α strain (manufactured by TOYOBO) was transformed. Each plasmid DNA was prepared from the clone of the transformant and confirmed by restriction enzyme treatment to obtain a plasmid into which the ApaI-NotI fragment of the target VH or the EcoRI-BSIWI fragment of VL was inserted.
　該プラスミドに関して、ＢｉｇＤｙｅ　Ｔｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　ＦＳ　Ｒｅａｄｙ　Ｒｅａｃｔｉｏｎ　Ｋｉｔ（ＰＥバイオシステムズ社製）を用いて、添付の説明書に従って反応後、同社のシーケンサーＡＢＩ　ＰＲＩＳＭ３７００により塩基配列を解析した。 The plasmid was reacted using the BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (manufactured by PE Biosystems) according to the attached instructions, and the base sequence was analyzed by the sequencer ABI PRISM 3700 of the company.
　その結果、ＫＭ４０７１～ＫＭ４０７５のＶＨをコードするｃＤＮＡがクローニングされたベクターと、ＫＭ４０７１～ＫＭ４０７５のＶＬをコードするｃＤＮＡがクローニングされたベクターを取得した。こうして得られた、各抗体のＶＨまたはＶＬが組み込まれたＰＫＴＸ９３／１１３ＦベクターにＥｃｏＲＩおよびＮｏｔＩを加えて制限酵素処理を行い、ＶＬのＥｃｏＲＩ－ＮｏｔＩ断片と、ＶＨが組み込まれたＰＫＴＸ９３／１１３ＦベクターのＮｏｔＩ－ＥｃｏＲＩ断片を取得した。 As a result, a vector in which a cDNA encoding KM4071 to KM4075 VH was cloned and a vector in which a cDNA encoding KM4071 to KM4075 VL was cloned were obtained. The thus obtained PKTX93 / 113F vector incorporating VH or VL of each antibody was subjected to restriction enzyme treatment by adding EcoRI and NotI, and the EcoRI-NotI fragment of VL and the PKTX93 / 113F vector containing VH were incorporated. A NotI-EcoRI fragment was obtained.
　これら２種類の断片をＬｉｇａｔｉｏｎ　ｈｉｇｈ（ＴＯＹＯＢＯ社製）を用いて連結し、得られた組換えプラスミドＤＮＡ溶液を用いて大腸菌ＤＨ５α株（ＴＯＹＯＢＯ社製）を形質転換した。形質転換株のクローンより各プラスミドＤＮＡを調製して制限酵素処理により確認し、目的のＶＬのＥｃｏＲＩ－ＮｏｔＩ断片が挿入されたプラスミドを取得した。 These two kinds of fragments were ligated using Ligation high (manufactured by TOYOBO), and Escherichia coli DH5α strain (manufactured by TOYOBO) was transformed with the obtained recombinant plasmid DNA solution. Each plasmid DNA was prepared from the clone of the transformant and confirmed by restriction enzyme treatment to obtain a plasmid into which the EcoRI-NotI fragment of the target VL was inserted.
　該プラスミドに関して、ＢｉｇＤｙｅ　Ｔｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　ＦＳ　Ｒｅａｄｙ　Ｒｅａｃｔｉｏｎ　Ｋｉｔ（ＰＥバイオシステムズ社製）を用いて、添付の説明書に従って反応後、同社のシーケンサーＡＢＩ　ＰＲＩＳＭ３７００により塩基配列を解析した。その結果、ＫＭ４０７１～ＫＭ４０７５のＶＨおよびＶＬをコードするｃＤＮＡがクローニングされた、抗ＣＤ３３キメラ抗体発現ベクターを取得した。ベクター構築の概略図を図５に示す。 The plasmid was reacted using the BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (manufactured by PE Biosystems) according to the attached instructions, and the base sequence was analyzed by the sequencer ABI PRISM 3700 of the company. As a result, an anti-CD33 chimeric antibody expression vector in which cDNAs encoding VH and VL of KM4071 to KM4075 were cloned was obtained. A schematic diagram of vector construction is shown in FIG.
（２）抗ＣＤ３３キメラ抗体の動物細胞での発現
　上記（１）で得られた抗ＣＤ３３キメラ抗体発現ベクターを用いて抗ＣＤ３３キメラ抗体の動物細胞での発現を、常法［Ａｎｔｉｂｏｄｙ　Ｅｎｇｉｎｅｅｒｉｎｇ，Ａ　Ｐｒａｃｔｉｃａｌ　Ｇｕｉｄｅ，Ｗ．Ｈ．Ｆｒｅｅｍａｎ　ａｎｄ　Ｃｏｍｐａｎｙ（１９９２）］により行い、抗ＣＤ３３キメラ抗体を産生する形質転換株ＫＭ４０５１～ＫＭ４０５５を取得した（表３）。 (2) Expression of anti-CD33 chimeric antibody in animal cells Using the anti-CD33 chimeric antibody expression vector obtained in (1) above, the expression of anti-CD33 chimeric antibody in animal cells was determined using conventional methods [Antibody Engineering, A Practical. Guide, W.M. H. Freeman and Company (1992)], transformants KM4051 to KM4055 producing anti-CD33 chimeric antibodies were obtained (Table 3).
　発現させる動物細胞株としてはα１，６－フコシルトランスフェラーゼ（ＦＵＴ８）の遺伝子をダブルノックアウトしたＣＨＯ／ＤＧ４４細胞株（以下、ＦＵＴ８ノックアウトＣＨＯ細胞またはＣＨＯ／Ｍｓ７０４と表記することがある）を用いた。この宿主細胞株で発現させた抗体のＮ－結合型複合型糖鎖のコア部分には、フコースが付加されないことが知られている（国際公開第２００２／３１１４０号）。 As an animal cell line to be expressed, a CHO / DG44 cell line (hereinafter sometimes referred to as FUT8 knockout CHO cell or CHO / Ms704) in which the α1,6-fucosyltransferase (FUT8) gene was double knocked out was used. It is known that fucose is not added to the core part of the N-linked complex sugar chain of the antibody expressed in this host cell line (WO 2002/31140).
　また、フコースが付加されていないヒトＩｇＧ１抗体は、付加されている場合に比べ抗体依存性細胞傷害活性（以下、ＡＤＣＣ活性と表記することがある）が高いことが知れている。以下、フコースが付加されていない抗体を高ＡＤＣＣ型抗体と表記することがある。 Further, it is known that the human IgG1 antibody to which fucose is not added has higher antibody-dependent cytotoxic activity (hereinafter sometimes referred to as ADCC activity) than the case where it is added. Hereinafter, an antibody to which fucose is not added may be referred to as a high ADCC type antibody.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
（３）精製キメラ抗体の取得
　上記（２）で得られたＫＭ４０５１～ＫＭ４０５５を、通常の培養法で培養した後、細胞懸濁液を回収し、３０００ｒｐｍ、４℃の条件で２０分間の遠心分離を行って培養上清を回収した後、０．２２μｍ孔径ＭｉｌｌｅｘＧＶフィルター（Ｍｉｌｌｉｐｏｒｅ社製）を通して濾過滅菌した。得られた培養上清よりＰｒｏｔｅｉｎ　Ａ　Ｈｉｇｈ－ｃａｐａｃｉｔｙレジン（Ｍｉｌｌｉｐｏｒｅ社製）カラムを用いて、添付の説明書に従い、ＫＭ４０５１～ＫＭ４０５５を精製した。 (3) Acquisition of purified chimeric antibody After KM4051 to KM4055 obtained in (2) above were cultured by a normal culture method, the cell suspension was recovered and centrifuged for 20 minutes at 3000 rpm and 4 ° C. After collecting the culture supernatant, the solution was sterilized by filtration through a 0.22 μm pore size Millex GV filter (manufactured by Millipore). KM4051 to KM4055 were purified from the obtained culture supernatant using a Protein A High-capacity resin (Millipore) column according to the attached instructions.
（４）抗ＣＤ３３キメラ抗体のフコース含量測定
　国際公開第２００２／３１１４０号に記載の方法に従い、各キメラ抗体のＦｃに結合するＮ結合型糖鎖の還元末端のＮ－アセチルグルコサミンの６位にフコースの１位がα結合していない糖鎖の割合を調べた。結果を表４に示す。 (4) Measurement of fucose content of anti-CD33 chimeric antibody According to the method described in International Publication No. 2002/31140, fucose at position 6 of N-acetylglucosamine at the reducing end of the N-linked sugar chain that binds to Fc of each chimeric antibody The proportion of sugar chains that were not α-bonded at the 1-position was examined. The results are shown in Table 4.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　表４に示すように、上記（３）で得られたキメラ抗体にはフコースが付加していないことが確認された。 As shown in Table 4, it was confirmed that fucose was not added to the chimeric antibody obtained in (3) above.
［実施例７］
抗ＣＤ３３キメラ抗体の活性評価
　以下の（１）から（６）では、実施例６で得られたＫＭ４０５１～ＫＭ４０５５、および下記実施例８で作製した抗ＣＤ３３抗体ＨＭ１９５のＩｇＧ１型抗体（以下、ＨＭ１９５－１と表記することがある）、ＨＭ１９５の高ＡＤＣＣ型抗体（以下、ＨＭ１９５－Ｐと表記することがある）、ＨＭ１９５の１１３Ｆ型であり、かつ高ＡＤＣＣ型である抗体（以下、ＨＭ１９５－１１３Ｆと表記することがある）の活性評価を行った。 [Example 7]
Evaluation of Activity of Anti-CD33 Chimeric Antibody In the following (1) to (6), KM4051 to KM4055 obtained in Example 6 and the anti-CD33 antibody HM195 IgG1 type antibody (hereinafter referred to as HM195) prepared in Example 8 HM195 high ADCC type antibody (hereinafter sometimes referred to as HM195-P), HM195 type 113F and high ADCC type antibody (hereinafter referred to as HM195-113F). Activity evaluation was performed.
（１）ＦＣＭにおけるヒトＣＤ３３陽性細胞株との反応性
　１～５×１０５細胞のＴＨＰ－１に、希釈したマウス血清（ＣＥＤＡＲＬＡＮＥ　ＬＡＢＯＲＡＴＯＲＩＥＳ社製）を加えブロッキングをし、ＫＭ４０５１～ＫＭ４０５５およびＨＭ１９５－１１３Ｆを上記のＦＣＭバッファーで適宜希釈したものを添加し、全量を１００μＬとした。これらの細胞懸濁液を氷上で６０分間反応させ，ＰＢＳで３回洗浄した。 (1) Reactivity with human CD33-positive cell line in FCM 1-5 × 10 5 cells of THP-1 were diluted with serum from a diluted mouse serum (CEDARLANE LABORATORIES) and blocked, and KM4051 to KM4055 and HM195-113F Was appropriately diluted with the above FCM buffer to make a total volume of 100 μL. These cell suspensions were reacted for 60 minutes on ice and washed 3 times with PBS.
　前記細胞にＦＣＭバッファーで希釈調製したＦＩＴＣ標識ヤギ抗ヒトＩｇＧ（Ｆｃ）抗体（Ａｃｒｉｓ　Ａｎｔｉｂｏｄｉｅｓ社製）を３０μＬ添加し、氷上で４０分間反応させた。ＰＢＳで３回洗浄後、ＦＣＭバッファーに懸濁してフローサイトメーター（ベックマン・コールター社製）用いて、蛍光強度を測定した。 30 μL of FITC-labeled goat anti-human IgG (Fc) antibody (manufactured by Acris Antibodies) diluted with FCM buffer was added to the cells and reacted on ice for 40 minutes. After washing with PBS three times, the suspension was suspended in FCM buffer and the fluorescence intensity was measured using a flow cytometer (manufactured by Beckman Coulter).
　図６に、実施例６で得られたＫＭ４０５１～ＫＭ４０５５を２μｇ／ｍＬから各濃度４倍希釈で段階的に希釈したものを反応させた場合のＭＦＩを示す。ＫＭ４０５１～ＫＭ４０５５およびＨＭ１９５－１１３Ｆはいずれも濃度依存的にＴＨＰ－１へ結合することが確認された。 FIG. 6 shows the MFI when the KM4051 to KM4055 obtained in Example 6 are reacted stepwise diluted from 2 μg / mL at a 4-fold dilution at each concentration. It was confirmed that KM4051 to KM4055 and HM195-113F all bind to THP-1 in a concentration-dependent manner.
（２）バインディングＥＬＩＳＡ法によるリコンビナントヒトＣＤ３３－Ｆｃへの結合活性評価
　実施例３（３）に記載の方法に従って行った。プレートにはリコンビナントヒトＣＤ３３－Ｆｃまたは、ヤギ抗ヒトＩｇＧ（Ｈ＋Ｌ）抗体（Ａｍｅｒｉｃａｎ　Ｑｕａｌｅｘ社製）を吸着させた。１次抗体には実施例６で得られたＫＭ４０５１～ＫＭ４０５５、および実施例８で得られたＨＭ１９５－１１３Ｆを、２μｇ／ｍＬから５倍希釈で段階的に希釈したものを用いた。 (2) Evaluation of binding activity to recombinant human CD33-Fc by binding ELISA was performed according to the method described in Example 3 (3). Recombinant human CD33-Fc or goat anti-human IgG (H + L) antibody (American Qualex) was adsorbed on the plate. As the primary antibody, KM4051 to KM4055 obtained in Example 6 and HM195-113F obtained in Example 8 were diluted stepwise from 2 μg / mL at a 5-fold dilution.
　２次抗体には、ペルオキシダーゼ標識ヤギ抗ヒトＩｇＧ（Ｈ＋Ｌ鎖）抗体（Ａｍｅｒｉｃａｎ　Ｑｕａｌｅｘ社製）またはペルオキシダーゼ標識ヤギ抗ヒトκ鎖（Ｓｏｕｔｈｅｒｎ　Ｂｉｏｔｅｃｈ社製）を用いた。リコンビナントヒトＣＤ３３－Ｆｃおよびヤギ抗ヒトＩｇＧ（Ｈ＋Ｌ）抗体の結果を、それぞれ図７（Ａ）および（Ｂ）に示す。 Peroxidase-labeled goat anti-human IgG (H + L chain) antibody (American Qualex) or peroxidase-labeled goat anti-human κ chain (Southern Biotech) was used as the secondary antibody. The results of recombinant human CD33-Fc and goat anti-human IgG (H + L) antibody are shown in FIGS. 7 (A) and (B), respectively.
　図７に示すように、ＫＭ４０５１～ＫＭ４０５５、ＨＭ１９５－１１３Ｆは、いずれもリコンビナントヒトＣＤ３３－Ｆｃに反応性を示した。 As shown in FIG. 7, KM4051 to KM4055 and HM195-113F all showed reactivity to recombinant human CD33-Fc.
（３）ＢｉａｃｏｒｅにおけるリコンビナントヒトＣＤ３３－Ｆｃとの結合活性
　ＫＭ４０５１～ＫＭ４０５５およびＨＭ１９５－１１３ＦのｈＣＤ３３－Ｈｉｓに対する結合活性を反応速度論的に解析するため、表面プラズモン共鳴法（ＳＰＲ法）を用いて結合活性測定を行った。 (3) Binding activity to recombinant human CD33-Fc in Biacore In order to analyze the binding activity of KM4051 to KM4055 and HM195-113F to hCD33-His, binding was performed using the surface plasmon resonance method (SPR method). Activity measurement was performed.
　以下の操作は全てＢｉａｃｏｒｅＴ－１００（ＧＥヘルスケアバイオサイエンス社製）を用いて行った。Ｈｕｍａｎ　Ａｎｔｉｂｏｄｙ　Ｃａｐｔｕｒｅ　Ｋｉｔ（ＧＥヘルスケアバイオサイエンス社製）を用い、添付のプロトコールに従い、抗ヒトＩｇＧ抗体をアミンカップリング法によりＣＭ５センサーチップ（ＧＥヘルスケアバイオサイエンス社製）に固層化した。 The following operations were all performed using Biacore T-100 (GE Healthcare Bioscience). Using an Human Antibody Capture Kit (manufactured by GE Healthcare Bioscience), the anti-human IgG antibody was solidified on a CM5 sensor chip (manufactured by GE Healthcare Bioscience) by the amine coupling method according to the attached protocol.
