WO2015076425A1 - New monoclonal antibody - Google Patents

New monoclonal antibody Download PDF

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Publication number
WO2015076425A1
WO2015076425A1 PCT/JP2014/081674 JP2014081674W WO2015076425A1 WO 2015076425 A1 WO2015076425 A1 WO 2015076425A1 JP 2014081674 W JP2014081674 W JP 2014081674W WO 2015076425 A1 WO2015076425 A1 WO 2015076425A1
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Prior art keywords
seq
amino acid
antibody
acid sequence
antigen
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PCT/JP2014/081674
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French (fr)
Japanese (ja)
Inventor
眞一郎 丹羽
林　秀美
大 小倉
孝之 進藤
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リンク・ジェノミクス株式会社
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Publication of WO2015076425A1 publication Critical patent/WO2015076425A1/en

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Definitions

  • the present invention belongs to the field of oncology.
  • the present invention relates to an antibody that specifically binds to CD44v9 and inhibits the function of cancer stem cells.
  • the invention further relates to antibody and / or immunoconjugate compositions and their use in treating, preventing and / or diagnosing tumors.
  • CD44 is one of adhesion molecules that adhere cell-cell and cell-cell matrix (ECM; extra cellular matrix). CD44 plays an important role as a hyaluronic acid receptor in cellular activities such as adhesion, migration, and T lymphocyte activation. In recent years, it has become clear that CD44 is also involved in angiogenesis and cancer invasion and metastasis.
  • CD44 is a type I membrane protein and consists of three domains, the N-terminal extracellular, transmembrane region, and cytoplasmic tail C-terminal. The extracellular region also has a linking module at the N-terminus and shows homology with other hyaluronic acid binding linking proteins. It is composed of 20 exons, among which exons 6 to 15 are variant exons inserted by alternative splicing. Due to the diversity of these insertion patterns, the existence of various isoforms is known.
  • Non-Patent Document 1 Report on the expression and regulation of CD44 isoforms in humans (Mackay et al., The Journal of Cell Biology, Volume 124, Numbers 1 & 2, January 1994, pp. 71-82: Non-Patent Document 1) There is a report that CD44v8-10 containing variant exons 8 to 10 is the most highly expressed isoform in cancer tissues (Sasaki et al., 1998: Non-Patent Document 2).
  • CD44v8-10 has attracted attention as a major cancer stem cell marker in various solid cancers such as breast cancer, stomach cancer, colon cancer and prostate cancer.
  • Cancer stem cells are present in tumors and are resistant to various stresses. Therefore, the presence of cancer stem cells is considered to be a cause of recurrence and metastasis even if the tumor shrinks temporarily due to cancer treatment with anticancer agents or radiotherapy.
  • CD44v8-10 has been reported to colocalize and interact with the amino acid transporter xCT on the cell membrane. It is considered that xCT is stabilized on the cell membrane due to high expression of CD44v8-10 in cancer stem cells.
  • xCT has the function of suppressing the accumulation of reactive oxygen species (ROS) in the cell and enhancing the resistance to oxidative stress by promoting the uptake of cystine into the cell and the generation of glutathione (GSH).
  • ROS reactive oxygen species
  • GSH glutathione
  • XCT is also a molecule responsible for drug excretion, and contributes to the survival and proliferation of cancer stem cells through these actions.
  • CD44v8-10 which has been considered as one of the markers so far, to maintain the characteristics of cancer stem cells through the control of active oxygenoma is that cancer stem cells are resistant to anticancer drugs and radiation therapy. It is thought that this is a molecular mechanism showing resistance and suggests that inhibition of CD44v8-10 may lead to the establishment of a new cancer radical treatment method targeting cancer stem cells.
  • the present invention provides an antibody that specifically binds to CD44v9 contained in CD44v8-10 isoform and inhibits the function of cancer stem cells.
  • the present invention further provides a hybridoma that produces the antibody of the present invention, a complex (immunoconjugate) of the antibody of the present invention and a compound having a cell-killing activity and / or antitumor activity, or a radioisotope.
  • the present inventors have succeeded in producing a novel anti-CD44v9 antibody having an amino acid sequence of a complementarity-determining region (CDR) different from the antibody disclosed in Patent Document 1, and the antibody is used in cancer stem cells. It was confirmed to have an inhibitory effect on the main properties, Sphere (Colony) formation ability and metastasis formation ability. Moreover, it was confirmed that the antibody has a remarkable cytotoxicity-inducing activity and internalization activity.
  • CDR complementarity-determining region
  • the present invention provides the following.
  • An antibody or antigen-binding fragment thereof that specifically binds to an extracellular region of CD44v9 of a cell that expresses CD44v9 (1) as light chain complementarity determining region 1 (CDRL1), light chain complementarity determining region 2 (CDRL2), and light chain complementarity determining region 3 (CDRL3), respectively, SEQ ID NO: 11, SEQ ID NO: 12, and sequence An amino acid sequence of No. 13, or an amino acid sequence having a mutation of one or several amino acid residues in the amino acid sequences of SEQ ID No. 11, SEQ ID No. 12 and SEQ ID No.
  • CDRH1 heavy chain complementarity determining region 1
  • CDRH2 heavy chain complementarity determining region 2
  • CDRH3 heavy chain complementarity determining region 3
  • An antibody or an antigen-binding fragment thereof comprising an amino acid sequence or an amino acid sequence each having a mutation of one or several amino acid residues in the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, (2) As CDRL1, CDRL2, and CDRL3, one or a number in the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, or the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively
  • An amino acid sequence each having a mutation of one amino acid residue, CDRH1, CDRH2, and CDRH3 are one or several amino acids in the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, or the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively.
  • An antibody or antigen-binding fragment thereof comprising an amino acid sequence each having a mutation of a residue, (3) As CDRL1, CDRL2, and CDRL3, one or more amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25, or amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25, respectively.
  • An amino acid sequence each having a mutation of one amino acid residue, CDRH1, CDRH2, and CDRH3 are one or several amino acids in the amino acid sequences of SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, or the amino acid sequences of SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, respectively.
  • An antibody or antigen-binding fragment thereof comprising an amino acid sequence each having a mutation of a residue, (4) As CDRL1, CDRL2, and CDRL3, one or more amino acid sequences of SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, or SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, respectively.
  • An amino acid sequence each having a mutation of one amino acid residue, CDRH1, CDRH2, and CDRH3 are one or several amino acids in the amino acid sequences of SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34, or the amino acid sequences of SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34, respectively.
  • An antibody or antigen-binding fragment thereof comprising an amino acid sequence each having a mutation of residues, or (5) the amino acid sequences of SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37, respectively, as CDRL1, CDRL2, and CDRL3, or Comprising amino acid sequences each having a mutation of one or several amino acid residues in the amino acid sequences of SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37; CDRH1, CDRH2, and CDRH3 are one or several amino acids in the amino acid sequences of SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, or the amino acid sequences of SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively.
  • An antibody or antigen-binding fragment thereof comprising an amino acid sequence each having a residue mutation.
  • An antibody or antigen-binding fragment thereof that specifically binds to an extracellular region of CD44v9 of a cell that expresses CD44v9 (1) Light chain complementarity determining region 1 (CDRL1) comprising the amino acid sequence of RS-X 1 -KX 2 -LLH-X 3 -NGN-X 4 -YLY (SEQ ID NO: 41), (2) a light chain complementarity determining region 2 (CDRL2) comprising the amino acid sequence of R-X 5 -SX 6 -LAS (SEQ ID NO: 42), (3) a light chain complementarity determining region 3 (CDRL3) comprising the amino acid sequence of X 7 -QHLEYP-X 8 -T (SEQ ID NO: 43), (4) heavy chain complementarity determining region 1 (CDRH1) comprising the amino acid sequence of GFN-X 9 -KDTY-X 10 -H (SEQ ID NO:
  • X 1 is S, X 2 is S, X 3 is S, X 4 is T, X 5 is V, X 6 is N, X 7 is L, X 8 is L, X 9 is I , X 10 is M, X 11 is G, X 12 is N, X 13 is T, X 14 is Q, X 15 is D, X 16 is D, X 17 is A, X 18 is Y, and X seq is HLGLP (SEQ ID NO: 46) (2) X 1 is S, X 2 is S, X 3 is S, X 4 is T, X 5 is M, X 6 is N, X 7 is M, X 8 is L, X 9 is I, X 10 Is M, X 11 is D, X 12 is N, X 13 is T, X 14 is E, X 15 is D, X 16 is G, X 17 is A, X 18 is Y, and X seq is QLGLP (seque
  • [5] The antibody or antigen-binding fragment thereof according to any one of [1] to [4] above, (1) including a light chain variable region (LH) comprising an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 1, and an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 2.
  • LH light chain variable region
  • Heavy chain variable region (2) LH including an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 3, and VH including an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 4, (3) LH including an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 5, and VH including an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 6, (4) LH including an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 7 and VH including an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 8, or (5) LH including an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 9, and VH including an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 10 Or an antigen-binding fragment thereof, comprising [6] The antibody or antigen-binding fragment thereof according to any one of [1] to [5] above, (1) a light chain
  • an antigen-binding fragment thereof comprising [7] The antibody or antigen-binding fragment thereof according to any one of [1] to [6] above, which has cytotoxic activity and / or internalization activity for cells expressing CD44v9. [8] The cell according to any one of the above [1] to [6], which specifically binds to CD44v9 of a cell expressing CD44v9 and has an activity of inhibiting an interaction between CD44v9 and the amino acid transporter xCT. An antibody or antigen-binding fragment thereof. [9] (I) The antibody or antigen-binding fragment thereof according to any one of [1] to [6], which suppresses a decrease in intracellular reactive oxygen species, or (ii) inhibits extracellular discharge of a drug.
  • any of the above [1] to [6], which specifically binds to CD44v9 of a cell expressing CD44v9 and has an activity of inhibiting the tumor-forming ability and metastasis-forming ability of cancer stem cells and / or cancer cells The antibody or antigen-binding fragment thereof according to claim 1.
  • the antigen-binding fragment is Fab, Fab ′, F (ab ′) 2 , scFv, dsFv, ds-scFv, their dimers, minibodies, diabodies, and multimers, or two The antibody or antigen-binding fragment thereof according to any one of [1] to [12] above, which is a multispecific antibody fragment.
  • [16] The antibody or antigen-binding fragment thereof according to any one of [1] to [14], which is a chimeric antibody or a humanized antibody.
  • An immunoconjugate comprising the antibody or antigen-binding fragment thereof according to any one of [1] to [16] above and at least one cytotoxic drug, antitumor agent, or radioisotope.
  • An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of [1] to [17].
  • An expression vector comprising the nucleic acid molecule according to [18].
  • a gene encoding an anti-CD44v9 monoclonal antibody is isolated from the cell described in [20] above, an expression vector containing the gene is constructed, the expression vector is introduced into a host, and the monoclonal antibody is expressed.
  • a method for producing a monoclonal antibody that specifically binds to CD44v9 which comprises collecting a monoclonal antibody that specifically binds to CD44v9 from the resulting host, the culture supernatant of the host or the secretion of the host.
  • a medicament for preventing or treating a tumor comprising the antibody or antigen-binding fragment thereof or immunoconjugate thereof according to any one of [1] to [17].
  • a method for detecting a tumor comprising reacting the composition according to [26] above with a sample collected from a living body and detecting a reacted signal.
  • a method for immunodetecting a tumor in a test tube comprising the step of contacting the composition according to [26] above with a cancer cell.
  • a method for imaging a tumor in vivo comprising the step of administering the composition of [26] to an individual and obtaining a detection image obtained by near-infrared light imaging, PET, MRI, or ultrasonic imaging.
  • a medicament for diagnosing a tumor comprising the antibody or antigen-binding fragment or immunoconjugate thereof according to any one of [1] to [17].
  • the tumor is colon cancer, colorectal cancer, lung cancer, breast cancer, brain tumor, melanoma, renal cell cancer, bladder cancer, leukemia, lymphoma, T cell lymphoma, multiple myeloma, gastric cancer, pancreatic cancer, cervix Cancer, endometrial cancer, ovarian cancer, esophageal cancer, liver cancer, squamous cell carcinoma of the head and neck, skin cancer, urinary tract cancer, prostate cancer, choriocarcinoma, pharyngeal cancer, laryngeal cancer, tongue cancer, oral cancer, gallbladder cancer, Thyroid cancer, mesothelioma, pleurioma, male embryo, endometrial hyperplasia, endometriosis, embryonal, fibrosarcoma, Kaposi's sarcoma, hemangioma, spongiform hemangioma, hemangioblastoma, retinoblastoma,
  • the alkylating agent is selected from the group consisting of ifosfamide, cyclophosphamide, dacarbacin, temozolomide, nimustine, busulfan, procarbazine, melphalan, and ranimustine,
  • the antimetabolite is enocitabine, capecitabine, carmofur, cladripine, gemcitabine, cytarabine, cytarabine ocphosate, tegafur, tegafur uracil, TS-1, doxyfluridine, nelarabine, hydroxycarbamide, fluorouracil, fludarabine, pemetrexed captostatin, pentostatin, pentostatin And selected from the group consisting of methotrexate,
  • the molecular targeted drug is selected from the group consisting of Zevalin, Imatinib, Erlotinib, Gefitinib, Sunitinib, Cetuximab,
  • CDR Complementarity determining region selected from the group consisting of the following (1) to (6): (1) CDRL1, comprising the amino acid sequences of SEQ ID NOs: 11, 17, 23, 29, and 35, (2) CDRL2, comprising the amino acid sequences of SEQ ID NOs: 12, 18, 24, 30, 36 (3) CDRL3 comprising the amino acid sequences of SEQ ID NOs: 13, 19, 25, 31, 37 (4) CDRH1, comprising the amino acid sequences of SEQ ID NOs: 14, 20, 26, 32, 38, (5) CDRH2 comprising the amino acid sequences of SEQ ID NOs: 15, 21, 27, 33, 39, and (6) CDRH3 comprising the amino acid sequences of SEQ ID NOs: 16, 22, 28, 34, 40.
  • CDRL1 comprising the amino acid sequences of SEQ ID NOs: 11, 17, 23, 29, and 35
  • CDRL2 comprising the amino acid sequences of SEQ ID NOs: 12, 18, 24, 30, 36
  • CDRL3 comprising the amino acid sequences of SEQ ID NOs: 13, 19, 25, 31, 37
  • the antibody or antigen-binding fragment thereof of the present invention is highly upregulated in cancer stem cells and specifically binds to an antigen that is hardly expressed in normal cells (ie, CD44v9). Since it has an inhibitory effect on nest-forming ability, it is particularly useful as a medicine for treatment targeting the function of cancer stem cells, which prevents recurrence and metastasis, which are the most important problems in cancer, and leads to the cure of cancer.
  • an isoform containing CD44v9 such as CD44v8-10 is expressed by an immunoconjugate in which a compound having a cell-killing activity and / or an antitumor activity or a radioisotope is bound to the antibody of the present invention or an antigen-binding fragment thereof. Induction and detection of cell death can be performed.
  • the antibody or antigen-binding fragment thereof of the present invention inhibits the xCT stabilization function of CD44v8-10, thereby increasing the intracellular ROS concentration and promoting cell death induction by an anticancer agent or radiation therapy.
  • the anticancer drug can be easily stored in the cells, and the effect of the drug can be enhanced.
  • Vertical axis Number of spheres formed in each well. It is a figure which shows the inhibitory effect of the Sphere formation ability by the ACSC002 monoclonal antibody, the ACSC005 monoclonal antibody, the ACSC009 monoclonal antibody, and the ACSC010 monoclonal antibody by the in vitro evaluation system. Vertical axis: Number of spheres formed in each well.
  • A It is a figure which shows the antibody-concentration-dependent metastatic focus formation inhibitory effect by the ACSC002 monoclonal antibody by an in vivo evaluation system.
  • B It is the figure which compared the metastasis formation inhibitory effect of ACSC002 monoclonal antibody and LGAdh01-01 monoclonal antibody.
  • Vertical axis number of engrafted colonies confirmed on the lung surface after dissection.
  • an antibody that specifically recognizes CD44v9 provides an antibody and an antigen-binding fragment thereof having an epitope of a part or all of the amino acid sequence of CD44v9.
  • CD44 is a type I membrane glycoprotein that penetrates the cell membrane once, has an N-terminal extracellular, and a C-terminal is intracellular. Currently, it has been confirmed that there are 20 exons in the CD44 gene. Various isoforms have been confirmed so far by inserting exons 6 to 15 (variant exons: v1 to v10) in various combinations through alternative splicing.
  • CD44v9 or “CD44 variant9” is a variant isoform of CD44 in which a v9 exon consisting of 31 amino acid residues is inserted.
  • Information such as amino acid sequence and mRNA sequence of v9 exon can be obtained from publicly accessible databases such as GenBank with accession numbers such as NM_001001390 and NP_001001390, respectively.
  • CD44v9 for mice and other mammals can be obtained from publicly accessible databases.
  • CD44v9 or “CD44 variant9” simply refers to CD44v9, and in particular, refers to CD44v9 protein.
  • the gene encoding the CD44v9 protein may be simply referred to as CD44v9, but in that case it will be clear to the skilled person that the term refers to the CD44v9 gene.
  • CD44v9 is typically human CD44v9, but may be CD44v9 of mammals other than humans (eg, mouse, rat, bovine, etc.).
  • the antibodies and antigen-binding fragments thereof of the present invention typically bind specifically to CD44v9 expressed in CD44v9-expressing cells and function to inhibit interaction with the amino acid transporter xCT ( Or active).
  • Human xCT (cationic amino acid transporter, y + system)
  • cystine / glutamate exchange transport system is a 12-transmembrane membrane protein consisting of 501 amino acid residues.
  • Information such as amino acid sequence and mRNA sequence of human xCT can be obtained from publicly accessible databases such as GenBank with accession numbers such as AAG35592, AC110804, and AF200708, respectively.
  • xCT of mice and other mammals eg, rats, cows, etc.
  • mice and other mammals eg, rats, cows, etc.
  • xCT simply refers to xCT protein.
  • the gene encoding the xCT protein may simply be referred to as xCT, in which case it will be clear to the skilled person to refer to the xCT gene from the context.
  • xCT is typically human xCT, but may be xCT of mammals other than humans (for example, mouse, rat, cow, etc.).
  • XCT which is one of amino acid transporters, releases glutamic acid from the inside of the cell, takes cystine from the outside of the cell, reduces it to cysteine, and generates GSH which is a kind of peptide.
  • GSH is a kind of peptide.
  • CD44R1 including CD44v9 promotes the synthesis of GSH, which is one of the main antioxidants in the body, by stabilizing xCT, and contributes to reducing intracellular ROS.
  • antibodies and antigen-binding fragments thereof that can inhibit the interaction between CD44v9 and xCT (i) suppress the reduction of intracellular reactive oxygen species, or (ii) inhibit the extracellular excretion of drugs. It is considered possible. Cells that have undergone such suppression or inhibition can lead to cell death.
  • an antibody or antigen-binding fragment thereof “specifically binds” to CD44v9 means that the antibody or antigen-binding fragment has a specific amino acid sequence of this protein rather than its affinity for other amino acid sequences.
  • substantially high affinity means high affinity that allows the specific amino acid sequence to be detected separately from other amino acid sequences by a desired measuring device.
  • antibody is intended to include all classes and subclasses of intact immunoglobulins.
  • antibodies of the invention are of the IgG subclass, and more preferably, is human IgG 1 subclass.
  • Antibodies include in particular monoclonal antibodies.
  • an “antigen-binding fragment” refers to a fragment having the antigen-binding or variable region of an intact and / or humanized and / or chimeric antibody, such as Fab, Fab ′, Includes F (ab ′) 2 , Fv, ScFv fragments.
  • fragments have been produced by proteolysis of intact antibodies, for example by papain degradation (see eg WO 94/29348), but can also be produced directly from transgenic host cells by genetic recombination. .
  • the method described in Bird et al. (1988) Science, 242, 423-426 can be used.
  • antibody fragments can be generated using the various genetic engineering techniques described below.
  • the Fv fragment appears to have a lower interaction energy between its two chains than the Fab fragment.
  • these domains are composed of peptides (Bird et al., (1988) Science 242, 423-426, Huston et al., PNAS, 85, 5879-5883), disulfide bridges (Glockshuber et al. (1990) Biochemistry, 29, 1362-1367), and the “knob in hole” mutation (Zhu et al. (1997), Protein Sci., 6, 781-788).
  • ScFv fragments can be generated by methods well known to those skilled in the art (Whitlow et al., (1991) Methods companion Methods Enzymol, 2, 97-105 and Huston et al., (1993) Int. Rev. Immunol 10, 195-217).
  • ScFv can be produced in bacterial cells such as E. coli, but is preferably produced in eukaryotic cells.
  • the disadvantage of ScFv is that the product is monovalent, thus making it impossible to increase the binding force due to multivalent bonds, and the short half-life. Attempts to overcome these problems include bivalent (ScFv ′) 2 , which can be obtained from a ScFv containing an additional C-terminal cysteine by chemical coupling (Adams et al.
  • ScFv can be multimerized by shortening the peptide linker to 3-12 residues to form a “diabody”. See Holliger et al., PNAS (1993), 90, 6444-6448. By further reducing the linker, ScFv trimers ("Triabodies", see Kortt et al.
  • Bispecific diabodies can be made by non-covalent attachment of two single-chain fusion products consisting of a VH domain from another antibody linked to a VL domain of one antibody by a short linker (Kipriyanov). (1998), Int. J. Can 77, 763-772).
  • the stability of such a bispecific diabody can be achieved by introducing a disulfide bridge, or by introducing the “knob in hole” mutation described above, or by connecting two hybrid ScFv fragments via a peptide linker. It can be enhanced by forming a chain diabody (ScDb) (see Kontermann et al. (1999) J. Immunol. Methods 226 179-188).
  • Tetravalent bispecific molecules are obtained, for example, by fusing a ScFv fragment to the CH3 domain of an IgG molecule or to a Fab fragment via the hinge region (Coloma et al. (1997) Nature Biotechnol. 15 , 159-163).
  • tetravalent bispecific molecules have been created by fusion of bispecific single stranded diabodies (see Alt et al. (1999) FEBS Lett 454, 90-94).
  • dimerization of ScFv-ScFv tandems with a helix-loop-helix motif-containing linker dimerization of ScFv-ScFv tandems with a helix-loop-helix motif-containing linker (DiBi miniantibody, Muller et al.
  • VH and VL antibody variable regions
  • Bispecific F (ab ′) 2 fragments can be generated by chemical coupling of Fab ′ fragments or by heterodimerization with leucine zippers (Shalaby et al. (1992) J. Exp. Med). 175, 217-225, and Kostelny et al. (1992), J. Immunol. 148, 1547-1553). Isolated VH and VL domains (Domantis plc) can also be used.
  • monoclonal antibody means an antibody (or antibody fragment) obtained from a substantially homogeneous population of antibodies, ie, a naturally occurring individual antibody that comprises the population may be present in small amounts. Identical except for possible mutations. Monoclonal antibodies are highly specific and are directed against a single antigenic site. In contrast to polyclonal antibody preparations that generally contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture and are free from contamination by other immunoglobulins.
  • monoclonal indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not meant to imply that production of the antibody by any particular method is required.
  • monoclonal antibodies used in accordance with the present invention may be made by the hybridoma method first described in Kohler et al., Nature, 256: 495 [1975], or by recombinant DNA methods (eg, US Patent No. 4 , 816, 567).
  • “Monoclonal antibodies” are also described in, for example, Clackson et al., Nature, 352: 624-628 [1991] and Marks et al., J. MoI. Mol. Biol. , 222: 581-597 (1991), including antigen recognition and binding site clones containing antibody fragments (Fv clones) isolated from phage antibody libraries.
  • “Monoclonal antibody” includes “chimeric” antibodies (immunoglobulins), which correspond to antibodies whose heavy and / or light chain portions are derived from a particular species or belong to a particular antibody class or subclass. Antibodies that are identical or homologous to the sequence, but the rest of the chain are derived from other species or belong to other antibody classes or subclasses, as long as they exhibit the desired biological activity Is identical or homologous to the corresponding sequence of the fragment (Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 [1984]).
  • “Humanized” forms of non-human (eg, murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (eg, Fv, Fab, Fab ′, etc.) that contain minimal sequence derived from non-human immunoglobulin. F (ab ′) 2 or other antigen-binding sequence of the antibody).
  • humanized antibodies are human immunoglobulins (recipient antibodies) whose residues from the recipient CDRs are of non-human species such as mice, rats, rabbits, primates (eg, monkeys). Substituted with residues with the desired specificity, affinity and capacity from the CDR (donor antibody).
  • Fv framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues.
  • humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the grafted CDR or framework sequences. These modifications are made to further refine and optimize antibody performance.
  • a humanized antibody has at least one, typically all, or substantially all of the CDR region corresponding to that of a non-human immunoglobulin and all or substantially all of the FR region is of a human immunoglobulin sequence, typically It can contain substantially all of the two variable domains.
  • An optimal humanized antibody may also contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • Examples of the antibodies of the present invention or antigen-binding fragments thereof include (1) light chain complementarity determining region 1 (CDRL1) comprising the amino acid sequence of SEQ ID NO: 41, or (ii) an amino acid sequence having a mutation of one or several amino acid residues at the corresponding position of SEQ ID NO: 41 , (2) Light chain complementarity determining region 2 comprising (i) the amino acid sequence of SEQ ID NO: 42, or (ii) an amino acid sequence having a mutation of one or several amino acid residues at the corresponding position of SEQ ID NO: 42 (CDRL2), (3) Light chain complementarity determining region 3 comprising (i) the amino acid sequence of SEQ ID NO: 43, or (ii) an amino acid sequence having a mutation of one or several amino acid residues at the corresponding position of SEQ ID NO: 43 (CDRL3), (4) Heavy chain complementarity determining region 1 comprising (i) the amino acid sequence of SEQ ID NO: 44, or (ii) an amino acid sequence having a mutation of one
  • mutant of one or several amino acid residues means one or several (for example, 2, 3, 4, 5) amino acids in the original amino acid sequence. It means that the residue has been deleted, substituted, inserted or added.
  • an antibody having an amino acid sequence having such a mutation at a corresponding position in the original amino acid sequence or an antigen-binding fragment thereof is equivalent to an antibody having the original amino acid sequence or an antigen-binding fragment thereof. Has biological activity.
  • “equivalent biological activity” includes (i) the ability to bind with specificity to an antigen to which an antibody having an original amino acid sequence or an antigen-binding fragment thereof specifically binds, (ii) Cytotoxic activity when bound to CD44v9 on CD44v9 expressing cells, or (iii) both. Most preferably, “equivalent biological activity” means (iii) above. In addition, the upper limit of the number of mutated amino acid residues in the above “mutation of one or several amino acid residues” is a criterion for whether or not such equivalent specificity can be maintained (criteria). Limited by.
  • amino acids constituting a naturally occurring protein can be grouped according to the characteristics of their side chains.
  • amino acids having similar characteristics include aromatic amino acids (tyrosine, phenylalanine). , Tryptophan), basic amino acids (lysine, arginine, histidine), acidic amino acids (aspartic acid, glutamic acid), neutral amino acids (serine, threonine, asparagine, glutamine), amino acids having a hydrocarbon chain (alanine, valine, leucine, It can be classified into groups such as isoleucine, proline) and others (glycine, methionine, cysteine).