　抗ヒトＩｇＧ抗体を固層化したチップに測定サンプル（ＫＭ４０５１～ＫＭ４０５５およびＨＭ１９５－１１３Ｆを添加し、約３００ＲＵ（レゾナンス　ユニット）になるようにキャプチャーさせた。その後１．２５μｇ／ｍＬから５段階に希釈したｈＣＤ３３－Ｈｉｓを３０μＬ／ｍｉｎの速度でチップ上に流し、各濃度におけるセンサーグラムを取得した。装置添付の解析ソフトを用い、１：１バインディングモデルを用いて解析し、各抗体のヒトＣＤ３３に対する結合速度定数ｋａ及び解離速度定数ｋｄを算出した。 Samples (KM4051 to KM4055 and HM195-113F) were added to the chip on which the anti-human IgG antibody was solidified, and captured to about 300 RU (resonance unit), and then diluted from 1.25 μg / mL in 5 steps The hCD33-His was flowed over the chip at a rate of 30 μL / min, and sensorgrams at each concentration were obtained, analyzed using a 1: 1 binding model using analysis software attached to the device, and each antibody against human CD33 The association rate constant ka and the dissociation rate constant kd were calculated.
　その結果得られた各抗体のｋａ、ｋｄおよび解離定数ＫＤを表５に示す。 The resulting ka of each antibody, the kd and dissociation constant, K D, shown in Table 5.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　表５に示すように、いずれのキメラ抗体もＨＭ１９５－１１３Ｆを上回る高いアフィニティーを示した。 As shown in Table 5, all of the chimeric antibodies showed high affinity exceeding HM195-113F.
（４）抗ＣＤ３３キメラ抗体のヒト血液癌細胞株に対するＡＤＣＣ活性の評価
　ＫＭ４０５１～ＫＭ４０５５、ＨＭ１９５－１およびＨＭ１９５－１１３ＦのＡＤＣＣ活性を、以下に示す方法に従って測定した。 (4) Evaluation of ADCC activity of anti-CD33 chimeric antibody against human hematological cancer cell line ADCC activity of KM4051 to KM4055, HM195-1, and HM195-113F was measured according to the following method.
（４）－１　標的細胞溶液の調製
　ＣＤ３３陽性であるヒト急性前骨髄球性白血病細胞株ＮＢ－４をＰＢＳで洗浄後、５％ウシ胎児血清（ｄＦＢＳ）を含むフェノールレッド不含有ＲＰＭＩ１６４０培地（以下、ＡＤＣＣ用培地と記することがある）で洗浄し、同培地で至適濃度に調製して標的細胞溶液とした。 (4) -1 Preparation of target cell solution After washing CD33-positive human acute promyelocytic leukemia cell line NB-4 with PBS, phenol red-free RPMI 1640 medium containing 5% fetal bovine serum (dFBS) , Sometimes referred to as ADCC medium), and adjusted to an optimal concentration with the same medium to obtain a target cell solution.
（４）－２　エフェクター細胞溶液の調製
　ＰＢＭＣは、健常人末梢血から以下に示した方法により分離した。ヘパリンナトリウム注Ｎ「シミズ」（清水製薬社製）を０．５ｍＬ含ませたシリンジで健常人末梢血５０ｍＬを採血した。採取した末梢血に同量の生理食塩水（大塚製薬製）を加えて希釈し、良く攪拌した。１５ｍＬチューブ（Ｇｒｅｉｎｅｒ社製）に４．７ｍＬずつ分注したＬｙｍｐｈｏｐｒｅｐ（Ａｘｉｓ－Ｓｈｉｅｌｄ社製）の上に、１０ｍＬの希釈末梢血を静かに重層し、２０００ｒｐｍ、ブレーキオフ、室温の条件で２０分間遠心して単核球層を分離した。こうして得られた単核球画分を、ＡＤＣＣ用培地を用いて２回洗浄し、同培地により至適濃度に調製してエフェクター細胞溶液とした。 (4) -2 Preparation of effector cell solution PBMCs were separated from healthy human peripheral blood by the method described below. 50 mL of healthy human peripheral blood was collected with a syringe containing 0.5 mL of heparin sodium injection N “Shimizu” (manufactured by Shimizu Pharmaceutical Co., Ltd.). The collected peripheral blood was diluted by adding the same amount of physiological saline (manufactured by Otsuka Pharmaceutical) and stirred well. On a Lymphoprep (Axis-Shield) dispensed 4.7 mL into a 15 mL tube (Greiner), 10 mL of diluted peripheral blood is gently layered, and then 20 minutes at 2000 rpm, brake off, and room temperature. Keep in mind to separate the mononuclear cell layer. The thus obtained mononuclear cell fraction was washed twice with an ADCC medium, adjusted to an optimal concentration with the same medium, and used as an effector cell solution.
（４）－３　ＡＤＣＣ活性の測定
　ＣｙｔｏＴｏｘ９６　Ｎｏｎ－Ｒａｄｉｏａｃｔｉｖｅ　Ｃｙｔｏｔｏｘｉｃｉｔｙ　Ａｓｓａｙ（Ｐｒｏｍｅｇａ社製）を用い、付属の説明書に従い以下の手順で測定した。 (4) -3 Measurement of ADCC activity CytoTox96 Non-Radioactive Cytotoxicity Assay (manufactured by Promega) was used and measured according to the following procedure.
　９６ウェルＵ底プレート（ＦＡＬＣＯＮ社製）の各ウェルに適当濃度に希釈した抗体液を５０μＬ分注しておき、（４）－１で調製した標的細胞溶液を５０μＬ、（４）－２で調製したエフェクター細胞溶液を５０μＬ添加して全量を１５０μＬとし、３７℃で４時間反応させた。ＡＤＣＣ活性は次式により求めた。 50 μL of antibody solution diluted to an appropriate concentration is dispensed into each well of a 96-well U-bottom plate (manufactured by FALCON), and 50 μL of the target cell solution prepared in (4) -1 is prepared in (4) -2. 50 μL of the effector cell solution was added to a total volume of 150 μL, and reacted at 37 ° C. for 4 hours. ADCC activity was determined by the following formula.
（式）
ＡＤＣＣ活性（％）＝｛（［検体の吸光度］－［標的細胞自然遊離の吸光度］－［エフェクター細胞自然遊離の吸光度］）／（［標的細胞全遊離の吸光度］－［標的細胞自然遊離の吸光度］）｝×１００ (formula)
ADCC activity (%) = {([Absorbance of specimen]-[Absorbance of spontaneous release of target cell]-[Absorption of spontaneous release of effector cell]) / ([Absorbance of total release of target cell]-[Absorbance of spontaneous release of target cell] ]) X 100
　結果を図８に示す。標的細胞は１．０×１０４細胞／ウェル、エフェクター細胞は２×１０５細胞／ウェルとした（エフェクター細胞（Ｅ）：標的細胞（Ｔ）＝２０：１とした）。抗体濃度は、１μｇ／ｍＬから段階的に１０倍希釈した。 The results are shown in FIG. The target cells were 1.0 × 10 4 cells / well, and the effector cells were 2 × 10 5 cells / well (effector cells (E): target cells (T) = 20: 1). The antibody concentration was diluted 10-fold in steps from 1 μg / mL.
　図８に示すように、ＫＭ４０５１～ＫＭ４０５５、およびＨＭ１９５－１、ＨＭ１９５－１１３Ｆは、ＮＢ－４に対して抗体濃度依存的なＡＤＣＣ活性を示した。また、ＫＭ４０５１～ＫＭ４０５５はＨＭ１９５－１１３Ｆよりも強いＡＤＣＣ活性を示した。 As shown in FIG. 8, KM4051 to KM4055, HM195-1, and HM195-113F showed antibody concentration-dependent ADCC activity against NB-4. In addition, KM4051 to KM4055 showed stronger ADCC activity than HM195-113F.
（５）抗ＣＤ３３キメラ抗体のＣＤ３３トランスフェクタントに対するＣＤＣ活性の評価
　ＫＭ４０５１～ＫＭ４０５５のＣＤＣ活性を、以下に示す方法に従って測定した。 (5) Evaluation of CDC activity of CD33 transfectant of anti-CD33 chimeric antibody The CDC activity of KM4051 to KM4055 was measured according to the method shown below.
　ＣＤ３３トランスフェクタントをＰＢＳで洗浄後、１０％ウシ胎児血清（ＦＢＳ）を含むＲＰＭＩ１６４０培地（和光純薬工業社製）で洗浄し、同培地で至適濃度に調製して標的細胞溶液とした。１ウェル当たり５ｘ１０４個となるよう、標的細胞溶液を９６ｗｅｌｌ平底プレート（住友ベークライト社製）に加え、更に、適当な濃度に調製した抗ＣＤ３３キメラ抗体液、及び、終濃度４倍希釈となるように調製したヒト補体（ＳＩＧＭＡ社製）を添加し、１ｗｅｌｌ当たり１００μＬとなるように調製した。 After washing the CD33 transfectant with PBS, the CD33 transfectant was washed with RPMI 1640 medium (Wako Pure Chemical Industries, Ltd.) containing 10% fetal bovine serum (FBS). . The target cell solution is added to a 96-well flat bottom plate (manufactured by Sumitomo Bakelite Co., Ltd.) so that the number of target cell solutions is 5 × 10 4 per well, and the anti-CD33 chimeric antibody solution prepared to an appropriate concentration and the final concentration is diluted 4-fold. Human complement (manufactured by SIGMA) was added to prepare 100 μL per well.
　また、ＣＤＣが惹起されない場合の対照として抗体を含まない反応ウェル（０％Ｌｙｓｉｓウェル）、ＣＤＣが惹起された場合の対照として細胞を含まない反応ウェル（１００％Ｌｙｓｉｓウェル）をそれぞれ用意した。３７℃インキュベーターにおいて３時間反応を行った。 Also, a reaction well containing no antibody (0% Lysis well) was prepared as a control when CDC was not induced, and a reaction well containing no cells (100% Lysis well) was prepared as a control when CDC was induced. The reaction was performed in a 37 ° C. incubator for 3 hours.
　反応終了後、各反応ウェルに１０μＬのＷＳＴ－１試薬（ＲＯＣＨＥ社製）を加え、３７℃で３時間程度反応させ、各ウェルにおけるＯＤ４５０を測定し、各ウェルの吸光度より以下の式を用いてＣＤＣ活性（細胞傷害活性［％］）を算出した。その結果を図９に示す。 After completion of the reaction, 10 μL of WST-1 reagent (manufactured by ROCHE) is added to each reaction well, reacted at 37 ° C. for about 3 hours, OD450 in each well is measured, and the following formula is used from the absorbance of each well. CDC activity (cytotoxic activity [%]) was calculated. The result is shown in FIG.
（式）
ＣＤＣ活性（細胞傷害活性［％］）＝１００ｘ｛１－（反応ウェルの吸光度―１００％Ｌｙｓｉｓウェルの吸光度）／（０％Ｌｙｓｉｓウェルの吸光度―１００％Ｌｙｓｉｓウェルの吸光度）｝ (formula)
CDC activity (cytotoxic activity [%]) = 100 × {1- (absorbance of reaction well−absorbance of 100% Lysis well) / (absorbance of 0% Lysis well−absorbance of 100% Lysis well)}
　その結果、図９に示すように、ＫＭ４０５１～ＫＭ４０５５はいずれのクローンもＣＤＣ活性を示した。 As a result, as shown in FIG. 9, KM4051 to KM4055 all exhibited CDC activity.
（６）抗ＣＤ３３キメラ抗体のＣＤ３３発現細胞株における細胞膜上での抗体結合量
　ＴＨＰ－１の細胞膜上での抗ＣＤ３３キメラ抗体およびＨＭ１９５－１１３Ｆの経時的な抗体結合量を以下の方法に従って測定した。 (6) Antibody binding amount on cell membrane of CD33-expressing cell line of anti-CD33 chimeric antibody The amount of antibody binding over time of anti-CD33 chimeric antibody and HM195-113F on the cell membrane of THP-1 was measured according to the following method. .
　１～５×１０５細胞のＴＨＰ－１に、希釈したマウス血清（ＣＥＤＡＲＬＡＮＥ　ＬＡＢＯＲＡＴＯＲＩＥＳ社製）を加えブロッキングをし、ＫＭ４０５１～ＫＭ４０５５および実施例８に示すＨＭ１９５－１１３Ｆを１０％ＦＣＳ－ＲＰＭＩで適宜希釈したものを添加し、全量を１００μＬとした。 Diluted mouse serum (CEDARLANE LABORATORIES) was added to 1-5 × 10 5 THP-1 for blocking, and KM4051-KM4055 and HM195-113F shown in Example 8 were appropriately diluted with 10% FCS-RPMI. Was added to make a total volume of 100 μL.
　これらの細胞懸濁液を氷上で４０分間反応後、３７℃で３０、６０および１２０分間培養した。その後、冷ＰＢＳで３回洗浄し、該細胞に上記ＦＣＭバッファーで希釈調製したＦＩＴＣ標識ヤギ抗ヒトＩｇＧ（Ｆｃ）抗体（Ａｃｒｉｓ　Ａｎｔｉｂｏｄｉｅｓ社製）を３０μＬ添加し、氷上で４０分間反応させた。ＰＢＳで３回洗浄後、ＦＣＭバッファーに懸濁してフローサイトメーター（ベックマン・コールター社製）用いて、蛍光強度を測定した。 These cell suspensions were reacted on ice for 40 minutes and then incubated at 37 ° C. for 30, 60 and 120 minutes. Thereafter, the cells were washed 3 times with cold PBS, and 30 μL of FITC-labeled goat anti-human IgG (Fc) antibody (manufactured by Acris Antibodies) diluted with the above FCM buffer was added to the cells and reacted on ice for 40 minutes. After washing with PBS three times, the suspension was suspended in FCM buffer and the fluorescence intensity was measured using a flow cytometer (manufactured by Beckman Coulter).
　得られた結果を図１０に示す［図１０の（Ａ）および（Ｂ）は、それぞれ抗体結合量および抗体残存率を示す］。図１０に示すように、ＫＭ４０５１～ＫＭ４０５５およびＨＭ１９５－１１３Ｆは時間依存的に細胞表面上での結合量が減少した。ＫＭ４０５１～ＫＭ４０５５はいずれのクローンも各時間においてＨＭ１９５－１１３Ｆに比べて、細胞表面上に結合した抗体の量および残存率ともに上回っていた。 The obtained results are shown in FIG. 10 ((A) and (B) in FIG. 10 show the amount of antibody binding and the antibody remaining rate, respectively). As shown in FIG. 10, KM4051 to KM4055 and HM195-113F decreased the amount of binding on the cell surface in a time-dependent manner. In each clone, KM4051 to KM4055 exceeded both HM195-113F and the amount of antibody bound to the cell surface and the residual ratio at each time.
［実施例８］
ＨＭ１９５のＩｇＧ１型抗体および１１３Ｆ型抗体の作製
（１）ＨＭ１９５－１およびＨＭ１９５－１１３Ｆ発現ベクターの作製
　米国特許第７０２２５００号明細書に記載の配列情報に基づき、ヒトＣＤ３３を特異的に認識するヒト抗体ＨＭ１９５のＶＨおよびＶＬをコードする遺伝子を、実施例６（１）に記載の方法により構築し、ベクターｐＫＡＮＴＥＸ９３およびベクターＰＫＴＸ９３／１１３Ｆに組み込んだ。ベクター構築の概略図を図１１に示す。 [Example 8]
Production of HM195 IgG1-type and 113F-type antibodies (1) Production of HM195-1 and HM195-113F expression vectors Human antibodies that specifically recognize human CD33 based on the sequence information described in US Pat. No. 7,022,500 Genes encoding HM195 VH and VL were constructed by the method described in Example 6 (1) and incorporated into vector pKANTEX93 and vector PKTX93 / 113F. A schematic diagram of vector construction is shown in FIG.
（２）ＨＭ１９５－１およびＨＭ１９５－１１３Ｆの動物細胞での発現
　上記（１）で取得したベクターを用いて、実施例６（２）に記載の方法により行った。
ＨＭ１９５－１を発現させる動物細胞としてはＣＨＯ／ＤＧ４４細胞およびＣＨＯ／Ｍｓ７０４細胞を用いて、それぞれフコース付加した通常抗体およびフコース付加していない抗体を発現させた。ＨＭ１９５－１１３Ｆを発現させる動物細胞としては、ＣＨＯ／Ｍｓ７０４細胞を用いた。 (2) Expression of HM195-1 and HM195-113F in animal cells The vector obtained in (1) above was used and the method described in Example 6 (2).
As animal cells to express HM195-1, CHO / DG44 cells and CHO / Ms704 cells were used to express normal and non-fucose-added antibodies. CHO / Ms704 cells were used as animal cells that express HM195-113F.
（３）ＨＭ１９５－１、ＨＭ１９５－Ｐ、およびＨＭ１９５－１１３Ｆ精製抗体の取得
　上記（２）で作製した動物細胞株を用いて、実施例６（３）に記載の方法により行った。 (3) Obtaining HM195-1, HM195-P, and HM195-113F Purified Antibody The animal cell line prepared in (2) above was used and the method described in Example 6 (3) was performed.
［実施例９］
可変領域にＮ結合型糖鎖が結合するコンセンサス配列を有さない抗ＣＤ３３キメラ抗体の作製
　可変領域にＮ結合型糖鎖が結合するコンセンサス配列を有さない抗ＣＤ３３キメラ抗体（以下、改変型抗ＣＤ３３キメラ抗体と表記することがある）は、実施例３でＫＭ４０７４を基にして以下のように作製した。該抗体は配列番号５３で示されるＶＨのＣＤＲ２のアミノ酸配列中に、Ｎ結合型糖鎖が結合するコンセンサス配列を有していた。 [Example 9]
Preparation of anti-CD33 chimeric antibody having no consensus sequence for binding N-linked sugar chain to variable region Anti-CD33 chimeric antibody having no consensus sequence for binding N-linked sugar chain to variable region (hereinafter referred to as modified anti-antibody) CD33 chimeric antibody) was prepared as described below in Example 3 based on KM4074. The antibody had a consensus sequence to which an N-linked sugar chain was bound in the amino acid sequence of CDR2 of VH represented by SEQ ID NO: 53.
（１）Ｖ領域にＮ結合型糖鎖が結合するコンセンサス配列を有さない遺伝子組換え抗体のアミノ酸配列の設計
　ＫＭ４０７４のＶＨに含まれるＮ結合型糖鎖が結合するコンセンサス配列は、配列番号２２で示されるアミノ酸配列の７１番目のＡｓｎ、７２番目のＳｅｒおよび７３番目のＳｅｒからなる配列である。配列番号２２で示されるアミノ酸配列において、ＣＤＲ２は配列番号５３で示されるアミノ酸配列を有する。配列番号５３で示されるアミノ酸配列において、Ｎ結合型糖鎖が結合するコンセンサス配列は、３番目のＡｓｎ、４番目のＳｅｒおよび５番目のＳｅｒからなる配列である。 (1) Design of amino acid sequence of recombinant antibody having no consensus sequence to which N-linked sugar chain binds to V region The consensus sequence to which the N-linked sugar chain contained in VH of KM4074 binds is SEQ ID NO: 22 Is a sequence consisting of 71st Asn, 72nd Ser, and 73rd Ser of the amino acid sequence shown in FIG. In the amino acid sequence shown in SEQ ID NO: 22, CDR2 has the amino acid sequence shown in SEQ ID NO: 53. In the amino acid sequence represented by SEQ ID NO: 53, the consensus sequence to which the N-linked sugar chain binds is a sequence composed of the third Asn, the fourth Ser, and the fifth Ser.
　Ｎ結合型糖鎖が結合するコンセンサス配列は、配列番号５３で示されるアミノ酸配列において、４番目のアミノ酸が任意であることから、３番目のＡｓｎと５番目のＳｅｒを改変候補残基とした。既存の抗体配列［Ｓｅｑｕｅｎｃｅｓ　ｏｆ　Ｐｒｏｔｅｉｎｓ　ｏｆ　Ｉｍｍｕｎｏｌｏｇｉｃａｌ　Ｉｎｔｅｒｅｓｔ，ＵＳ　Ｄｅｐｔ．Ｈｅａｌｔｈ　ａｎｄ　Ｈｕｍａｎ　Ｓｅｒｖｉｃｅｓ（１９９１）］との比較やアミノ酸の性質および側鎖構造を勘案し、抗体の結合活性に影響を与えないと考えられるアミノ酸改変として、３番目のＡｓｎをＴｙｒ、Ｓｅｒ、またはＧｌｎのいずれかに置換するアミノ酸改変、または５番目のＳｅｒをＡｌａまたはＧｌｙのいずれかに置換するアミノ酸改変を選択した。 The consensus sequence to which the N-linked sugar chain binds is that the fourth amino acid is arbitrary in the amino acid sequence represented by SEQ ID NO: 53, and therefore the third Asn and the fifth Ser were used as candidate modification residues. Existing antibody sequences [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)] and the amino acid modification and side chain structure in consideration of the amino acid modification and the third Asn are considered as Tyr, Ser, or Gln. The amino acid modification which substitutes either of these, or the amino acid modification which substitutes 5th Ser to either Ala or Gly was selected.
　具体的には、３番目のＡｓｎをＴｙｒに置換したｖｅｒ．１、３番目のＡｓｎをＳｅｒに置換したｖｅｒ．２、３番目のＡｓｎをＧｌｎに置換したｖｅｒ．３、５番目のＳｅｒをＡｌａに置換したｖｅｒ．４、および５番目のＳｅｒをＧｌｙに置換したｖｅｒ．５の５種類の改変型ＣＤ３３キメラ抗体のＶＨを設計した。Ｖｅｒ．１～ｖｅｒ．５の各ＶＨのアミノ酸配列は、それぞれ配列番号１０３、１０４、１０５、９３、および１０６で示される。また、各Ｖｅｒ．のＶＨのＣＤＲ２のアミノ酸配列を、それぞれ配列番号９９、１００、１０１、９６および１０２に示す。以下、ｖｅｒ．１を有する抗体を、単にｖｅｒ．１と表記することがある。 Specifically, ver.3 in which the third Asn is replaced with Tyr. 1 and 3 where Asn is replaced with Ser. The second and third Asn were replaced with Gln ver. 3 and 5 where Ser was replaced with Ala. 4th and 5th Ser were replaced with Gly. Five VHs of 5 types of modified CD33 chimeric antibodies were designed. Ver. 1 to ver. The amino acid sequences of each VH of 5 are shown in SEQ ID NOs: 103, 104, 105, 93, and 106, respectively. Each Ver. The amino acid sequences of CDR2 of VH are shown in SEQ ID NOs: 99, 100, 101, 96 and 102, respectively. Hereinafter, ver. Antibody with 1 is simply ver. Sometimes referred to as 1.
　このようにして、抗体のＶＨが配列番号１０３、１０４、１０５、９３、１０６のいずれか１つで表されるアミノ酸配列であり、かつ抗体のＶＬが配列番号９５（配列番号３２で表されるアミノ酸配列からシグナル配列を除いたもの）で表されるアミノ酸配列である改変型ＣＤ３３キメラ抗体を設計した。 Thus, the VH of the antibody is an amino acid sequence represented by any one of SEQ ID NOs: 103, 104, 105, 93, and 106, and the VL of the antibody is SEQ ID NO: 95 (represented by SEQ ID NO: 32). A modified CD33 chimeric antibody having an amino acid sequence represented by the amino acid sequence minus the signal sequence was designed.
（２）改変型ＣＤ３３キメラ抗体の発現ベクターの作製
　実施例６で作製した、ＫＭ４０７４と同一のＶ領域を有するＫＭ４０５４発現ベクターを鋳型として、アミノ酸改変を導入した。アミノ酸改変を導入にはそれぞれ、
Ｖｅｒ．１の作製には、配列番号１０７および１０８で示される塩基配列を有するプライマーを、
Ｖｅｒ．２の作製には、配列番号１０９および１１０で示される塩基配列を有するプライマーを、
Ｖｅｒ．３の作製には、配列番号１１１および１１２で示される塩基配列を有するプライマーを、
Ｖｅｒ．４の作製には、配列番号９７および９８で示される塩基配列を有するプライマーを、
Ｖｅｒ．５の作製には、配列番号１１３および１１４で示される塩基配列を有するプライマーを用い、ＱｕｉｃｋＣｈａｎｇｅ　Ｓｉｔｅ－Ｄｉｒｅｃｔｅｄ　Ｍｕｔａｇｅｎｅｓｉｓ　Ｋｉｔ（Ｓｔｒａｔａｇｅｎｅ社製）を用いて、添付の使用説明書に従い、目的のアミノ酸置換を実施したプラスミドＤＮＡを取得した。 (2) Production of expression vector of modified CD33 chimeric antibody Amino acid modification was introduced using KM4054 expression vector having the same V region as KM4074 produced in Example 6 as a template. For introducing amino acid modifications,
Ver. For the preparation of 1, primers having the base sequences shown in SEQ ID NOs: 107 and 108 are used.
Ver. 2 is prepared by using primers having the nucleotide sequences represented by SEQ ID NOs: 109 and 110,
Ver. 3 is prepared by using primers having the nucleotide sequences represented by SEQ ID NOs: 111 and 112,
Ver. For the preparation of 4, primers having the base sequences shown in SEQ ID NOs: 97 and 98 are used.
Ver. 5 was prepared using primers having the nucleotide sequences shown in SEQ ID NOs: 113 and 114, and using the QuickChange Site-Directed Mutagenesis Kit (manufactured by Stratagene) according to the attached instruction manual. The obtained plasmid DNA was obtained.
　得られたベクターは、ＢｉｇＤｙｅ　Ｔｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　ＦＳ　Ｒｅａｄｙ　Ｒｅａｃｔｉｏｎ　Ｋｉｔ（ＰＥバイオシステムズ社製）を用い添付の説明書に従って反応後、同社のシーケンサーＡＢＩ　ＰＲＩＳＭ３７００によりクローニングしたＰＣＲ産物の塩基配列を解析し、目的の改変が施された、改変型抗体発現ベクターが得られたことを確認した。 The obtained vector was analyzed using the BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (manufactured by PE Biosystems) according to the attached instructions, and the base sequence of the PCR product cloned by its sequencer ABI PRISM3700 was analyzed. It was confirmed that a modified antibody expression vector having been modified was obtained.
（３）改変型ＣＤ３３キメラ抗体の動物細胞での発現
　上記（２）で得られた改変型抗体発現ベクターを用い、実施例６－（２）と同様の手法により形質転換株を取得した。 (3) Expression of modified CD33 chimeric antibody in animal cells Using the modified antibody expression vector obtained in (2) above, a transformant was obtained by the same method as in Example 6- (2).
（４）精製された改変型ＣＤ３３キメラ抗体の取得
　上記（３）で得られた各形質転換株を用い、実施例６－（３）と同様の手法により、精製された改変型ＣＤ３３キメラ抗体を取得した。 (4) Obtaining purified modified CD33 chimeric antibody Using each transformed strain obtained in (3) above, purifying the modified CD33 chimeric antibody purified in the same manner as in Example 6- (3). I got it.
［実施例１０］
改変型ＣＤ３３キメラ抗体の活性評価
　実施例６で得られたＫＭ４０５４、および実施例９で作製した改変型ＣＤ３３キメラ抗体の活性評価を行った。 [Example 10]
Activity evaluation of the modified CD33 chimeric antibody The activity of the KM4054 obtained in Example 6 and the modified CD33 chimeric antibody prepared in Example 9 were evaluated.
（１）ＢｉａｃｏｒｅにおけるｈＣＤ３３－Ｈｉｓとの結合活性
　ＫＭ４０５４および実施例９で作製した改変型ＣＤ３３キメラ抗体の、ｈＣＤ３３－Ｈｉｓに対する結合活性を反応速度論的に解析するため、表面プラズモン共鳴法（ＳＰＲ法）を用いて結合活性測定を実施例７－（３）と同様の手法により行った。 (1) Binding activity with hCD33-His in Biacore In order to analyze the binding activity of the modified CD33 chimeric antibody produced in KM4054 and Example 9 to hCD33-His kinetically, surface plasmon resonance method (SPR method) ) Was used to measure the binding activity by the same method as in Example 7- (3).
　その結果得られた各抗体のｋａ、ｋｄおよび解離定数ＫＤを表６に示す。 The resulting ka of each antibody, the kd and dissociation constant, K D, shown in Table 6.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
　表６に示すように、改変型ＣＤ３３キメラ抗体ｖｅｒ．１～５は、ＫＭ４０５４と同等のアフィニティーを有しており、このうちｖｅｒ．４を　ＫＭ４０８４と命名した。 As shown in Table 6, modified CD33 chimeric antibody ver. 1 to 5 have an affinity equivalent to that of KM4054. 4 was named KM4084.
　ＫＭ４０８４のＶＨのＣＤＲ１、ＣＤＲ２およびＣＤＲ３のアミノ酸配列は、それぞれ配列番号５２、９６および５４に、ＶＬのＣＤＲ１、ＣＤＲ２およびＣＤＲ３のアミノ酸配列は、それぞれ配列番号５５、５６および５７に示す。 The amino acid sequences of CDR1, CDR2 and CDR3 of VH of KM4084 are shown in SEQ ID NOs: 52, 96 and 54, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 of VL are shown in SEQ ID NOs: 55, 56 and 57, respectively.
　また、配列番号９３で表されるＫＭ４０８４のＶＨのアミノ酸配列をコードするＤＮＡの塩基配列を配列番号９２に、配列番号９５で表されるＫＭ４０８４のＶＬのアミノ酸配列をコードするＤＮＡの塩基配列を配列番号９４に示す。 In addition, the nucleotide sequence of the DNA encoding the amino acid sequence of KM4084 represented by SEQ ID NO: 93 is represented by SEQ ID NO: 92, and the nucleotide sequence of the DNA encoding the amino acid sequence of VL of KM4084 represented by SEQ ID NO: 95 is sequenced. The number 94 is shown.
［実施例１１］
抗ＣＤ３３ヒト化抗体の作製
（１）抗ＣＤ３３ヒト化抗体のＶＨおよびＶＬのアミノ酸配列の設計
　抗ＣＤ３３ヒト化抗体のＶＨのアミノ酸配列を以下のようにして設計した。初めに、ＫＭ４０８４のＶＨのＣＤＲ１～３のアミノ酸配列（配列番号５２、９６、５４）を移植するために、ヒト抗体のＶＨのフレームワーク領域（以下、ＦＲと表記することがある）のアミノ酸配列を選択した。 [Example 11]
Preparation of anti-CD33 humanized antibody (1) Design of amino acid sequences of VH and VL of anti-CD33 humanized antibody The amino acid sequence of VH of anti-CD33 humanized antibody was designed as follows. First, in order to transplant the amino acid sequence of CDR1 to 3 of VH of KM4084 (SEQ ID NO: 52, 96, 54), the amino acid sequence of the VH framework region of human antibody (hereinafter sometimes referred to as FR) Selected.
　カバットらは、既知の様々なヒト抗体のＶＨをそのアミノ酸配列の相同性からサブグループ（ＨＳＧ　Ｉ～ＩＩＩ）に分類し、それらのサブグループ毎に共通配列を報告している［Ｓｅｑｕｅｎｃｅｓ　ｏｆ　Ｐｒｏｔｅｉｎｓ　ｏｆ　Ｉｍｍｕｎｏｌｏｇｉｃａｌ　Ｉｎｔｅｒｅｓｔ，ＵＳ　Ｄｅｐｔ．Ｈｅａｌｔｈ　ａｎｄ　Ｈｕｍａｎ　Ｓｅｒｖｉｃｅｓ（１９９１）］。そこで、ヒト抗体のＶＨのサブグループＩ～ＩＩＩの共通配列のＦＲのアミノ酸配列とＫＭ４０８４ＶＨのＦＲのアミノ酸配列との相同性検索を実施した。 Kabat et al. Classify VHs of various known human antibodies into subgroups (HSG I-III) based on their amino acid sequence homology, and report a common sequence for each subgroup [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)]. Therefore, a homology search was performed between the FR amino acid sequence of the consensus sequence of VH subgroups I to III of the human antibody and the FR amino acid sequence of KM4084VH.
　相同性を検索した結果、ＨＳＧＩ、ＨＳＧＩＩ、およびＨＳＧＩＩＩの相同性はそれぞれ５０．６％、６７．１％、５４．１％であった。従って、ＫＭ４０８４ＶＨのＦＲのアミノ酸配列はサブグループＩＩと最も高い相同性を有していた。 As a result of searching for homology, the homology of HSGI, HSGII, and HSGIII was 50.6%, 67.1%, and 54.1%, respectively. Therefore, the FR amino acid sequence of KM4084VH had the highest homology with subgroup II.