  • amino acid residues which can be substituted with each other including non-natural amino acids include the following groups, and amino acid residues contained in the same group can be substituted with 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,
  • the amino acid sequences of two types of proteins to be compared are arranged, and the number of portions that are the same amino acid residues is visually counted. The total number of amino acid residues) ⁇ 100 (%) ”can also be obtained.
  • CDRL1 light chain complementarity determining region 1
  • CDRL2 light chain complementarity determining region 2
  • CDRL3 light chain complementarity determining region 3
  • SEQ ID NO: 11 SEQ ID NO: 12
  • sequence An amino acid sequence of No. 13 or an amino acid sequence having a mutation of one or several amino acid residues in the amino acid sequences of SEQ ID No. 11, SEQ ID No. 12 and SEQ ID No.
  • CDRH1 heavy chain complementarity determining region 1
  • CDRH2 heavy chain complementarity determining region 2
  • CDRH3 heavy chain complementarity determining region 3
  • the extracellular region of CD44v9 of cells expressing CD44v9 comprising an amino acid sequence or an amino acid sequence having a mutation of one or several amino acid residues in the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively An antibody or antigen-binding fragment thereof that specifically binds to (2) As CDRL1, CDRL2, and CDRL3, one or a number in the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, or the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively
  • An amino acid sequence each having a mutation of one amino acid residue, CDRH1, CDRH2, and CDRH3 are one or several amino acids in the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, or the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively.
  • An amino acid sequence each having a mutation of one amino acid residue, CDRH1, CDRH2, and CDRH3 are one or several amino acids in the amino acid sequences of SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, or the amino acid sequences of SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, respectively.
  • amino acid sequence each having a mutation of one amino acid residue, CDRH1, CDRH2, and CDRH3 are one or several amino acids in the amino acid sequences of SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34, or the amino acid sequences of SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34, respectively.
  • An antibody or antigen-binding fragment thereof that specifically binds to the extracellular region of CD44v9 of cells expressing CD44v9, each comprising an amino acid sequence having a residue mutation, or (5) CDRL1, CDRL2, and CDRL3, respectively,
  • Including CDRH1, CDRH2, and CDRH3 are one or several amino acids in the amino acid sequences of SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, or the amino acid sequences of SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively.
  • Examples thereof include an antibody or an antigen-binding fragment thereof that specifically binds to the extracellular region of CD44v9 of cells expressing CD44v9, each of which contains an amino acid sequence having a residue mutation.
  • preferred examples of the antibody of the present invention or an antigen-binding fragment thereof include (1) a light chain variable region (LH) comprising an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 1, and an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 2 Heavy chain variable region (VH), (2) LH containing an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 3, and VH containing an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 4.
  • LH light chain variable region
  • VH Heavy chain variable region
  • the percentage of the above-mentioned identity is, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. possible.
  • the present invention in another embodiment, provides isolated nucleic acid molecules that encode antibodies and antigen-binding fragments thereof that specifically bind to CD44v9.
  • the nucleic acid molecule is RNA or DNA.
  • the nucleic acid molecule of the present invention can be used to produce the antibody of the present invention or an antigen-binding fragment thereof.
  • a gene encoding an anti-CD44v9 monoclonal antibody is isolated from the hybridoma cell of the present invention, an expression vector containing the gene is constructed, and the expression vector is introduced into a host A monoclonal antibody that specifically binds to CD44v9, wherein said monoclonal antibody is expressed and the monoclonal antibody that specifically binds to CD44v9 is collected from the obtained host, host culture supernatant or host secretion.
  • a manufacturing method is provided.
  • the DNA encoding the antibody of the present invention or an antigen-binding fragment thereof can be obtained using a conventional method (for example, by using an oligonucleotide probe capable of specifically binding to a gene encoding the heavy and light chains of a mouse antibody. ) Easily separated and sequenced.
  • Hybridoma cells producing monoclonal antibodies are a preferred source of such DNA. Once isolated, this DNA is inserted into an expression vector to synthesize monoclonal antibodies in recombinant host cells, which are then used to produce E. coli cells, human HEK293 fetal kidney-derived cells, monkeys that do not produce antibody proteins in other circumstances.
  • telomeres can be transfected into host cells such as COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells.
  • host cells such as COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells.
  • homologous mouse sequences with sequences of human heavy and light chain constant domains (Morrison et al., Proc. Nat. Cad. Sci., USA, 81: 6851 [1984]
  • immunoglobulin coding This DNA may be modified by covalently linking all or part of the coding sequence of the non-immunoglobulin polypeptide to the coding sequence.
  • “Chimeric” or “hybrid” antibodies are prepared so as to have the binding specificity of the anti-CD44v9 monoclonal antibodies of the invention.
  • the present invention provides a hybridoma cell that produces an antibody that specifically binds to CD44v9 and an antigen-binding fragment thereof.
  • the invention also provides a hybridoma containing a nucleic acid molecule encoding an antibody that specifically binds to CD44v9 and an antigen-binding fragment thereof.
  • the preparation of the hybridoma cell of the present invention can be specifically performed as follows, but is not limited to this method.
  • Preparation of antigen Antigen necessary for producing the hybridoma cell of the present invention includes a cell producing CD44v9 or a fraction thereof, a vector incorporating a DNA encoding the CD44 gene containing CD44v9, or CD44v9.
  • a full-length or partial fragment of cDNA encoding CD44v9 is known in host cells transformed with the encoding DNA, ie, prokaryotic host cells such as E. coli and eukaryotic host cells such as insect cells and mammalian cells.
  • prokaryotic host cells such as E. coli and eukaryotic host cells such as insect cells and mammalian cells.
  • a protein incorporated by a method and expressed or purified as it is or as a fusion protein, or a CD44v9 partial peptide synthesized using a peptide synthesizer can be used.
  • a mouse or rat whose serum showed a sufficient antibody titer against the antigen cells used for immunization is used as a source of antibody-producing cells.
  • the spleen cells are used for fusion with myeloma cells, the spleen is removed from the immunized mouse or rat 3 to 4 days after the final administration of the antigen substance, and the spleen cells are collected.
  • the spleen is shredded in a basal medium without serum (hereinafter referred to as a washing medium), centrifuged to collect the cells, and then treated with Tris-ammonium chloride buffer (pH 7.65) for 2 to 3 minutes. Erythrocytes are removed, washed with washing medium, and then provided as fusion splenocytes.
  • myeloma cells cell lines obtained from mice are used.
  • 8-azaguanine-resistant mouse BALB / c-derived myeloma cell line P3-X63Ag8-U1 (P3-U1) [Current Topics in Microbiology and Immunology-1, European J. Biol. Immunology, 6, 511-519 (1976)]
  • SP2 / O-Ag14 SP2 / O-Ag14
  • SP-2 [Nature 276, 269-270 (1978)]
  • P3-X63-Ag8653 653 [J.
  • HAT medium hypoxanthine (10 ⁇ 4 M), thymidine (1.5 ⁇ 10 ⁇ 5 M) and aminopterin (4 ⁇ 10 ⁇ 7 M) to the normal medium.
  • HAT medium hypoxanthine (10 ⁇ 4 M), thymidine (1.5 ⁇ 10 ⁇ 5 M) and aminopterin (4 ⁇ 10 ⁇ 7 M)
  • an antibody that specifically reacts with the CD44v9 protein is selected using, for example, the enzyme immunoassay described in (5) or FACS (abbreviation of Fluorescence-Activated Cell Sorting) .
  • cloning was repeated twice by the limiting dilution method (the first time was HT medium (a medium obtained by removing aminopterin from HAT medium), the second time was using a normal medium), and a stable and strong antibody titer was observed. Is selected as an anti-CD44v9 monoclonal antibody-producing hybridoma strain.
  • a monoclonal antibody that specifically binds CD44v9 comprising culturing hybridoma cells and collecting an antibody that specifically binds CD44v9 from the resulting culture.
  • a manufacturing method is provided.
  • the present invention also provides an immunoconjugate in which an antibody of the present invention and an antigen-binding fragment thereof are combined with a compound having a cell killing activity and / or an antitumor activity or a radioisotope. A conjugate is provided.
  • the antibody of the present invention has excellent internalization activity into target tumor cells that express CD44v9. Therefore, an immunoconjugate to which a compound having cell killing activity and / or antitumor activity is bound can cause these compounds to act directly and selectively on tumor cells.
  • the present invention also provides a pharmaceutical composition for preventing or treating or diagnosing a tumor, comprising as an active ingredient the antibody of the present invention or an antigen-binding fragment thereof or an immunoconjugate thereof. Offer things.
  • the pharmaceutical composition of the present invention further contains a pharmaceutically acceptable carrier.
  • the antibody of the present invention or an antigen-binding fragment thereof specifically binds to CD44v9 of cells expressing CD44v9 (eg, tumor cells), and is a CD44v9 and amino acid transporter xCT.
  • CD44v9 eg, tumor cells
  • an immunoconjugate to which a compound having cytocidal activity and / or antitumor activity is bound can directly and selectively act these compounds on tumor cells.
  • Such compounds include, for example, cytotoxic drugs, antitumor agents, or radioisotopes. Therefore, the pharmaceutical composition of the present invention is for killing tumor cells expressing CD44v9 on the cell surface, or for the prevention or treatment of tumors characterized by such cells and diseases caused by similar mechanisms. Can be used.
  • cytotoxic activity means the ability to cause cytotoxicity to cells.
  • the antibody of the present invention or an antigen-binding fragment thereof specifically binds to a CD44v9-expressing cell.
  • cytotoxic activity can be measured, for example, by the method described in Example 2 of the present invention and evaluated as a cytotoxic rate.
  • cell death means “apoptosis”.
  • Apoptosis is a function induced by various physiological and pathological factors such as ontogeny programs, death factor stimulation, severe chromosomal DNA damage due to radiation, severe prescription stress due to abnormal protein accumulation, etc. It is a typical example of active cell death. Contrary to necrosis (necrosis) involving swelling of cell bodies and nuclei, contraction and fragmentation of cell bodies and nuclei occur.
  • the antibody of the present invention and an antibody-binding fragment thereof bind to CD44v9 expressed in a cancer stem cell with enhanced expression of CD44v9 fraction, and have the Sphere (colony) formation ability and metastasis characteristic of cancer stem cells. It can be used for treatment and diagnosis that inhibits the formation ability and targets cancer stem cells.
  • tumor cancer examples include colon cancer, colorectal cancer, lung cancer, breast cancer, brain tumor, melanoma, renal cell cancer, bladder cancer, leukemia, lymphoma, T cell lymphoma, multiple myeloma, gastric cancer, pancreatic cancer, Cervical cancer, endometrial cancer, ovarian cancer, esophageal cancer, liver cancer, squamous cell carcinoma of the head and neck, skin cancer, urinary tract cancer, prostate cancer, choriocarcinoma, pharyngeal cancer, laryngeal cancer, tongue cancer, oral cancer, gallbladder Cancer, thyroid cancer, mesothelioma, pleuromatous, male embryo, endometrial hyperplasia, endometriosis, embryonal, fibrosarcoma, Kaposi's sarcoma, hemangioma, cavernous hemangioma, hemangioblastoma, retinoblast Tumors, astro
  • colon cancer colorectal cancer
  • lung cancer breast cancer
  • breast cancer brain tumor, bladder cancer, lymphoma
  • gastric cancer pancreatic cancer
  • pancreatic cancer liver cancer, and prostate cancer
  • breast cancer triple negative breast cancer (HER2 negative, ER negative, PR negative) is mentioned.
  • the antibody of the present invention or an antigen-binding fragment thereof is used as a medicine, it can be formulated according to conventional means.
  • the antibody or antigen-binding fragment thereof is converted into IgA and bound to the secretory component, and then orally as tablets, capsules, elixirs, microcapsules, etc. with sugar coating as necessary, or water or other
  • It can be used parenterally in the form of an injectable solution such as a sterile solution with a pharmaceutically acceptable liquid or a suspension.
  • the unit dose required for the formulation practice generally accepted together with known physiologically recognized carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binding agents, etc. It can be produced by mixing in the form.
  • the amount of active ingredient in these preparations is such that an appropriate dose within the indicated range can be obtained.
  • the antibody or antigen-binding fragment thereof of the present invention can be used together with other cell growth inhibitors.
  • the antibody or antigen-binding fragment thereof of the present invention requires treatment simultaneously, sequentially or separately with a pharmaceutically acceptable cytostatic agent in order to further enhance the therapeutic effect as an anticancer agent. It can be administered to a subject or patient.
  • cytostatic agents examples include, but are not limited to, alkylating agents, antimetabolites, molecular targeted drugs, plant alkaloids, anticancer antibiotics, platinum preparations and the like. .
  • alkylating agents include, but are not limited to, ifosfamide, cyclophosphamide, dacarbacin, temozolomide, nimustine, busulfan, procarbazine, melphalan, ranimustine, and the like.
  • antimetabolites include eninotabine, capecitabine, carmofur, cladripine, gemcitabine, cytarabine, cytarabine ocphosate, tegafur, tegafur uracil, TS-1, doxyfluridine, nelarabine, hydroxycarbamide, fluorouracil, fludarabine, pemetrexed, pemetrexed
  • molecular target drugs include, but are not limited to, Zevalin, Imatinib, Erlotinib, Gefitinib, Sunitinib, Cetuximab, Sorafenib, Dasatinib, Trastuzumab, Tretinoin, Panitumumab, Bevacizumab, Bortezomib, Rituximab, etc.
  • plant alkaloids include, but are not limited to, irinotecan, etoposide, sobuzoxan, docetaxel, nogitecan, paclitaxel, vinorelbine, vincristine, vindesine, vinblastine and the like.
  • anti-cancer antibiotics examples include actinomycin D, aclarubicin, amrubicin, idarubicin, epirubicin, smux, daunorubicin, doxorubicin, pirarubicin, bleomycin, peomycin, mitomycin C, mitoxantrone, doxil, etc. It is not limited.
  • platinum preparations include, but are not limited to, oxaliplatin, cisplatin, carboplatin, nedaplatin and the like.
  • pharmaceutically acceptable has a reasonable benefit / risk ratio within the scope of normal medical judgment, and does not cause excessive toxicity, irritation, allergic responses, or other problems or complications. Utilized herein to refer to compounds, materials, compositions, and / or dosage forms suitable for use in contact with human and animal tissue, not shown.
  • the route of administration of the pharmaceutical composition for the prevention or treatment of the tumor of the present invention can be determined by well-known methods such as intravenous, intraperitoneal, intracerebral, subcutaneous, intramuscular, intraocular, intraarterial, intracerebral spinal cord, or By intralesional injection or infusion, or by controlled release system. Furthermore, as a means for directly administering the antibody or antigen-binding fragment thereof to a tumor site, administration by a catheter or the like is also possible.
  • the medicament for preventing or treating tumors of the present invention includes, for example, a buffer (for example, phosphate buffer, sodium acetate buffer), a soothing agent (for example, benzalkonium chloride, procaine hydrochloride, etc.). , Stabilizers (eg, human serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc.
  • a buffer for example, phosphate buffer, sodium acetate buffer
  • a soothing agent for example, benzalkonium chloride, procaine hydrochloride, etc.
  • Stabilizers eg, human serum albumin, polyethylene glycol, etc.
  • preservatives eg, benzyl alcohol, phenol, etc.
  • antioxidants e.g, antioxidants, etc.
  • the prepared injection solution is usually filled in a suitable ampoule. Since the preparation thus obtained is safe and has low toxicity, it can be administered to mammals including humans.
  • the dose of the antibody or antigen-binding fragment thereof or a salt thereof varies depending on the administration subject, symptoms, administration method, etc., but in the case of oral administration, generally, for example, endometriosis patients or uterine adenomyosis patients In (as 60 kg), it is about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day.
  • the dose varies depending on the subject of administration, symptoms, administration method, etc. For example, in the form of an injection, it is usually about 0.01 per day for a 60 kg patient, for example.
  • About 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg can be administered by intravenous injection.
  • the present invention provides a method for diagnosing a tumor by using a diagnostic immunoconjugate in which at least one diagnostic or detection agent is bound to the above-described antibody of the present invention or an antigen-binding fragment thereof. It is characterized by detecting.
  • the diagnostic or detection agent is selected from the group consisting of a radionuclide, a contrast agent, a fluorescent agent, a chemiluminescent agent, a bioluminescent agent, a paramagnetic ion, an enzyme, and a photoactive diagnostic agent.
  • a tumor can be detected by reacting a diagnostic immunoconjugate with a sample collected from a living body, such as a tissue fragment or blood, and detecting a signal.
  • the measurement method examples include ELISA method, EI method, RIA method, fluorescence immunoassay method, chemiluminescence immunoassay method and the like. In yet another embodiment, it can be used for PET imaging with a diagnostic immunoconjugate to which a diagnostic radionuclide is bound.
  • Example 1 Preparation of immunogen A mammalian cell expression vector (pRC-CMV) incorporating CD44R1 was prepared as an antigen necessary for preparing an anti-CD44v9 monoclonal antibody. The full length of cDNA encoding CD44R1 was incorporated into a pRc-CMV vector using a known method.
  • pRC-CMV mammalian cell expression vector
  • mice (Balb / c) were immunized with the antigen prepared by the method shown in (1), and antibody-producing cells were collected from their spleens. Immunization is performed by administering 20 ug of pRc-CMV vector incorporating CD44R1 as an antigen into the tail vein of rats, or 20 ug of pRc-CMV vector incorporating CD44R1 and an adjuvant (pCACC-CD40L or pBOOST2-wtmIRF1). went. Antigen administration was performed twice 3 weeks and 6 weeks after the first administration.
  • the spleen When the spleen cells were used for fusion with myeloma cells, the spleen was removed from the immunized mouse and the spleen cells were collected. The spleen is shredded in a serum-free basal medium (hereinafter referred to as a washing medium), centrifuged, and the cells are collected, treated with Tris-ammonium chloride buffer (pH 7.65) for 2 to 3 minutes and treated with erythrocytes. After washing with a washing medium, it was used as a splenocyte for fusion.
  • a serum-free basal medium hereinafter referred to as a washing medium
  • Tris-ammonium chloride buffer pH 7.65
  • mice splenocytes were added at a rate of 1 to 2 ml of DMEM medium several times every 1 to 2 minutes, and then DMEM medium was added to make the total volume 50 ml. After centrifugation (900 rpm, 5 minutes), the supernatant was discarded and the cells were gently suspended in 100 ml of HAT medium. This suspension was dispensed at 100 ⁇ l / well into a 96-well culture plate and cultured at 37 ° C. for 10 to 14 days in a 5% CO 2 incubator.
  • the obtained hybridoma culture supernatant was reacted with a GFP-CD44v8-10 transfectant, and FACS analysis was performed to screen whether or not a CD44v8-v10 specific antibody was produced. .
  • the FACS reaction was performed in a 96-well plate. Transfectants were dispensed into tubes at 50 ⁇ l each so as to give 2.5 ⁇ 10 5 to 3 ⁇ 10 6 cells / well. 50 ⁇ l of the hybridoma culture supernatant was added to the cell suspension and allowed to react for 45 minutes under ice cooling. After adding 100 ⁇ l of 0.1% BSA-PBS to the tube, centrifuging at 500 g at 4 ° C.
  • ACSC002 monoclonal antibody After passing through a prefilter and a membrane filter (0.22 ⁇ m), purification was performed using Protein G Sepharose (GE Healthcare Bioscience), followed by dialysis with PBS (3 hours or more ⁇ 4 times).
  • the resulting antibodies were designated as ACSC002 monoclonal antibody, ACSC005 monoclonal antibody, ACSC007 monoclonal antibody, ACSC009 monoclonal antibody, ACSC010 monoclonal antibody.
  • Example 2 (1) Examination of binding ability of monoclonal antibody to tumor cell line Using 3 kinds of human tumor cell lines, binding ability of ACSC002 monoclonal antibody, ACSC005 monoclonal antibody, ACSC007 monoclonal antibody, ACSC009 monoclonal antibody, ACSC010 monoclonal antibody was determined by FACS method. It was measured. The FACS reaction was performed in a 96-well plate. The human tumor cell line was dispensed into tubes at 50 ⁇ l so as to be 1.0 ⁇ 10 5 / well. 50 ⁇ l of each monoclonal antibody adjusted to 40 ug / mL was added to the cell suspension, and the mixture was reacted for 45 minutes under ice cooling.
  • ACSC002 monoclonal antibody, ACSC005 monoclonal antibody, ACSC007 monoclonal antibody, ACSC009 monoclonal antibody, ACSC010 monoclonal antibody are human breast cancer cell lines (MDA-MB468 and MDA-MB231) and human colon cancer cell line (HCT116). In particular, higher binding activity was observed for HCT-116.
  • Rinse was performed 3 times with PBS for 3 minutes, and blocking was performed with 3% BSA / PBS for 30 minutes at room temperature. After removing the blocking solution, a monoclonal antibody diluted with 0.2% BSA / PBS was added as a primary antibody and allowed to react at room temperature for 1 hour. After rinsing with PBS for 3 minutes three times, Alexa Fluor 488-labeled anti-mouse IgG antibody diluted with 0.2% BSA / PBS was added as a secondary antibody and allowed to react for 1 hour at room temperature in the dark.
  • Example 3 (1) Preparation of human chimeric antibody
  • Total RNA was isolated from the established anti-CD44v9 antibody-producing hybridoma strain, and SMARTer TM RACE cDNA.
  • CDNA was synthesized using Amplification Kit (Clontech).
  • cDNA encoding heavy chain and light chain variable regions was amplified by the polymerase chain reaction (PCR) method and subcloned into a cloning vector.
  • PCR polymerase chain reaction
  • the nucleotide sequence of the obtained cDNA was analyzed, and the amino acid sequences (sequences) of the variable regions of the heavy and light chains of each antibody were determined by a conventional method (described in ⁇ http://www.bioinf.org.uk/abs/>).
  • CDR sequence-specific primers were synthesized and used to amplify CDRs from the extracted plasmid DNA and incorporated into a human chimeric antibody heavy chain expression vector or a human chimeric antibody light chain expression vector.
  • the obtained plasmid was ligated using Ligase to prepare a human chimeric antibody production plasmid.
  • FreeStyle TM MAX Reagent a human chimeric antibody-producing plasmid is introduced into FreeStyle 293F cells (Invitrogen), and cultured after 96 hours of swirling culture (FreeStyle TM 293 medium, 37 ° C, 8% CO 2 , 135 rpm) The supernatant was obtained. The culture supernatant was purified using Protein G Sepharose (GE Healthcare Bioscience) and then dialyzed with PBS (3 hours or more x 4 times) to obtain a human chimeric CD44v9 antibody.
  • PBS Protein G Sepharose
  • human breast cancer cell line MDA-MB468, human normal mammary gland-derived cell line (Hs617.Mg), and human normal breast skin-derived cell line (Hs714.Sk) are set to 2 ⁇ 10 5 cells / mL as target cells. Adjusted with medium. 50 ⁇ L of ACSC010 human chimeric antibody was dispensed in advance into a round bottom 96-well plate, and 50 ⁇ L of the target cell solution was added. After 15 minutes of incubation at 4 ° C., the mixture was centrifuged at 1500 rpm for 3 minutes, and the supernatant was discarded.
  • Cell damage rate (%) (sample release ⁇ spontaneous release) / (total release ⁇ spontaneous release) ⁇ 100.
  • the ADCC activity of the ACSC010 human chimeric antibody against the human breast cancer cell line (MDA-MB468) was detected.
  • a human normal mammary gland-derived cell line (Hs617.Mg) and a human normal milk skin-derived cell line (Hs714.Sk) were not found, indicating cancer cell-specific ADCC activity.
  • the internalization activity of anti-CD44v9 monoclonal antibody was measured by FCM analysis using a human tumor cell line.
  • a group of high expression cells of CD44v9 concentrated from the human prostate cancer cell line PC3 by the method of (7) is suspended in 0.1% BSA-PBS, and 50 ⁇ L is dispensed on a U-bottom 96-well plate so that 1 ⁇ 10 5 cells / well. Noted.
  • 50 ⁇ L of each monoclonal antibody prepared to 20 ⁇ g / mL was added and reacted at 4 ° C. for 45 minutes.
  • 150 ⁇ L of 0.1% BSA-PBS was added, and washing was performed by centrifuging at 500 g for 3 minutes at 4 ° C. three times to obtain a cell pellet bound with a monoclonal antibody.
  • 100 ⁇ L of 0.1% BSA-PBS was added and incubated at 37 ° C. (temperature at which internalization was induced) or 4 ° C. (temperature at which internalization was not induced), respectively, for 1 hour. Washing by adding 150 ⁇ L of 0.1% BSA-PBS and centrifuging at 500 g for 3 minutes at 4 ° C. was performed three times.
  • ROS reactive oxygen species
  • the CD44 gene was knocked down with siRNA and the same ROS was evaluated, and a strong rise in ROS was detected.
  • the action of raising active oxygenoma (ROS) was confirmed by the addition of the ACSC002 monoclonal antibody, the ACSC005 monoclonal antibody, and the ACSC010 monoclonal antibody.
  • ROS active oxygenoma
  • Human prostate cancer cell lines (PC3-CD44v9High and PC3-CD44v9Low) are cultured and then collected by trypsin treatment, suspended in a serum-free medium, and then pipetted 10 to 20 times to obtain a single cell suspension. It was. 24-well Ultra Low attachment Plate (Costar # 3473) was seeded at 1250 cells / well, 2500 cells / well, 5000 cells / well, bFGF (final concentration 10 ng / mL), EGF (final concentration 20 ng) / ML) was added and culture was performed. Evaluation was performed 3 to 5 days later by counting the number of Sphere (Colony) formed under a microscope. As shown in FIG.
  • PC3-CD44v9High has significantly higher Sphere-forming ability than PC3-CD44v9Low, indicating that it possesses the characteristics of cancer stem cells. From this result, it was confirmed that cancer stem cells were concentrated in PC3-CD44v9High.
  • the number of Sphere (Colony) formed was evaluated by counting under a microscope.
  • the addition of the ACSC002 monoclonal antibody, the ACSC005 monoclonal antibody, the ACSC009 monoclonal antibody, and the ACSC010 monoclonal antibody showed a Sphere formation inhibitory effect of 20% or more compared to the mouse IgG antibody as a control.
  • the ACSC002 antibody showed a higher Sphere formation inhibitory effect.
  • Dissection was performed 42 days after transplantation, and the effect of antibodies on the formation of lung metastases was evaluated by observation of lung tissue. As shown in FIG. 10 (A), a decrease in the number of colonies engrafted on the dissected lung surface in an antibody concentration-dependent manner was confirmed as compared with PBS as a control, and the effect of inhibiting the formation of metastasis by the ACSC002 monoclonal antibody was shown. It was.
  • the present invention is particularly useful as a pharmaceutical or the like for treatment or diagnosis targeting the function of cancer stem cells.

Abstract

Provided is a drug for preventing or treating various malignant tumors such as solid tumors that are presently intractable, and that comprises, as an active ingredient, new antibodies that are able to bind with human CD44v9 and that specifically induce antibody-dependent cellular cytotoxicity in cancer cells. Provided is an antibody to human CD44v9 and a drug for preventing or treating tumours that contains said antibody.