　以上の結果から、ヒト抗体のＶＨのサブグループＩＩの共通配列のＦＲのアミノ酸配列の適切な位置に、ＫＭ４０８４ＶＨのＣＤＲ１～３のアミノ酸配列（配列番号５２、９６、５４）を移植した。このようにして、ＫＭ４０８４ヒト化抗体のＶＨのアミノ酸配列ＨＶ０（配列番号８４）を設計した。 Based on the above results, the amino acid sequence of CDRs 1 to 3 (SEQ ID NOs: 52, 96, and 54) of KM4084VH was transplanted to the appropriate position of the FR amino acid sequence of the consensus sequence of VH subgroup II of the human antibody. In this way, the amino acid sequence HV0 (SEQ ID NO: 84) of VH of the KM4084 humanized antibody was designed.
　次に、ＫＭ４０８４ヒト化抗体のＶＬのアミノ酸配列を以下のようにして設計した。ＫＭ４０８４のＶＬのＣＤＲ１～３のアミノ酸配列（配列番号５５～５７）を移植するために、ヒト抗体のＶＬのＦＲのアミノ酸配列を選択した。カバットらは、既知の様々なヒト抗体のＶＬをそのアミノ酸配列の相同性からサブグループ（ＨＳＧ　Ｉ～ＩＶ）に分類し、それらのサブグループ毎に共通配列を報告している［Ｓｅｑｕｅｎｃｅｓ　ｏｆ　Ｐｒｏｔｅｉｎｓ　ｏｆ　Ｉｍｍｕｎｏｌｏｇｉｃａｌ　Ｉｎｔｅｒｅｓｔ，ＵＳ　Ｄｅｐｔ．Ｈｅａｌｔｈ　ａｎｄ　Ｈｕｍａｎ　Ｓｅｒｖｉｃｅｓ（１９９１）］。そこで、ヒト抗体のＶＬのサブグループＩ～ＩＶの共通配列のＦＲのアミノ酸配列とＫＭ４０８４ＶＬのＦＲのアミノ酸配列との相同性検索を実施した。 Next, the amino acid sequence of VL of the KM4084 humanized antibody was designed as follows. In order to graft the amino acid sequence of CDR1-3 of VL of KM4084 (SEQ ID NOs: 55-57), the amino acid sequence of FR of VL of human antibody was selected. Kabat et al. Classify VL of various known human antibodies into subgroups (HSG I-IV) based on their amino acid sequence homology, and report common sequences for each subgroup [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)]. Therefore, a homology search was performed between the FR amino acid sequence of the consensus sequence of subgroups I to IV of human antibody VL and the FR amino acid sequence of KM4084VL.
　相同性を検索した結果、ＨＳＧＩ、ＨＳＧＩＩ、ＨＳＧＩＩＩ、およびＨＳＧＩＶの相同性はそれぞれ６０．０％、７７．５％、６５．０％、６５．０％であった。従って、ＫＭ４０８４ＶＬのＦＲのアミノ酸配列はサブグループＩＩと最も高い相同性を有していた。 As a result of searching for homology, the homology of HSGI, HSGII, HSGIII, and HSGIV was 60.0%, 77.5%, 65.0%, and 65.0%, respectively. Therefore, the amino acid sequence of FR of KM4084VL had the highest homology with subgroup II.
　以上の結果から、ヒト抗体のＶＬのサブグループＩＩの共通配列のＦＲのアミノ酸配列の適切な位置に、ＫＭ４０８４ＶＬのＣＤＲ１～３のアミノ酸配列（配列番号５５～５７）を移植した。しかし、ＫＭ４０８４ＶＬのアミノ酸配列（配列番号９５）中の４番目のＬｅｕ、および１０９番目のＬｅｕは、カバットらがあげるヒト抗体ＦＲのアミノ酸配列の相当する部位において、最も使用される頻度が高いアミノ酸残基ではないが、比較的高い頻度で使用されるアミノ酸残基であるため、上記のＫＭ４０８４のアミノ酸配列で認められるアミノ酸残基を用いることとした。このようにして、ＫＭ４０８４ヒト化抗体のＶＬのアミノ酸配列ＬＶ０（配列番号８５）を設計した。 Based on the above results, the amino acid sequence of CDRs 1 to 3 (SEQ ID NOs: 55 to 57) of KM4084VL was transplanted to the appropriate position of the FR amino acid sequence of the consensus sequence of subgroup II of the VL of the human antibody. However, the 4th Leu and 109th Leu in the amino acid sequence of KM4084VL (SEQ ID NO: 95) are the most frequently used amino acid residues at the site corresponding to the amino acid sequence of human antibody FR mentioned by Kabat et al. Although it is not a group, since it is an amino acid residue that is used at a relatively high frequency, the amino acid residue recognized in the amino acid sequence of KM4084 is used. In this way, the amino acid sequence LV0 (SEQ ID NO: 85) of the VL of the KM4084 humanized antibody was designed.
　上記で設計したＫＭ４０８４ヒト化抗体のＶＨのアミノ酸配列ＨＶ０およびＶＬのアミノ酸配列ＬＶ０は、選択したヒト抗体のＦＲのアミノ酸配列にＫＭ４０８４のＣＤＲのアミノ酸配列のみを移植した配列である。しかし、一般に、ヒト化抗体を作製する場合には、単なるヒト抗体のＦＲへのマウス抗体のＣＤＲのアミノ酸配列の移植のみでは結合活性が低下してしまうことが多い。 The VH amino acid sequence HV0 and VL amino acid sequence LV0 of the KM4084 humanized antibody designed above are sequences in which only the amino acid sequence of the CDR of KM4084 is grafted to the FR amino acid sequence of the selected human antibody. However, in general, when a humanized antibody is prepared, the binding activity often decreases only by transplanting the amino acid sequence of the CDR of the mouse antibody to the FR of the human antibody.
　このため、結合活性の低下を回避するため、ＣＤＲのアミノ酸配列の移植とともに、ヒト抗体とマウス抗体で異なっているＦＲのアミノ酸残基のうち、結合活性に影響を与えると考えられるアミノ酸残基を改変することが行われている。そこで、結合活性に影響を与えると考えられるＦＲのアミノ酸残基を以下のようにして同定した。 For this reason, in order to avoid a decrease in binding activity, along with the grafting of CDR amino acid sequences, among amino acid residues of FR that differ between human and mouse antibodies, amino acid residues that are thought to affect binding activity Modifications are being made. Therefore, FR amino acid residues that are thought to affect the binding activity were identified as follows.
　まず、上記で設計したＫＭ４０８４ヒト化抗体のＶＨのアミノ酸配列ＨＶ０およびＶＬのアミノ酸配列ＬＶ０よりなる抗体Ｖ領域（以下、ＨＶ０ＬＶ０と表記することがある）の三次元構造をコンピューターモデリングの手法を用いて構築した。三次元構造座標作製および三次元構造の表示には、Ｄｉｓｃｏｖｅｒｙ　Ｓｔｕｄｉｏ（アクセルリス社製）を添付の使用説明書に従い、用いた。 First, using a computer modeling technique, the three-dimensional structure of the antibody V region (hereinafter sometimes referred to as HV0LV0) consisting of the VH amino acid sequence HV0 and the VL amino acid sequence LV0 of the KM4084 humanized antibody designed above is used. It was constructed. Discovery Studio (manufactured by Accelrys Co., Ltd.) was used according to the attached instruction manual for preparing the three-dimensional structure coordinates and displaying the three-dimensional structure.
　また、ＫＭ４０８４のＶ領域の三次元構造のコンピューターモデルも同様にして構築した。更に、ＨＶ０ＬＶ０のＶＨおよびＶＬのＦＲのアミノ酸配列の中で、ＫＭ４０８４と異なっているアミノ酸残基を選択し、ＫＭ４０８４のアミノ酸残基へ改変したアミノ酸配列を作製し、同様に三次元構造モデルを構築した。これら作製したＫＭ４０８４、ＨＶ０ＬＶ０および改変体のＶ領域の三次元構造を比較し、抗体の結合活性に影響を与えると予測されるアミノ酸残基を同定した。 The computer model of the three-dimensional structure of the V region of KM4084 was also constructed in the same manner. Furthermore, in the amino acid sequence of HV0LV0 VH and VL FR, an amino acid residue different from KM4084 is selected, an amino acid sequence modified to KM4084 amino acid residue is prepared, and a three-dimensional structure model is constructed in the same manner. did. The three-dimensional structures of the V regions of these prepared KM4084, HV0LV0 and the variant were compared, and amino acid residues predicted to affect the binding activity of the antibody were identified.
　その結果、ＨＶ０ＬＶ０のＦＲのアミノ酸残基の中で抗原結合部位の三次元構造を変化させ、抗体の結合活性に影響を与えると考えられるアミノ酸残基として、ＨＶ０では２５番目のＳｅｒ、２７番目のＧｌｙ、２８番目のＳｅｒ、２９番目のＶａｌ、３０番目のＳｅｒ、４０番目のＧｌｎ、４５番目のＧｌｙ、４６番目のＬｅｕ、４９番目のＩｌｅ、７２番目のＶａｌ、９３番目のＶａｌ、９５番目のＴｙｒ、９７番目のＡｌａ、および１０６番目のＴｈｒを、ＬＶ０では２番目のＩｌｅ、８番目のＰｒｏ、１１番目のＬｅｕ、１５番目のＰｒｏ、および９０番目のＶａｌをそれぞれ選択した。 As a result, among the amino acid residues of FR of HV0LV0, the three-dimensional structure of the antigen binding site is changed, and the amino acid residues that are thought to affect the binding activity of the antibody are 25th Ser, 27th in HV0. Gly, 28th Ser, 29th Val, 30th Ser, 40th Gln, 45th Gly, 46th Leu, 49th Ile, 72nd Val, 93th Val, 95th Tyr, 97th Ala, and 106th Thr were selected for LV0 as 2nd Ile, 8th Pro, 11th Leu, 15th Pro, and 90th Val, respectively.
　これらの選択したアミノ酸残基のうち、少なくとも１つ以上のアミノ酸配列をＫＭ４０８４の同じ部位に存在するアミノ酸残基へ改変し、様々な改変を有するヒト化抗体のＶＨおよびＶＬを設計した。具体的には、ＶＨについては、配列番号８４のアミノ酸配列２５番目のＳｅｒをＴｈｒに、２７番目のＧｌｙをＴｙｒに、２８番目のＳｅｒをＴｈｒに、２９番目のＶａｌをＩｌｅに、３０番目のＳｅｒをＴｈｒに、４０番目のＧｌｎをＬｙｓに、４５番目のＧｌｙをＡｒｇに、４６番目のＬｅｕをＭｅｔに、４９番目のＩｌｅをＭｅｔに、７２番目のＶａｌをＡｒｇに、９３番目のＶａｌをＴｈｒに、９５番目のＴｙｒをＰｈｅに、９７番目のＡｌａをＴｈｒに、および１０６番目のＴｈｒをＶａｌに置換するアミノ酸改変のうち、少なくとも１つの改変を導入した。 Among these selected amino acid residues, at least one amino acid sequence was modified to an amino acid residue present at the same site of KM4084, and VH and VL of humanized antibodies having various modifications were designed. Specifically, for VH, the 25th Ser of the amino acid sequence of SEQ ID NO: 84 is Thr, the 27th Gly is Tyr, the 28th Ser is Thr, the 29th Val is Ile, and the 30th Ser to Thr, 40th Gln to Lys, 45th Gly to Arg, 46th Leu to Met, 49th Ile to Met, 72nd Val to Arg, 93th Val At least one of the amino acid modifications was introduced into Thr, replacing 95th Tyr with Phe, 97th Ala with Thr, and 106th Thr with Val.
　また、ＶＬについては、配列番号８５のアミノ酸配列の２番目のＩｌｅをＶａｌに、８番目のＰｒｏをＬｅｕに、１１番目のＬｅｕをＧｌｎに、１５番目のＰｒｏをＬｅｕに、および９０番目のＶａｌがＬｅｕに置換するアミノ酸改変のうち、少なくとも１つの改変を導入した。 For VL, the second Ile of the amino acid sequence of SEQ ID NO: 85 is Val, the 8th Pro is Leu, the 11th Leu is Gln, the 15th Pro is Leu, and the 90th Val. At least one modification among the amino acid modifications in which is substituted for Leu was introduced.
　ＨＶ０ＬＶ０のＦＲに存在する、少なくとも１つのアミノ酸残基を改変した抗ＣＤ３３ヒト化抗体の抗体Ｖ領域として、ＨＶ０ＬＶ０、ＨＶ７ＬＶ０、ＨＶ１４ＬＶ０をそれぞれ設計した。Ｈ鎖可変領域ＨＶ７およびＨＶ１４のアミノ酸配列を、それぞれ配列番号８６、８７に示した。 HV0LV0, HV7LV0, and HV14LV0 were each designed as an antibody V region of an anti-CD33 humanized antibody in which at least one amino acid residue was present in the FR of HV0LV0. The amino acid sequences of the heavy chain variable regions HV7 and HV14 are shown in SEQ ID NOs: 86 and 87, respectively.
（２）抗ＣＤ３３ヒト化抗体の作製
　抗ＣＤ３３ヒト化抗体の可変領域のアミノ酸配列をコードするＤＮＡは、ＫＭ４０８４ＶＨおよびＫＭ４０８４ＶＬのアミノ酸配列をコードするＤＮＡ（配列番号９２、９４）で用いられているコドンを利用し、アミノ酸改変を行う場合は、哺乳動物細胞において高頻度で使用されるコドンを用いて作製した。 (2) Preparation of anti-CD33 humanized antibody The DNA encoding the variable region amino acid sequence of the anti-CD33 humanized antibody is the codon used in the DNA encoding the amino acid sequences of KM4084VH and KM4084VL (SEQ ID NOs: 92 and 94). When amino acid modification is carried out using a codon, it was prepared using codons frequently used in mammalian cells.
　抗ＣＤ３３ヒト化抗体のＨＶ０およびＬＶ０のアミノ酸配列をコードするＤＮＡ配列を配列番号８８、８９にそれぞれ示し、またアミノ酸改変を行った可変領域ＨＶ７、およびＨＶ１４のアミノ酸配列をコードするＤＮＡ配列を、配列番号９０、および９１にそれぞれ示した。 The DNA sequences encoding the amino acid sequences of HV0 and LV0 of the anti-CD33 humanized antibody are shown in SEQ ID NOs: 88 and 89, respectively, and the DNA sequences encoding the amino acid sequences of the variable regions HV7 and HV14 subjected to amino acid modification are The numbers 90 and 91 are shown respectively.
　実施例６と同様にして、各可変領域をコードするＤＮＡを作製し、抗体発現ベクターｐＫＡＮＴＥＸ９３に挿入して、抗ＣＤ３３ヒト化抗体発現ベクターを作製した。すなわち、実施例６（２）および（３）の方法に従って、作製した抗ＣＤ３３ヒト化抗体発現ベクターを動物細胞に導入し高ＡＤＣＣ型抗体としてヒト化抗体を発現させ、ＣＨとしてｈＩｇＧ１を有する抗ＣＤ３３ヒト化抗体ＨＶ０ＬＶ０、ＨＶ７ＬＶ０、およびＨＶ１４ＬＶ０を作製した。 In the same manner as in Example 6, DNA encoding each variable region was prepared and inserted into the antibody expression vector pKANTEX93 to prepare an anti-CD33 humanized antibody expression vector. That is, according to the method of Example 6 (2) and (3), the prepared anti-CD33 humanized antibody expression vector is introduced into animal cells to express the humanized antibody as a high ADCC type antibody, and anti-CD33 having hIgG1 as CH. Humanized antibodies HV0LV0, HV7LV0, and HV14LV0 were generated.
（３）ＢｉａｃｏｒｅにおけるｈＣＤ３３－Ｈｉｓとの結合活性
　上記（２）で取得した各抗ＣＤ３３ヒト化抗体の、ｈＣＤ３３－Ｈｉｓに対する結合活性を反応速度論的に解析するため、表面プラズモン共鳴法（ＳＰＲ法）を用いて結合活性測定を実施例７－（３）と同様の手法により行った。 (3) Binding activity with hCD33-His in Biacore In order to analyze the binding activity of each anti-CD33 humanized antibody obtained in (2) above with respect to hCD33-His, the surface plasmon resonance method (SPR method) is used. The binding activity was measured by the same method as in Example 7- (3).
　その結果得られた各抗体のｋａ、ｋｄおよび解離定数ＫＤを表７に示す。 The resulting ka of each antibody, the kd and dissociation constant, K D, shown in Table 7.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
　表７に示すように、作製した抗ＣＤ３３ヒト化抗体のうち、ＨＶ０ＬＶ０に結合活性は認められなかった。一方で、ＨＶ７ＬＶ０、およびＨＶ１４ＬＶ０はいずれも、ｈＣＤ３３－Ｈｉｓに対し、高いアフィニティーを有していた。また、そのアフィニティーはＫＭ４０５４と同等以上であった。よって、高い結合活性を有する抗ＣＤ３３ヒト化抗体が作製された。 As shown in Table 7, binding activity was not recognized in HV0LV0 among the prepared anti-CD33 humanized antibodies. On the other hand, both HV7LV0 and HV14LV0 had high affinity for hCD33-His. Further, the affinity was equal to or higher than that of KM4054. Therefore, an anti-CD33 humanized antibody having high binding activity was produced.
［実施例１２］
抗ＣＤ３３ヒト化抗体の活性評価
　以下の（１）から（４）では、実施例１１で作製したＫＭ４０８４のＨＶ７ＬＶ０の高ＡＤＣＣ型抗体であるＫＭ８０８４および実施例８で作製したＨＭ１９５－１、ＨＭ１９５－Ｐの活性評価を行った。陰性対照抗体として、ジニトロフェノール（ＤＮＰ）に対する高ＡＤＣＣ型抗体（ａｎｔｉ－ＤＮＰ）を用いた。また、抗体非添加サンプルを－Ａｂと示した。 [Example 12]
Evaluation of activity of anti-CD33 humanized antibody In the following (1) to (4), KM8084 which is a high ADCC type antibody of HV7LV0 of KM4084 produced in Example 11 and HM195-1, HM1955-P produced in Example 8 The activity was evaluated. As a negative control antibody, a high ADCC type antibody (anti-DNP) against dinitrophenol (DNP) was used. In addition, the antibody-free sample was shown as -Ab.
（１）ヒト血液癌細胞株に対するＡＤＣＣ活性の評価
　実施例７（４）に記載の方法に従って行った。標的細胞として、ＴＨＰ－１、ＮＢ－４、ヒト赤白血病細胞株ＴＦ－１、ヒト急性巨核球性白血病細胞株ＣＭＫ１１－５を用いた。標的細胞は１．０×１０４細胞／ウェル、エフェクター細胞は２．５×１０５細胞／ウェルとした（エフェクター細胞（Ｅ）：標的細胞（Ｔ）＝２５：１とした）。抗体濃度は、０．５μｇ／ｍＬから段階的に４倍希釈した。結果を図１２に示す。 (1) Evaluation of ADCC activity against human blood cancer cell line It was performed according to the method described in Example 7 (4). As target cells, THP-1, NB-4, human erythroleukemia cell line TF-1, and human acute megakaryocytic leukemia cell line CMK11-5 were used. Target cells were 1.0 × 10 4 cells / well, and effector cells were 2.5 × 10 5 cells / well (effector cells (E): target cells (T) = 25: 1). The antibody concentration was diluted 4-fold stepwise from 0.5 μg / mL. The results are shown in FIG.
　図１２に示すように、ＫＭ８０８４、ＨＭ１９５－１およびＨＭ１９５－Ｐは、各細胞株に対して抗体濃度依存的なＡＤＣＣ活性を示した。また、ＫＭ８０８４はＨＭ１９５－Ｐよりも強いＡＤＣＣ活性を示した。 As shown in FIG. 12, KM8084, HM195-1, and HM195-P exhibited an antibody concentration-dependent ADCC activity against each cell line. KM8084 showed stronger ADCC activity than HM195-P.
（２）ＣＤ３３発現細胞株における細胞膜上での抗体結合量
　実施例７（６）方法に従って行った。細胞としてＴＨＰ－１を用いた。１次抗体としてＫＭ８０８４およびＨＭ１９５－Ｐを用いた。 (2) Amount of antibody binding on cell membrane in CD33-expressing cell line This was performed according to the method of Example 7 (6). THP-1 was used as the cell. KM8084 and HM195-P were used as primary antibodies.
　得られた結果を図１３に示す［図１３の（Ａ）および（Ｂ）は、それぞれ抗体結合量および抗体残存率を示す］。図１３に示すように、ＫＭ８０８４およびＨＭ１９５－Ｐは時間依存的に細胞表面上での結合量が減少した。また、ＫＭ８０８４は各時間においてＨＭ１９５－Ｐに比べて、細胞表面上に結合した抗体の量および残存率ともに上回っていた。 The obtained results are shown in FIG. 13 [(A) and (B) of FIG. 13 show the amount of antibody binding and the antibody remaining rate, respectively]. As shown in FIG. 13, KM8084 and HM195-P decreased the amount of binding on the cell surface in a time-dependent manner. In addition, KM8084 exceeded both HM195-P and the amount of antibody bound to the cell surface and the residual rate at each time.
（３）ヒト末梢血中における細胞除去活性
　ＫＭ８０８４、ＨＭ１９５－１およびＨＭ１９５－Ｐの末梢血中におけるＮＢ－４およびＴＦ－１に対する細胞除去活性を以下の方法により測定した。 (3) Cell removal activity in human peripheral blood Cell removal activity of KM8084, HM195-1, and HM195-P against NB-4 and TF-1 in the peripheral blood was measured by the following method.
　ＰＫＨ２６　Ｒｅｄ　Ｆｌｕｏｒｅｓｃｅｎｔ　Ｃｅｌｌ　Ｌｉｎｋｅｒ　Ｋｉｔ（シグマ社製）に添付の説明書に従い、ＮＢ－４およびＴＦ－１を蛍光標識し、ＲＰＭＩ１６４０（インビトロジェン社製）にて希釈調製した。 NB-4 and TF-1 were fluorescently labeled according to the instructions attached to PKH26 Red Fluorescent Cell Linker Kit (manufactured by Sigma) and diluted with RPMI 1640 (manufactured by Invitrogen).
　２４ウェルプレート（ＮＵＮＣ社製）にヘパリンナトリウム注　Ｎ（清水製薬社製）を１％含む、健常人末梢血を５００μＬ／ウェルにて分注した。ＲＰＭＩ１６４０にて希釈調製した各抗体を５０μＬ、蛍光標識した細胞溶液を５０μＬ添加し全量を６００μＬとし、５％ＣＯ２インキュベーター中３７℃にて、１６時間、振とうして攪拌しながら反応させた。 Healthy human peripheral blood containing 1% of heparin sodium injection N (manufactured by Shimizu Pharmaceutical Co., Ltd.) in a 24-well plate (manufactured by NUNC) was dispensed at 500 μL / well. 50 μL of each antibody diluted with RPMI1640 and 50 μL of the fluorescently labeled cell solution were added to make a total volume of 600 μL, and the reaction was carried out with shaking and stirring at 37 ° C. for 16 hours in a 5% CO 2 incubator.
　反応後、内部標準粒子であるＦｌｏｗ　Ｂｅａｓ（ＦＢ）としてＦｌｏｗ－Ｃｏｕｎｔ（ベックマン・コールター社製）を１００μＬ／ウェル加え、混和後、各ウェルから４００μＬを１５ｍＬ遠心チューブ（ＮＵＮＣ社製）にそれぞれ移した。該チューブを４００×ｇ、５分間遠心後、上清を取り除き、各チューブにＡＣＫ　Ｌｙｓｉｎｇ　Ｂｕｆｆｅｒ（Ｌｏｎｚａ社製）を１ｍＬ添加し、３７℃、１０分間、振とう、反応させた。 After the reaction, 100 μL / well of Flow-Count (manufactured by Beckman Coulter) was added as an internal standard particle Flow Beas (FB), and after mixing, 400 μL of each well was transferred to a 15 mL centrifuge tube (manufactured by NUNC). . After centrifuging the tube at 400 × g for 5 minutes, the supernatant was removed, 1 mL of ACK Lysing Buffer (manufactured by Lonza) was added to each tube, and the mixture was reacted by shaking at 37 ° C. for 10 minutes.
　該チューブを１％ＢＳＡ－ＰＢＳにて２回洗浄し、５００μＬの１％ＢＳＡ－ＰＢＳに懸濁し、フローサイトメーター（ベックマン・コールター社製）用いて、ＦＢ数、蛍光標識した細胞数を測定した。得られた結果より、ＦＢ１０００個あたりの蛍光標識した細胞数を算出した。 The tube was washed twice with 1% BSA-PBS, suspended in 500 μL of 1% BSA-PBS, and the number of FB and the number of fluorescently labeled cells were measured using a flow cytometer (manufactured by Beckman Coulter). . From the obtained results, the number of fluorescently labeled cells per 1000 FBs was calculated.
　得られた結果を図１４に示す。図１４に示すように、ＨＭ１９５－１に対して、同抗体の高ＡＤＣＣ型抗体であるＨＭ１９５－Ｐは１０倍程度の高い活性を示した。また、ＫＭ８０８４はＨＭ１９５－１に対して１００倍以上、ＨＭ１９５－Ｐに対して数１０倍以上強い細胞除去活性を示した。 The obtained results are shown in FIG. As shown in FIG. 14, HM195-P, which is a high ADCC type antibody of the same antibody, showed about 10 times higher activity than HM195-1. In addition, KM8084 exhibited a cell removal activity 100 times or more stronger than HM195-1, and several tens of times stronger than HM195-P.
　ヒト末梢血中に白血病細胞を添加した本評価系は、急性骨髄性白血病（ＡＭＬ）患者血中の環境をモデル化した系であり、実施例１２（１）のＡＤＣＣ評価系に比べて、より臨床における薬効を反映していると考えられる。このため、ＫＭ８０８４は急性骨髄性白血病などの血液癌や骨髄細胞の病変に伴う骨髄異形性症候群に対して、ＨＭ１９５を上回る効果を示すことが示唆される。 This evaluation system in which leukemia cells are added to human peripheral blood is a system that models the environment in the blood of an acute myeloid leukemia (AML) patient, and is more compared to the ADCC evaluation system of Example 12 (1). It is thought to reflect clinical efficacy. For this reason, it is suggested that KM8084 exhibits an effect higher than that of HM195 for blood cancer such as acute myeloid leukemia and myelodysplastic syndrome accompanying bone marrow cell lesions.
（４）ヒト、カニクイザルおよびチンパンジーＣＤ３３に対する反応性
　実施例７（１）に記載の方法に従って行った。細胞には実施例１にて作製したヒトＣＤ３３発現細胞株、カニクイザルＣＤ３３発現細胞株およびチンパンジーＣＤ３３発現細胞株を用いた。１次抗体にはＫＭ８０８４、ＨＭ１９５－１およびＨＭ１９５－Ｐを段階的に希釈したもの用いた。２次抗体にはＦＩＴＣ標識ヤギ抗ヒトＩｇＧ（Ｆｃ）抗体（Ａｃｒｉｓ　Ａｎｔｉｂｏｄｉｅｓ社製）を用いた。 (4) Reactivity to human, cynomolgus monkey and chimpanzee CD33 The reaction was performed according to the method described in Example 7 (1). The cells used were the human CD33-expressing cell line, cynomolgus monkey CD33-expressing cell line, and chimpanzee CD33-expressing cell line prepared in Example 1. As primary antibodies, KM8084, HM195-1, and HM195-P were diluted serially. A FITC-labeled goat anti-human IgG (Fc) antibody (manufactured by Acris Antibodies) was used as the secondary antibody.
　図１５に、ＫＭ８０８４、ＨＭ１９５－１およびＨＭ１９５－Ｐを５０μｇ／ｍＬから各濃度１０倍希釈で段階的に希釈したものを反応させた場合のＭＦＩを示す。図１５に示すように、ＫＭ８０８４、ＨＭ１９５－１およびＨＭ１９５－Ｐはいずれも濃度依存的にヒトＣＤ３３発現細胞株へ反応し、反応性の強さに差は無かった。一方、カニクイザルＣＤ３３発現細胞株およびチンパンジーＣＤ３３発現細胞株に対して、ＫＭ８０８４は濃度依存的に反応したが、ＨＭ１９５－１およびＨＭ１９５－Ｐはほとんど反応しなかった。 FIG. 15 shows the MFI when KM8084, HM195-1, and HM195-P are reacted stepwise diluted from 50 μg / mL at 10-fold dilutions. As shown in FIG. 15, KM8084, HM195-1, and HM195-P all reacted with human CD33-expressing cell lines in a concentration-dependent manner, and there was no difference in the strength of reactivity. On the other hand, KM8084 reacted in a concentration-dependent manner to cynomolgus monkey CD33-expressing cell line and chimpanzee CD33-expressing cell line, but HM195-1 and HM195-P hardly reacted.