Description

新規モノクローナル抗体New monoclonal antibody
　本発明は腫瘍学分野に属する。本発明は、ＣＤ４４ｖ９に特異的に結合し、癌幹細胞の機能を阻害する抗体に関する。本発明はさらに、抗体および／もしくはイムノコンジュゲート組成物ならびに腫瘍を治療すること、予防することおよび／もしくは診断することにおけるそれらの使用に関する。 The present invention belongs to the field of oncology. The present invention relates to an antibody that specifically binds to CD44v9 and inhibits the function of cancer stem cells. The invention further relates to antibody and / or immunoconjugate compositions and their use in treating, preventing and / or diagnosing tumors.
　ＣＤ４４は細胞−細胞、細胞−細胞間基質（ＥＣＭ；ｅｘｔｒａ　ｃｅｌｌｕｌａｒ　ｍａｔｒｉｘ）を接着させる接着分子のひとつである。また、ＣＤ４４はヒアルロン酸の受容体として、接着や遊走、Ｔリンパ球活性化などの細胞活動に重要な役割を果たしている。近年では、ＣＤ４４は血管新生や癌の浸潤転移にも関与していることが明らかとなってきている。ＣＤ４４はＩ型膜蛋白質であり、Ｎ末端細胞外、膜貫通領域、ならびに細胞質尾部Ｃ末端の３つのドメインからなる。細胞外領域はＮ末端に連結モジュールも有し、他のヒアルロン酸結合連結蛋白質と相同性を示す。２０のエキソンから構成されるが、その中のエキソン６~１５は選択的スプライシングによって挿入されるバリアントエキソンである。これらの挿入パターンの多様性によって様々なアイソフォームの存在が知られている。 CD44 is one of adhesion molecules that adhere cell-cell and cell-cell matrix (ECM; extra cellular matrix). CD44 plays an important role as a hyaluronic acid receptor in cellular activities such as adhesion, migration, and T lymphocyte activation. In recent years, it has become clear that CD44 is also involved in angiogenesis and cancer invasion and metastasis. CD44 is a type I membrane protein and consists of three domains, the N-terminal extracellular, transmembrane region, and cytoplasmic tail C-terminal. The extracellular region also has a linking module at the N-terminus and shows homology with other hyaluronic acid binding linking proteins. It is composed of 20 exons, among which exons 6 to 15 are variant exons inserted by alternative splicing. Due to the diversity of these insertion patterns, the existence of various isoforms is known.
　ヒトにおけるＣＤ４４アイソフォームの発現およびその調節に関する報告（Ｍａｃｋａｙ　ｅｔ　ａｌ．，　Ｔｈｅ　Ｊｏｕｒｎａｌ　ｏｆ　Ｃｅｌｌ　Ｂｉｏｌｏｇｙ，　Ｖｏｌｕｍｅ　１２４，　Ｎｕｍｂｅｒｓ　１　＆　２，　Ｊａｎｕａｒｙ　１９９４，　ｐｐ．７１−８２：非特許文献１）、また、ヒト癌組織においてバリアントエキソン８~１０を含むＣＤ４４ｖ８−１０が最も高発現しているアイソフォームであるという報告がある（Ｓａｓａｋｉ　ｅｔ　ａｌ．，１９９８：非特許文献２）。 Report on the expression and regulation of CD44 isoforms in humans (Mackay et al., The Journal of Cell Biology, Volume 124, Numbers 1 & 2, January 1994, pp. 71-82: Non-Patent Document 1) There is a report that CD44v8-10 containing variant exons 8 to 10 is the most highly expressed isoform in cancer tissues (Sasaki et al., 1998: Non-Patent Document 2).
　近年、ＣＤ４４ｖ８−１０は、乳癌、胃癌、大腸癌、前立腺癌などの様々な固形癌における主要な癌幹細胞のマーカーとしても注目されている。癌幹細胞は腫瘍の中に存在し、様々なストレスに対する抵抗性を有している。そのため、癌幹細胞の存在は抗癌剤や放射線治療によるがん治療によって一時的に腫瘍が縮小しても再発や転移が生じる一因として考えられている。 In recent years, CD44v8-10 has attracted attention as a major cancer stem cell marker in various solid cancers such as breast cancer, stomach cancer, colon cancer and prostate cancer. Cancer stem cells are present in tumors and are resistant to various stresses. Therefore, the presence of cancer stem cells is considered to be a cause of recurrence and metastasis even if the tumor shrinks temporarily due to cancer treatment with anticancer agents or radiotherapy.
　ＣＤ４４ｖ８−１０は、アミノ酸トランスポーターｘＣＴと細胞膜上で共局在し、相互作用することが報告されている。癌幹細胞においてＣＤ４４ｖ８−１０が高発現することにより、ｘＣＴが細胞膜上で安定化していると考えられている。ｘＣＴは細胞内へシスチンの取り込み、グルタチオン（ＧＳＨ）生成を促進することで細胞内での活性酸素種（ＲＯＳ；ｒｅａｃｔｉｖｅ　ｏｘｙｇｅｎ　ｓｐｅｃｉｅｓ）の蓄積を抑制し、酸化ストレスへの抵抗性を高める働きを持つ。またｘＣＴは薬剤排出を担う分子でもあり、これらの作用によって、癌幹細胞の生存・増殖に寄与している。これまで単なるマーカーのひとつと考えられていたＣＤ４４ｖ８−１０が活性酸素腫の制御を介して癌幹細胞としての特性を維持する為に機能的に働くことは、癌幹細胞が抗癌剤や放射線治療に対して抵抗性を示す分子機構であると考えられると共に、ＣＤ４４ｖ８−１０の阻害が癌幹細胞を標的とした新たな癌の根治療法の確立につながる可能性を示唆している。 CD44v8-10 has been reported to colocalize and interact with the amino acid transporter xCT on the cell membrane. It is considered that xCT is stabilized on the cell membrane due to high expression of CD44v8-10 in cancer stem cells. xCT has the function of suppressing the accumulation of reactive oxygen species (ROS) in the cell and enhancing the resistance to oxidative stress by promoting the uptake of cystine into the cell and the generation of glutathione (GSH). . XCT is also a molecule responsible for drug excretion, and contributes to the survival and proliferation of cancer stem cells through these actions. The function of CD44v8-10, which has been considered as one of the markers so far, to maintain the characteristics of cancer stem cells through the control of active oxygenoma is that cancer stem cells are resistant to anticancer drugs and radiation therapy. It is thought that this is a molecular mechanism showing resistance and suggests that inhibition of CD44v8-10 may lead to the establishment of a new cancer radical treatment method targeting cancer stem cells.
再表２０１１／００７８５３Table 2011/007853
　癌の根治を目的とした癌幹細胞の機能を阻害する薬剤が強く求められている。 There is a strong demand for drugs that inhibit the function of cancer stem cells for the purpose of cancer cure.
　本発明は、ＣＤ４４ｖ８−１０アイソフォームに含まれるＣＤ４４ｖ９に特異的に結合し、癌幹細胞の機能を阻害する抗体を提供する。本発明は、さらに、本発明の抗体を産生するハイブリドーマ、本発明の抗体と殺細胞活性および／または抗腫瘍活性を有する化合物あるいは放射性同位体との複合体（イムノコンジュゲート）を提供する。 The present invention provides an antibody that specifically binds to CD44v9 contained in CD44v8-10 isoform and inhibits the function of cancer stem cells. The present invention further provides a hybridoma that produces the antibody of the present invention, a complex (immunoconjugate) of the antibody of the present invention and a compound having a cell-killing activity and / or antitumor activity, or a radioisotope.
　これまでにも、ＣＤ４４ｖ９に特異的に結合する抗体の報告があるが（再表２０１１／００７８５３：特許文献１）、癌幹細胞に対する機能阻害の活性は示されていない。 So far, there has been a report of an antibody that specifically binds to CD44v9 (see Table 2011/007853: Patent Document 1), but no function-inhibiting activity against cancer stem cells has been shown.
　本発明者らは、特許文献１に開示される抗体とは異なる相補性決定領域（ＣＤＲ；ｃｏｍｐｌｅｍｅｎｔａｒｉｔｙ−ｄｅｔｅｒｍｉｎｉｎｇ　ｒｅｇｉｏｎ）のアミノ酸配列有する新規の抗ＣＤ４４ｖ９抗体の作製に成功し、該抗体が癌幹細胞の主要な特性であるＳｐｈｅｒｅ（Ｃｏｌｏｎｙ）形成能および転移巣形成能に対して阻害効果を持つことを確認した。また、該抗体が顕著な細胞障害誘導活性、およびインターナリゼーション活性を有することを確認した。 The present inventors have succeeded in producing a novel anti-CD44v9 antibody having an amino acid sequence of a complementarity-determining region (CDR) different from the antibody disclosed in Patent Document 1, and the antibody is used in cancer stem cells. It was confirmed to have an inhibitory effect on the main properties, Sphere (Colony) formation ability and metastasis formation ability. Moreover, it was confirmed that the antibody has a remarkable cytotoxicity-inducing activity and internalization activity.
　したがって、本発明は、以下を提供する。
［１］ＣＤ４４ｖ９を発現する細胞のＣＤ４４ｖ９の細胞外領域に特異的に結合する抗体またはその抗原結合断片であって、
　（１）軽鎖相補性決定領域１（ＣＤＲＬ１）、軽鎖相補性決定領域２（ＣＤＲＬ２）、および軽鎖相補性決定領域３（ＣＤＲＬ３）として、それぞれ、配列番号１１、配列番号１２、および配列番号１３のアミノ酸配列、または配列番号１１、配列番号１２、および配列番号１３のアミノ酸配列に１個もしくは数個のアミノ酸残基の変異をそれぞれ有するアミノ酸配列を含み、
　重鎖相補性決定領域１（ＣＤＲＨ１）、重鎖相補性決定領域２（ＣＤＲＨ２）、および重鎖相補性決定領域３（ＣＤＲＨ３）として、それぞれ、配列番号１４、配列番号１５、および配列番号１６のアミノ酸配列、もしくは配列番号１４、配列番号１５、および配列番号１６のアミノ酸配列に１個もしくは数個のアミノ酸残基の変異をそれぞれ有するアミノ酸配列を含む、抗体またはその抗原結合断片、
　（２）ＣＤＲＬ１、ＣＤＲＬ２、およびＣＤＲＬ３として、それぞれ、配列番号１７、配列番号１８、および配列番号１９のアミノ酸配列、または配列番号１７、配列番号１８、および配列番号１９のアミノ酸配列に１個もしくは数個のアミノ酸残基の変異をそれぞれ有するアミノ酸配列を含み、
　ＣＤＲＨ１、ＣＤＲＨ２、およびＣＤＲＨ３として、それぞれ、配列番号２０、配列番号２１、および配列番号２２のアミノ酸配列、もしくは配列番号２０、配列番号２１、および配列番号２２のアミノ酸配列に１個もしくは数個のアミノ酸残基の変異をそれぞれ有するアミノ酸配列を含む、抗体またはその抗原結合断片、
　（３）ＣＤＲＬ１、ＣＤＲＬ２、およびＣＤＲＬ３として、それぞれ、配列番号２３、配列番号２４、および配列番号２５のアミノ酸配列、または配列番号２３、配列番号２４、および配列番号２５のアミノ酸配列に１個もしくは数個のアミノ酸残基の変異をそれぞれ有するアミノ酸配列を含み、
　ＣＤＲＨ１、ＣＤＲＨ２、およびＣＤＲＨ３として、それぞれ、配列番号２６、配列番号２７、および配列番号２８のアミノ酸配列、もしくは配列番号２６、配列番号２７、および配列番号２８のアミノ酸配列に１個もしくは数個のアミノ酸残基の変異をそれぞれ有するアミノ酸配列を含む、抗体またはその抗原結合断片、
　（４）ＣＤＲＬ１、ＣＤＲＬ２、およびＣＤＲＬ３として、それぞれ、配列番号２９、配列番号３０、および配列番号３１のアミノ酸配列、または配列番号２９、配列番号３０、および配列番号３１のアミノ酸配列に１個もしくは数個のアミノ酸残基の変異をそれぞれ有するアミノ酸配列を含み、
　ＣＤＲＨ１、ＣＤＲＨ２、およびＣＤＲＨ３として、それぞれ、配列番号３２、配列番号３３、および配列番号３４のアミノ酸配列、もしくは配列番号３２、配列番号３３、および配列番号３４のアミノ酸配列に１個もしくは数個のアミノ酸残基の変異をそれぞれ有するアミノ酸配列を含む、抗体またはその抗原結合断片、または
　（５）ＣＤＲＬ１、ＣＤＲＬ２、およびＣＤＲＬ３として、それぞれ、配列番号３５、配列番号３６、および配列番号３７のアミノ酸配列、または配列番号３５、配列番号３６、および配列番号３７のアミノ酸配列に１個もしくは数個のアミノ酸残基の変異をそれぞれ有するアミノ酸配列を含み、
　ＣＤＲＨ１、ＣＤＲＨ２、およびＣＤＲＨ３として、それぞれ、配列番号３８、配列番号３９、および配列番号４０のアミノ酸配列、もしくは配列番号３８、配列番号３９、および配列番号４０のアミノ酸配列に１個もしくは数個のアミノ酸残基の変異をそれぞれ有するアミノ酸配列を含む、抗体またはその抗原結合断片。
［２］ＣＤ４４ｖ９を発現する細胞のＣＤ４４ｖ９の細胞外領域に特異的に結合する抗体またはその抗原結合断片であって、
　（１）ＲＳ−Ｘ１−Ｋ−Ｘ２−ＬＬＨ−Ｘ３−ＮＧＮ−Ｘ４−ＹＬＹ（配列番号４１）のアミノ酸配列を含む軽鎖相補性決定領域１（ＣＤＲＬ１）、
　（２）Ｒ−Ｘ５−Ｓ−Ｘ６−ＬＡＳ（配列番号４２）のアミノ酸配列を含む軽鎖相補性決定領域２（ＣＤＲＬ２）、
　（３）Ｘ７−ＱＨＬＥＹＰ−Ｘ８−Ｔ（配列番号４３）のアミノ酸配列を含む軽鎖相補性決定領域３（ＣＤＲＬ３）、
　（４）ＧＦＮ−Ｘ９−ＫＤＴＹ−Ｘ１０−Ｈ（配列番号４４）のアミノ酸配列を含む重鎖相補性決定領域１（ＣＤＲＨ１）、
　（５）ＲＩＤＰＡＮ−Ｘ１１−Ｘ１２−Ｘ１３−Ｘ１４−Ｙ−Ｘ１５−ＰＫＦＱ−Ｘ１６（配列番号４５）のアミノ酸配列を含む重鎖相補性決定領域２（ＣＤＲＨ２）、および
　（６）Ｒ−Ｘｓｅｑ−Ｆ−Ｘ１７−Ｘ１８のアミノ酸配列を含む重鎖相補性決定領域３（ＣＤＲＨ３）
（但し、Ｘ１−Ｘ１８は、任意の天然に存在するアミノ酸残基であり、Ｘｓｅｑは、以下からなる群：
　ＨＬＧＬＰ（配列番号４６）
　ＱＬＧＬＰ（配列番号４７）
　ＤＶＳＳＭＩＰＴＧ（配列番号４８）
　ＳＧＶＧ（配列番号４９）、および
　ＤＱＴＲＡＴＧ（配列番号５０）
から選択されるアミノ酸配列である）
を含む、抗体またはその抗原結合断片。
［３］Ｘ１が、ＳまたはＮであり、
　Ｘ２が、ＳまたはＴであり、
　Ｘ３が、ＳまたはＴであり、
　Ｘ４が、ＴまたはＩであり、
　Ｘ５が、ＶまたはＭであり、
　Ｘ６が、ＮまたはＳであり、
　Ｘ７が、ＬまたはＭであり、
　Ｘ８が、Ｌ、ＦまたはＹであり、
　Ｘ９が、ＩまたはＶであり、
　Ｘ１０が、ＭまたはＩであり、
　Ｘ１１が、ＧまたはＤであり、
　Ｘ１２が、ＮまたはＳであり、
　Ｘ１３が、ＴまたはＣであり、
　Ｘ１４が、Ｑ、Ｅ、またはＫであり、
　Ｘ１５が、ＤまたはＮであり、
　Ｘ１６が、ＤまたはＧであり、
　Ｘ１７が、ＡまたはＧであり、
　Ｘ１８が、ＹまたはＤである、
　上記［２］に記載の抗体またはその抗原結合断片。
［４］（１）Ｘ１がＳ、Ｘ２がＳ、Ｘ３がＳ、Ｘ４がＴ、Ｘ５がＶ、Ｘ６がＮ、Ｘ７がＬ、Ｘ８がＬ、Ｘ９がＩ、Ｘ１０がＭ、Ｘ１１がＧ、Ｘ１２がＮ、Ｘ１３がＴ、Ｘ１４がＱ、Ｘ１５がＤ、Ｘ１６がＤ、Ｘ１７がＡ、Ｘ１８がＹ、およびＸｓｅｑがＨＬＧＬＰ（配列番号４６）であるか、
　（２）Ｘ１がＳ、Ｘ２がＳ、Ｘ３がＳ、Ｘ４がＴ、Ｘ５がＭ、Ｘ６がＮ、Ｘ７がＭ、Ｘ８がＬ、Ｘ９がＩ、Ｘ１０がＭ、Ｘ１１がＤ、Ｘ１２がＮ、Ｘ１３がＴ、Ｘ１４がＥ、Ｘ１５がＤ、Ｘ１６がＧ、Ｘ１７がＡ、Ｘ１８がＹ、およびＸｓｅｑがＱＬＧＬＰ（配列番号４７）であるか、
　（３）Ｘ１がＳ、Ｘ２がＳ、Ｘ３がＳ、Ｘ４がＩ、Ｘ５がＭ、Ｘ６がＮ、Ｘ７がＭ、Ｘ８がＦ、Ｘ９がＩ、Ｘ１０がＭ、Ｘ１１がＤ、Ｘ１２がＮ、Ｘ１３がＴ、Ｘ１４がＫ、Ｘ１５がＮ、Ｘ１６がＧ、Ｘ１７がＡ、Ｘ１８がＹ、およびＸｓｅｑがＤＶＳＳＭＩＰＴＧ（配列番号４８）であるか、
　（４）Ｘ１がＮ、Ｘ２がＴ、Ｘ３がＴ、Ｘ４がＴ、Ｘ５がＭ、Ｘ６がＮ、Ｘ７がＭ、Ｘ８がＦ、Ｘ９がＩ、Ｘ１０がＭ、Ｘ１１がＧ、Ｘ１２がＮ、Ｘ１３がＣ、Ｘ１４がＫ、Ｘ１５がＤ、Ｘ１６がＧ、Ｘ１７がＧ、Ｘ１８がＹ、およびＸｓｅｑがＳＧＶＧ（配列番号４９）であるか、または
　（５）Ｘ１がＳ、Ｘ２がＳ、Ｘ３がＳ、Ｘ４がＴ、Ｘ５がＶ、Ｘ６がＳ、Ｘ７がＭ、Ｘ８がＹ、Ｘ９がＶ、Ｘ１０がＩ、Ｘ１１がＧ、Ｘ１２がＳ、Ｘ１３がＴ、Ｘ１４がＫ、Ｘ１５がＤ、Ｘ１６がＧ、Ｘ１７がＡ、Ｘ１８がＤ、およびＸｓｅｑがＤＱＴＲＡＴＧ（配列番号５０）である、項［２］に記載の抗体またはその抗原結合断片。
［５］上記［１］~［４］のいずれか一項に記載の抗体またはその抗原結合断片であって、
（１）配列番号１のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含む軽鎖可変領域（ＬＨ）、および配列番号２のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含む重鎖可変領域（ＶＨ）、
（２）配列番号３のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含むＬＨ、および配列番号４のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含むＶＨ、
（３）配列番号５のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含むＬＨ、および配列番号６のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含むＶＨ、
（４）配列番号７のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含むＬＨ、および配列番号８のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含むＶＨ、または
（５）配列番号９のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含むＬＨ、および配列番号１０のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含むＶＨ
を含有する、抗体もしくはその抗原結合断片。
［６］上記［１］~［５］のいずれか一項に記載の抗体もしくはその抗原結合断片であって、
（１）配列番号１のアミノ酸配列を含む軽鎖可変領域（ＶＬ）、および配列番号２のアミノ酸配列を含む重鎖可変領域（ＶＨ）、
（２）配列番号３のアミノ酸配列を含むＶＬ、および配列番号４のアミノ酸配列を含むＶＨ、
（３）配列番号５のアミノ酸配列を含むＶＬ、および配列番号６のアミノ酸配列を含むＶＨ、
（４）配列番号７のアミノ酸配列を含むＶＬ、および配列番号８のアミノ酸配列を含むＶＨ、または
（５）配列番号９のアミノ酸配列を含むＶＬ、および配列番号１０のアミノ酸配列を含むＶＨ
を含有する、抗体もしくはその抗原結合断片。
［７］ＣＤ４４ｖ９を発現する細胞に対する細胞障害活性および／またはインターナリゼーション活性を有する、上記［１］~［６］のいずれか一項に記載の抗体またはその抗原結合断片。
［８］ＣＤ４４ｖ９を発現する細胞のＣＤ４４ｖ９に特異的に結合し、ＣＤ４４ｖ９とアミノ酸トランスポーターｘＣＴとの相互作用を阻害する活性を有する、上記［１］~［６］のいずれか一項に記載の抗体またはその抗原結合断片。
［９］
（ｉ）細胞内活性酸素種の低下を抑制するか、または（ｉｉ）薬剤の細胞外排出を阻害する、上記［１］~［６］のいずれか一項に記載の抗体またはその抗原結合断片。
［１０］ＣＤ４４ｖ９を発現する細胞のＣＤ４４ｖ９に特異的に結合し、癌幹細胞および／または癌細胞の腫瘍形成能および転移巣形成能を阻害する活性を有する、上記［１］~［６］のいずれか一項に記載の抗体またはその抗原結合断片。
［１１］サブクラスがＩｇＧである、上記［１］~［１０］のいずれか一項に記載の抗体またはその抗原結合断片。
［１２］上記ＩｇＧが、ＩｇＧ２ｂまたはＩｇＧ１である、上記［１１］に記載の抗体またはその抗原結合断片。
［１３］上記抗原結合断片が、Ｆａｂ、Ｆａｂ’、Ｆ（ａｂ’）２、ｓｃＦｖ、ｄｓＦｖ、ｄｓ−ｓｃＦｖ、それらのダイマー、ミニボディ（ｍｉｎｏｂｏｄｉｅｓ）、ダイアボディ（ｄｉａｂｏｄｉｅｓ）、およびマルチマー、または二重特異性抗体断片である、上記［１］~［１２］のいずれか一項に記載の抗体またはその抗原結合断片。
［１４］モノクローナル抗体である、上記［１］~［１３］のいずれか一項に記載の抗体またはその抗原結合断片。
［１５］ラット、マウス、霊長類、またはヒト抗体である、上記［１］~［１４］のいずれか一項に記載の抗体またはその抗原結合断片。
［１６］キメラ抗体またはヒト化抗体である、上記［１］~［１４］のいずれか一項に記載の抗体またはその抗原結合断片。
［１７］上記［１］~［１６］のいずれか一項に記載の抗体またはその抗原結合断片と少なくとも１つの細胞毒性薬、抗腫瘍剤または放射性同位体を含んでなるイムノコンジュゲート。
［１８］上記［１］~［１７］のいずれか一項に記載の抗体またはその抗原結合断片をコードする単離された核酸分子。
［１９］上記［１８］に記載の核酸分子を含む発現ベクター。
［２０］上記［１８］に記載の核酸分子を含むかまたは該核酸分子を導入した、上記［１］~［１６］のいずれか一項に記載の抗体を産生する、ハイブリドーマ細胞または遺伝子導入細胞。
［２１］上記［２０］に記載の細胞を培養し、得られる培養物からＣＤ４４ｖ９特異的に結合する抗体を採取することを含む、ＣＤ４４ｖ９に特異的に結合するモノクローナル抗体の製造方法。
［２２］上記［２０］に記載の細胞から抗ＣＤ４４ｖ９モノクローナル抗体をコードする遺伝子を単離し、該遺伝子を含む発現ベクターを構築し、該発現ベクターを宿主に導入して上記モノクローナル抗体を発現せしめ、得られる宿主、宿主の培養上清または宿主の分泌物からＣＤ４４ｖ９に特異的に結合するモノクローナル抗体を採取することを含む、ＣＤ４４ｖ９に特異的に結合するモノクローナル抗体の製造方法。
［２３］上記宿主が、大腸菌、酵母細胞、昆虫細胞、哺乳動物細胞、植物細胞、または哺乳動物である、上記［２２］に記載の製造方法。
［２４］上記［１］~［１７］のいずれか一項に記載の抗体またはその抗原結合断片またはイムノコンジュゲートを含有する組成物であって、ＣＤ４４ｖ９を発現する細胞に適用して該細胞のアポトーシスを誘導するために使用する、組成物。
［２５］上記［１］~［１７］のいずれか一項に記載の抗体またはその抗原結合断片またはイムノコンジュゲートを含有する、腫瘍の予防または治療のための医薬。
［２６］上記［１］~［１７］のいずれか一項に記載の抗体またはその抗原結合断片またはイムノコンジュゲートを含有する組成物であって、ＣＤ４４ｖ９を発現する細胞を検出するために使用する、組成物。
［２７］上記［２６の組成物と、生体から採取された試料とを反応させ、反応したシグナルを検出することを特徴とする、腫瘍の検出方法。
［２８］上記［２６の組成物を癌細胞と接触する工程を含む試験管内の腫瘍の免疫検出方法。
［２９］上記［２６の組成物を個体に投与する工程および近赤外光イメージング、ＰＥＴ、ＭＲＩまたは超音波イメージングによって得られる検出映像を得る工程を含む生体内で腫瘍の映像化方法。
［３０］上記［１］~［１７］のいずれか一項に記載の抗体またはその抗原結合断片またはイムノコンジュゲートを含有する、腫瘍の診断のための医薬。
［３１］上記腫瘍が、大腸癌、結腸直腸癌、肺癌、乳癌、脳腫瘍、黒色腫、腎細胞癌、膀胱癌、白血病、リンパ腫、Ｔ細胞リンパ腫、多発性骨髄腫、胃癌、膵臓癌、子宮頸癌、子宮内膜癌、卵巣癌、食道癌、肝臓癌、頭頸部扁平上皮癌、皮膚癌、尿路癌、前立腺癌、絨毛癌、咽頭癌、喉頭癌、舌癌、口腔癌、胆嚢癌、甲状腺癌、中皮腫、胸膜腫、男性胚腫、子宮内膜過形成、子宮内膜症、胚芽腫、線維肉腫、カポジ肉腫、血管腫、海綿状血管腫、血管芽腫、網膜芽腫、星状細胞腫、神経線維腫、稀突起謬腫、髄芽腫、神経芽腫、神経膠腫、横紋筋肉腫、謬芽腫、骨原性肉腫、平滑筋肉腫、甲状肉腫、およびウィルムス腫瘍からなる群から選択される少なくとも１つである、上記［２５］または［３０］に記載の医薬。
［３２］上記腫瘍が、大腸癌、結腸直腸癌、肺癌、乳癌、脳腫瘍、膀胱癌、リンパ腫、胃癌、膵臓癌、肝臓癌、または前立腺癌である、上記［２５］または［３０］に記載の医薬。
［３３］薬学的に許容し得る細胞増殖抑制剤と同時、個別、又は逐次投与するための、上記［３０］~［３２］のいずれか一項に記載の医薬。
［３４］上記細胞増殖抑制剤が、アルキル化剤、代謝拮抗剤、分子標的薬、植物アルカロイド、抗癌性抗生物質、およびプラチナ製剤からなる群から選択される少なくとも１つである、上記［３３］に記載の医薬。
［３５］上記アルキル化剤が、イホスファミド、シクロフォスファミド、ダカルバシン、テモゾロミド、ニムスチン、ブスルファン、プロカルバジン、メルファラン、およびラニムスチンからなる群から選択され、
　上記代謝拮抗剤が、エノシタビン、カペシタビン、カルモフール、クラドリピン、ゲムシタビン、シタラビン、シタラビンオクホスファート、テガフール、テガフールウラシル、ＴＳ−１、ドキシフルリジン、ネララビン、ヒドロキシカルバミド、フルオロウラシル、フルダラビン、ペメトレキセド、ペントスタチン、メルカプトプリン、およびメトトレキサートからなる群から選択され、
　上記分子標的薬が、ゼヴァリン、イマチニブ、エルロチニブ、ゲフィチニブ、スニチニブ、セツキシマブ、ソラフェニブ、ダサチニブ、トラスツズマブ、トレチノイン、パニツムマブ、ベバシズマブ、ボルテゾミブ、およびリツキシマブからなる群から選択され、
　植物アルカロイドが、イリノテカン、エトポシド、ソブゾキサン、ドセタキセル、ノギテカン、パクリタキセル、ビノレルビン、ビンクリスチン、ビンデシン、およびビンブラスチンからなる群から選択され、
　上記抗癌性抗生物質が、アクチノマイシンＤ、アクラルビシン、アムルビシン、イダルビシン、エピルビシン、スマンクス、ダウノルビシン、ドキソルビシン、ピラルビシン、ブレオマイシン、ペプロマイシン、マイトマイシンＣ、ミトキサントロン、およびドキシルからなる群から選択され、
　上記プラチナ製剤が、オキサリプラチン、シスプラチン、カルボプラチン、およびネダプラチンからなる群から選択される少なくとも１つである、上記［３３］に記載の医薬。
［３６］以下の（１）~（６）からなる群から選択される相補性決定領域（ＣＤＲ）：
　（１）配列番号１１、１７、２３、２９、３５のアミノ酸配列を含むＣＤＲＬ１、
　（２）配列番号１２、１８、２４、３０、３６のアミノ酸配列を含むＣＤＲＬ２、
　（３）配列番号１３、１９、２５、３１、３７のアミノ酸配列を含むＣＤＲＬ３、
　（４）配列番号１４、２０、２６、３２、３８のアミノ酸配列を含むＣＤＲＨ１、
　（５）配列番号１５、２１、２７、３３、３９のアミノ酸配列を含むＣＤＲＨ２、および
　（６）配列番号１６、２２、２８、３４、４０のアミノ酸配列を含むＣＤＲＨ３。 Accordingly, the present invention provides the following.