　各配列番号の配列は以下の通りである。
配列番号１：ヒトＣＤ３３発現ベクター作製のプライマーの塩基配列
配列番号２：ヒトＣＤ３３発現ベクター作製のプライマーの塩基配列
配列番号３：ヒトＣＤ３３のアミノ酸配列
配列番号４：ヒトＣＤ３３遺伝子の塩基配列
配列番号５：ｈＣＤ３３－Ｈｉｓ発現ベクター作製のプライマーの塩基配列
配列番号６：ｈＣＤ３３－Ｈｉｓのアミノ酸配列
配列番号７：ｈＣＤ３３－ｈｉｓ遺伝子の塩基配列
配列番号８：ラットＩｇＧ１特異的プライマーの塩基配列
配列番号９：ラットＩｇＧ２ａ特異的プライマーの塩基配列
配列番号１０：マウスＩｇＧ１特異的プライマーの塩基配列
配列番号１１：マウスＩｇＧ２ａ特異的プライマーの塩基配列
配列番号１２：ラットＩｇ（κ）特異的プライマーの塩基配列
配列番号１３：マウスＩｇ（κ）特異的プライマーの塩基配列
配列番号１４：ＫＭ４０７１　ＶＨの塩基配列
配列番号１５：ＫＭ４０７２　ＶＨの塩基配列
配列番号１６：ＫＭ４０７３　ＶＨの塩基配列
配列番号１７：ＫＭ４０７４　ＶＨの塩基配列
配列番号１８：ＫＭ４０７５　ＶＨの塩基配列
配列番号１９：ＫＭ４０７１　ＶＨのアミノ酸配列
配列番号２０：ＫＭ４０７２　ＶＨのアミノ酸配列
配列番号２１：ＫＭ４０７３　ＶＨのアミノ酸配列
配列番号２２：ＫＭ４０７４　ＶＨのアミノ酸配列
配列番号２３：ＫＭ４０７５　ＶＨのアミノ酸配列
配列番号２４：ＫＭ４０７１　ＶＬの塩基配列
配列番号２５：ＫＭ４０７２　ＶＬの塩基配列
配列番号２６：ＫＭ４０７３　ＶＬの塩基配列
配列番号２７：ＫＭ４０７４　ＶＬの塩基配列
配列番号２８：ＫＭ４０７５　ＶＬの塩基配列
配列番号２９：ＫＭ４０７１　ＶＬのアミノ酸配列
配列番号３０：ＫＭ４０７２　ＶＬのアミノ酸配列
配列番号３１：ＫＭ４０７３　ＶＬのアミノ酸配列
配列番号３２：ＫＭ４０７４　ＶＬのアミノ酸配列
配列番号３３：ＫＭ４０７５　ＶＬのアミノ酸配列
配列番号３４：ＫＭ４０７１　ＶＨ　ＣＤＲ１のアミノ酸配列
配列番号３５：ＫＭ４０７１　ＶＨ　ＣＤＲ２のアミノ酸配列
配列番号３６：ＫＭ４０７１　ＶＨ　ＣＤＲ３のアミノ酸配列
配列番号３７：ＫＭ４０７１　ＶＬ　ＣＤＲ１のアミノ酸配列
配列番号３８：ＫＭ４０７１　ＶＬ　ＣＤＲ２のアミノ酸配列
配列番号３９：ＫＭ４０７１　ＶＬ　ＣＤＲ３のアミノ酸配列
配列番号４０：ＫＭ４０７２　ＶＨ　ＣＤＲ１のアミノ酸配列
配列番号４１：ＫＭ４０７２　ＶＨ　ＣＤＲ２のアミノ酸配列
配列番号４２：ＫＭ４０７２　ＶＨ　ＣＤＲ３のアミノ酸配列
配列番号４３：ＫＭ４０７２　ＶＬ　ＣＤＲ１のアミノ酸配列
配列番号４４：ＫＭ４０７２　ＶＬ　ＣＤＲ２のアミノ酸配列
配列番号４５：ＫＭ４０７２　ＶＬ　ＣＤＲ３のアミノ酸配列
配列番号４６：ＫＭ４０７３　ＶＨ　ＣＤＲ１のアミノ酸配列
配列番号４７：ＫＭ４０７３　ＶＨ　ＣＤＲ２のアミノ酸配列
配列番号４８：ＫＭ４０７３　ＶＨ　ＣＤＲ３のアミノ酸配列
配列番号４９：ＫＭ４０７３　ＶＬ　ＣＤＲ１のアミノ酸配列
配列番号５０：ＫＭ４０７３　ＶＬ　ＣＤＲ２のアミノ酸配列
配列番号５１：ＫＭ４０７３　ＶＬ　ＣＤＲ３のアミノ酸配列
配列番号５２：ＫＭ４０７４およびＫＭ４０８４　ＶＨ　ＣＤＲ１のアミノ酸配列
配列番号５３：ＫＭ４０７４　ＶＨ　ＣＤＲ２のアミノ酸配列
配列番号５４：ＫＭ４０７４およびＫＭ４０８４　ＶＨ　ＣＤＲ３のアミノ酸配列
配列番号５５：ＫＭ４０７４およびＫＭ４０８４　ＶＬ　ＣＤＲ１のアミノ酸配列
配列番号５６：ＫＭ４０７４およびＫＭ４０８４　ＶＬ　ＣＤＲ２のアミノ酸配列
配列番号５７：ＫＭ４０７４およびＫＭ４０８４　ＶＬ　ＣＤＲ３のアミノ酸配列
配列番号５８：ＫＭ４０７５　ＶＨ　ＣＤＲ１のアミノ酸配列
配列番号５９：ＫＭ４０７５　ＶＨ　ＣＤＲ２のアミノ酸配列
配列番号６０：ＫＭ４０７５　ＶＨ　ＣＤＲ３のアミノ酸配列
配列番号６１：ＫＭ４０７５　ＶＬ　ＣＤＲ１のアミノ酸配列
配列番号６２：ＫＭ４０７５　ＶＬ　ＣＤＲ２のアミノ酸配列
配列番号６３：ＫＭ４０７５　ＶＬ　ＣＤＲ３のアミノ酸配列
配列番号６４：ＫＭ４０７１　ＶＨのキメラ化プライマーの塩基配列
配列番号６５：ＫＭ４０７１　ＶＨのキメラ化プライマーの塩基配列
配列番号６６：ＫＭ４０７１　ＶＬのキメラ化プライマー
配列番号６７：ＫＭ４０７１　ＶＬのキメラ化プライマーの塩基配列
配列番号６８：ＫＭ４０７２　ＶＨのキメラ化プライマーの塩基配列
配列番号６９：ＫＭ４０７２　ＶＨのキメラ化プライマーの塩基配列
配列番号７０：ＫＭ４０７２　ＶＬのキメラ化プライマーの塩基配列
配列番号７１：ＫＭ４０７２　ＶＬのキメラ化プライマーの塩基配列
配列番号７２：ＫＭ４０７３　ＶＨのキメラ化プライマーの塩基配列
配列番号７３：ＫＭ４０７３　ＶＨのキメラ化プライマーの塩基配列
配列番号７４：ＫＭ４０７３　ＶＬのキメラ化プライマーの塩基配列
配列番号７５：ＫＭ４０７３　ＶＬのキメラ化プライマーの塩基配列
配列番号７６：ＫＭ４０７４　ＶＨのキメラ化プライマーの塩基配列
配列番号７７：ＫＭ４０７４　ＶＨのキメラ化プライマーの塩基配列
配列番号７８：ＫＭ４０７４　ＶＬのキメラ化プライマーの塩基配列
配列番号７９：ＫＭ４０７４　ＶＬのキメラ化プライマーの塩基配列
配列番号８０：ＫＭ４０７５　ＶＨのキメラ化プライマーの塩基配列
配列番号８１：ＫＭ４０７５　ＶＨのキメラ化プライマーの塩基配列
配列番号８２：ＫＭ４０７５　ＶＬのキメラ化プライマーの塩基配列
配列番号８３：ＫＭ４０７５　ＶＬのキメラ化プライマーの塩基配列
配列番号８４：ＫＭ４０８４ヒト化抗体のＶＨのアミノ酸配列ＨＶ０
配列番号８５：ＫＭ４０８４ヒト化抗体のＶＬのアミノ酸配列ＬＶ０
配列番号８６：ＫＭ４０８４ヒト化抗体のＶＨのアミノ酸配列ＨＶ７
配列番号８７：ＫＭ４０８４ヒト化抗体のＶＨのアミノ酸配列ＨＶ１４
配列番号８８：ＫＭ４０８４ヒト化抗体　ＨＶ０の塩基配列　
配列番号８９：ＫＭ４０８４ヒト化抗体　ＬＶ０の塩基配列
配列番号９０：ＫＭ４０８４ヒト化抗体　ＨＶ７の塩基配列
配列番号９１：ＫＭ４０８４ヒト化抗体　ＨＶ１４の塩基配列
配列番号９２：Ｖｅｒ．４　ＶＨの塩基配列　／　ＫＭ４０８４　ＶＨの塩基酸配列
配列番号９３：Ｖｅｒ．４　ＶＨのアミノ酸配列　／　ＫＭ４０８４　ＶＨのアミノ酸配列　
配列番号９４：ＫＭ４０８４　ＶＬの塩基配列
配列番号９５：ＫＭ４０８４　ＶＬのアミノ酸配列
配列番号９６：Ｎ結合型糖鎖結合コンセンサス配列を有さないアミノ酸改変抗体Ｖｅｒ．４　ＣＤＲ２配列　／　ＫＭ４０８４　ＶＨ　ＣＤＲ２のアミノ酸配列
配列番号９７：ＶＨ　ＣＤＲ２アミノ酸改変抗体　ｖｅｒ．４作製用プライマー（センス鎖）の塩基配列　
配列番号９８：ＶＨ　ＣＤＲ２アミノ酸改変抗体　ｖｅｒ．４作製用プライマー（アンチセンス鎖）の塩基配列　
配列番号９９：Ｎ結合型糖鎖結合コンセンサス配列を有さないアミノ酸改変抗体Ｖｅｒ．１　ＣＤＲ２のアミノ酸配列
配列番号１００：Ｎ結合型糖鎖結合コンセンサス配列を有さないアミノ酸改変抗体Ｖｅｒ．２　ＣＤＲ２のアミノ酸配列
配列番号１０１：Ｎ結合型糖鎖結合コンセンサス配列を有さないアミノ酸改変抗体Ｖｅｒ．３　ＣＤＲ２のアミノ酸配列
配列番号１０２：Ｎ結合型糖鎖結合コンセンサス配列を有さないアミノ酸改変抗体Ｖｅｒ．５　ＣＤＲ２のアミノ酸配列
配列番号１０３：Ｖｅｒ．１　ＶＨのアミノ酸配列　
配列番号１０４：Ｖｅｒ．２　ＶＨのアミノ酸配列　
配列番号１０５：Ｖｅｒ．３　ＶＨのアミノ酸配列　
配列番号１０６：Ｖｅｒ．５　ＶＨのアミノ酸配列　
配列番号１０７：ＶＨ　ＣＤＲ２アミノ酸改変抗体　ｖｅｒ．１作製用プライマー（センス鎖）の塩基配列
配列番号１０８：ＶＨ　ＣＤＲ２アミノ酸改変抗体　ｖｅｒ．１作製用プライマー（アンチセンス鎖）の塩基配列
配列番号１０９：ＶＨ　ＣＤＲ２アミノ酸改変抗体　ｖｅｒ．２作製用プライマー（センス鎖）の塩基配列　
配列番号１１０：ＶＨ　ＣＤＲ２アミノ酸改変抗体　ｖｅｒ．２作製用プライマー（アンチセンス鎖）の塩基配列　
配列番号１１１：ＶＨ　ＣＤＲ２アミノ酸改変抗体　ｖｅｒ．３作製用プライマー（センス鎖）の塩基配列　
配列番号１１２：ＶＨ　ＣＤＲ２アミノ酸改変抗体　ｖｅｒ．３作製用プライマー（アンチセンス鎖）の塩基配列　
配列番号１１３：ＶＨ　ＣＤＲ２アミノ酸改変抗体　ｖｅｒ．５作製用プライマー（センス鎖）の塩基配列　
配列番号１１４：ＶＨ　ＣＤＲ２アミノ酸改変抗体　ｖｅｒ．５作製用プライマー（アンチセンス鎖）の塩基配列　
配列番号１１５：カニクイザルＣＤ３３発現ベクター作製のプライマーの塩基配列
配列番号１１６：カニクイザルＣＤ３３発現ベクター作製のプライマーの塩基配列
配列番号１１７：カニクイザルＣＤ３３のアミノ酸配列
配列番号１１８：カニクイザルＣＤ３３の塩基配列
配列番号１１９：チンパンジーＣＤ３３全合成遺伝子の塩基配列
配列番号１２０：チンパンジーＣＤ３３のアミノ酸配列
配列番号１２１：チンパンジーＣＤ３３の塩基配列
配列番号１２２：ＫＭ４０７１　ＶＨの塩基配列（シグナル配列なし）
配列番号１２３：ＫＭ４０７２　ＶＨの塩基配列（シグナル配列なし）
配列番号１２４：ＫＭ４０７３　ＶＨの塩基配列（シグナル配列なし）
配列番号１２５：ＫＭ４０７４　ＶＨの塩基配列（シグナル配列なし）
配列番号１２６：ＫＭ４０７５　ＶＨの塩基配列（シグナル配列なし）
配列番号１２７：ＫＭ４０７１　ＶＬの塩基配列（シグナル配列なし）
配列番号１２８：ＫＭ４０７２　ＶＬの塩基配列（シグナル配列なし）
配列番号１２９：ＫＭ４０７３　ＶＬの塩基配列（シグナル配列なし）
配列番号１３０：ＫＭ４０７４　ＶＬの塩基配列（シグナル配列なし）
配列番号１３１：ＫＭ４０７５　ＶＬの塩基配列（シグナル配列なし）
配列番号１３２：ＫＭ４０７１　ＶＨのアミノ酸配列（シグナル配列なし）
配列番号１３３：ＫＭ４０７２　ＶＨのアミノ酸配列（シグナル配列なし）
配列番号１３４：ＫＭ４０７３　ＶＨのアミノ酸配列（シグナル配列なし）
配列番号１３５：ＫＭ４０７４　ＶＨのアミノ酸配列（シグナル配列なし）
配列番号１３６：ＫＭ４０７５　ＶＨのアミノ酸配列（シグナル配列なし）
配列番号１３７：ＫＭ４０７１　ＶＬのアミノ酸配列（シグナル配列なし）
配列番号１３８：ＫＭ４０７２　ＶＬのアミノ酸配列（シグナル配列なし）
配列番号１３９：ＫＭ４０７３　ＶＬのアミノ酸配列（シグナル配列なし）
配列番号１４０：ＫＭ４０７４　ＶＬのアミノ酸配列（シグナル配列なし）
配列番号１４１：ＫＭ４０７５　ＶＬのアミノ酸配列（シグナル配列なし） The sequence of each SEQ ID NO is as follows.
SEQ ID NO: 1: nucleotide sequence of primer for preparing human CD33 expression vector SEQ ID NO: 2: nucleotide sequence of primer for preparing human CD33 expression vector SEQ ID NO: 3: amino acid sequence of human CD33 SEQ ID NO: 4: nucleotide sequence of human CD33 gene SEQ ID NO: 5 : Nucleotide sequence of primer for preparing hCD33-His expression vector SEQ ID NO: 6: amino acid sequence of hCD33-His SEQ ID NO: 7: nucleotide sequence of hCD33-his sequence SEQ ID NO: 8: nucleotide sequence of rat IgG1-specific primer SEQ ID NO: 9: rat Base sequence of IgG2a-specific primer SEQ ID NO: 10: Base sequence of mouse IgG1-specific primer SEQ ID NO: 11: Base sequence of mouse IgG2a-specific primer SEQ ID NO: 12: Base sequence of rat Ig (κ) -specific primer SEQ ID NO: 13: Mouse Ig (κ) specific Primer nucleotide sequence SEQ ID NO: 14: KM4071 VH nucleotide sequence SEQ ID NO: 15: KM4072 VH nucleotide sequence SEQ ID NO: 16: KM4073 VH nucleotide sequence SEQ ID NO: 17: KM4074 VH nucleotide sequence SEQ ID NO: 18: KM4075 VH nucleotide sequence No. 19: KM4071 VH amino acid sequence SEQ ID NO: 20: KM4072 VH amino acid sequence SEQ ID NO: 21: KM4073 VH amino acid sequence SEQ ID NO: 22: KM4074 VH amino acid sequence SEQ ID NO: 23: KM4075 VH amino acid sequence SEQ ID NO: 24: KM4071 VL Nucleotide sequence SEQ ID NO: 25: KM4072 VL nucleotide sequence SEQ ID NO: 26: KM4073 VL nucleotide sequence SEQ ID NO: 27: KM4074 VL nucleotide sequence SEQ ID NO: 28: KM4075 VL nucleotide sequence SEQ ID NO: 29: K 4071 VL amino acid sequence SEQ ID NO: 30: KM4072 VL amino acid sequence SEQ ID NO: 31: KM4073 VL amino acid sequence SEQ ID NO: 32: KM4074 VL amino acid sequence SEQ ID NO: 33: KM4075 VL amino acid sequence SEQ ID NO: 34: KM4071 VH CDR1 amino acid SEQ ID NO: 35: amino acid sequence of KM4071 VH CDR2 SEQ ID NO: 36: amino acid sequence of KM4071 VH CDR3 SEQ ID NO: 37: amino acid sequence of KM4071 VL CDR1 SEQ ID NO: 38: amino acid sequence of KM4071 VL CDR2 SEQ ID NO: 39: amino acid of KM4071 VL CDR3 SEQ ID NO: 40: KM4072 VH CDR1 amino acid sequence SEQ ID NO: 41: KM4072 VH CDR2 amino acid sequence SEQ ID NO: 42: KM4072 VHC DR3 amino acid sequence SEQ ID NO: 43: KM4072 VL CDR1 amino acid sequence SEQ ID NO: 44: KM4072 VL CDR2 amino acid sequence SEQ ID NO: 45: KM4072 VL CDR3 amino acid sequence SEQ ID NO: 46: KM4073 VH CDR1 amino acid sequence SEQ ID NO: 47: KM4073 VH CDR2 amino acid sequence SEQ ID NO: 48: KM4073 VH CDR3 amino acid sequence SEQ ID NO: 49: KM4073 VL CDR1 amino acid sequence SEQ ID NO: 50: KM4073 VL CDR2 amino acid sequence SEQ ID NO: 51: KM4073 VL CDR3 amino acid sequence SEQ ID NO: 52: KM4074 and Amino acid sequence of KM4084 VH CDR1 SEQ ID NO: 53: Amino acid sequence of KM4074 VH CDR2 SEQ ID NO: 54: KM4074 and KM408 4 VH CDR3 amino acid sequence SEQ ID NO: 55: KM4074 and KM4084 VL CDR1 amino acid sequence SEQ ID NO: 56: KM4074 and KM4084 VL CDR2 amino acid sequence SEQ ID NO: 57: KM4074 and KM4084 VL CDR3 amino acid sequence SEQ ID NO: 58: KM4075 VH CDR1 Amino acid sequence SEQ ID NO: 59: KM4075 VH CDR2 amino acid sequence SEQ ID NO: 60: KM4075 VH CDR3 amino acid sequence SEQ ID NO: 61: KM4075 VL CDR1 amino acid sequence SEQ ID NO: 62: KM4075 VL CDR2 amino acid sequence SEQ ID NO: 63: KM4075 VL CDR3 Amino acid sequence SEQ ID NO: 64: KM4071 VH chimerization primer base sequence SEQ ID NO: 65: KM4071 VH chimerization sequence Limer base sequence SEQ ID NO: 66: chimerization primer of KM4071 VL SEQ ID NO: 67: base sequence of chimerization primer of KM4071 VL SEQ ID NO: 68: base sequence of chimerization primer of KM4072 VH SEQ ID NO: 69: chimerization primer of KM4072 VH Nucleotide sequence SEQ ID NO: 70: nucleotide sequence of KM4072 VL chimerization primer SEQ ID NO: 71: nucleotide sequence of KM4072 VL chimerization primer SEQ ID NO: 72: nucleotide sequence of KM4073 VH chimerization primer SEQ ID NO: 73: chimera of KM4073 VH SEQ ID NO: 74: nucleotide sequence of chimerized primer of KM4073 VL SEQ ID NO: 75: nucleotide sequence of chimerized primer of KM4073 VL SEQ ID NO: 76: chimerized primer of KM4074 VH Base sequence SEQ ID NO: 77: Base sequence of chimerization primer of KM4074 VH SEQ ID NO: 78: Base sequence of chimerization primer of KM4074 VL SEQ ID NO: 79: Base sequence of chimerization primer of KM4074 VL SEQ ID NO: 80: Chimerization of KM4075 VH Primer base sequence SEQ ID NO: 81: KM4075 VH chimerized primer base sequence SEQ ID NO: 82: KM4075 VL chimerized primer base sequence SEQ ID NO: 83: KM4075 VL chimerized primer base sequence SEQ ID NO: 84: KM4084 humanized Antibody VH amino acid sequence HV0
SEQ ID NO: 85: VL amino acid sequence LV0 of the KM4084 humanized antibody
SEQ ID NO: 86: Amino acid sequence HV7 of the VH of the KM4084 humanized antibody
SEQ ID NO: 87: VH amino acid sequence HV14 of KM4084 humanized antibody
SEQ ID NO: 88: base sequence of KM4084 humanized antibody HV0
SEQ ID NO: 89: KM4084 humanized antibody LV0 nucleotide sequence SEQ ID NO: 90: KM4084 humanized antibody HV7 nucleotide sequence SEQ ID NO: 91: KM4084 humanized antibody HV14 nucleotide sequence SEQ ID NO: 92: Ver. 4 VH base sequence / KM4084 VH base acid sequence SEQ ID NO: 93: Ver. 4 VH amino acid sequence / KM4084 VH amino acid sequence
SEQ ID NO: 94: Base sequence of KM4084 VL SEQ ID NO: 95: Amino acid sequence of KM4084 VL SEQ ID NO: 96: Amino acid-modified antibody without the N-linked sugar chain binding consensus sequence Ver. 4 CDR2 sequence / KM4084 VH CDR2 amino acid sequence SEQ ID NO: 97: VH CDR2 amino acid modified antibody ver. 4. Base sequence of primer for preparation (sense strand)
SEQ ID NO: 98: VH CDR2 amino acid modified antibody ver. 4. Base sequence of primer for preparation (antisense strand)
SEQ ID NO: 99: Amino acid-modified antibody Ver. 1 amino acid sequence of CDR2 SEQ ID NO: 100: amino acid-modified antibody Ver. 2 CDR2 amino acid sequence SEQ ID NO: 101: Amino acid-modified antibody Ver. 3 amino acid sequence of CDR2 SEQ ID NO: 102: amino acid-modified antibody Ver.1 having no N-linked sugar chain-binding consensus sequence 5 amino acid sequence of CDR2 SEQ ID NO: 103: Ver. 1 VH amino acid sequence
SEQ ID NO: 104: Ver. 2 VH amino acid sequence
SEQ ID NO: 105: Ver. 3 VH amino acid sequence
SEQ ID NO: 106: Ver. 5 VH amino acid sequence
SEQ ID NO: 107: VH CDR2 amino acid modified antibody ver. 1. Base sequence of primer for preparation (sense strand) SEQ ID NO: 108: VH CDR2 amino acid-modified antibody ver. 1 Base sequence of primer for preparation (antisense strand) SEQ ID NO: 109: VH CDR2 amino acid modified antibody ver. 2 Base sequence of primer for preparation (sense strand)
SEQ ID NO: 110: VH CDR2 amino acid modified antibody ver. 2 Base sequence of primer for preparation (antisense strand)
SEQ ID NO: 111: VH CDR2 amino acid modified antibody ver. 3. Base sequence of primer for preparation (sense strand)
SEQ ID NO: 112: VH CDR2 amino acid modified antibody ver. 3 Base sequence of primer for preparation (antisense strand)
SEQ ID NO: 113: VH CDR2 amino acid modified antibody ver. 5 Base sequence of primer for preparation (sense strand)
SEQ ID NO: 114: VH CDR2 amino acid modified antibody ver. 5 Base sequence of primer for preparation (antisense strand)
SEQ ID NO: 115: base sequence of primer for preparing cynomolgus monkey CD33 expression vector SEQ ID NO: 116: base sequence of primer for preparing cynomolgus monkey CD33 expression vector SEQ ID NO: 117: amino acid sequence of cynomolgus monkey CD33 SEQ ID NO: 118: base sequence of cynomolgus monkey CD33 Base sequence of chimpanzee CD33 total synthetic gene SEQ ID NO: 120: amino acid sequence of chimpanzee CD33 SEQ ID NO: 121: base sequence of chimpanzee CD33 SEQ ID NO: 122: base sequence of KM4071 VH (no signal sequence)
SEQ ID NO: 123: KM4072 VH base sequence (no signal sequence)
SEQ ID NO: 124: Base sequence of KM4073 VH (no signal sequence)
SEQ ID NO: 125: KM4074 VH base sequence (no signal sequence)
SEQ ID NO: 126: KM4075 VH base sequence (no signal sequence)
SEQ ID NO: 127: KM4071 VL base sequence (no signal sequence)
SEQ ID NO: 128: KM4072 VL base sequence (no signal sequence)
SEQ ID NO: 129: Base sequence of KM4073 VL (no signal sequence)
SEQ ID NO: 130: KM4074 VL base sequence (no signal sequence)
SEQ ID NO: 131: KM4075 VL base sequence (no signal sequence)
SEQ ID NO: 132: amino acid sequence of KM4071 VH (no signal sequence)
SEQ ID NO: 133: KM4072 VH amino acid sequence (no signal sequence)
SEQ ID NO: 134: amino acid sequence of KM4073 VH (no signal sequence)
SEQ ID NO: 135: amino acid sequence of KM4074 VH (no signal sequence)
SEQ ID NO: 136: KM4075 VH amino acid sequence (no signal sequence)
SEQ ID NO: 137: Amino acid sequence of KM4071 VL (no signal sequence)
SEQ ID NO: 138: Amino acid sequence of KM4072 VL (no signal sequence)
SEQ ID NO: 139: amino acid sequence of KM4073 VL (no signal sequence)
SEQ ID NO: 140: amino acid sequence of KM4074 VL (no signal sequence)
SEQ ID NO: 141: amino acid sequence of KM4075 VL (no signal sequence)
　本発明を特定の態様を用いて詳細に説明したが、本発明の意図と範囲を離れることなく様々な変更および変形が可能であることは、当業者にとって明らかである。なお、本出願は、２０１０年１２月３日付けで出願された米国仮出願（６１／４１９３４２号）に基づいており、その全体が引用により援用される。 Although the present invention has been described in detail using specific embodiments, it will be apparent to those skilled in the art that various modifications and variations can be made without departing from the spirit and scope of the invention. In addition, this application is based on the US provisional application (61/419342) for which it applied on December 3, 2010, The whole is used by reference.

Claims (33)

  1. （ｉ）配列番号５２で示されるアミノ酸配列を含むＣＤＲ１、配列番号９９で示されるアミノ酸配列を含むＣＤＲ２、および配列番号５４で示されるアミノ酸配列を含むＣＤＲ３を含む重鎖可変領域、並びに配列番号５５で示されるアミノ酸配列を含むＣＤＲ１、配列番号５６で示されるアミノ酸配列を含むＣＤＲ２、および配列番号５７で示されるアミノ酸配列を含むＣＤＲ３を含む軽鎖可変領域を含む、ヒトＣＤ３３に結合するモノクローナル抗体または該抗体の断片、
    （ｉｉ）配列番号５２で示されるアミノ酸配列を含むＣＤＲ１、配列番号１００で示されるアミノ酸配列を含むＣＤＲ２、および配列番号５４で示されるアミノ酸配列を含むＣＤＲ３を含む重鎖可変領域、並びに配列番号５５で示されるアミノ酸配列を含むＣＤＲ１、配列番号５６で示されるアミノ酸配列を含むＣＤＲ２、および配列番号５７で示されるアミノ酸配列を含むＣＤＲ３を含む軽鎖可変領域を含む、ヒトＣＤ３３に結合するモノクローナル抗体または該抗体の断片、
    （ｉｉｉ）配列番号５２で示されるアミノ酸配列を含むＣＤＲ１、配列番号１０１で示されるアミノ酸配列を含むＣＤＲ２、および配列番号５４で示されるアミノ酸配列を含むＣＤＲ３を含む重鎖可変領域、並びに配列番号５５で示されるアミノ酸配列を含むＣＤＲ１、配列番号５６で示されるアミノ酸配列を含むＣＤＲ２、および配列番号５７で示されるアミノ酸配列を含むＣＤＲ３を含む軽鎖可変領域を含む、ヒトＣＤ３３に結合するモノクローナル抗体または該抗体の断片、または
    （ｉｖ）配列番号５２で示されるアミノ酸配列を含むＣＤＲ１、配列番号１０２で示されるアミノ酸配列を含むＣＤＲ２、および配列番号５４で示されるアミノ酸配列を含むＣＤＲ３を含む重鎖可変領域、並びに配列番号５５で示されるアミノ酸配列を含むＣＤＲ１、配列番号５６で示されるアミノ酸配列を含むＣＤＲ２、および配列番号５７で示されるアミノ酸配列を含むＣＤＲ３を含む軽鎖可変領域を含む、ヒトＣＤ３３に結合するモノクローナル抗体または該抗体の断片。     (I) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 52, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 99, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 54, and SEQ ID NO: 55 A monoclonal antibody that binds to human CD33, comprising a light chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 56, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 56, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 57, or A fragment of the antibody,
    (Ii) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 52, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 100, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 54, and SEQ ID NO: 55 A monoclonal antibody that binds to human CD33, comprising a light chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 56, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 56, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 57, or A fragment of the antibody,
    (Iii) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 52, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 101, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 54, and SEQ ID NO: 55 A monoclonal antibody that binds to human CD33, comprising a light chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 56, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 56, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 57, or A heavy chain variable comprising a fragment of said antibody, or (iv) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 52, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 102, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 54 Region and the amino acid sequence shown in SEQ ID NO: 55. CDR1, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 56 and a light chain variable region comprising a CDR3 comprising an amino acid sequence shown in SEQ ID NO: 57, a fragment of a monoclonal antibody or the antibody that binds human CD33.
  2. 　配列番号５２で示されるアミノ酸配列を含むＣＤＲ１、配列番号９６で示されるアミノ酸配列を含むＣＤＲ２、および配列番号５４で示されるアミノ酸配列を含むＣＤＲ３を含む重鎖可変領域、並びに配列番号５５で示されるアミノ酸配列を含むＣＤＲ１、配列番号５６で示されるアミノ酸配列を含むＣＤＲ２、および配列番号５７で示されるアミノ酸配列を含むＣＤＲ３を有する軽鎖可変領域を含む、ヒトＣＤ３３に結合するモノクローナル抗体または該抗体の断片。 A heavy chain variable region comprising CDR1, comprising the amino acid sequence represented by SEQ ID NO: 52, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 96, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 54, and represented by SEQ ID NO: 55 A monoclonal antibody that binds to human CD33, comprising a CDR1 comprising an amino acid sequence, a CDR2 comprising an amino acid sequence represented by SEQ ID NO: 56, and a light chain variable region having a CDR3 comprising an amino acid sequence represented by SEQ ID NO: 57 fragment.
  3. 　配列番号８６で示されるアミノ酸配列を含む重鎖可変領域および配列番号８５で示されるアミノ酸配列を含む軽鎖可変領域を含む、請求項２に記載のモノクローナル抗体または該抗体の断片。 The monoclonal antibody or fragment of the antibody according to claim 2, comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 86 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 85.
  4. 　配列番号８７で示されるアミノ酸配列を含む重鎖可変領域および配列番号８５で示されるアミノ酸配列を含む軽鎖可変領域を含む、請求項２に記載のモノクローナル抗体または該抗体の断片。 The monoclonal antibody or fragment of the antibody according to claim 2, comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 87 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 85.
  5. （ｉ）配列番号３４で示されるアミノ酸配列を含むＣＤＲ１、配列番号３５で示されるアミノ酸配列を含むＣＤＲ２、および配列番号３６で示されるアミノ酸配列を含むＣＤＲ３を含む重鎖可変領域、並びに配列番号３７で示されるアミノ酸配列を含むＣＤＲ１、配列番号３８で示されるアミノ酸配列を含むＣＤＲ２、および配列番号３９で示されるアミノ酸配列を含むＣＤＲ３を含む軽鎖可変領域を含む、ヒトＣＤ３３に結合するモノクローナル抗体または該抗体の断片、
    （ｉｉ）配列番号４０で示されるアミノ酸配列を含むＣＤＲ１、配列番号４１で示されるアミノ酸配列を含むＣＤＲ２、および配列番号４２で示されるアミノ酸配列を含むＣＤＲ３を含む重鎖可変領域、並びに配列番号４３で示されるアミノ酸配列を含むＣＤＲ１、配列番号４４で示されるアミノ酸配列を含むＣＤＲ２、および配列番号４５で示されるアミノ酸配列を含むＣＤＲ３を含む軽鎖可変領域を含む、ヒトＣＤ３３に結合するモノクローナル抗体または該抗体の断片、
    （ｉｉｉ）配列番号４６で示されるアミノ酸配列を含むＣＤＲ１、配列番号４７で示されるアミノ酸配列を含むＣＤＲ２、および配列番号４８で示されるアミノ酸配列を含むＣＤＲ３を含む重鎖可変領域、並びに配列番号４９示されるアミノ酸配列を含むＣＤＲ１、配列番号５０で示されるアミノ酸配列を含むＣＤＲ２、および配列番号５１で示されるアミノ酸配列を含むＣＤＲ３を含む軽鎖可変領域を含む、ヒトＣＤ３３に結合するモノクローナル抗体または該抗体の断片、
    （ｉｖ）配列番号５２で示されるアミノ酸配列を含むＣＤＲ１、配列番号５３で示されるアミノ酸配列を含むＣＤＲ２、および配列番号５４で示されるアミノ酸配列を含むＣＤＲ３を含む重鎖可変領域、並びに配列番号５５示されるアミノ酸配列を含むＣＤＲ１、配列番号５６で示されるアミノ酸配列を含むＣＤＲ２、および配列番号５７で示されるアミノ酸配列を含むＣＤＲ３を含む軽鎖可変領域を含む、ヒトＣＤ３３に結合するモノクローナル抗体または該抗体の断片、または
    （ｖ）配列番号５８で示されるアミノ酸配列を含むＣＤＲ１、配列番号５９で示されるアミノ酸配列を含むＣＤＲ２、および配列番号６０で示されるアミノ酸配列を含むＣＤＲ３を含む重鎖可変領域、並びに配列番号６１示されるアミノ酸配列を含むＣＤＲ１、配列番号６２で示されるアミノ酸配列を含むＣＤＲ２、および配列番号６３で示されるアミノ酸配列を含むＣＤＲ３を含む軽鎖可変領域を含む、ヒトＣＤ３３に結合するモノクローナル抗体または該抗体の断片。     (I) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36, and SEQ ID NO: 37 A monoclonal antibody that binds to human CD33, comprising a light chain variable region comprising CDR1, comprising the amino acid sequence represented by SEQ ID NO: 38, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 38, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 39; A fragment of the antibody,
    (Ii) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 40, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 41, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 42, and SEQ ID NO: 43 A monoclonal antibody that binds to human CD33, comprising a light chain variable region comprising CDR1, comprising the amino acid sequence represented by SEQ ID NO: 44, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 44, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 45, or A fragment of the antibody,
    (Iii) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 46, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 47, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 48, and SEQ ID NO: 49 A monoclonal antibody that binds to human CD33, comprising a CDR1 comprising the amino acid sequence shown, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 50, and a light chain variable region comprising CDR3 comprising the amino acid sequence represented by SEQ ID NO: 51, or Antibody fragments,
    (Iv) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 52, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 53, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 54, and SEQ ID NO: 55 A monoclonal antibody that binds to human CD33 comprising a CDR1 comprising the amino acid sequence shown, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 56, and a light chain variable region comprising CDR3 comprising the amino acid sequence represented by SEQ ID NO: 57 or A heavy chain variable region comprising a fragment of an antibody, or (v) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 58, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 59, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 60 As well as a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 61, CDR2 comprising an amino acid sequence represented by sequence number 62, and comprises a light chain variable region comprising a CDR3 comprising an amino acid sequence represented by SEQ ID NO: 63, a fragment of a monoclonal antibody or the antibody that binds human CD33.