[1] An antibody or antigen-binding fragment thereof that specifically binds to an extracellular region of CD44v9 of a cell that expresses CD44v9,
(1) as light chain complementarity determining region 1 (CDRL1), light chain complementarity determining region 2 (CDRL2), and light chain complementarity determining region 3 (CDRL3), respectively, SEQ ID NO: 11, SEQ ID NO: 12, and sequence An amino acid sequence of No. 13, or an amino acid sequence having a mutation of one or several amino acid residues in the amino acid sequences of SEQ ID No. 11, SEQ ID No. 12 and SEQ ID No. 13, respectively;
As heavy chain complementarity determining region 1 (CDRH1), heavy chain complementarity determining region 2 (CDRH2), and heavy chain complementarity determining region 3 (CDRH3), SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively. An antibody or an antigen-binding fragment thereof comprising an amino acid sequence or an amino acid sequence each having a mutation of one or several amino acid residues in the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16,
(2) As CDRL1, CDRL2, and CDRL3, one or a number in the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, or the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively An amino acid sequence each having a mutation of one amino acid residue,
CDRH1, CDRH2, and CDRH3 are one or several amino acids in the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, or the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively. An antibody or antigen-binding fragment thereof comprising an amino acid sequence each having a mutation of a residue,
(3) As CDRL1, CDRL2, and CDRL3, one or more amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25, or amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25, respectively An amino acid sequence each having a mutation of one amino acid residue,
CDRH1, CDRH2, and CDRH3 are one or several amino acids in the amino acid sequences of SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, or the amino acid sequences of SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, respectively. An antibody or antigen-binding fragment thereof comprising an amino acid sequence each having a mutation of a residue,
(4) As CDRL1, CDRL2, and CDRL3, one or more amino acid sequences of SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, or SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, respectively. An amino acid sequence each having a mutation of one amino acid residue,
CDRH1, CDRH2, and CDRH3 are one or several amino acids in the amino acid sequences of SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34, or the amino acid sequences of SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34, respectively. An antibody or antigen-binding fragment thereof comprising an amino acid sequence each having a mutation of residues, or (5) the amino acid sequences of SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37, respectively, as CDRL1, CDRL2, and CDRL3, or Comprising amino acid sequences each having a mutation of one or several amino acid residues in the amino acid sequences of SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37;
CDRH1, CDRH2, and CDRH3 are one or several amino acids in the amino acid sequences of SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, or the amino acid sequences of SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively. An antibody or antigen-binding fragment thereof comprising an amino acid sequence each having a residue mutation.
[2] An antibody or antigen-binding fragment thereof that specifically binds to an extracellular region of CD44v9 of a cell that expresses CD44v9,
(1) Light chain complementarity determining region 1 (CDRL1) comprising the amino acid sequence of RS-X 1 -KX 2 -LLH-X 3 -NGN-X 4 -YLY (SEQ ID NO: 41),
(2) a light chain complementarity determining region 2 (CDRL2) comprising the amino acid sequence of R-X 5 -SX 6 -LAS (SEQ ID NO: 42),
(3) a light chain complementarity determining region 3 (CDRL3) comprising the amino acid sequence of X 7 -QHLEYP-X 8 -T (SEQ ID NO: 43),
(4) heavy chain complementarity determining region 1 (CDRH1) comprising the amino acid sequence of GFN-X 9 -KDTY-X 10 -H (SEQ ID NO: 44),
(5) RIDPAN-X 11 -X 12 -X 13 -X 14 -Y-X 15 -PKFQ-X 16 ( SEQ ID NO: 45) heavy chain complementarity determining region 2 comprising an amino acid sequence of (CDRH2), and (6 ) Heavy chain complementarity determining region 3 (CDRH3) comprising the amino acid sequence of R-X seq -F-X 17 -X 18
(Where X 1 -X 18 are any naturally occurring amino acid residue and X seq is a group consisting of:
HLGLP (SEQ ID NO: 46)
QLGLP (SEQ ID NO: 47)
DVSSMIPTG (SEQ ID NO: 48)
SGVG (SEQ ID NO: 49), and DQTRATG (SEQ ID NO: 50)
Is an amino acid sequence selected from
Or an antigen-binding fragment thereof.
[3] X 1 is S or N;
X 2 is S or T;
X 3 is S or T;
X 4 is T or I;
X 5 is V or M;
X 6 is N or S;
X 7 is L or M;
X 8 is L, F or Y,
X 9 is I or V;
X 10 is M or I;
X 11 is G or D;
X 12 is N or S;
X 13 is T or C;
X 14 is Q, E, or K;
X 15 is D or N;
X 16 is D or G;
X 17 is A or G;
X 18 is Y or D,
The antibody or antigen-binding fragment thereof according to [2] above.
[4] (1) X 1 is S, X 2 is S, X 3 is S, X 4 is T, X 5 is V, X 6 is N, X 7 is L, X 8 is L, X 9 is I , X 10 is M, X 11 is G, X 12 is N, X 13 is T, X 14 is Q, X 15 is D, X 16 is D, X 17 is A, X 18 is Y, and X seq is HLGLP (SEQ ID NO: 46)
(2) X 1 is S, X 2 is S, X 3 is S, X 4 is T, X 5 is M, X 6 is N, X 7 is M, X 8 is L, X 9 is I, X 10 Is M, X 11 is D, X 12 is N, X 13 is T, X 14 is E, X 15 is D, X 16 is G, X 17 is A, X 18 is Y, and X seq is QLGLP (sequence Number 47)
(3) X 1 is S, X 2 is S, X 3 is S, X 4 is I, X 5 is M, X 6 is N, X 7 is M, X 8 is F, X 9 is I, X 10 Is M, X 11 is D, X 12 is N, X 13 is T, X 14 is K, X 15 is N, X 16 is G, X 17 is A, X 18 is Y, and X seq is DVSS MIPTG (array Number 48),
(4) X 1 is N, X 2 is T, X 3 is T, X 4 is T, X 5 is M, X 6 is N, X 7 is M, X 8 is F, X 9 is I, X 10 Is M, X 11 is G, X 12 is N, X 13 is C, X 14 is K, X 15 is D, X 16 is G, X 17 is G, X 18 is Y, and X seq is SGVG (array (5) X 1 is S, X 2 is S, X 3 is S, X 4 is T, X 5 is V, X 6 is S, X 7 is M, X 8 is Y , X 9 is V, X 10 is I, X 11 is G, X 12 is S, X 13 is T, X 14 is K, X 15 is D, X 16 is G, X 17 is A, X 18 is D And the antibody or antigen-binding fragment thereof according to Item [2], wherein X seq is DQTRATG (SEQ ID NO: 50).
[5] The antibody or antigen-binding fragment thereof according to any one of [1] to [4] above,
(1) including a light chain variable region (LH) comprising an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 1, and an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 2. Heavy chain variable region (VH),
(2) LH including an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 3, and VH including an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 4,
(3) LH including an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 5, and VH including an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 6,
(4) LH including an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 7 and VH including an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 8, or (5) LH including an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 9, and VH including an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 10
Or an antigen-binding fragment thereof, comprising
[6] The antibody or antigen-binding fragment thereof according to any one of [1] to [5] above,
(1) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 2,
(2) VL comprising the amino acid sequence of SEQ ID NO: 3 and VH comprising the amino acid sequence of SEQ ID NO: 4,
(3) a VL comprising the amino acid sequence of SEQ ID NO: 5, and a VH comprising the amino acid sequence of SEQ ID NO: 6,
(4) VL including the amino acid sequence of SEQ ID NO: 7 and VH including the amino acid sequence of SEQ ID NO: 8, or (5) VL including the amino acid sequence of SEQ ID NO: 9 and VH including the amino acid sequence of SEQ ID NO: 10.
Or an antigen-binding fragment thereof, comprising
[7] The antibody or antigen-binding fragment thereof according to any one of [1] to [6] above, which has cytotoxic activity and / or internalization activity for cells expressing CD44v9.
[8] The cell according to any one of the above [1] to [6], which specifically binds to CD44v9 of a cell expressing CD44v9 and has an activity of inhibiting an interaction between CD44v9 and the amino acid transporter xCT. An antibody or antigen-binding fragment thereof.
[9]
(I) The antibody or antigen-binding fragment thereof according to any one of [1] to [6], which suppresses a decrease in intracellular reactive oxygen species, or (ii) inhibits extracellular discharge of a drug. .
[10] Any of the above [1] to [6], which specifically binds to CD44v9 of a cell expressing CD44v9 and has an activity of inhibiting the tumor-forming ability and metastasis-forming ability of cancer stem cells and / or cancer cells The antibody or antigen-binding fragment thereof according to claim 1.
[11] The antibody or antigen-binding fragment thereof according to any one of [1] to [10], wherein the subclass is IgG.
[12] The antibody or antigen-binding fragment thereof according to [11] above, wherein the IgG is IgG 2b or IgG 1 .
[13] The antigen-binding fragment is Fab, Fab ′, F (ab ′) 2 , scFv, dsFv, ds-scFv, their dimers, minibodies, diabodies, and multimers, or two The antibody or antigen-binding fragment thereof according to any one of [1] to [12] above, which is a multispecific antibody fragment.
[14] The antibody or antigen-binding fragment thereof according to any one of [1] to [13], which is a monoclonal antibody.
[15] The antibody or antigen-binding fragment thereof according to any one of [1] to [14] above, which is a rat, mouse, primate, or human antibody.
[16] The antibody or antigen-binding fragment thereof according to any one of [1] to [14], which is a chimeric antibody or a humanized antibody.
[17] An immunoconjugate comprising the antibody or antigen-binding fragment thereof according to any one of [1] to [16] above and at least one cytotoxic drug, antitumor agent, or radioisotope.
[18] An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of [1] to [17].
[19] An expression vector comprising the nucleic acid molecule according to [18].
[20] A hybridoma cell or a gene-introduced cell that produces the antibody according to any one of [1] to [16] above, which contains the nucleic acid molecule according to [18] or into which the nucleic acid molecule has been introduced. .
[21] A method for producing a monoclonal antibody that specifically binds to CD44v9, comprising culturing the cell according to [20] above and collecting an antibody that specifically binds to CD44v9 from the obtained culture.
[22] A gene encoding an anti-CD44v9 monoclonal antibody is isolated from the cell described in [20] above, an expression vector containing the gene is constructed, the expression vector is introduced into a host, and the monoclonal antibody is expressed. A method for producing a monoclonal antibody that specifically binds to CD44v9, which comprises collecting a monoclonal antibody that specifically binds to CD44v9 from the resulting host, the culture supernatant of the host or the secretion of the host.
[23] The production method according to [22], wherein the host is Escherichia coli, yeast cells, insect cells, mammalian cells, plant cells, or mammals.
[24] A composition containing the antibody or antigen-binding fragment thereof or immunoconjugate thereof according to any one of [1] to [17] above, which is applied to a cell expressing CD44v9, A composition used for inducing apoptosis.
[25] A medicament for preventing or treating a tumor, comprising the antibody or antigen-binding fragment thereof or immunoconjugate thereof according to any one of [1] to [17].
[26] A composition containing the antibody or antigen-binding fragment thereof or immunoconjugate thereof according to any one of [1] to [17], which is used for detecting a cell expressing CD44v9. ,Composition.
[27] A method for detecting a tumor, comprising reacting the composition according to [26] above with a sample collected from a living body and detecting a reacted signal.
[28] A method for immunodetecting a tumor in a test tube, comprising the step of contacting the composition according to [26] above with a cancer cell.
[29] A method for imaging a tumor in vivo, comprising the step of administering the composition of [26] to an individual and obtaining a detection image obtained by near-infrared light imaging, PET, MRI, or ultrasonic imaging.
[30] A medicament for diagnosing a tumor, comprising the antibody or antigen-binding fragment or immunoconjugate thereof according to any one of [1] to [17].
[31] The tumor is colon cancer, colorectal cancer, lung cancer, breast cancer, brain tumor, melanoma, renal cell cancer, bladder cancer, leukemia, lymphoma, T cell lymphoma, multiple myeloma, gastric cancer, pancreatic cancer, cervix Cancer, endometrial cancer, ovarian cancer, esophageal cancer, liver cancer, squamous cell carcinoma of the head and neck, skin cancer, urinary tract cancer, prostate cancer, choriocarcinoma, pharyngeal cancer, laryngeal cancer, tongue cancer, oral cancer, gallbladder cancer, Thyroid cancer, mesothelioma, pleurioma, male embryo, endometrial hyperplasia, endometriosis, embryonal, fibrosarcoma, Kaposi's sarcoma, hemangioma, spongiform hemangioma, hemangioblastoma, retinoblastoma, Astrocytoma, neurofibromatosis, oligodendroma, medulloblastoma, neuroblastoma, glioma, rhabdomyosarcoma, glioblastoma, osteogenic sarcoma, leiomyosarcoma, thyroid sarcoma, and Wilms tumor The medicament according to [25] or [30] above, which is at least one selected from the group consisting of:
[32] The above-mentioned [25] or [30], wherein the tumor is colon cancer, colorectal cancer, lung cancer, breast cancer, brain tumor, bladder cancer, lymphoma, gastric cancer, pancreatic cancer, liver cancer, or prostate cancer. Medicine.
[33] The medicament according to any one of [30] to [32] above, which is administered simultaneously, separately or sequentially with a pharmaceutically acceptable cytostatic agent.
[34] The above-mentioned [33], wherein the cytostatic agent is at least one selected from the group consisting of an alkylating agent, an antimetabolite, a molecular target drug, a plant alkaloid, an anticancer antibiotic, and a platinum preparation. ] The medicine as described in].
[35] The alkylating agent is selected from the group consisting of ifosfamide, cyclophosphamide, dacarbacin, temozolomide, nimustine, busulfan, procarbazine, melphalan, and ranimustine,
The antimetabolite is enocitabine, capecitabine, carmofur, cladripine, gemcitabine, cytarabine, cytarabine ocphosate, tegafur, tegafur uracil, TS-1, doxyfluridine, nelarabine, hydroxycarbamide, fluorouracil, fludarabine, pemetrexed captostatin, pentostatin, pentostatin And selected from the group consisting of methotrexate,
The molecular targeted drug is selected from the group consisting of Zevalin, Imatinib, Erlotinib, Gefitinib, Sunitinib, Cetuximab, Sorafenib, Dasatinib, Trastuzumab, Tretinoin, Panitumumab, Bevacizumab, Bortezomib, and Rituximab
The plant alkaloid is selected from the group consisting of irinotecan, etoposide, sobuzoxan, docetaxel, nogitecan, paclitaxel, vinorelbine, vincristine, vindesine, and vinblastine;
The anticancer antibiotic is selected from the group consisting of actinomycin D, aclarubicin, amrubicin, idarubicin, epirubicin, smux, daunorubicin, doxorubicin, pirarubicin, bleomycin, peomycin, mitomycin C, mitoxantrone, and doxil;
The medicament according to [33] above, wherein the platinum preparation is at least one selected from the group consisting of oxaliplatin, cisplatin, carboplatin, and nedaplatin.
[36] Complementarity determining region (CDR) selected from the group consisting of the following (1) to (6):
(1) CDRL1, comprising the amino acid sequences of SEQ ID NOs: 11, 17, 23, 29, and 35,
(2) CDRL2, comprising the amino acid sequences of SEQ ID NOs: 12, 18, 24, 30, 36
(3) CDRL3 comprising the amino acid sequences of SEQ ID NOs: 13, 19, 25, 31, 37
(4) CDRH1, comprising the amino acid sequences of SEQ ID NOs: 14, 20, 26, 32, 38,
(5) CDRH2 comprising the amino acid sequences of SEQ ID NOs: 15, 21, 27, 33, 39, and (6) CDRH3 comprising the amino acid sequences of SEQ ID NOs: 16, 22, 28, 34, 40.
　本発明の抗体またはその抗原結合断片は、癌幹細胞において高度に発現亢進し、正常細胞にはほとんど発現しない抗原（すなわち、ＣＤ４４ｖ９）に対して特異的に結合し、癌幹細胞の腫瘍形成能および転移巣形成能の抑制効果を有するため、癌における最も重要な課題である再発や転移を防ぎ癌の根治につながる、癌幹細胞の機能を標的とした治療のための医薬等として特に有用である。 The antibody or antigen-binding fragment thereof of the present invention is highly upregulated in cancer stem cells and specifically binds to an antigen that is hardly expressed in normal cells (ie, CD44v9). Since it has an inhibitory effect on nest-forming ability, it is particularly useful as a medicine for treatment targeting the function of cancer stem cells, which prevents recurrence and metastasis, which are the most important problems in cancer, and leads to the cure of cancer.
　さらに、本発明の抗体またはその抗原結合断片に殺細胞活性および／または抗腫瘍活性を有する化合物あるいは放射性同位体を結合させたイムノコンジュゲートにより、ＣＤ４４ｖ８−１０などのＣＤ４４ｖ９を含むアイソフォームを発現する細胞の細胞死誘導、および検出を行うができる。 Furthermore, an isoform containing CD44v9 such as CD44v8-10 is expressed by an immunoconjugate in which a compound having a cell-killing activity and / or an antitumor activity or a radioisotope is bound to the antibody of the present invention or an antigen-binding fragment thereof. Induction and detection of cell death can be performed.
　さらに、本発明の抗体またはその抗原結合断片は、ＣＤ４４ｖ８−１０のｘＣＴ安定化機能を阻害することで、細胞内ＲＯＳ濃度を上昇させ抗癌剤や放射線治療による細胞死誘導を促進する、また、ｘＣＴが関与する薬剤排出機能を阻害することにより細胞内に抗癌剤が貯留しやすくなり、薬剤の効果を増強することができる。 Furthermore, the antibody or antigen-binding fragment thereof of the present invention inhibits the xCT stabilization function of CD44v8-10, thereby increasing the intracellular ROS concentration and promoting cell death induction by an anticancer agent or radiation therapy. By inhibiting the drug discharge function involved, the anticancer drug can be easily stored in the cells, and the effect of the drug can be enhanced.
ＣＤ４４ｖ８−１０強制発現細胞のＦＡＣＳ解析によるＡＣＳＣ００２モノクローナル抗体、ＡＣＳＣ００５モノクローナル抗体、ＡＣＳＣ００７モノクローナル抗体、ＡＣＳＣ００９モノクローナル抗体、ＡＣＳＣ０１０モノクローナル抗体の特異性を示す図である。横軸：強制発現させた遺伝子の発現量、縦軸：モノクローナル抗体の反応性。It is a figure which shows the specificity of ACSC002 monoclonal antibody, ACSC005 monoclonal antibody, ACSC007 monoclonal antibody, ACSC009 monoclonal antibody, ACSC010 monoclonal antibody by FACS analysis of CD44v8-10 forced expression cell. Horizontal axis: expression level of forcedly expressed gene, vertical axis: reactivity of monoclonal antibody. ウエスタンブロット法によるＡＣＳＣ００２モノクローナル抗体、ＡＣＳＣ００５モノクローナル抗体、ＡＣＳＣ００７モノクローナル抗体、ＡＣＳＣ００９モノクローナル抗体、ＡＣＳＣ０１０モノクローナル抗体の結合部位を示す図である。ＣＤ４４ｖ８、ＣＤ４４ｖ９、ＣＤ４４ｖ１０の組換え蛋白質に対する反応性を検出した。It is a figure which shows the binding site of ACSC002 monoclonal antibody, ACSC005 monoclonal antibody, ACSC007 monoclonal antibody, ACSC009 monoclonal antibody, ACSC010 monoclonal antibody by Western blotting. Reactivity with CD44v8, CD44v9 and CD44v10 recombinant proteins was detected. ヒト乳癌細胞株およびヒト大腸癌細胞株へのＡＣＳＣ００２モノクローナル抗体、ＡＣＳＣ００５モノクローナル抗体、ＡＣＳＣ００７モノクローナル抗体、ＡＣＳＣ００９モノクローナル抗体、ＡＣＳＣ０１０モノクローナル抗体の結合を示す図である。値は抗体反応時のＭＦＩからＣｏｎｔｒｏｌとして抗体を含まないときのＭＦＩを引いた値を示す。It is a figure which shows the coupling | bonding of ACSC002 monoclonal antibody, ACSC005 monoclonal antibody, ACSC007 monoclonal antibody, ACSC009 monoclonal antibody, ACSC010 monoclonal antibody to a human breast cancer cell line and a human colon cancer cell line. The value indicates a value obtained by subtracting MFI when no antibody is contained as Control from MFI at the time of antibody reaction. Ｉｍｍｕｎｏｃｙｔｏｃｈｅｍｉｓｔｒｙ法によるＡＣＳＣ００２モノクローナル抗体、ＡＣＳＣ００５モノクローナル抗体、ＡＣＳＣ００７モノクローナル抗体、ＡＣＳＣ００９モノクローナル抗体、ＡＣＳＣ０１０モノクローナル抗体の細胞膜に局在するＣＤ４４ｖ９への結合を示す図である。緑色はモノクローナル抗体のシグナル、青色は核を示す。It is a figure which shows the coupling | bonding to CD44v9 localized in the cell membrane of ACSC002 monoclonal antibody, ACSC005 monoclonal antibody, ACSC007 monoclonal antibody, ACSC009 monoclonal antibody, and ACSC010 monoclonal antibody by Immunocytochemistry method. Green indicates the monoclonal antibody signal, and blue indicates the nucleus. ＡＣＳＣ０１０キメラ化抗体を用いたＡＤＣＣ活性を示す図である。値は細胞障害率（％）を示す。（Ａ）ヒト乳癌細胞ＭＤＡ−ＭＢ４６８、（Ｂ）コントロールである正常細胞株Ｈｓ６１７．ＭｇおよびＨｓ７４２．Ｓｋ。It is a figure which shows ADCC activity using an ACSC010 chimerized antibody. The value indicates the cytotoxic rate (%). (A) Human breast cancer cell MDA-MB468, (B) Normal cell line Hs617. Mg and Hs742. Sk. ＡＣＳＣ００２モノクローナル抗体、ＡＣＳＣ００５モノクローナル抗体、ＡＣＳＣ００７モノクローナル抗体、ＡＣＳＣ００９モノクローナル抗体、ＡＣＳＣ０１０モノクローナル抗体のインターナリゼーション活性を示す図である。値は３７℃（インターナリゼーションが誘導される温度）および４℃（インターナリゼーションが誘導されない温度）の各条件下においてモノクローナル抗体を反応させたときのＭＦＩからマウスＩｇＧを反応させたＣｏｎｔｒｏｌのＭＦＩを引いた値（ΔＭＦＩ）を示す。It is a figure which shows the internalization activity of an ACSC002 monoclonal antibody, an ACSC005 monoclonal antibody, an ACSC007 monoclonal antibody, an ACSC009 monoclonal antibody, and an ACSC010 monoclonal antibody. Values are 37 ° C. (temperature at which internalization is induced) and 4 ° C. (temperature at which internalization is not induced). The MFI of the control reacted with mouse IgG from the MFI when the monoclonal antibody was reacted. The value obtained by subtracting (ΔMFI) is shown. ＡＣＳＣ００２モノクローナル抗体、ＡＣＳＣ００５モノクローナル抗体、ＡＣＳＣ０１０モノクローナル抗体によるＲＯＳ上昇効果を示す図である。青色はＨｏｅｃｈｓｔ３３３４２による核染色、緑色はＤＣＦによるＲＯＳの検出を示す。It is a figure which shows the ROS raise effect by an ACSC002 monoclonal antibody, an ACSC005 monoclonal antibody, and an ACSC010 monoclonal antibody. Blue indicates nuclear staining with Hoechst 33342, and green indicates detection of ROS with DCF. （Ａ）セルソーティングによる前立腺癌細胞株ＰＣ３におけるＣＤ４４ｖ９高発現群とＣＤ４４ｖ９低発現群の分取を示す図である。値は抗体反応時のＭＦＩからＣｏｎｔｒｏｌとして抗体を含まないときのＭＦＩを引いた値を示す。横軸：ＣＤ４４ｖ９の発現量。（Ｂ）ｉｎ　ｖｉｔｒｏ評価系による前立腺癌細胞株ＰＣ３のＣＤ４４ｖ９高発現群のＳｐｈｅｒｅ（Ｃｏｌｏｎｙ）形成能を示す図である。縦軸：各ウェルのＳｐｈｅｒｅ形成数。(A) Sorting of CD44v9 high expression group and CD44v9 low expression group in prostate cancer cell line PC3 by cell sorting. The value indicates a value obtained by subtracting MFI when no antibody is contained as Control from MFI at the time of antibody reaction. Horizontal axis: CD44v9 expression level. (B) It is a figure which shows the Sphere (Colony) formation ability of the CD44v9 high expression group of prostate cancer cell line PC3 by an in vitro evaluation system. Vertical axis: Number of spheres formed in each well. ｉｎ　ｖｉｔｒｏ評価系によるＡＣＳＣ００２モノクローナル抗体、ＡＣＳＣ００５モノクローナル抗体、ＡＣＳＣ００９モノクローナル抗体、ＡＣＳＣ０１０モノクローナル抗体によるＳｐｈｅｒｅ形成能の抑制効果を示す図である。縦軸：各ウェルのＳｐｈｅｒｅ形成数。It is a figure which shows the inhibitory effect of the Sphere formation ability by the ACSC002 monoclonal antibody, the ACSC005 monoclonal antibody, the ACSC009 monoclonal antibody, and the ACSC010 monoclonal antibody by the in vitro evaluation system. Vertical axis: Number of spheres formed in each well. （Ａ）ｉｎ　ｖｉｖｏ評価系によるＡＣＳＣ００２モノクローナル抗体による抗体濃度依存的な転移巣形成抑制効果を示す図である。（Ｂ）ＡＣＳＣ００２モノクローナル抗体とＬＧａｄｈ０１−０１モノクローナル抗体の転移巣形成抑制効果を比較した図である。縦軸：解剖後の肺表面上に確認された生着コロニー数。(A) It is a figure which shows the antibody-concentration-dependent metastatic focus formation inhibitory effect by the ACSC002 monoclonal antibody by an in vivo evaluation system. (B) It is the figure which compared the metastasis formation inhibitory effect of ACSC002 monoclonal antibody and LGAdh01-01 monoclonal antibody. Vertical axis: number of engrafted colonies confirmed on the lung surface after dissection.