  6. （ｉ）配列番号１３２で示されるアミノ酸配列を含む重鎖可変領域および配列番号１３７で示されるアミノ酸配列を含む軽鎖可変領域を含むヒトＣＤ３３に結合するモノクローナル抗体、
    （ｉｉ）配列番号１３３で示されるアミノ酸配列を含む重鎖可変領域および配列番号１３８で示されるアミノ酸配列を含む軽鎖可変領域を含むヒトＣＤ３３に結合するモノクローナル抗体、
    （ｉｉｉ）配列番号１３４で示されるアミノ酸配列を含む重鎖可変領域および配列番号１３９で示されるアミノ酸配列を含む軽鎖可変領域を含むヒトＣＤ３３に結合するモノクローナル抗体、
    （ｉｖ）配列番号１３５で示されるアミノ酸配列を含む重鎖可変領域および配列番号１４０で示されるアミノ酸配列を含む軽鎖可変領域を含むヒトＣＤ３３に結合するモノクローナル抗体、または
    （ｖ）配列番号１３６で示されるアミノ酸配列を含む重鎖可変領域および配列番号１４１で示されるアミノ酸配列を含む軽鎖可変領域を含むヒトＣＤ３３に結合するモノクローナル抗体が結合するヒトＣＤ３３上のエピトープに結合する、ヒトＣＤ３３に結合するモノクローナル抗体または該抗体の断片。     (I) a monoclonal antibody that binds to human CD33 comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 132 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 137;
    (Ii) a monoclonal antibody that binds to human CD33 comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 133 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 138;
    (Iii) a monoclonal antibody that binds to human CD33 comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 134 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 139,
    (Iv) a monoclonal antibody that binds to human CD33 comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 135 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 140, or (v) in SEQ ID NO: 136 Binds to human CD33, which binds to an epitope on human CD33 to which a monoclonal antibody that binds to human CD33, which comprises a heavy chain variable region comprising the amino acid sequence shown and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 141 A monoclonal antibody or a fragment of said antibody.
  7. （ｉ）配列番号１３２で示されるアミノ酸配列を含む重鎖可変領域および配列番号１３７で示されるアミノ酸配列を含む軽鎖可変領域を含むヒトＣＤ３３に結合するモノクローナル抗体、
    （ｉｉ）配列番号１３３で示されるアミノ酸配列を含む重鎖可変領域および配列番号１３８で示されるアミノ酸配列を含む軽鎖可変領域を含むヒトＣＤ３３に結合するモノクローナル抗体、
    （ｉｉｉ）配列番号１３４で示されるアミノ酸配列を含む重鎖可変領域および配列番号１３９で示されるアミノ酸配列を含む軽鎖可変領域を含むヒトＣＤ３３に結合するモノクローナル抗体、
    （ｉｖ）配列番号１３５で示されるアミノ酸配列を含む重鎖可変領域および配列番号１４０で示されるアミノ酸配列を含む軽鎖可変領域を含むヒトＣＤ３３に結合するモノクローナル抗体、または
    （ｖ）配列番号１３６で示されるアミノ酸配列を含む重鎖可変領域および配列番号１４１で示されるアミノ酸配列を含む軽鎖可変領域を含むヒトＣＤ３３に結合するモノクローナル抗体と競合してヒトＣＤ３３に結合するモノクローナル抗または該抗体の断片。     (I) a monoclonal antibody that binds to human CD33 comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 132 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 137;
    (Ii) a monoclonal antibody that binds to human CD33 comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 133 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 138;
    (Iii) a monoclonal antibody that binds to human CD33 comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 134 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 139,
    (Iv) a monoclonal antibody that binds to human CD33 comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 135 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 140, or (v) in SEQ ID NO: 136 Monoclonal anti or a fragment of said antibody that binds to human CD33 in competition with a monoclonal antibody that binds to human CD33 comprising a heavy chain variable region comprising the amino acid sequence shown and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 141 .
  8. 　ヒトＣＤ３３に対する解離定数ＫＤが４．０×１０－９Ｍ以下である、請求項１から７のいずれか１項に記載のモノクローナル抗体または該抗体の断片。 Dissociation constant K D for human CD33 is 4.0 × 10 -9 M or less, a monoclonal antibody or fragment of the antibody according to any one of claims 1 to 7.
  9. 　ヒトＣＤ３３に対する解離定数ＫＤが２．０×１０－９Ｍ以下である、請求項８に記載のモノクローナル抗体または該抗体の断片。 Dissociation constant K D for human CD33 is the 2.0 × 10 -9 M or less, a monoclonal antibody or fragment of the antibody of claim 8.
  10. 　重鎖定常領域を含む請求項１から９のいずれか１項に記載のモノクローナル抗体または該抗体の断片であって、重鎖定常領域のクラスがヒトＩｇＧであるモノクローナル抗体または該抗体の断片。 The monoclonal antibody or fragment of the antibody according to any one of claims 1 to 9, comprising a heavy chain constant region, wherein the heavy chain constant region class is human IgG or a fragment of the antibody.
  11. 　ヒトＩｇＧのサブクラスがヒトＩｇＧ１である、請求項１０に記載のモノクローナル抗体または該抗体の断片。 The monoclonal antibody or fragment of the antibody according to claim 10, wherein the subclass of human IgG is human IgG1.
  12. 　重鎖定常領域を含む請求項１から１１のいずれか１項に記載のモノクローナル抗体または該抗体の断片であって、重鎖定常領域のＦｃに結合しているＮ結合複合型糖鎖にフコースが結合していないモノクローナル抗体または該抗体の断片。 The monoclonal antibody according to any one of claims 1 to 11, or a fragment of the antibody, comprising a heavy chain constant region, wherein fucose is present in an N-linked complex type sugar chain that is bound to Fc of the heavy chain constant region. Unbound monoclonal antibody or fragment of the antibody.
  13. 　軽鎖定常領域を含む請求項１から１２のいずれか１項に記載のモノクローナル抗体または該抗体の断片であって、軽鎖定常領域のクラスがヒトκであるモノクローナル抗体または該抗体の断片。 The monoclonal antibody or fragment of the antibody according to any one of claims 1 to 12, which comprises a light chain constant region, wherein the light chain constant region class is human κ.
  14. 　Ｆａｂ、Ｆ（ａｂ’）２、Ｆａｂ’、ｓｃＦｖ、ｄｉａｂｏｄｙ、ｄｓＦｖまたはＣＤＲを含むペプチドである、請求項１から１３のいずれか１項に記載の抗体の断片。 14. The antibody fragment according to any one of claims 1 to 13, which is a peptide comprising Fab, F (ab ') 2 , Fab', scFv, diabody, dsFv or CDR.
  15. 　請求項１から１４のいずれか１項に記載のモノクローナル抗体または該抗体の断片をコードするＤＮＡ。 DNA encoding the monoclonal antibody or fragment of the antibody according to any one of claims 1 to 14.
  16. 　請求項１５に記載のＤＮＡを含有する組換えベクター。 A recombinant vector containing the DNA according to claim 15.
  17. （ｉ）配列番号８９で示されるアミノ酸配列を含む軽鎖可変領域をコードするＤＮＡを含む組換えベクター、
    （ｉｉ）配列番号９０で示されるアミノ酸配列を含む重鎖可変領域をコードするＤＮＡを含む組換えベクター、
    （ｉｉｉ）配列番号９１で示されるアミノ酸配列を含む重鎖可変領域をコードするＤＮＡを含む組換えベクター、
    （ｉｖ）配列番号９０で示されるアミノ酸配列を含む重鎖可変領域をコードするＤＮＡおよび配列番号８９で示されるアミノ酸配列を含む軽鎖可変領域をコードするＤＮＡを含む組換えベクター、
    または
    （ｖ）配列番号９１で示されるアミノ酸配列を含む重鎖可変領域をコードするＤＮＡおよび配列番号８９で示されるアミノ酸配列を含む軽鎖可変領域をコードするＤＮＡを含む組換えベクター。     (I) a recombinant vector comprising DNA encoding a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 89;
    (Ii) a recombinant vector comprising DNA encoding a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 90;
    (Iii) a recombinant vector comprising DNA encoding a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 91;
    (Iv) a recombinant vector comprising DNA encoding a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 90 and DNA encoding a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 89;
    Or (v) a recombinant vector comprising DNA encoding a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 91 and DNA encoding a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 89.
  18. 　請求項１６および１７に記載の組換えベクターから選ばれる少なくとも１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体。 A transformant obtained by introducing at least one recombinant vector selected from the recombinant vectors according to claims 16 and 17 into a host cell.
  19. 　以下の（ｉ）または（ｉｉ）の組換えベクターを宿主細胞に導入して得られる請求項１８に記載の形質転換体。
    （ｉ）配列番号８９で示されるアミノ酸配列を含む軽鎖可変領域をコードするＤＮＡを含む組換えベクターおよび配列番号９０で示されるアミノ酸配列を含む重鎖可変領域をコードするＤＮＡを含む組換えベクター
    （ｉｉ）配列番号８９で示されるアミノ酸配列を含む軽鎖可変領域をコードするＤＮＡを含む組換えベクターおよび配列番号９１で示されるアミノ酸配列を含む重鎖可変領域をコードするＤＮＡを含む組換えベクター     The transformant according to claim 18, which is obtained by introducing the following recombinant vector (i) or (ii) into a host cell.
    (I) a recombinant vector comprising a DNA encoding a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 89 and a recombinant vector comprising a DNA encoding a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 90 (Ii) a recombinant vector comprising a DNA encoding a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 89 and a recombinant vector comprising a DNA encoding a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 91
  20. 　請求項１８または１９に記載の形質転換体を培地に培養し、培養物中に請求項１から１４のいずれか１項に記載のモノクローナル抗体または該抗体の断片を生成蓄積させ、該培養物から該モノクローナル抗体または該抗体の断片を採取することを特徴とする、請求項１から１４のいずれか１項に記載のモノクローナル抗体または該抗体の断片の製造方法。 The transformant according to claim 18 or 19 is cultured in a medium, the monoclonal antibody according to any one of claims 1 to 14 or a fragment of the antibody is produced and accumulated in the culture, and the culture is used. The method for producing a monoclonal antibody or a fragment of the antibody according to any one of claims 1 to 14, wherein the monoclonal antibody or a fragment of the antibody is collected.
  21. 　請求項１から１４のいずれか１項に記載のモノクローナル抗体または該抗体の断片を有効成分として含有する医薬組成物。 A pharmaceutical composition comprising the monoclonal antibody according to any one of claims 1 to 14 or a fragment of the antibody as an active ingredient.
  22. 　請求項１から１４のいずれか１項に記載のモノクローナル抗体または該抗体の断片を有効成分として含有する、ＣＤ３３陽性細胞が関与する疾患の治療剤。 A therapeutic agent for a disease involving CD33-positive cells, comprising the monoclonal antibody according to any one of claims 1 to 14 or a fragment of the antibody as an active ingredient.
  23. 　ＣＤ３３陽性細胞が関与する疾患が、癌、自己免疫疾患またはアレルギー性疾患である、請求項２２に記載の治療剤。 The therapeutic agent according to claim 22, wherein the disease involving CD33 positive cells is cancer, autoimmune disease or allergic disease.
  24. 　癌が、血液癌、乳癌、子宮癌、大腸癌、食道癌、胃癌、卵巣癌、肺癌、腎臓癌、直腸癌、甲状腺癌、子宮頸癌、小腸癌、前立腺癌または膵臓癌である、請求項２３に記載の治療剤。 The cancer is blood cancer, breast cancer, uterine cancer, colon cancer, esophageal cancer, stomach cancer, ovarian cancer, lung cancer, kidney cancer, rectal cancer, thyroid cancer, cervical cancer, small intestine cancer, prostate cancer or pancreatic cancer. 24. The therapeutic agent according to 23.
  25. 　血液癌が、急性骨髄性白血病、未分化大細胞型リンパ腫、急性リンパ性白血病、骨髄異形成症候群、多発性骨髄腫、ホジキンリンパ腫、または非ホジキンリンパ腫である、請求項２４に記載の治療剤。 The therapeutic agent according to claim 24, wherein the blood cancer is acute myeloid leukemia, anaplastic large cell lymphoma, acute lymphocytic leukemia, myelodysplastic syndrome, multiple myeloma, Hodgkin lymphoma, or non-Hodgkin lymphoma.
  26. 　請求項１から１４のいずれか１項に記載のモノクローナル抗体または該抗体の断片を哺乳動物に投与する工程を含む、ＣＤ３３陽性細胞が関与する疾患を治療する方法。 A method for treating a disease involving CD33-positive cells, comprising a step of administering the monoclonal antibody or fragment of the antibody according to any one of claims 1 to 14 to a mammal.
  27. 　ＣＤ３３陽性細胞が関与する疾患が、癌、自己免疫疾患またはアレルギー性疾患である、請求項２６に記載の方法。 The method according to claim 26, wherein the disease involving CD33-positive cells is cancer, autoimmune disease or allergic disease.
  28. 　癌が、血液癌、乳癌、子宮癌、大腸癌、食道癌、胃癌、卵巣癌、肺癌、腎臓癌、直腸癌、甲状腺癌、子宮頸癌、小腸癌、前立腺癌または膵臓癌である、請求項２７に記載の方法。 The cancer is blood cancer, breast cancer, uterine cancer, colon cancer, esophageal cancer, stomach cancer, ovarian cancer, lung cancer, kidney cancer, rectal cancer, thyroid cancer, cervical cancer, small intestine cancer, prostate cancer or pancreatic cancer. 28. The method according to 27.
  29. 　血液癌が、急性骨髄性白血病、未分化大細胞型リンパ腫、急性リンパ性白血病、骨髄異形成症候群、多発性骨髄腫、ホジキンリンパ腫、または非ホジキンリンパ腫である、請求項２８に記載の方法。 The method according to claim 28, wherein the blood cancer is acute myeloid leukemia, anaplastic large cell lymphoma, acute lymphocytic leukemia, myelodysplastic syndrome, multiple myeloma, Hodgkin lymphoma, or non-Hodgkin lymphoma.
  30. 　ＣＤ３３陽性細胞が関与する疾患を治療するための、請求項１から１４のいずれか１項に記載のモノクローナル抗体または該抗体の断片の使用。 Use of the monoclonal antibody according to any one of claims 1 to 14 or a fragment of the antibody for treating a disease involving CD33-positive cells.
  31. 　ＣＤ３３陽性細胞が関与する疾患が、癌、自己免疫疾患またはアレルギー性疾患である、請求項３０に記載の使用。 The use according to claim 30, wherein the disease involving CD33 positive cells is cancer, autoimmune disease or allergic disease.
  32. 　癌が、血液癌、乳癌、子宮癌、大腸癌、食道癌、胃癌、卵巣癌、肺癌、腎臓癌、直腸癌、甲状腺癌、子宮頸癌、小腸癌、前立腺癌または膵臓癌である、請求項３１に記載の使用。 The cancer is blood cancer, breast cancer, uterine cancer, colon cancer, esophageal cancer, stomach cancer, ovarian cancer, lung cancer, kidney cancer, rectal cancer, thyroid cancer, cervical cancer, small intestine cancer, prostate cancer or pancreatic cancer. 31. Use according to 31.
  33. 　血液癌が、急性骨髄性白血病、未分化大細胞型リンパ腫、急性リンパ性白血病、骨髄異形成症候群、多発性骨髄腫、ホジキンリンパ腫または非ホジキンリンパ腫である、請求項３２に記載の使用。 The use according to claim 32, wherein the blood cancer is acute myeloid leukemia, anaplastic large cell lymphoma, acute lymphocytic leukemia, myelodysplastic syndrome, multiple myeloma, Hodgkin lymphoma or non-Hodgkin lymphoma.
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Cited By (9)

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WO2016201389A2 (en) 2015-06-12 2016-12-15 Alector Llc Anti-cd33 antibodies and methods of use thereof
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WO2020047374A1 (en) 2018-08-31 2020-03-05 Alector Llc Anti-cd33 antibodies and methods of use thereof
US20210292413A1 (en) * 2018-09-25 2021-09-23 Academia Sinica Anti-siglec antibody, pharmaceutical composition comprising the same, and uses thereof
WO2021138407A2 (en) 2020-01-03 2021-07-08 Marengo Therapeutics, Inc. Multifunctional molecules that bind to cd33 and uses thereof
WO2023081898A1 (en) 2021-11-08 2023-05-11 Alector Llc Soluble cd33 as a biomarker for anti-cd33 efficacy
CN115806622A (en) * 2022-11-25 2023-03-17 厦门康基生物科技有限公司 Monoclonal antibody R8F3 of Taq DNA polymerase and application thereof
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