１．ＣＤ４４ｖ９を特異的に認識する抗体
　本発明は、１つの実施形態において、ＣＤ４４ｖ９に特異的に結合する抗体（本明細書中、「抗ＣＤ４４ｖ９抗体」と呼ぶこともある。）およびその抗原結合断片を提供する。すなわち、本発明は、ＣＤ４４ｖ９のアミノ酸配列の一部または全部をエピトープとする抗体およびその抗原結合断片を提供する。 1. Antibody that specifically recognizes CD44v9 In one embodiment of the present invention, an antibody that specifically binds to CD44v9 (sometimes referred to herein as an “anti-CD44v9 antibody”) and an antigen-binding fragment thereof. provide. That is, the present invention provides an antibody and an antigen-binding fragment thereof having an epitope of a part or all of the amino acid sequence of CD44v9.
　「ＣＤ４４」は、細胞膜を１回貫通し、Ｎ末端が細胞外、Ｃ末端が細胞内にあるＩ型膜糖蛋白質である。現在、ＣＤ４４遺伝子には２０個のエキソンが存在することが確認されている。選択的スプライシングを受けて、エキソン６~１５（バリアントエキソン：ｖ１~ｖ１０）が種々な組合せで挿入されることにより、これまでに多様なアイソフォームが確認されている。 “CD44” is a type I membrane glycoprotein that penetrates the cell membrane once, has an N-terminal extracellular, and a C-terminal is intracellular. Currently, it has been confirmed that there are 20 exons in the CD44 gene. Various isoforms have been confirmed so far by inserting exons 6 to 15 (variant exons: v1 to v10) in various combinations through alternative splicing.
　本明細書中、「ＣＤ４４ｖ９」または「ＣＤ４４　ｖａｒｉａｎｔ９」は、３１アミノ酸残基からなるｖ９エキソンが挿入されているＣＤ４４のバリアントアイソフォームである。ｖ９エキソンのアミノ酸配列、ｍＲＮＡ配列等の情報は、それぞれＮＭ＿００１００１３９０、ＮＰ＿００１００１３９０等のアクセッション番号でＧｅｎＢａｎｋ等の公にアクセス可能なデータベースから入手することができる。また、マウスや他の哺乳動物（例えば、ラット、ウシなど）のＣＤ４４ｖ９も同様に、公にアクセス可能なデータベースから入手することが出来る。 In the present specification, “CD44v9” or “CD44 variant9” is a variant isoform of CD44 in which a v9 exon consisting of 31 amino acid residues is inserted. Information such as amino acid sequence and mRNA sequence of v9 exon can be obtained from publicly accessible databases such as GenBank with accession numbers such as NM_001001390 and NP_001001390, respectively. Similarly, CD44v9 for mice and other mammals (eg, rats, cows, etc.) can be obtained from publicly accessible databases.
　本明細書中、単に「ＣＤ４４ｖ９」または「ＣＤ４４　ｖａｒｉａｎｔ９」という場合、ＣＤ４４ｖ９のことを指すものとし、特に、ＣＤ４４ｖ９蛋白質のことを指すものとする。場合によっては、ＣＤ４４ｖ９蛋白質をコードする遺伝子（または単にＣＤ４４ｖ９遺伝子）を単にＣＤ４４ｖ９と呼ぶ場合もありうるが、その場合には当業者にとっては文脈からＣＤ４４ｖ９遺伝子を指すことが明らかであろう。本明細書中、ＣＤ４４ｖ９は、典型的には、ヒトＣＤ４４ｖ９であるが、ヒト以外の哺乳動物（例えば、マウス、ラット、ウシなど）のＣＤ４４ｖ９であってもよい。 In this specification, “CD44v9” or “CD44 variant9” simply refers to CD44v9, and in particular, refers to CD44v9 protein. In some cases, the gene encoding the CD44v9 protein (or simply the CD44v9 gene) may be simply referred to as CD44v9, but in that case it will be clear to the skilled person that the term refers to the CD44v9 gene. In this specification, CD44v9 is typically human CD44v9, but may be CD44v9 of mammals other than humans (eg, mouse, rat, bovine, etc.).
　さらなる実施形態において、本発明の抗体およびその抗原結合断片は、典型的には、ＣＤ４４ｖ９発現細胞に発現しているＣＤ４４ｖ９に特異的に結合し、アミノ酸トランスポーターｘＣＴとの相互作用を阻害する機能（または活性）を有する。 In further embodiments, the antibodies and antigen-binding fragments thereof of the present invention typically bind specifically to CD44v9 expressed in CD44v9-expressing cells and function to inhibit interaction with the amino acid transporter xCT ( Or active).
　シスチン・グルタミン酸交換輸送系である「ヒトｘＣＴ（ｃａｔｉｏｎｉｃ　ａｍｉｎｏ　ａｃｉｄ　ｔｒａｎｓｐｏｒｔｅｒ，　ｙ＋　ｓｙｓｔｅｍ）」は、５０１アミノ酸残基からなる１２回膜貫通型の膜タンパク質である。ヒトｘＣＴのアミノ酸配列、ｍＲＮＡ配列等の情報は、それぞれＡＡＧ３５５９２、ＡＣ１１０８０４、ＡＦ２００７０８等のアクセッション番号でＧｅｎＢａｎｋ等の公にアクセス可能なデータベースから入手することができる。また、マウスや他の哺乳動物（例えば、ラット、ウシなど）のｘＣＴも同様に、公にアクセス可能なデータベースから入手することができる。 “Human xCT (cationic amino acid transporter, y + system)”, which is a cystine / glutamate exchange transport system, is a 12-transmembrane membrane protein consisting of 501 amino acid residues. Information such as amino acid sequence and mRNA sequence of human xCT can be obtained from publicly accessible databases such as GenBank with accession numbers such as AAG35592, AC110804, and AF200708, respectively. Similarly, xCT of mice and other mammals (eg, rats, cows, etc.) can be obtained from publicly accessible databases.
　本明細書中、単に「ｘＣＴ」という場合、ｘＣＴタンパク質のことを指すものとする。場合によっては、ｘＣＴタンパク質をコードする遺伝子（または単にｘＣＴ遺伝子）を単にｘＣＴと呼ぶ場合もありうるが、その場合には当業者にとっては文脈からｘＣＴ遺伝子を指すことが明らかであろう。本明細書中、ｘＣＴは、典型的には、ヒトｘＣＴであるが、ヒト以外の哺乳動物（例えば、マウス、ラット、ウシなど）のｘＣＴであってもよい。 In this specification, “xCT” simply refers to xCT protein. In some cases, the gene encoding the xCT protein (or simply the xCT gene) may simply be referred to as xCT, in which case it will be clear to the skilled person to refer to the xCT gene from the context. In the present specification, xCT is typically human xCT, but may be xCT of mammals other than humans (for example, mouse, rat, cow, etc.).
　アミノ酸トランスポーターの一つであるｘＣＴは、細胞内からグルタミン酸を放出し、細胞外からシスチンを取り込んでシステインに還元して、ペプチドの一種であるＧＳＨを生成させる。細胞内ＧＳＨの役割は２つあり、１つは細胞内のＲＯＳを還元することで、細胞死から回避させることである。もう１つは、グルタチオン抱合により薬剤を細胞外に排出することである。ＣＤ４４ｖ９を含むＣＤ４４Ｒ１はｘＣＴを安定化させることによって、生体内の主要な抗酸化物質のひとつであるＧＳＨの合成を促進し、細胞内のＲＯＳを還元するのに貢献している。したがって、ＣＤ４４ｖ９とｘＣＴとの相互作用を阻害することができる抗体およびその抗原結合断片は、（ｉ）細胞内活性酸素種の低下を抑制するか、または（ｉｉ）薬剤の細胞外排出を阻害することができると考えられる。このような抑制または阻害を受けた細胞は、細胞死へと導かれうる。 XCT, which is one of amino acid transporters, releases glutamic acid from the inside of the cell, takes cystine from the outside of the cell, reduces it to cysteine, and generates GSH which is a kind of peptide. There are two roles of intracellular GSH, and one is to avoid intracellular death by reducing intracellular ROS. The other is to drain the drug out of the cell by glutathione conjugation. CD44R1 including CD44v9 promotes the synthesis of GSH, which is one of the main antioxidants in the body, by stabilizing xCT, and contributes to reducing intracellular ROS. Therefore, antibodies and antigen-binding fragments thereof that can inhibit the interaction between CD44v9 and xCT (i) suppress the reduction of intracellular reactive oxygen species, or (ii) inhibit the extracellular excretion of drugs. It is considered possible. Cells that have undergone such suppression or inhibition can lead to cell death.
　本明細書中、抗体またはその抗原結合断片が、ＣＤ４４ｖ９に「特異的に結合する」とは、その抗体または抗原結合断片が他のアミノ酸配列に対するその親和性よりも、この蛋白質の特定のアミノ酸配列に対して実質的に高い親和性で結合することを意味する。ここで、「実質的に高い親和性」とは、所望の測定装置によって、その特定のアミノ酸配列を他のアミノ酸配列から区別して検出することが可能なほどに高い親和性を意味し、典型的には、結合定数（Ｋａ）が少なくとも１０７Ｍ−１、好ましくは、少なくとも１０８Ｍ−１、より好ましくは、１０９Ｍ−１、さらにより好ましくは、１０１０Ｍ−１、１０１１Ｍ−１、１０１２Ｍ−１またはそれより高い、例えば、最高で１０１３Ｍ−１またはそれより高いものであるような結合親和性を意味する。 As used herein, an antibody or antigen-binding fragment thereof “specifically binds” to CD44v9 means that the antibody or antigen-binding fragment has a specific amino acid sequence of this protein rather than its affinity for other amino acid sequences. Means binding with substantially high affinity. Here, “substantially high affinity” means high affinity that allows the specific amino acid sequence to be detected separately from other amino acid sequences by a desired measuring device. Has a binding constant (K a ) of at least 10 7 M −1 , preferably at least 10 8 M −1 , more preferably 10 9 M −1 , even more preferably 10 10 M −1 , 10 11. It means a binding affinity such that it is M −1 , 10 12 M −1 or higher, for example up to 10 13 M −1 or higher.
　本明細書中、「抗体」は、インタクトな免疫グロブリンの全てのクラスおよびサブクラスを含むものとする。好ましくは、本発明の抗体は、ＩｇＧサブクラスのものであり、より好ましくは、ヒトＩｇＧ１サブクラスのものである。「抗体」は、特に、モノクローナル抗体を含む。 As used herein, “antibody” is intended to include all classes and subclasses of intact immunoglobulins. Preferably, antibodies of the invention are of the IgG subclass, and more preferably, is human IgG 1 subclass. “Antibodies” include in particular monoclonal antibodies.
　本明細書中、「抗原結合断片」は、インタクトな、および／またはヒト化された、および／またはキメラな抗体の抗原結合領域または可変領域を有するフラグメント、たとえば、上記抗体のＦａｂ、Ｆａｂ’、Ｆ（ａｂ’）２、Ｆｖ、ＳｃＦｖフラグメントを含む。従来、こうしたフラグメントは、インタクトな抗体の蛋白質分解によって、たとえばパパイン分解によって（たとえば、国際公開ＷＯ９４／２９３４８を参照）作製されているが、遺伝子組換えによる形質転換宿主細胞から直接産生させることもできる。ＳｃＦｖの作製については、Ｂｉｒｄら、（１９８８）　Ｓｃｉｅｎｃｅ，　２４２，　４２３−４２６に記載の方法が使用できる。さらに、抗体フラグメントは、下記のさまざまな遺伝子工学技術を用いて作製することができる。 As used herein, an “antigen-binding fragment” refers to a fragment having the antigen-binding or variable region of an intact and / or humanized and / or chimeric antibody, such as Fab, Fab ′, Includes F (ab ′) 2 , Fv, ScFv fragments. Traditionally, such fragments have been produced by proteolysis of intact antibodies, for example by papain degradation (see eg WO 94/29348), but can also be produced directly from transgenic host cells by genetic recombination. . For the production of ScFv, the method described in Bird et al. (1988) Science, 242, 423-426 can be used. Furthermore, antibody fragments can be generated using the various genetic engineering techniques described below.
　Ｆｖフラグメントは、その２つの鎖の相互作用エネルギーがＦａｂフラグメントより低いと思われる。ＶＨおよびＶＬドメインの結合を安定化するために、これらのドメインは、ペプチド（Ｂｉｒｄら、（１９８８）　Ｓｃｉｅｎｃｅ　２４２，　４２３−４２６、Ｈｕｓｔｏｎら、ＰＮＡＳ，　８５，　５８７９−５８８３）、ジスルフィド架橋（Ｇｌｏｃｋｓｈｕｂｅｒら、（１９９０）　Ｂｉｏｃｈｅｍｉｓｔｒｙ，　２９，　１３６２−１３６７）、および「ｋｎｏｂ　ｉｎ　ｈｏｌｅ」変異（Ｚｈｕら、（１９９７），　Ｐｒｏｔｅｉｎ　Ｓｃｉ．，６，　７８１−７８８）によって連結されている。ＳｃＦｖフラグメントは、当業者によく知られている方法によって作製することができる（Ｗｈｉｔｌｏｗら、（１９９１）　Ｍｅｔｈｏｄｓ　ｃｏｍｐａｎｉｏｎ　Ｍｅｔｈｏｄｓ　Ｅｎｚｙｍｏｌ，　２，　９７−１０５およびＨｕｓｔｏｎら、（１９９３）　Ｉｎｔ．Ｒｅｖ．Ｉｍｍｕｎｏｌ　１０，　１９５−２１７を参照）。ＳｃＦｖは、大腸菌（Ｅ．　ｃｏｌｉ）などの細菌細胞内で産生させることができるが、真核細胞内で産生させる方が好ましい。ＳｃＦｖの不利な点は、産物が一価であること、そのために多価結合による結合力の増加が不可能になること、ならびに半減期が短いことである。こうした問題を克服するための試みには、二価（ＳｃＦｖ’）２があるが、これは、追加のＣ末端システインを含有するＳｃＦｖから、化学的カップリングによって（Ａｄａｍｓら、（１９９３）　Ｃａｎ．Ｒｅｓ　５３，４０２６−４０３４、およびＭｃＣａｒｔｎｅｙら、（１９９５）　Ｐｒｏｔｅｉｎ　Ｅｎｇ．　８，　３０１−３１４）、または不対Ｃ末端システイン残基を含有するＳｃＦｖの、自然発生的な部位特異的二量体化によって（Ｋｉｐｒｉｙａｎｏｖら、（１９９５）　Ｃｅｌｌ．　Ｂｉｏｐｈｙｓ　２６，　１８７−２０４を参照）作製される。あるいはまた、ペプチドリンカーを３~１２残基に短縮して「ダイアボディ（ｄｉａｂｏｄｙ）」を形成することによって、ＳｃＦｖに多量体を形成させることができる。Ｈｏｌｌｉｇｅｒら、ＰＮＡＳ　（１９９３），　９０，　６４４４−６４４８を参照。リンカーをさらに小さくすることで、ＳｃＦｖ三量体（「トリアボディ」、Ｋｏｒｔｔら、（１９９７）　Ｐｒｏｔｅｉｎ　Ｅｎｇ，　１０，　４２３−４３３を参照）および四量体（「テトラボディ」、Ｌｅ　Ｇａｌｌら、（１９９９）　ＦＥＢＳ　Ｌｅｔｔ，　４５３，　１６４−１６８を参照）をもたらすことができる。二価ＳｃＦｖ分子の構築は、「ミニ抗体（ｍｉｎｉａｎｔｉｂｏｄｙ）」（Ｐａｃｋら、（１９９２）　Ｂｉｏｃｈｅｍｉｓｔｒｙ　３１，　１５７９−１５８４を参照）および「ミニボディ」（Ｈｕら、（１９９６），　Ｃａｎｃｅｒ　Ｒｅｓ．　５６，　３０５５−３０６１を参照）を形成することができる蛋白質二量体化モチーフとの遺伝子融合によっても、達成することができる。ＳｃＦｖ−ＳｃＦｖタンデム（（ＳｃＦｖ）２）は、第３のペプチドリンカーによって２つのＳｃＦｖ単位を連結することによって作製することもできる（Ｋｕｒｕｃｚら、（１９９５）　Ｊ．Ｉｍｍｏｌ．１５４，　４５７６−４５８２を参照）。二重特異性ダイアボディは、ある抗体のＶＬドメインに短いリンカーで連結された、別の抗体由来のＶＨドメインからなる、２つの一本鎖融合産物の非共有結合によって作製することができる（Ｋｉｐｒｉｙａｎｏｖら、（１９９８），　Ｉｎｔ．　Ｊ．　Ｃａｎ　７７，　７６３−７７２を参照）。このような二重特異性ダイアボディの安定性は、ジスルフィド架橋、もしくは上記の「ｋｎｏｂ　ｉｎ　ｈｏｌｅ」変異を導入することによって、または２つのハイブリッドＳｃＦｖフラグメントがペプチドリンカーを介して連結される、一本鎖ダイアボディ（ＳｃＤｂ）を形成することによって、高めることができる（Ｋｏｎｔｅｒｍａｎｎら、（１９９９）　Ｊ．　Ｉｍｍｕｎｏｌ．　Ｍｅｔｈｏｄｓ　２２６　１７９−１８８を参照）。四価の二重特異性分子は、たとえば、ＳｃＦｖフラグメントを、ＩｇＧ分子のＣＨ３ドメインに、またはヒンジ領域を介してＦａｂフラグメントに、融合することによって得られる（Ｃｏｌｏｍａら、（１９９７）　Ｎａｔｕｒｅ　Ｂｉｏｔｅｃｈｎｏｌ．　１５，　１５９−１６３を参照）。あるいはまた、四価の二重特異性分子は、二重特異性一本鎖ダイアボディの融合によって作製されている（Ａｌｔら、（１９９９）　ＦＥＢＳ　Ｌｅｔｔ　４５４，　９０−９４を参照）。より小さい四価の二重特異性分子は、ヘリックス−ループ−ヘリックスモチーフ含有リンカーによるＳｃＦｖ−ＳｃＦｖタンデムの二量体化（ＤｉＢｉミニ抗体、Ｍｕｌｌｅｒら、（１９９８）　ＦＥＢＳ　Ｌｅｔｔ　４３２，　４５−４９を参照）、または分子内対合を妨げる配向で４つの抗体可変領域（ＶＨおよびＶＬ）を含む一本鎖分子の二量体化（タンデムダイアボディ、Ｋｉｐｒｉｙａｎｏｖら、（１９９９）Ｊ．Ｍｏｌ．Ｂｉｏｌ．　２９３，　４１−５６を参照）のいずれかによって、作製することもできる。二重特異性Ｆ（ａｂ’）２フラグメントは、Ｆａｂ’フラグメントの化学的カップリングによって、またはロイシンジッパーによるヘテロ二量体化によって作製することができる（Ｓｈａｌａｂｙら、（１９９２）Ｊ．Ｅｘｐ．Ｍｅｄ．　１７５，　２１７−２２５、およびＫｏｓｔｅｌｎｙら、（１９９２），　Ｊ．Ｉｍｍｕｎｏｌ．　１４８，　１５４７−１５５３を参照）。また、単離されたＶＨおよびＶＬドメイン（Ｄｏｍａｎｔｉｓ　ｐｌｃ）も利用できる。 The Fv fragment appears to have a lower interaction energy between its two chains than the Fab fragment. In order to stabilize the binding of VH and VL domains, these domains are composed of peptides (Bird et al., (1988) Science 242, 423-426, Huston et al., PNAS, 85, 5879-5883), disulfide bridges (Glockshuber et al. (1990) Biochemistry, 29, 1362-1367), and the “knob in hole” mutation (Zhu et al. (1997), Protein Sci., 6, 781-788). ScFv fragments can be generated by methods well known to those skilled in the art (Whitlow et al., (1991) Methods companion Methods Enzymol, 2, 97-105 and Huston et al., (1993) Int. Rev. Immunol 10, 195-217). ScFv can be produced in bacterial cells such as E. coli, but is preferably produced in eukaryotic cells. The disadvantage of ScFv is that the product is monovalent, thus making it impossible to increase the binding force due to multivalent bonds, and the short half-life. Attempts to overcome these problems include bivalent (ScFv ′) 2 , which can be obtained from a ScFv containing an additional C-terminal cysteine by chemical coupling (Adams et al. (1993) Can. Res 53, 4026-4034, and McCartney et al. (1995) Protein Eng. 8, 301-314), or by spontaneous site-specific dimerization of ScFv containing unpaired C-terminal cysteine residues. (See Kipriyanov et al. (1995) Cell. Biophys 26, 187-204). Alternatively, ScFv can be multimerized by shortening the peptide linker to 3-12 residues to form a “diabody”. See Holliger et al., PNAS (1993), 90, 6444-6448. By further reducing the linker, ScFv trimers ("Triabodies", see Kortt et al. (1997) Protein Eng, 10, 423-433) and tetramers ("Tetrabodies", Le Gall et al., ( 1999) FEBS Lett, 453, 164-168). The construction of bivalent ScFv molecules is described in “miniantibody” (see Pack et al., (1992) Biochemistry 31, 1579-1584) and “minibody” (Hu et al. (1996), Cancer Res. 56, 3055. It can also be achieved by gene fusion with a protein dimerization motif that can form -3061). ScFv-ScFv tandem ((ScFv) 2 ) can also be made by linking two ScFv units by a third peptide linker (see Kurucz et al. (1995) J. Immol. 154, 4576-4582. ). Bispecific diabodies can be made by non-covalent attachment of two single-chain fusion products consisting of a VH domain from another antibody linked to a VL domain of one antibody by a short linker (Kipriyanov). (1998), Int. J. Can 77, 763-772). The stability of such a bispecific diabody can be achieved by introducing a disulfide bridge, or by introducing the “knob in hole” mutation described above, or by connecting two hybrid ScFv fragments via a peptide linker. It can be enhanced by forming a chain diabody (ScDb) (see Kontermann et al. (1999) J. Immunol. Methods 226 179-188). Tetravalent bispecific molecules are obtained, for example, by fusing a ScFv fragment to the CH3 domain of an IgG molecule or to a Fab fragment via the hinge region (Coloma et al. (1997) Nature Biotechnol. 15 , 159-163). Alternatively, tetravalent bispecific molecules have been created by fusion of bispecific single stranded diabodies (see Alt et al. (1999) FEBS Lett 454, 90-94). For smaller tetravalent bispecific molecules, dimerization of ScFv-ScFv tandems with a helix-loop-helix motif-containing linker (DiBi miniantibody, Muller et al. (1998) FEBS Lett 432, 45-49. ), Or dimerization of single chain molecules containing four antibody variable regions (VH and VL) in an orientation that prevents intramolecular pairing (tandem diabodies, Kipriyanov et al. (1999) J. Mol. Biol. 293). , 41-56). Bispecific F (ab ′) 2 fragments can be generated by chemical coupling of Fab ′ fragments or by heterodimerization with leucine zippers (Shalaby et al. (1992) J. Exp. Med). 175, 217-225, and Kostelny et al. (1992), J. Immunol. 148, 1547-1553). Isolated VH and VL domains (Domantis plc) can also be used.
　本明細書中、「モノクローナル抗体」は、実質的に均一な抗体の集団から得られる抗体（または抗体断片）を意味する、すなわち、集団を構成する個々の抗体が、少量存在しうる自然に生じる可能な突然変異を除いて同一である。モノクローナル抗体は、高度に特異的であり、単一の抗原部位に対するものである。概して異なる決定基（エピトープ）に対する異なる抗体を含むポリクローナル抗体調製物とは対照的に、各モノクローナル抗体は、抗原上の単一の決定基に対するものである。それらの特異性の他に、ハイブリドーマ培養によって合成され、他の免疫グロブリンによる混入がないという点で、モノクローナル抗体は有利である。「モノクローナル」という修飾語は、実質的に均一な抗体の集団から得られる抗体の特徴を示すものであって、ある特定の方法による抗体の産生を必要とすることを意味するためのものではない。例えば、本発明に従って使用されるモノクローナル抗体は、Ｋｏｈｌｅｒ等，　Ｎａｔｕｒｅ，　２５６：　４９５　［１９７５］に最初に記載されたハイブリドーマ法によって作成してもよいし、組換えＤＮＡ法（例えば、米国特許第４，８１６，５６７号参照）によって作成してもよい。また「モノクローナル抗体」は、例えば、Ｃｌａｃｋｓｏｎ等，　Ｎａｔｕｒｅ，　３５２：　６２４−６２８　［１９９１］およびＭａｒｋｓ等，　Ｊ．Ｍｏｌ．　Ｂｉｏｌ．，　２２２：　５８１−５９７　（１９９１）　に記載された技術を用いて、ファージ抗体ライブラリから単離した抗体断片（Ｆｖクローン）を含む抗原認識および結合部位のクローンを含む。 As used herein, “monoclonal antibody” means an antibody (or antibody fragment) obtained from a substantially homogeneous population of antibodies, ie, a naturally occurring individual antibody that comprises the population may be present in small amounts. Identical except for possible mutations. Monoclonal antibodies are highly specific and are directed against a single antigenic site. In contrast to polyclonal antibody preparations that generally contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture and are free from contamination by other immunoglobulins. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not meant to imply that production of the antibody by any particular method is required. . For example, monoclonal antibodies used in accordance with the present invention may be made by the hybridoma method first described in Kohler et al., Nature, 256: 495 [1975], or by recombinant DNA methods (eg, US Patent No. 4 , 816, 567). “Monoclonal antibodies” are also described in, for example, Clackson et al., Nature, 352: 624-628 [1991] and Marks et al., J. MoI. Mol. Biol. , 222: 581-597 (1991), including antigen recognition and binding site clones containing antibody fragments (Fv clones) isolated from phage antibody libraries.
　「モノクローナル抗体」は、「キメラ」抗体（免疫グロブリン）を含み、それは、重鎖および／または軽鎖の一部が特定の種から誘導されたまたは特定の抗体クラスまたはサブクラスに属する抗体の対応する配列と同一または相同であるが、鎖の残りの部分は他の種から誘導されたまたは他の抗体クラスまたはサブクラスに属する抗体、並びにそれらが所望の生物学的活性を示す限りにおいて、それらの抗体の断片の対応する配列と同一または相同である（Ｍｏｒｒｉｓｏｎ等，Ｐｒｏｃ．　Ｎａｔｌ．　Ａｃａｄ．　Ｓｃｉ．　ＵＳＡ，　８１：　６８５１−６８５５　［１９８４］）。 “Monoclonal antibody” includes “chimeric” antibodies (immunoglobulins), which correspond to antibodies whose heavy and / or light chain portions are derived from a particular species or belong to a particular antibody class or subclass. Antibodies that are identical or homologous to the sequence, but the rest of the chain are derived from other species or belong to other antibody classes or subclasses, as long as they exhibit the desired biological activity Is identical or homologous to the corresponding sequence of the fragment (Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 [1984]).
　非ヒト（例えばマウス）抗体の「ヒト化」型は、非ヒト免疫グロブリンから誘導された最小配列を含有する、キメラ免疫グロブリン、免疫グロブリン鎖またはそれらの断片（例えば、Ｆｖ、Ｆａｂ、Ｆａｂ’、Ｆ（ａｂ’）２あるいは抗体の他の抗原結合性配列）である。大部分において、ヒト化抗体はヒト免疫グロブリン（レシピエント抗体）であって、そのレシピエントＣＤＲ由来の残基が、マウス、ラット、ウサギ、霊長類（例えば、サル）などのヒト以外の種のＣＤＲ（ドナー抗体）に由来する所望の特異性、親和性および容量を持つ残基で置換されている。ある場合は、ヒト免疫グロブリンのＦｖフレームワーク領域（ＦＲ）残基が対応する非ヒト残基で置換される。さらに、ヒト化抗体は、レシピエント抗体にも、移植されるＣＤＲまたはフレームワーク配列にも見られない残基を含んでもよい。これらの修飾は、抗体の性能をさらに精密かつ最適化するために施される。一般にヒト化抗体は、ＣＤＲ領域の全てまたは実質上全てが非ヒト免疫グロブリンのものに対応し、ＦＲ領域の全てまたは実質上全てがヒト免疫グロブリン配列のものである少なくとも１つ、典型的には２つの可変ドメインの実質的に全部を含有しうる。また、最適なヒト化抗体は、免疫グロブリン定常領域（Ｆｃ）、典型的にはヒト免疫グロブリンのものの少なくとも一部も含有しうる。 “Humanized” forms of non-human (eg, murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (eg, Fv, Fab, Fab ′, etc.) that contain minimal sequence derived from non-human immunoglobulin. F (ab ′) 2 or other antigen-binding sequence of the antibody). For the most part, humanized antibodies are human immunoglobulins (recipient antibodies) whose residues from the recipient CDRs are of non-human species such as mice, rats, rabbits, primates (eg, monkeys). Substituted with residues with the desired specificity, affinity and capacity from the CDR (donor antibody). In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the grafted CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In general, a humanized antibody has at least one, typically all, or substantially all of the CDR region corresponding to that of a non-human immunoglobulin and all or substantially all of the FR region is of a human immunoglobulin sequence, typically It can contain substantially all of the two variable domains. An optimal humanized antibody may also contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
　本発明の抗体またはその抗原結合断片の例としては、
　（１）配列番号４１のアミノ酸配列、または（ｉｉ）１個もしくは数個のアミノ酸残基の変異を配列番号４１の対応する位置に有するアミノ酸配列、を含む軽鎖相補性決定領域１（ＣＤＲＬ１）、
　（２）（ｉ）配列番号４２のアミノ酸配列、または（ｉｉ）１個もしくは数個のアミノ酸残基の変異を配列番号４２の対応する位置に有するアミノ酸配列、を含む軽鎖相補性決定領域２（ＣＤＲＬ２）、
　（３）（ｉ）配列番号４３のアミノ酸配列、または（ｉｉ）１個もしくは数個のアミノ酸残基の変異を配列番号４３の対応する位置に有するアミノ酸配列、を含む軽鎖相補性決定領域３（ＣＤＲＬ３）、
　（４）（ｉ）配列番号４４のアミノ酸配列、または（ｉｉ）１個もしくは数個のアミノ酸残基の変異を配列番号４４の対応する位置に有するアミノ酸配列、を含む重鎖相補性決定領域１（ＣＤＲＨ１）、
　（５）（ｉ）配列番号４５のアミノ酸配列、または（ｉｉ）１個もしくは数個のアミノ酸残基の変異を配列番号４５の対応する位置に有するアミノ酸配列、を含む重鎖相補性決定領域２（ＣＤＲＨ２）、および
　（６）（ｉ）配列番号４６のアミノ酸配列、または（ｉｉ）１個もしくは数個のアミノ酸残基の変異を配列番号４６の対応する位置に有するアミノ酸配列、を含む重鎖相補性決定領域３（ＣＤＲＨ３）、
のうちの少なくとも１つを含む、ＣＤ４４ｖ９を発現する細胞のＣＤ４４ｖ９の細胞外領域に結合する抗体またはその抗原結合断片が挙げられる。 Examples of the antibodies of the present invention or antigen-binding fragments thereof include
(1) light chain complementarity determining region 1 (CDRL1) comprising the amino acid sequence of SEQ ID NO: 41, or (ii) an amino acid sequence having a mutation of one or several amino acid residues at the corresponding position of SEQ ID NO: 41 ,
(2) Light chain complementarity determining region 2 comprising (i) the amino acid sequence of SEQ ID NO: 42, or (ii) an amino acid sequence having a mutation of one or several amino acid residues at the corresponding position of SEQ ID NO: 42 (CDRL2),
(3) Light chain complementarity determining region 3 comprising (i) the amino acid sequence of SEQ ID NO: 43, or (ii) an amino acid sequence having a mutation of one or several amino acid residues at the corresponding position of SEQ ID NO: 43 (CDRL3),
(4) Heavy chain complementarity determining region 1 comprising (i) the amino acid sequence of SEQ ID NO: 44, or (ii) an amino acid sequence having a mutation of one or several amino acid residues at the corresponding position of SEQ ID NO: 44 (CDRH1),
(5) heavy chain complementarity determining region 2 comprising (i) the amino acid sequence of SEQ ID NO: 45, or (ii) an amino acid sequence having a mutation of one or several amino acid residues at the corresponding position of SEQ ID NO: 45 (CDRH2) and (6) a heavy chain comprising (i) the amino acid sequence of SEQ ID NO: 46, or (ii) an amino acid sequence having a mutation of one or several amino acid residues at the corresponding position of SEQ ID NO: 46 Complementarity determining region 3 (CDRH3),
An antibody or antigen-binding fragment thereof that binds to the extracellular region of CD44v9 of cells that express CD44v9, including at least one of the above.
　本明細書中、「１個または数個のアミノ酸残基の変異」とは、元となるアミノ酸配列中の１個または数個（例えば、２個、３個、４個、５個）のアミノ酸残基が、欠失、置換、挿入、または付加されていることを意味する。本明細書中、そのような変異を元となるアミノ酸配列中の対応する位置に有するアミノ酸配列を有する抗体またはその抗原結合断片は、元となるアミノ酸配列を有する抗体またはその抗原結合断片と同等の生物学的活性を有する。ここで、「同等の生物学的活性」としては、（ｉ）元となるアミノ酸配列を有する抗体またはその抗原結合断片が特異的に結合する抗原に対して特異性をもって結合する能力、（ｉｉ）ＣＤ４４ｖ９発現細胞上のＣＤ４４ｖ９に結合した場合における細胞障害活性、または（ｉｉｉ）これらの両方が挙げられる。最も好ましくは、「同等の生物学的活性」とは、上記（ｉｉｉ）を意味する。また、上記の「１個または数個のアミノ酸残基の変異」における変異したアミノ酸残基の数の上限は、このような同等の特異性を保持することができるか否かという基準（ｃｒｉｔｅｒｉａ）によって制限される。 In the present specification, “mutation of one or several amino acid residues” means one or several (for example, 2, 3, 4, 5) amino acids in the original amino acid sequence. It means that the residue has been deleted, substituted, inserted or added. In the present specification, an antibody having an amino acid sequence having such a mutation at a corresponding position in the original amino acid sequence or an antigen-binding fragment thereof is equivalent to an antibody having the original amino acid sequence or an antigen-binding fragment thereof. Has biological activity. Here, “equivalent biological activity” includes (i) the ability to bind with specificity to an antigen to which an antibody having an original amino acid sequence or an antigen-binding fragment thereof specifically binds, (ii) Cytotoxic activity when bound to CD44v9 on CD44v9 expressing cells, or (iii) both. Most preferably, “equivalent biological activity” means (iii) above. In addition, the upper limit of the number of mutated amino acid residues in the above “mutation of one or several amino acid residues” is a criterion for whether or not such equivalent specificity can be maintained (criteria). Limited by.
　一般的には、天然に存在するタンパク質を構成しているアミノ酸は、それらの側鎖の特性によって群分け可能であり、例えば、同様な特性を有するアミノ酸群としては、芳香族アミノ酸（チロシン、フェニルアラニン、トリプトファン）、塩基性アミノ酸（リジン、アルギニン、ヒスチジン）、酸性アミノ酸（アスパラギン酸、グルタミン酸）、中性アミノ酸（セリン、トレオニン、アスパラギン、グルタミン）、炭化水素鎖を有するアミノ酸（アラニン、バリン、ロイシン、イソロイシン、プロリン）、およびその他（グリシン、メチオニン、システイン）の群などに分類できる。 In general, amino acids constituting a naturally occurring protein can be grouped according to the characteristics of their side chains. For example, amino acids having similar characteristics include aromatic amino acids (tyrosine, phenylalanine). , Tryptophan), basic amino acids (lysine, arginine, histidine), acidic amino acids (aspartic acid, glutamic acid), neutral amino acids (serine, threonine, asparagine, glutamine), amino acids having a hydrocarbon chain (alanine, valine, leucine, It can be classified into groups such as isoleucine, proline) and others (glycine, methionine, cysteine).
　非天然型のアミノ酸も含めた相互に置換可能なアミノ酸残基の例としては、下記の様な群わけもあり、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、トリプトファン。 Examples of amino acid residues which can be substituted with each other including non-natural amino acids include the following groups, and amino acid residues contained in the same group can be substituted with 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, tryptophan.
　なお、アミノ酸配列や塩基配列の同一性は、カーリンおよびアルチュールによるアルゴリズムＢＬＡＳＴ（ＰＮＡＳ，１９９０（ｖｏｌ．８７）ｐ２２６４；ＰＮＡＳ，１９９３（ｖｏｌ．９０）ｐ５８７３）を用いて決定できる。ＢＬＡＳＴのアルゴリズムに基づいたＢＬＡＳＴＮやＢＬＡＳＴＸと呼ばれるプログラムが開発されている（Ｊ　Ｍｏｌ　Ｂｉｏｌ，１９９０（ｖｏｌ．２１５）ｐ４０３）。ＢＬＡＳＴＮを用いて塩基配列を解析する場合は、パラメーターは、例えばｓｃｏｒｅ＝１００、ｗｏｒｄｌｅｎｇｔｈ＝１２とする。また、ＢＬＡＳＴＸを用いてアミノ酸配列を解析する場合は、パラメーターは、例えばｓｃｏｒｅ＝５０、ｗｏｒｄｌｅｎｇｔｈ＝３とする。ＢＬＡＳＴとＧａｐｐｅｄ　ＢＬＡＳＴプログラムを用いる場合は、各プログラムのデフォルトパラメーターを用いる。もしくは、タンパク質のアミノ酸配列の同一性を求める際、比較する２種類のタンパク質のアミノ酸配列を並べ、目視で同じアミノ酸残基である部分の数を数えることにより「（同一なアミノ酸残基数／タンパク質全長のアミノ酸残基数）×１００（％）」により求めることもできる。 The identity of amino acid sequences and base sequences can be determined using the algorithm BLAST (PNAS, 1990 (vol. 87) p2264; PNAS, 1993 (vol. 90) p5873) by Carlin and Arthur. Programs called BLASTN and BLASTX based on the BLAST algorithm have been developed (J Mol Biol, 1990 (vol. 215) p403). When analyzing a base sequence using BLASTN, parameters are set to, for example, score = 100 and wordlength = 12. When analyzing an amino acid sequence using BLASTX, parameters are set to score = 50 and wordlength = 3, for example. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Alternatively, when determining the identity of amino acid sequences of proteins, the amino acid sequences of two types of proteins to be compared are arranged, and the number of portions that are the same amino acid residues is visually counted. The total number of amino acid residues) × 100 (%) ”can also be obtained.
　本発明の抗体またはその抗原結合断片のより好ましい例としては、
　（１）軽鎖相補性決定領域１（ＣＤＲＬ１）、軽鎖相補性決定領域２（ＣＤＲＬ２）、および軽鎖相補性決定領域３（ＣＤＲＬ３）として、それぞれ、配列番号１１、配列番号１２、および配列番号１３のアミノ酸配列、または配列番号１１、配列番号１２、および配列番号１３のアミノ酸配列に１個もしくは数個のアミノ酸残基の変異をそれぞれ有するアミノ酸配列を含み、
　重鎖相補性決定領域１（ＣＤＲＨ１）、重鎖相補性決定領域２（ＣＤＲＨ２）、および重鎖相補性決定領域３（ＣＤＲＨ３）として、それぞれ、配列番号１４、配列番号１５、および配列番号１６のアミノ酸配列、もしくは配列番号１４、配列番号１５、および配列番号１６のアミノ酸配列に１個もしくは数個のアミノ酸残基の変異をそれぞれ有するアミノ酸配列を含む、ＣＤ４４ｖ９を発現する細胞のＣＤ４４ｖ９の細胞外領域に特異的に結合する抗体またはその抗原結合断片、
　（２）ＣＤＲＬ１、ＣＤＲＬ２、およびＣＤＲＬ３として、それぞれ、配列番号１７、配列番号１８、および配列番号１９のアミノ酸配列、または配列番号１７、配列番号１８、および配列番号１９のアミノ酸配列に１個もしくは数個のアミノ酸残基の変異をそれぞれ有するアミノ酸配列を含み、
　ＣＤＲＨ１、ＣＤＲＨ２、およびＣＤＲＨ３として、それぞれ、配列番号２０、配列番号２１、および配列番号２２のアミノ酸配列、もしくは配列番号２０、配列番号２１、および配列番号２２のアミノ酸配列に１個もしくは数個のアミノ酸残基の変異をそれぞれ有するアミノ酸配列を含む、ＣＤ４４ｖ９を発現する細胞のＣＤ４４ｖ９の細胞外領域に特異的に結合する抗体またはその抗原結合断片、
　（３）ＣＤＲＬ１、ＣＤＲＬ２、およびＣＤＲＬ３として、それぞれ、配列番号２３、配列番号２４、および配列番号２５のアミノ酸配列、または配列番号２３、配列番号２４、および配列番号２５のアミノ酸配列に１個もしくは数個のアミノ酸残基の変異をそれぞれ有するアミノ酸配列を含み、
　ＣＤＲＨ１、ＣＤＲＨ２、およびＣＤＲＨ３として、それぞれ、配列番号２６、配列番号２７、および配列番号２８のアミノ酸配列、もしくは配列番号２６、配列番号２７、および配列番号２８のアミノ酸配列に１個もしくは数個のアミノ酸残基の変異をそれぞれ有するアミノ酸配列を含む、ＣＤ４４ｖ９を発現する細胞のＣＤ４４ｖ９の細胞外領域に特異的に結合する抗体またはその抗原結合断片、
　（４）ＣＤＲＬ１、ＣＤＲＬ２、およびＣＤＲＬ３として、それぞれ、配列番号２９、配列番号３０、および配列番号３１のアミノ酸配列、または配列番号２９、配列番号３０、および配列番号３１のアミノ酸配列に１個もしくは数個のアミノ酸残基の変異をそれぞれ有するアミノ酸配列を含み、
　ＣＤＲＨ１、ＣＤＲＨ２、およびＣＤＲＨ３として、それぞれ、配列番号３２、配列番号３３、および配列番号３４のアミノ酸配列、もしくは配列番号３２、配列番号３３、および配列番号３４のアミノ酸配列に１個もしくは数個のアミノ酸残基の変異をそれぞれ有するアミノ酸配列を含む、ＣＤ４４ｖ９を発現する細胞のＣＤ４４ｖ９の細胞外領域に特異的に結合する抗体またはその抗原結合断片、または
　（５）ＣＤＲＬ１、ＣＤＲＬ２、およびＣＤＲＬ３として、それぞれ、配列番号３５、配列番号３６、および配列番号３７のアミノ酸配列、または配列番号３５、配列番号３６、および配列番号３７のアミノ酸配列に１個もしくは数個のアミノ酸残基の変異をそれぞれ有するアミノ酸配列を含み、
　ＣＤＲＨ１、ＣＤＲＨ２、およびＣＤＲＨ３として、それぞれ、配列番号３８、配列番号３９、および配列番号４０のアミノ酸配列、もしくは配列番号３８、配列番号３９、および配列番号４０のアミノ酸配列に１個もしくは数個のアミノ酸残基の変異をそれぞれ有するアミノ酸配列を含む、ＣＤ４４ｖ９を発現する細胞のＣＤ４４ｖ９の細胞外領域に特異的に結合する抗体またはその抗原結合断片が挙げられる。 As a more preferred example of the antibody of the present invention or an antigen-binding fragment thereof,
(1) as light chain complementarity determining region 1 (CDRL1), light chain complementarity determining region 2 (CDRL2), and light chain complementarity determining region 3 (CDRL3), respectively, SEQ ID NO: 11, SEQ ID NO: 12, and sequence An amino acid sequence of No. 13, or an amino acid sequence having a mutation of one or several amino acid residues in the amino acid sequences of SEQ ID No. 11, SEQ ID No. 12 and SEQ ID No. 13, respectively;
As heavy chain complementarity determining region 1 (CDRH1), heavy chain complementarity determining region 2 (CDRH2), and heavy chain complementarity determining region 3 (CDRH3), SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively. The extracellular region of CD44v9 of cells expressing CD44v9, comprising an amino acid sequence or an amino acid sequence having a mutation of one or several amino acid residues in the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively An antibody or antigen-binding fragment thereof that specifically binds to
(2) As CDRL1, CDRL2, and CDRL3, one or a number in the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, or the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively An amino acid sequence each having a mutation of one amino acid residue,
CDRH1, CDRH2, and CDRH3 are one or several amino acids in the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, or the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively. An antibody or antigen-binding fragment thereof that specifically binds to the extracellular region of CD44v9 of a cell expressing CD44v9, comprising an amino acid sequence each having a residue mutation;
(3) As CDRL1, CDRL2, and CDRL3, one or more amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25, or amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25, respectively An amino acid sequence each having a mutation of one amino acid residue,
CDRH1, CDRH2, and CDRH3 are one or several amino acids in the amino acid sequences of SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, or the amino acid sequences of SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, respectively. An antibody or antigen-binding fragment thereof that specifically binds to the extracellular region of CD44v9 of a cell expressing CD44v9, comprising an amino acid sequence each having a residue mutation;
(4) As CDRL1, CDRL2, and CDRL3, one or more amino acid sequences of SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, or SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, respectively. An amino acid sequence each having a mutation of one amino acid residue,
CDRH1, CDRH2, and CDRH3 are one or several amino acids in the amino acid sequences of SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34, or the amino acid sequences of SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34, respectively. An antibody or antigen-binding fragment thereof that specifically binds to the extracellular region of CD44v9 of cells expressing CD44v9, each comprising an amino acid sequence having a residue mutation, or (5) CDRL1, CDRL2, and CDRL3, respectively, An amino acid sequence having SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37, or an amino acid sequence having one or several amino acid residue mutations in the amino acid sequences of SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37, respectively. Including
CDRH1, CDRH2, and CDRH3 are one or several amino acids in the amino acid sequences of SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, or the amino acid sequences of SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively. Examples thereof include an antibody or an antigen-binding fragment thereof that specifically binds to the extracellular region of CD44v9 of cells expressing CD44v9, each of which contains an amino acid sequence having a residue mutation.
　あるいは、本発明の抗体またはその抗原結合断片の好ましい例としては、
（１）配列番号１のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含む軽鎖可変領域（ＬＨ）、および
　配列番号２のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含む重鎖可変領域（ＶＨ）、
（２）配列番号３のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含むＬＨ、および
　配列番号４のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含むＶＨ、
（３）配列番号５のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含むＬＨ、および
　配列番号６のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含むＶＨ、
（４）配列番号７のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含むＬＨ、および
　配列番号８のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含むＶＨ、または
（５）配列番号９のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含むＬＨ、および
　配列番号１０のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含むＶＨ
を含有する、ＣＤ４４ｖ９を発現する細胞のＣＤ４４ｖ９の細胞外領域に特異的に結合する抗体もしくはその抗原結合断片が挙げられる。 Alternatively, preferred examples of the antibody of the present invention or an antigen-binding fragment thereof include
(1) a light chain variable region (LH) comprising an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 1, and an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 2 Heavy chain variable region (VH),
(2) LH containing an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 3, and VH containing an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 4.
(3) LH containing an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 5, and VH containing an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 6,
(4) LH containing an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 7, and VH containing an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 8, or (5) LH including an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 9, and VH including an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 10
Or an antigen-binding fragment thereof that specifically binds to the extracellular region of CD44v9 of cells expressing CD44v9.
　なお、上記同一性のパーセンテージは、具体的には、例えば、９０％、９１％、９２％、９３％、９４％、９５％、９６％、９７％、９８％、９９％、または１００％であり得る。 Specifically, the percentage of the above-mentioned identity is, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. possible.
２．本発明の抗体をコードする核酸
　本発明は、別の実施形態において、ＣＤ４４ｖ９に特異的に結合する抗体およびその抗原結合断片をコードする単離された核酸分子を提供する。核酸分子は、ＲＮＡまたはＤＮＡである。本発明の核酸分子は、本発明の抗体またはその抗原結合断片を作製するために使用することができる。 2. Nucleic Acids Encoding Antibodies of the Invention The present invention, in another embodiment, provides isolated nucleic acid molecules that encode antibodies and antigen-binding fragments thereof that specifically bind to CD44v9. The nucleic acid molecule is RNA or DNA. The nucleic acid molecule of the present invention can be used to produce the antibody of the present invention or an antigen-binding fragment thereof.
　したがって、これに関連する本発明の別の実施形態では、本発明のハイブリドーマ細胞から抗ＣＤ４４ｖ９モノクローナル抗体をコードする遺伝子を単離し、該遺伝子を含む発現ベクターを構築し、該発現ベクターを宿主に導入して前記モノクローナル抗体を発現せしめ、得られる宿主、宿主の培養上清または宿主の分泌物からＣＤ４４ｖ９に特異的に結合するモノクローナル抗体を採取することを含む、ＣＤ４４ｖ９に特異的に結合するモノクローナル抗体の製造方法が提供される。 Therefore, in another embodiment of the present invention related thereto, a gene encoding an anti-CD44v9 monoclonal antibody is isolated from the hybridoma cell of the present invention, an expression vector containing the gene is constructed, and the expression vector is introduced into a host A monoclonal antibody that specifically binds to CD44v9, wherein said monoclonal antibody is expressed and the monoclonal antibody that specifically binds to CD44v9 is collected from the obtained host, host culture supernatant or host secretion. A manufacturing method is provided.
　本発明の抗体またはその抗原結合断片をコードするＤＮＡは、常法を用いて（例えば、マウス抗体の重鎖および軽鎖をコードしている遺伝子に特異的に結合できるオリゴヌクレオチドプローブを用いることによって）容易に分離されて、配列決定される。モノクローナル抗体を産生するハイブリドーマ細胞は、このようなＤＮＡの好ましい供給源となる。一旦分離されると、組換え宿主細胞でモノクローナル抗体を合成するために、このＤＮＡを発現ベクターへ挿入し、それをこの状況以外では抗体蛋白質を産生しない大腸菌細胞、ヒトＨＥＫ２９３胎児腎由来細胞、サルＣＯＳ細胞、チャイニーズハムスター卵巣（ＣＨＯ）細胞、または骨髄腫細胞のような宿主細胞へトランスフェクトしうる。例えば、相同的なマウス配列をヒト重鎖および軽鎖定常ドメインの配列で置換することによって（Ｍｏｒｒｉｓｏｎら，　Ｐｒｏｃ．　Ｎａｔ．　ｃａｄ．　Ｓｃｉ．，　ＵＳＡ，８１：６８５１［１９８４］）、または免疫グロブリンコード化配列に非免疫グロブリンポリペプチドのコード配列の全部または一部を共有結合させることによって、このＤＮＡを修飾しうる。本発明の抗ＣＤ４４ｖ９モノクローナル抗体の結合特異性を有するように、「キメラ」または「ハイブリッド」抗体は調製される。 The DNA encoding the antibody of the present invention or an antigen-binding fragment thereof can be obtained using a conventional method (for example, by using an oligonucleotide probe capable of specifically binding to a gene encoding the heavy and light chains of a mouse antibody. ) Easily separated and sequenced. Hybridoma cells producing monoclonal antibodies are a preferred source of such DNA. Once isolated, this DNA is inserted into an expression vector to synthesize monoclonal antibodies in recombinant host cells, which are then used to produce E. coli cells, human HEK293 fetal kidney-derived cells, monkeys that do not produce antibody proteins in other circumstances. It can be transfected into host cells such as COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells. For example, by replacing homologous mouse sequences with sequences of human heavy and light chain constant domains (Morrison et al., Proc. Nat. Cad. Sci., USA, 81: 6851 [1984]), or immunoglobulin coding This DNA may be modified by covalently linking all or part of the coding sequence of the non-immunoglobulin polypeptide to the coding sequence. “Chimeric” or “hybrid” antibodies are prepared so as to have the binding specificity of the anti-CD44v9 monoclonal antibodies of the invention.
３．本発明の抗体を産生するハイブリドーマ細胞
　本発明はさらに別の実施形態において、ＣＤ４４ｖ９に特異的に結合する抗体およびその抗原結合断片を産生するハイブリドーマ細胞を提供する。本発明はまた、ＣＤ４４ｖ９に特異的に結合する抗体およびその抗原結合断片をコードする核酸分子を含有するハイブリドーマを提供する。 3. Hybridoma Cells Producing the Antibody of the Present Invention In yet another embodiment, the present invention provides a hybridoma cell that produces an antibody that specifically binds to CD44v9 and an antigen-binding fragment thereof. The invention also provides a hybridoma containing a nucleic acid molecule encoding an antibody that specifically binds to CD44v9 and an antigen-binding fragment thereof.
　本発明のハイブリドーマ細胞の調製は、具体的には、以下のように行うことができるが、この方法に限定されるわけではない。 The preparation of the hybridoma cell of the present invention can be specifically performed as follows, but is not limited to this method.
（１）抗原の調製
　本発明のハイブリドーマ細胞を作製するために必要な抗原としては、ＣＤ４４ｖ９を産生する細胞あるいはその細胞画分、ＣＤ４４ｖ９を含むＣＤ４４遺伝子をコードするＤＮＡを組み込んだベクター、またはＣＤ４４ｖ９をコードするＤＮＡにより形質転換された宿主細胞、すなわち、大腸菌などの原核性宿主細胞および昆虫細胞、哺乳動物細胞等の真核性宿主細胞中に、ＣＤ４４ｖ９をコードするｃＤＮＡの全長または部分断片を公知の方法を用いて組み込み、そのままあるいは融合蛋白として発現、精製された蛋白質、さらには、ペプチド合成機を用いて合成されたＣＤ４４ｖ９の部分ペプチド等を用いることができる。 (1) Preparation of antigen Antigen necessary for producing the hybridoma cell of the present invention includes a cell producing CD44v9 or a fraction thereof, a vector incorporating a DNA encoding the CD44 gene containing CD44v9, or CD44v9. A full-length or partial fragment of cDNA encoding CD44v9 is known in host cells transformed with the encoding DNA, ie, prokaryotic host cells such as E. coli and eukaryotic host cells such as insect cells and mammalian cells. A protein incorporated by a method and expressed or purified as it is or as a fusion protein, or a CD44v9 partial peptide synthesized using a peptide synthesizer can be used.
（２）動物の免疫と抗体産生細胞の調製
　３~２０週令のマウスまたはラットに、（１）に示した方法で作製された抗原を免疫して、その動物の脾、リンパ節、末梢血より抗体産生細胞を採取する。免疫は、動物の皮下あるいは静脈内あるいは腹腔内に、抗原を投与することにより行う。抗原の投与は、１回目の投与の後２~３週間おきに３~７回行う。各投与後５~１０日目に眼底静脈叢より採血し、その血清が抗原と反応することをＦＡＣＳ法や酵素免疫測定法〔酵素免疫測定法（ＥＬＩＳＡ法）：医学書院刊　１９７６年〕などで調べる。免疫に用いた抗原細胞に対し、その血清が十分な抗体価を示したマウスまたはラットを抗体産生細胞の供給源として供する。脾細胞を骨髄腫細胞との融合に供するにあたって、抗原物質の最終投与後３~４日目に、免疫したマウスまたはラットより脾臓を摘出し、脾細胞を採取する。脾臓を血清無添加の基礎培地（以下洗浄用培地という。）中で細断し、遠心分離して細胞を回収後、トリス−塩化アンモニウム緩衝液（ｐＨ　７．６５）で２~３分間処理し赤血球の除去を行い、洗浄用培地で洗浄した後に融合用脾細胞として提供する。 (2) Immunization of animals and preparation of antibody-producing cells 3 to 20-week-old mice or rats are immunized with the antigen prepared by the method described in (1), and the spleen, lymph nodes, peripheral blood of the animals are immunized. Collect antibody-producing cells. Immunization is performed by administering an antigen subcutaneously, intravenously, or intraperitoneally in an animal. The antigen is administered 3 to 7 times every 2 to 3 weeks after the first administration. On the 5th to 10th day after each administration, blood is collected from the fundus venous plexus, and the serum reacts with the antigen by FACS method or enzyme immunoassay [enzyme immunoassay (ELISA method): 1976, published by Medical Shoin). Investigate. A mouse or rat whose serum showed a sufficient antibody titer against the antigen cells used for immunization is used as a source of antibody-producing cells. When the spleen cells are used for fusion with myeloma cells, the spleen is removed from the immunized mouse or rat 3 to 4 days after the final administration of the antigen substance, and the spleen cells are collected. The spleen is shredded in a basal medium without serum (hereinafter referred to as a washing medium), centrifuged to collect the cells, and then treated with Tris-ammonium chloride buffer (pH 7.65) for 2 to 3 minutes. Erythrocytes are removed, washed with washing medium, and then provided as fusion splenocytes.
（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使用する。たとえば、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−１、Ｅｕｒｏｐｅａｎ　Ｊ．Ｉｍｍｕｎｏｌｏｇｙ，　６，　５１１−５１９（１９７６）〕、ＳＰ２／Ｏ−Ａｇ１４（ＳＰ−２）〔Ｎａｔｕｒｅ　２７６，　２６９−２７０　（１９７８）〕、Ｐ３−Ｘ６３−Ａｇ８６５３（６５３）〔Ｊ．Ｉｍｍｕｎｏｌｏｇｙ　１２３，１５４８−１５５０　（１９７９）〕、Ｐ３−Ｘ６３−Ａｇ８（Ｘ６３）〔Ｎａｔｕｒｅ　２５６，　４９５−４９７　（１９７５）〕などが用いられる。これらの細胞株は、８−アザグアニン培地〔ＲＰＭＩ−１６４０培地にグルタミン（１．５ｍＭ）、２−メルカプトエタノール（５×１０−５Ｍ）、ジェンタマイシン（１０μｇ／ｍｌ）および牛胎児血清（ＦＣＳ）を１０％加えた培地（以下、正常培地という。）に、さらに８−アザグアニン（１５μｇ／ｍｌ）を加えた培地〕で継代するが、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。 (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-1, European J. Biol. Immunology, 6, 511-519 (1976)], SP2 / O-Ag14 (SP-2) [Nature 276, 269-270 (1978)], P3-X63-Ag8653 (653) [J. Immunology 123, 1548-1550 (1979)], P3-X63-Ag8 (X63) [Nature 256, 495-497 (1975)] and the like are used. These cell lines consisted of 8-azaguanine medium [RPMI-1640 medium with glutamine (1.5 mM), 2-mercaptoethanol (5 × 10 −5 M), gentamicin (10 μg / ml) and fetal calf serum (FCS). Is further subcultured with a medium supplemented with 10% of the above (hereinafter referred to as a normal medium) and further added with 8-azaguanine (15 μg / ml)], but subcultured into a normal medium 3 to 4 days before cell fusion. On the day of fusion, secure a cell count of 2 × 10 7 or more.
（４）細胞融合
　（２）で免疫した抗体産生細胞と（３）で得られた骨髄腫細胞を洗浄用培地またはＰＢＳ（リン酸二ナトリウム１．８３ｇ、リン酸−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、抗体産生細胞：骨髄腫細胞＝５~１０：１になるよう混合させる。細胞回収後、細胞をよくほぐし、攪拌しながら、３７℃で、ポリエチレングライコール−１，５００（ＰＥＧ−１，５００）２ｇ、洗浄用培地２ｍｌおよびジメチルスルホキシド０．７ｍｌの混液０．２~１ｍｌ／１０８抗体産生細胞を加え、１~２分間毎に洗浄用培地１~２ｍｌを数回加え、全量が５０ｍｌになるまで洗浄する。細胞回収後、ゆるやかに細胞をほぐしながら、ＨＡＴ培地〔正常培地にヒポキサンチン（１０−４Ｍ）、チミジン（１．５×１０−５Ｍ）およびアミノプテリン（４×１０−７Ｍ）を加えた培地〕１００ｍｌ中に懸濁する。この懸濁液を９６ウェル培養用プレートに１００μｌ／ウェルずつ分注し、５％ＣＯ２インキュベーター中、３７℃で７~１４日間培養する。培養後、培養上清の一部をとり、例えば（５）に述べる酵素免疫測定法あるいはＦＡＣＳ（Ｆｌｕｏｒｅｓｃｅｎｃｅ−Ａｃｔｉｖａｔｅｄ　Ｃｅｌｌ　Ｓｏｒｔｉｎｇの略）などを用いて、ＣＤ４４ｖ９蛋白質に特異的に反応する抗体を選択する。ついで、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抗ＣＤ４４ｖ９モノクローナル抗体産生ハイブリドーマ株として選択する。 (4) Cell fusion The antibody-producing cells immunized in (2) and the myeloma cells obtained in (3) are washed medium or PBS (1.83 g of disodium phosphate, 0.21 g of phosphate-potassium, salt 7 Wash well with .65 g, 1 liter of distilled water, pH 7.2), and mix so that the number of cells is antibody-producing cells: myeloma cells = 5 to 10: 1. After recovering the cells, loosen the cells well and agitate the mixture at 37 ° C., 2 to 1 ml of polyethylene glycol-1,500 (PEG-1,500), 2 ml of washing medium and 0.7 ml of dimethyl sulfoxide. / 108 antibody-producing cells are added, and 1-2 ml of washing medium is added several times every 1-2 minutes, and the whole is washed to 50 ml. After collecting the cells, gently loosen the cells and add HAT medium [hypoxanthine (10 −4 M), thymidine (1.5 × 10 −5 M) and aminopterin (4 × 10 −7 M) to the normal medium]. Suspend in 100 ml. This suspension is dispensed at 100 μl / well into a 96-well culture plate and cultured at 37 ° C. for 7 to 14 days in a 5% CO 2 incubator. After culturing, a portion of the culture supernatant is taken, and an antibody that specifically reacts with the CD44v9 protein is selected using, for example, the enzyme immunoassay described in (5) or FACS (abbreviation of Fluorescence-Activated Cell Sorting) . Next, cloning was repeated twice by the limiting dilution method (the first time was HT medium (a medium obtained by removing aminopterin from HAT medium), the second time was using a normal medium), and a stable and strong antibody titer was observed. Is selected as an anti-CD44v9 monoclonal antibody-producing hybridoma strain.
　したがって、これに関連する本発明の別の実施形態では、ハイブリドーマ細胞を培養し、得られる培養物からＣＤ４４ｖ９に特異的に結合する抗体を採取することを含む、ＣＤ４４ｖ９に特異的に結合するモノクローナル抗体の製造方法が提供される。 Accordingly, in another related embodiment of the invention, a monoclonal antibody that specifically binds CD44v9, comprising culturing hybridoma cells and collecting an antibody that specifically binds CD44v9 from the resulting culture. A manufacturing method is provided.
４．本発明の抗体のイムノコンジュゲート
　本発明はまた、別の実施形態において、本発明の抗体およびその抗原結合断片に殺細胞活性および／または抗腫瘍活性を有する化合物あるいは放射性同位体を結合させたイムノコンジュゲートを提供する。 4). Immunoconjugate of antibody of the present invention In another embodiment, the present invention also provides an immunoconjugate in which an antibody of the present invention and an antigen-binding fragment thereof are combined with a compound having a cell killing activity and / or an antitumor activity or a radioisotope. A conjugate is provided.
　本発明の抗体は、ＣＤ４４ｖ９を発現する標的腫瘍細胞内へのインターナリゼーション活性に優れたものである。そのため、殺細胞活性および／または抗腫瘍活性を有する化合物を結合させたイムノコンジュゲートは、これら化合物を腫瘍細胞に直接かつ選択的に作用させることができる。 The antibody of the present invention has excellent internalization activity into target tumor cells that express CD44v9. Therefore, an immunoconjugate to which a compound having cell killing activity and / or antitumor activity is bound can cause these compounds to act directly and selectively on tumor cells.
５．医薬組成物
　本発明はまた、さらに別の実施形態において、本発明の抗体またはその抗原結合断片またはそれらとのイムノコンジュゲートを有効成分として含有する、腫瘍の予防または治療または診断のための医薬組成物を提供する。本発明の医薬組成物は、さらに薬学的に許容し得る担体を含有する。 5. Pharmaceutical composition In yet another embodiment, the present invention also provides a pharmaceutical composition for preventing or treating or diagnosing a tumor, comprising as an active ingredient the antibody of the present invention or an antigen-binding fragment thereof or an immunoconjugate thereof. Offer things. The pharmaceutical composition of the present invention further contains a pharmaceutically acceptable carrier.
　後述の実施例に示されるように、本発明の抗体またはその抗原結合断片は、ＣＤ４４ｖ９を発現する細胞（例：腫瘍細胞）のＣＤ４４ｖ９に特異的に結合して、ＣＤ４４ｖ９とアミノ酸トランスポーターであるｘＣＴとの相互作用を阻害するか、または細胞障害因子を誘導し、該細胞を細胞死に至らしめることができる。さらに、インターナリゼーション活性を有するため、殺細胞活性および／または抗腫瘍活性を有する化合物を結合させたイムノコンジュゲートは、これら化合物を腫瘍細胞に直接かつ選択的に作用させることができる。そのような化合物としては、例えば、細胞毒性薬、抗腫瘍剤、または放射性同位体が挙げられる。したがって、本発明の医薬組成物は、ＣＤ４４ｖ９を細胞表面に発現する腫瘍細胞を死滅させるため、またはそのような細胞で特徴付けられる腫瘍および、同様の機序により生ずる疾患の予防または治療のために使用することができる。 As shown in Examples described later, the antibody of the present invention or an antigen-binding fragment thereof specifically binds to CD44v9 of cells expressing CD44v9 (eg, tumor cells), and is a CD44v9 and amino acid transporter xCT. Can be inhibited, or cytotoxic factors can be induced to cause the cells to die. Furthermore, since it has internalization activity, an immunoconjugate to which a compound having cytocidal activity and / or antitumor activity is bound can directly and selectively act these compounds on tumor cells. Such compounds include, for example, cytotoxic drugs, antitumor agents, or radioisotopes. Therefore, the pharmaceutical composition of the present invention is for killing tumor cells expressing CD44v9 on the cell surface, or for the prevention or treatment of tumors characterized by such cells and diseases caused by similar mechanisms. Can be used.
　本明細書中、「細胞障害活性」とは、細胞に対して細胞障害を引き起こす能力を意味し、本発明の場合、ＣＤ４４ｖ９発現細胞に本発明の抗体またはその抗原結合断片が特異的に結合して、該細胞に細胞障害性因子を誘導して、該細胞を細胞死またはアポトーシスに至らしめる能力をいうものとする。細胞障害活性は、例えば、本発明の実施例２に記載される方法で測定し、細胞障害率として評価することができる。 In the present specification, “cytotoxic activity” means the ability to cause cytotoxicity to cells. In the present invention, the antibody of the present invention or an antigen-binding fragment thereof specifically binds to a CD44v9-expressing cell. Thus, it refers to the ability to induce cytotoxic factors in the cells, leading to cell death or apoptosis. The cytotoxic activity can be measured, for example, by the method described in Example 2 of the present invention and evaluated as a cytotoxic rate.
　本明細書中、「細胞死」は「アポトーシス」を意味する。「アポトーシス」は、個体発生のプログラム、デス因子刺激、放射線などによる染色体ＤＮＡの重度の損傷、異常蛋白質の蓄積などによる重度の処方体ストレスなどさまざまな生理的、病理的要因により誘導される機能的、能動的な細胞死の典型であり、細胞体や核の膨潤を伴う壊死（ネクローシス）とは逆に、細胞体や核の収縮や断片化が起こる。 In this specification, “cell death” means “apoptosis”. Apoptosis is a function induced by various physiological and pathological factors such as ontogeny programs, death factor stimulation, severe chromosomal DNA damage due to radiation, severe prescription stress due to abnormal protein accumulation, etc. It is a typical example of active cell death. Contrary to necrosis (necrosis) involving swelling of cell bodies and nuclei, contraction and fragmentation of cell bodies and nuclei occur.
　さらなる実施形態において、本発明の抗体およびその抗体結合断片は、ＣＤ４４ｖ９画発現亢進している癌幹細胞に発現しているＣＤ４４ｖ９に結合し、癌幹細胞の特性であるＳｐｈｅｒｅ（ｃｏｌｏｎｙ）形成能および転移巣形成能を阻害し、癌幹細胞を標的とした治療および診断等のために使用することができる。 In a further embodiment, the antibody of the present invention and an antibody-binding fragment thereof bind to CD44v9 expressed in a cancer stem cell with enhanced expression of CD44v9 fraction, and have the Sphere (colony) formation ability and metastasis characteristic of cancer stem cells. It can be used for treatment and diagnosis that inhibits the formation ability and targets cancer stem cells.
　上記「腫瘍」の例としては、大腸癌、結腸直腸癌、肺癌、乳癌、脳腫瘍、黒色腫、腎細胞癌、膀胱癌、白血病、リンパ腫、Ｔ細胞リンパ腫、多発性骨髄腫、胃癌、膵臓癌、子宮頸癌、子宮内膜癌、卵巣癌、食道癌、肝臓癌、頭頸部扁平上皮癌、皮膚癌、尿路癌、前立腺癌、絨毛癌、咽頭癌、喉頭癌、舌癌、口腔癌、胆嚢癌、甲状腺癌、中皮腫、胸膜腫、男性胚腫、子宮内膜過形成、子宮内膜症、胚芽腫、線維肉腫、カポジ肉腫、血管腫、海綿状血管腫、血管芽腫、網膜芽腫、星状細胞腫、神経線維腫、稀突起謬腫、髄芽腫、神経芽腫、神経膠腫、横紋筋肉腫、謬芽腫、平滑筋肉腫、およびウィルムス腫瘍等が挙げられるが、これらに限定されない。より好ましくは、大腸癌、結腸直腸癌、肺癌、乳癌、脳腫瘍、膀胱癌、リンパ腫、胃癌、膵臓癌、肝臓癌、および前立腺癌が挙げられる。さらに乳癌においては、トリプルネガティブ乳癌（ＨＥＲ２陰性、ＥＲ陰性、ＰＲ陰性）が挙げられる。 Examples of the “tumor” include colon cancer, colorectal cancer, lung cancer, breast cancer, brain tumor, melanoma, renal cell cancer, bladder cancer, leukemia, lymphoma, T cell lymphoma, multiple myeloma, gastric cancer, pancreatic cancer, Cervical cancer, endometrial cancer, ovarian cancer, esophageal cancer, liver cancer, squamous cell carcinoma of the head and neck, skin cancer, urinary tract cancer, prostate cancer, choriocarcinoma, pharyngeal cancer, laryngeal cancer, tongue cancer, oral cancer, gallbladder Cancer, thyroid cancer, mesothelioma, pleuromatous, male embryo, endometrial hyperplasia, endometriosis, embryonal, fibrosarcoma, Kaposi's sarcoma, hemangioma, cavernous hemangioma, hemangioblastoma, retinoblast Tumors, astrocytomas, neurofibromas, oligodendroma, medulloblastomas, neuroblastomas, gliomas, rhabdomyosarcomas, glioblastomas, leiomyosarcomas, and Wilms tumors, etc. It is not limited to these. More preferably, colon cancer, colorectal cancer, lung cancer, breast cancer, brain tumor, bladder cancer, lymphoma, gastric cancer, pancreatic cancer, liver cancer, and prostate cancer are mentioned. Furthermore, in breast cancer, triple negative breast cancer (HER2 negative, ER negative, PR negative) is mentioned.
　本発明の抗体またはその抗原結合断片を医薬として使用する場合は、常套手段に従って製剤化することができる。例えば、該抗体またはその抗原結合断片は、ＩｇＡ化し分泌成分を結合した後、必要に応じて糖衣を施した錠剤、カプセル剤、エリキシル剤、マイクロカプセル剤などとして経口的に、あるいは水もしくはそれ以外の薬学的に許容し得る液との無菌性溶液、または懸濁液剤などの注射剤の形で非経口的に使用できる。例えば、該抗体またはその抗原結合断片を生理学的に認められる公知の担体、香味剤、賦形剤、ベヒクル、防腐剤、安定剤、結合剤などとともに一般に認められた製剤実施に要求される単位用量形態で混和することによって製造することができる。これら製剤における有効成分量は指示された範囲の適当な用量が得られるようにするものである。 When the antibody of the present invention or an antigen-binding fragment thereof is used as a medicine, it can be formulated according to conventional means. For example, the antibody or antigen-binding fragment thereof is converted into IgA and bound to the secretory component, and then orally as tablets, capsules, elixirs, microcapsules, etc. with sugar coating as necessary, or water or other It can be used parenterally in the form of an injectable solution such as a sterile solution with a pharmaceutically acceptable liquid or a suspension. For example, the unit dose required for the formulation practice generally accepted together with known physiologically recognized carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binding agents, etc. It can be produced by mixing in the form. The amount of active ingredient in these preparations is such that an appropriate dose within the indicated range can be obtained.
　また、本発明の抗体またはその抗原結合断片は、他の細胞増殖抑制剤とともに使用され得る。例えば、本発明の抗体またはその抗原結合断片は、抗癌剤としての治療効果をより高めるために、薬学的に許容し得る細胞増殖抑制剤と同時に、逐次的に、または個別に、治療を必要とする対象または患者に投与されることができる。 In addition, the antibody or antigen-binding fragment thereof of the present invention can be used together with other cell growth inhibitors. For example, the antibody or antigen-binding fragment thereof of the present invention requires treatment simultaneously, sequentially or separately with a pharmaceutically acceptable cytostatic agent in order to further enhance the therapeutic effect as an anticancer agent. It can be administered to a subject or patient.
　本発明において使用され得る「細胞増殖抑制剤」の例としては、アルキル化剤、代謝拮抗剤、分子標的薬、植物アルカロイド、抗癌性抗生物質、プラチナ製剤等が挙げられるが、これらに限定されない。 Examples of “cytostatic agents” that can be used in the present invention include, but are not limited to, alkylating agents, antimetabolites, molecular targeted drugs, plant alkaloids, anticancer antibiotics, platinum preparations and the like. .
　アルキル化剤の例としては、イホスファミド、シクロフォスファミド、ダカルバシン、テモゾロミド、ニムスチン、ブスルファン、プロカルバジン、メルファラン、ラニムスチン等が挙げられるが、これらに限定されない。 Examples of alkylating agents include, but are not limited to, ifosfamide, cyclophosphamide, dacarbacin, temozolomide, nimustine, busulfan, procarbazine, melphalan, ranimustine, and the like.
　代謝拮抗剤の例としては、エノシタビン、カペシタビン、カルモフール、クラドリピン、ゲムシタビン、シタラビン、シタラビンオクホスファート、テガフール、テガフールウラシル、ＴＳ−１、ドキシフルリジン、ネララビン、ヒドロキシカルバミド、フルオロウラシル、フルダラビン、ペメトレキセド、ペントスタチン、メルカプトプリン、メトトレキサート等が挙げられるが、これらに限定されない。 Examples of antimetabolites include eninotabine, capecitabine, carmofur, cladripine, gemcitabine, cytarabine, cytarabine ocphosate, tegafur, tegafur uracil, TS-1, doxyfluridine, nelarabine, hydroxycarbamide, fluorouracil, fludarabine, pemetrexed, pemetrexed Examples include, but are not limited to, mercaptopurine and methotrexate.
　分子標的薬の例としては、ゼヴァリン、イマチニブ、エルロチニブ、ゲフィチニブ、スニチニブ、セツキシマブ、ソラフェニブ、ダサチニブ、トラスツズマブ、トレチノイン、パニツムマブ、ベバシズマブ、ボルテゾミブ、リツキシマブ等が挙げられるが、これらに限定されない。 Examples of molecular target drugs include, but are not limited to, Zevalin, Imatinib, Erlotinib, Gefitinib, Sunitinib, Cetuximab, Sorafenib, Dasatinib, Trastuzumab, Tretinoin, Panitumumab, Bevacizumab, Bortezomib, Rituximab, etc.
　植物アルカロイドの例としては、イリノテカン、エトポシド、ソブゾキサン、ドセタキセル、ノギテカン、パクリタキセル、ビノレルビン、ビンクリスチン、ビンデシン、ビンブラスチン等が挙げられるが、これらに限定されない。 Examples of plant alkaloids include, but are not limited to, irinotecan, etoposide, sobuzoxan, docetaxel, nogitecan, paclitaxel, vinorelbine, vincristine, vindesine, vinblastine and the like.
　抗癌性抗生物質の例としては、アクチノマイシンＤ、アクラルビシン、アムルビシン、イダルビシン、エピルビシン、スマンクス、ダウノルビシン、ドキソルビシン、ピラルビシン、ブレオマイシン、ペプロマイシン、マイトマイシンＣ、ミトキサントロン、ドキシル等が挙げられるが、これらに限定されない。 Examples of anti-cancer antibiotics include actinomycin D, aclarubicin, amrubicin, idarubicin, epirubicin, smux, daunorubicin, doxorubicin, pirarubicin, bleomycin, peomycin, mitomycin C, mitoxantrone, doxil, etc. It is not limited.
　プラチナ製剤の例としては、オキサリプラチン、シスプラチン、カルボプラチン、ネダプラチン等が挙げられるが、これらに限定されない。 Examples of platinum preparations include, but are not limited to, oxaliplatin, cisplatin, carboplatin, nedaplatin and the like.
　「薬学的に許容し得る」という用語は、正常な医学的判断の範囲内で、合理的な利益／リスク比を有し、過度の毒性、刺激、アレルギー応答、またはその他の問題もしくは合併症を示すことのない、ヒトおよび動物の組織と接触させて使用するのに適した、化合物、材料、組成物、および／または剤形を指すために、本明細書において利用される。 The term “pharmaceutically acceptable” has a reasonable benefit / risk ratio within the scope of normal medical judgment, and does not cause excessive toxicity, irritation, allergic responses, or other problems or complications. Utilized herein to refer to compounds, materials, compositions, and / or dosage forms suitable for use in contact with human and animal tissue, not shown.
　本発明の腫瘍の予防または治療のための医薬組成物の投与経路は、周知の方法、例えば、静脈内、腹膜内、脳内、皮下、筋肉内、眼内、動脈内、脳内脊髄、または病巣内経路での注射または注入、または徐放系による。またさらに、該抗体またはその抗原結合断片を直接腫瘍部位へ投与する手段として、カテーテル等による投与等も可能である。 The route of administration of the pharmaceutical composition for the prevention or treatment of the tumor of the present invention can be determined by well-known methods such as intravenous, intraperitoneal, intracerebral, subcutaneous, intramuscular, intraocular, intraarterial, intracerebral spinal cord, or By intralesional injection or infusion, or by controlled release system. Furthermore, as a means for directly administering the antibody or antigen-binding fragment thereof to a tumor site, administration by a catheter or the like is also possible.
　また、本発明の腫瘍の予防または治療のための医薬は、例えば、緩衝剤（例えば、リン酸塩緩衝液、酢酸ナトリウム緩衝液）、無痛化剤（例えば、塩化ベンザルコニウム、塩酸プロカインなど）、安定剤（例えば、ヒト血清アルブミン、ポリエチレングリコールなど）、保存剤（例えば、ベンジルアルコール、フェノールなど）、酸化防止剤などと配合してもよい。調製された注射液は通常、適当なアンプルに充填される。このようにして得られる製剤は安全で低毒性であるので、ヒトを含む哺乳動物に対して投与することができる。該抗体またはその抗原結合断片またはその塩の投与量は、投与対象、症状、投与方法などにより差異はあるが、経口投与の場合、一般的に例えば、子宮内膜症患者または子宮腺筋症患者（６０ｋｇとして）においては、一日につき約０．１~１００ｍｇ、好ましくは約１．０~５０ｍｇ、より好ましくは約１．０~２０ｍｇである。非経口的に投与する場合は、その投与量は投与対象、症状、投与方法などによっても異なるが、例えば注射剤の形では、通常、例えば６０ｋｇの患者に対して、一日につき約０．０１~３０ｍｇ程度、好ましくは約０．１~２０ｍｇ程度、より好ましくは約０．１~１０ｍｇ程度を静脈注射により投与することができる。 The medicament for preventing or treating tumors of the present invention includes, for example, a buffer (for example, phosphate buffer, sodium acetate buffer), a soothing agent (for example, benzalkonium chloride, procaine hydrochloride, etc.). , Stabilizers (eg, human serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc. The prepared injection solution is usually filled in a suitable ampoule. Since the preparation thus obtained is safe and has low toxicity, it can be administered to mammals including humans. The dose of the antibody or antigen-binding fragment thereof or a salt thereof varies depending on the administration subject, symptoms, administration method, etc., but in the case of oral administration, generally, for example, endometriosis patients or uterine adenomyosis patients In (as 60 kg), it is about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day. When administered parenterally, the dose varies depending on the subject of administration, symptoms, administration method, etc. For example, in the form of an injection, it is usually about 0.01 per day for a 60 kg patient, for example. About 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg can be administered by intravenous injection.
６．本発明の腫瘍の診断方法
　本発明はまた、さらに別の実施形態において、上記の本発明の抗体またはその抗原結合断片に少なくとも１つの診断または検出剤を結合した診断用イムノコンジュゲートにより、腫瘍を検出することを特徴とする。好ましくは、診断または検出剤は、放射性核種、造影剤、蛍光剤、化学発光剤、生物発光剤、常磁性イオン、酵素、および光活性診断剤からなる群から選択される。１つの実施形態では、診断用イムノコンジュゲートと生体から採取された試料、例えば組織片または血液等と反応させ、シグナルを検出することにより、腫瘍を検出することができる。測定法としては、例えばＥＬＩＳＡ法、ＥＩ法、ＲＩＡ法、蛍光免疫測定法、化学発光免疫測定法等が挙げられる。また別の実施形態では、診断用放射性核種を結合させた診断用イムノコンジュゲートによるＰＥＴイメージングに使用することができる。 6). In still another embodiment, the present invention provides a method for diagnosing a tumor by using a diagnostic immunoconjugate in which at least one diagnostic or detection agent is bound to the above-described antibody of the present invention or an antigen-binding fragment thereof. It is characterized by detecting. Preferably, the diagnostic or detection agent is selected from the group consisting of a radionuclide, a contrast agent, a fluorescent agent, a chemiluminescent agent, a bioluminescent agent, a paramagnetic ion, an enzyme, and a photoactive diagnostic agent. In one embodiment, a tumor can be detected by reacting a diagnostic immunoconjugate with a sample collected from a living body, such as a tissue fragment or blood, and detecting a signal. Examples of the measurement method include ELISA method, EI method, RIA method, fluorescence immunoassay method, chemiluminescence immunoassay method and the like. In yet another embodiment, it can be used for PET imaging with a diagnostic immunoconjugate to which a diagnostic radionuclide is bound.
　以下、実施例により本発明をより具体的に記載するが、本発明の範囲はこれらの実施例に限定されない。 Hereinafter, the present invention will be described more specifically by way of examples. However, the scope of the present invention is not limited to these examples.
［実施例１］
（１）免疫原の調製
　抗ＣＤ４４ｖ９モノクローナル抗体を作製するために必要な抗原として、ＣＤ４４Ｒ１を組み込んだ哺乳動物細胞発現ベクター（ｐＲＣ−ＣＭＶ）を作製した。ＣＤ４４Ｒ１をコードするｃＤＮＡの全長を公知の方法を用いて、ｐＲｃ−ＣＭＶベクターに組み込んだ。 [Example 1]
(1) Preparation of immunogen A mammalian cell expression vector (pRC-CMV) incorporating CD44R1 was prepared as an antigen necessary for preparing an anti-CD44v9 monoclonal antibody. The full length of cDNA encoding CD44R1 was incorporated into a pRc-CMV vector using a known method.
（２）動物の免疫と抗体産生細胞の調製
　マウス（Ｂａｌｂ／ｃ）７匹に、（１）に示した方法で作製された抗原を免疫して、その脾臓より抗体産生細胞を採取した。免疫は、ラットの尾静脈に抗原としてＣＤ４４Ｒ１を組み込んだｐＲｃ−ＣＭＶベクター２０ｕｇを投与すること、またはＣＤ４４Ｒ１を組み込んだｐＲｃ−ＣＭＶベクター２０ｕｇおよびアジュバント（ｐＣＡＣＣ−ＣＤ４０ＬまたはｐＢＯＯＳＴ２−ｗｔｍＩＲＦ１）を投与することにより行った。抗原の投与は、１回目の投与の３週間後および６週間後に２回行った。脾細胞を骨髄腫細胞との融合に供するにあたって、免疫したマウスより脾臓を摘出し脾細胞を採取した。脾臓を血清無添加の基礎培地（以下洗浄用培地という。）中で細断し、遠心分離して細胞を回収後、トリス−塩化アンモニウム緩衝液（ｐＨ７．６５）で２~３分間処理し赤血球の除去を行い、洗浄用培地で洗浄した後に融合用脾細胞として用いた。 (2) Immunization of animals and preparation of antibody-producing cells Seven mice (Balb / c) were immunized with the antigen prepared by the method shown in (1), and antibody-producing cells were collected from their spleens. Immunization is performed by administering 20 ug of pRc-CMV vector incorporating CD44R1 as an antigen into the tail vein of rats, or 20 ug of pRc-CMV vector incorporating CD44R1 and an adjuvant (pCACC-CD40L or pBOOST2-wtmIRF1). went. Antigen administration was performed twice 3 weeks and 6 weeks after the first administration. When the spleen cells were used for fusion with myeloma cells, the spleen was removed from the immunized mouse and the spleen cells were collected. The spleen is shredded in a serum-free basal medium (hereinafter referred to as a washing medium), centrifuged, and the cells are collected, treated with Tris-ammonium chloride buffer (pH 7.65) for 2 to 3 minutes and treated with erythrocytes. After washing with a washing medium, it was used as a splenocyte for fusion.
（３）ハイブリドーマの作製
　実施例１（２）で得られたマウス脾細胞とマウス骨髄腫細胞株Ｘ６３もしくはＰ３Ｕ１とを４：１の比率で混合し、遠心分離（１，２００ｒｐｍ、５分）した後、上清を捨て、沈澱した細胞群をよくほぐした後、攪拌しながら、３７℃で、ポリエチレングライコール−１５００（ＰＥＧ−１５００）２ｇ、ＤＭＥＭ培地２ｍｌおよびジメチルスルホキシド０．７ｍｌの混液を０．２~１ｍｌ／１０８個マウス脾細胞の割合で加え、さらに１~２分間毎にＤＭＥＭ培地１~２ｍｌを数回加えた後、ＤＭＥＭ培地を加えて全量が５０ｍｌになるようにした。遠心分離（９００ｒｐｍ、５分）後、上清を捨てＨＡＴ培地１００ｍｌ中に細胞をゆるやかに懸濁した。この懸濁液を９６ウェル培養用プレートに１００μｌ／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。 (3) Production of hybridoma The mouse spleen cells obtained in Example 1 (2) and the mouse myeloma cell line X63 or P3U1 were mixed at a ratio of 4: 1 and centrifuged (1,200 rpm, 5 minutes). Thereafter, the supernatant was discarded, and the precipitated cell group was thoroughly loosened. Then, with stirring, a mixed solution of 2 g of polyethylene glycol-1500 (PEG-1500), 2 ml of DMEM medium and 0.7 ml of dimethyl sulfoxide was added to the mixture at 0 ° C. .2 to 1 ml / 10 8 mouse splenocytes were added at a rate of 1 to 2 ml of DMEM medium several times every 1 to 2 minutes, and then DMEM medium was added to make the total volume 50 ml. After centrifugation (900 rpm, 5 minutes), the supernatant was discarded and the cells were gently suspended in 100 ml of HAT medium. This suspension was dispensed at 100 μl / well into a 96-well culture plate and cultured at 37 ° C. for 10 to 14 days in a 5% CO 2 incubator.
（４）抗体スクリーニング
　得られたハイブリドーマ培養上清をＧＦＰ−ＣＤ４４ｖ８−１０トランスフェクタントと反応させ、ＦＡＣＳ解析を行うことによりＣＤ４４ｖ８−ｖ１０特異的抗体を産生しているか否かのスクリーニングを行った。ＦＡＣＳの反応は９６ウェルプレートにて行った。トランスフェクタントを２．５×１０５~３×１０６個／ウェルになるように５０μｌずつチューブに分注した。この細胞浮遊液にハイブリドーマの培養上清５０μｌを加え、氷冷下で４５分反応させた。チューブに０．１％ＢＳＡ−ＰＢＳ　１００μｌを加え、５００ｇ、４℃で３分間遠心し上清を捨てる操作を３回繰り返して洗浄した後、２次抗体としてＰＥ−標識抗マウス抗体を添加し遮光氷冷下で４５分間反応させた。０．１％ＢＳＡ−ＰＢＳ１００μＬを加え、５００ｇ、４℃で３分間遠心し上清を捨てる操作を３回繰り返して洗浄した後、ＰＢＳ５００μＬに懸濁し、ＦＣＭチューブに移した。死細胞を除くために測定直前にＰＩ１００μＬを添加し、ＦＡＣＳＣａｌｉｂｅｒ（Ｂｅｃｔｏｎ　Ｄｉｃｋｉｎｓｏｎ）にて蛍光強度を測定した。図１に示すように、ＧＦＰ−ＣＤ４４ｖ８−１０トランスフェクタントに対し、ＣＤ４４ｖ８−１０の発現量に依存的に陽性反応を示す抗体を産生するクローンを選別し、ハイブリドーマ株を樹立した。 (4) Antibody screening The obtained hybridoma culture supernatant was reacted with a GFP-CD44v8-10 transfectant, and FACS analysis was performed to screen whether or not a CD44v8-v10 specific antibody was produced. . The FACS reaction was performed in a 96-well plate. Transfectants were dispensed into tubes at 50 μl each so as to give 2.5 × 10 5 to 3 × 10 6 cells / well. 50 μl of the hybridoma culture supernatant was added to the cell suspension and allowed to react for 45 minutes under ice cooling. After adding 100 μl of 0.1% BSA-PBS to the tube, centrifuging at 500 g at 4 ° C. for 3 minutes and discarding the supernatant, washing was repeated 3 times, and then PE-labeled anti-mouse antibody was added as a secondary antibody to block light. The reaction was allowed to proceed for 45 minutes under ice cooling. An operation of adding 100 μL of 0.1% BSA-PBS, centrifuging at 500 g at 4 ° C. for 3 minutes and discarding the supernatant was washed three times, then suspended in 500 μL of PBS and transferred to an FCM tube. To remove dead cells, 100 μL of PI was added immediately before the measurement, and the fluorescence intensity was measured with a FACSCaliber (Becton Dickinson). As shown in FIG. 1, clones producing antibodies that show a positive reaction depending on the expression level of CD44v8-10 against GFP-CD44v8-10 transfectants were selected to establish a hybridoma strain.
（５）モノクローナル抗体の認識部位の同定
　得られた抗体の認識部位を同定するために、大腸菌により発現させたＣＤ４４ｖ８、ＣＤ４４ｖ９、ＣＤ４４ｖ１０の精製蛋白質を用いたＷｅｓｔｅｒｎ　Ｂｌｏｔ法を行った。ライセートサンプル３０μｌ（総タンパク質量４０μｇ）をｅ・パジェル１０％（ＡＴＴＯ　）を用いて、２０ｍＡ、９０分間で電気泳動を行った。電気泳動後、メタノールに浸した後にＴｒａｎｓｆｅｒ　Ｂｕｆｆｅｒ（２５ｍＭ　Ｔｒｉｓ、１９０ｍＭ　Ｇｌｙｃｉｎｅ、５％　メタノール）で１５分間処理を行ったＰＶＤＦ膜へ、ＴＲＡＮＳ−ＢＬＯＴ　ＳＤ　Ｃｅｌｌ（ＢＩＯ−ＲＡＤ）を用いて、１５Ｖ、６０分間でトランスファーした。５％スキムミルク／ＰＢＳ−Ｔに浸し、室温で１時間ブロッキングを行った後、Ｃａｎ　Ｇｅｔ　Ｓｉｇｎａｌ　Ｓｏｌｕｔｉｏｎ　Ｉで希釈した抗体を添加し、４℃、１晩で１次抗体反応を行った。ＰＢＳ−Ｔで５分間洗浄を３回繰り返した後、Ｃａｎ　Ｇｅｔ　Ｓｉｇｎａｌ　Ｓｏｌｕｔｉｏｎ　ＩＩで希釈したＨＲＰ標識抗マウス抗体を加え、３７℃　３０分間で２次抗体反応を行った。ＰＢＳ−Ｔで５分間洗浄を３回繰り返した後、等量のＳｔａｂｌｅ　Ｐｅｒｏｘｉｄｅ　ＢｕｆｆｅｒとＬｕｍｉｎｏｌ／Ｅｎｈａｎｃｅｒ　Ｓｏｌｕｔｉｏｎの混合液を加え、室温、５分間インキュベートし、ＦｌｕｏｒＣｈｅｍ　８９００　（Ｒｏｐｅｒ）を用いて検出を行った。図２に示すように、各抗体はＣＤ４４ｖ９の断片蛋白質と反応し、ＣＤ４４ｖ８およびＣＤ４４ｖ１０の断片蛋白質とは反応しなかったことから、ＣＤ４４ｖ９特異的に結合する抗体であると結論付けられた。 (5) Identification of recognition site of monoclonal antibody In order to identify the recognition site of the obtained antibody, Western Blot method using purified proteins of CD44v8, CD44v9 and CD44v10 expressed by E. coli was performed. A 30 μl lysate sample (total protein amount: 40 μg) was electrophoresed at 20 mA for 90 minutes using e-pagel 10% (ATTO). After electrophoresis, PVDF membrane treated with Transfer Buffer (25 mM Tris, 190 mM Glycine, 5% methanol) for 15 minutes after soaking in methanol was transferred to 15 V, 60 using TRANS-BLOT SD Cell (BIO-RAD). Transferred in minutes. After soaking in 5% skim milk / PBS-T and blocking at room temperature for 1 hour, an antibody diluted with Can Get Signal Solution I was added, and a primary antibody reaction was performed at 4 ° C. overnight. After washing with PBS-T for 5 minutes three times, an HRP-labeled anti-mouse antibody diluted with Can Get Signal Solution II was added, and a secondary antibody reaction was performed at 37 ° C. for 30 minutes. After washing with PBS-T for 5 minutes three times, an equal volume of Stable Peroxide Buffer and Luminol / Enhancer Solution was added, incubated at room temperature for 5 minutes, and detection was performed using FluorChem 8900 (Roper). . As shown in FIG. 2, each antibody reacted with the fragment protein of CD44v9 and did not react with the fragment protein of CD44v8 and CD44v10. Therefore, it was concluded that the antibodies were specifically bound to CD44v9.
（６）モノクローナル抗体の精製
　プリスタン処理した８週令ヌード雄マウス（ＫＳＮ）に実施例１（４）で得られたハイブリドーマ株を１×１０７細胞／匹それぞれ腹腔内注射した。１０~１５日後に、ハイブリドーマは腹水癌化した。腹水のたまったマウスから腹水を採取し、遠心分離（２０００ｒｐｍ、５分、４℃）した後５０％飽和硫酸アンモニウムにて塩析し、沈渣を溶かして１００００ｇ、４℃で１０分間遠心した。プレフィルター、メンブランフィルター（０．２２μｍ）を通過させた後、Ｐｒｏｔｅｉｎ　Ｇ　Ｓｅｐｈａｒｏｓｅ（ＧＥヘルスケア　バイオサイエンス）を用いて精製した後、ＰＢＳで透析（３時間以上×４回）を行った。その結果得られた抗体を、ＡＣＳＣ００２モノクローナル抗体、ＡＣＳＣ００５モノクローナル抗体、ＡＣＳＣ００７モノクローナル抗体、ＡＣＳＣ００９モノクローナル抗体、ＡＣＳＣ０１０モノクローナル抗体とした。 (6) Purification of monoclonal antibody Pristane-treated 8-week-old nude male mice (KSN) were injected intraperitoneally with 1 × 10 7 cells / mouse of the hybridoma strain obtained in Example 1 (4). After 10 to 15 days, the hybridoma developed ascites tumor. Ascites was collected from the mouse with ascites collected, centrifuged (2000 rpm, 5 minutes, 4 ° C.), salted out with 50% saturated ammonium sulfate, the sediment was dissolved, and centrifuged at 10,000 g, 4 ° C. for 10 minutes. After passing through a prefilter and a membrane filter (0.22 μm), purification was performed using Protein G Sepharose (GE Healthcare Bioscience), followed by dialysis with PBS (3 hours or more × 4 times). The resulting antibodies were designated as ACSC002 monoclonal antibody, ACSC005 monoclonal antibody, ACSC007 monoclonal antibody, ACSC009 monoclonal antibody, ACSC010 monoclonal antibody.
［実施例２］
（１）腫瘍細胞株に対するモノクローナル抗体の結合性の検討
　ヒト腫瘍細胞株３種を用いて、ＡＣＳＣ００２モノクローナル抗体、ＡＣＳＣ００５モノクローナル抗体、ＡＣＳＣ００７モノクローナル抗体、ＡＣＳＣ００９モノクローナル抗体、ＡＣＳＣ０１０モノクローナル抗体の結合性をＦＡＣＳ法により測定した。ＦＡＣＳの反応は９６ウェルプレートにて行った。ヒト腫瘍細胞株を１．０×１０５／ウェルになるように５０μｌずつチューブに分注した。この細胞浮遊液に４０ｕｇ／ｍＬに調整した各モノクローナル抗体５０μｌを加え、氷冷下で４５分反応させた。チューブに０．１％ＢＳＡ−ＰＢＳ　１００μｌを加え、５００ｇ、４℃で３分間遠心し上清を捨てる操作を３回繰り返して洗浄した後、２次抗体としてＰＥ−標識抗マウス抗体を添加し遮光氷冷下で４５分間反応させた。０．１％ＢＳＡ−ＰＢＳ１００μＬを加え、５００ｇ、４℃で３分間遠心し上清を捨てる操作を３回繰り返して洗浄した後、ＰＢＳ５００μＬに懸濁し、ＦＣＭチューブに移した。死細胞を除くために測定直前にＰＩ１００μＬを添加し、ＦＡＣＳＣａｌｉｂｅｒ（Ｂｅｃｔｏｎ　Ｄｉｃｋｉｎｓｏｎ）にて蛍光強度を測定した。図３に示すように、ＡＣＳＣ００２モノクローナル抗体、ＡＣＳＣ００５モノクローナル抗体、ＡＣＳＣ００７モノクローナル抗体、ＡＣＳＣ００９モノクローナル抗体、ＡＣＳＣ０１０モノクローナル抗体は、ヒト乳癌細胞株（ＭＤＡ−ＭＢ４６８およびＭＤＡ−ＭＢ２３１）およびヒト大腸癌細胞株（ＨＣＴ１１６）に対して結合活性を示し、特にＨＣＴ−１１６に対してより高い結合活性が認められた。 [Example 2]
(1) Examination of binding ability of monoclonal antibody to tumor cell line Using 3 kinds of human tumor cell lines, binding ability of ACSC002 monoclonal antibody, ACSC005 monoclonal antibody, ACSC007 monoclonal antibody, ACSC009 monoclonal antibody, ACSC010 monoclonal antibody was determined by FACS method. It was measured. The FACS reaction was performed in a 96-well plate. The human tumor cell line was dispensed into tubes at 50 μl so as to be 1.0 × 10 5 / well. 50 μl of each monoclonal antibody adjusted to 40 ug / mL was added to the cell suspension, and the mixture was reacted for 45 minutes under ice cooling. After adding 100 μl of 0.1% BSA-PBS to the tube, centrifuging at 500 g at 4 ° C. for 3 minutes and discarding the supernatant, washing was repeated 3 times, and then PE-labeled anti-mouse antibody was added as a secondary antibody to block light. The reaction was allowed to proceed for 45 minutes under ice cooling. An operation of adding 100 μL of 0.1% BSA-PBS, centrifuging at 500 g at 4 ° C. for 3 minutes and discarding the supernatant was washed three times, then suspended in 500 μL of PBS and transferred to an FCM tube. To remove dead cells, 100 μL of PI was added immediately before the measurement, and the fluorescence intensity was measured with a FACSCaliber (Becton Dickinson). As shown in FIG. 3, ACSC002 monoclonal antibody, ACSC005 monoclonal antibody, ACSC007 monoclonal antibody, ACSC009 monoclonal antibody, ACSC010 monoclonal antibody are human breast cancer cell lines (MDA-MB468 and MDA-MB231) and human colon cancer cell line (HCT116). In particular, higher binding activity was observed for HCT-116.
（２）抗体の細胞膜のＣＤ４４ｖ９への結合活性の確認
　ヒト腫瘍細胞株細を用いて、ＡＣＳＣ００２モノクローナル抗体、ＡＣＳＣ００５モノクローナル抗体、ＡＣＳＣ００７モノクローナル抗体、ＡＣＳＣ００９モノクローナル抗体、ＡＣＳＣ０１０モノクローナル抗体の結合性の細胞膜上に発現するＣＤ４４ｖ９への結合活性を、Ｉｍｍｕｎｏｆｌｕｏｒｅｓｃｅｎｃｅにより検討した。ヒト乳癌細胞株（ＭＤＡ−ＭＢ４６８およびＭＤＡ−ＭＢ２３１）およびヒト大腸癌細胞株（ＨＣＴ１１６）をＣｅｌｌ　ｃｈａｍｂｅｒ　Ｓｌｉｄｅ上で培養し、ＰＢＳでリンスを行い、４％ｐａｒａｆｏｒｍａｌｄｅｈｙｄｅ／ＰＢＳで１５分間、室温で固定した。ＰＢＳで３分間リンスを３回行い、３％ＢＳＡ／ＰＢＳで３０分間、室温でブロッキングを行った。ブロッキング溶液を取り除いた後、１次抗体として、０．２％ＢＳＡ／ＰＢＳで希釈したモノクローナル抗体を添加し、１時間、室温で反応させた。ＰＢＳで３分間リンスを３回行い、２次抗体として、０．２％ＢＳＡ／ＰＢＳで希釈したＡｌｅｘａ　Ｆｌｕｏｒ　４８８標識抗マウスＩｇＧ抗体を添加し、１時間、室温、暗所で反応させた。ＰＢＳで３分間リンスを３回行い、ＳｌｏｗＦａｄｅ　Ｇｏｌｄ　Ａｎｔｉｆａｄｅ　Ｒｅａｇｅｎｔ　Ｗｉｔｈ　ＤＡＰＩ　（Ｉｎｖｉｔｒｏｇｅｎ）を用いて封入し、顕微鏡による観察を行った。図４に示すように、ヒト乳癌細胞株（ＭＤＡ−ＭＢ４６８）およびヒト大腸癌細胞株（ＨＣＴ１１６）の細胞膜にシグナル（緑色）が認められ、ＡＣＳＣ００２モノクローナル抗体、ＡＣＳＣ００５モノクローナル抗体、ＡＣＳＣ００７モノクローナル抗体、ＡＣＳＣ００９モノクローナル抗体、ＡＣＳＣ０１０モノクローナル抗体の細胞膜上のＣＤ４４ｖ９への結合活性が確認された。 (2) Confirmation of binding activity of antibody cell membrane to CD44v9 Using human tumor cell line, expressed on binding cell membrane of ACSC002 monoclonal antibody, ACSC005 monoclonal antibody, ACSC007 monoclonal antibody, ACSC009 monoclonal antibody, ACSC010 monoclonal antibody The binding activity to CD44v9 was examined by Immunofluorescence. Human breast cancer cell lines (MDA-MB468 and MDA-MB231) and human colon cancer cell line (HCT116) were cultured on Cell chamber Slide, rinsed with PBS, and fixed with 4% paraformaldehyde / PBS for 15 minutes at room temperature. . Rinse was performed 3 times with PBS for 3 minutes, and blocking was performed with 3% BSA / PBS for 30 minutes at room temperature. After removing the blocking solution, a monoclonal antibody diluted with 0.2% BSA / PBS was added as a primary antibody and allowed to react at room temperature for 1 hour. After rinsing with PBS for 3 minutes three times, Alexa Fluor 488-labeled anti-mouse IgG antibody diluted with 0.2% BSA / PBS was added as a secondary antibody and allowed to react for 1 hour at room temperature in the dark. After rinsing with PBS three times for 3 minutes, it was encapsulated using SlowFade Gold Antifade Reagent With DAPI (Invitrogen) and observed with a microscope. As shown in FIG. 4, signals (green) were observed in the cell membranes of human breast cancer cell line (MDA-MB468) and human colon cancer cell line (HCT116), and ACSC002 monoclonal antibody, ACSC005 monoclonal antibody, ACSC007 monoclonal antibody, ACSC009 monoclonal antibody. The binding activity of the antibody, ACSC010 monoclonal antibody, to CD44v9 on the cell membrane was confirmed.
［実施例３］
（１）ヒトキメラ抗体の作製
　樹立した抗ＣＤ４４ｖ９抗体産生ハイブリドーマ株からｔｏｔａｌ　ＲＮＡを分離し、ＳＭＡＲＴｅｒＴＭ　ＲＡＣＥ　ｃＤＮＡ．Ａｍｐｌｉｆｉｃａｔｉｏｎ　Ｋｉｔ　（クロンテック）を使ってｃＤＮＡを合成した。得られたｃＤＮＡよりｐｏｌｙｍｅｒａｓｅ　ｃｈａｉｎ　ｒｅａｃｔｉｏｎ（ＰＣＲ）法により重鎖および軽鎖可変領域をコードするｃＤＮＡを増幅し、クローニングベクターにサブクローニングした。得られたｃＤＮＡの塩基配列を解析し、常法（＜ｈｔｔｐ：／／ｗｗｗ．ｂｉｏｉｎｆ．ｏｒｇ．ｕｋ／ａｂｓ／＞に記載）により各抗体の重鎖及び軽鎖の可変領域のアミノ酸配列（配列番号１−１０）及びＣｏｍｐｌｅｍｅｎｔａｒｉｔｙ　ｄｅｔｅｒｍｉｎｉｎｇ　ｒｅｇｉｏｎ（ＣＤＲ）のアミノ酸配列（配列番号１１−４０）を決定した。ＣＤＲ配列特異的プライマーを合成し、それを用いて、抽出したプラスミドＤＮＡからＣＤＲを増幅し、ヒトキメラ抗体重鎖発現ベクターもしくはヒトキメラ抗体軽鎖発現ベクターに組み込んだ。得られたプラスミドはＬｉｇａｓｅを用いて結合し、ヒトキメラ抗体産生プラスミドを作製した。ＦｒｅｅＳｔｙｌｅＴＭ　ＭＡＸ　Ｒｅａｇｅｎｔ（インビトロジェン）を用いて、ヒトキメラ抗体産生プラスミドをＦｒｅｅＳｔｙｌｅ　２９３Ｆ細胞（インビトロジェン）に遺伝子導入し、９６時間　旋回培養（ＦｒｅｅＳｔｙｌｅＴＭ　２９３培地，３７℃，８％ＣＯ２，１３５ｒｐｍ）後の培養上清を取得した。培養上清はＰｒｏｔｅｉｎ　Ｇ　Ｓｅｐｈａｒｏｓｅ（ＧＥヘルスケア　バイオサイエンス）を用いて精製した後、ＰＢＳで透析（３時間以上ｘ４回）を行い、ヒトキメラＣＤ４４ｖ９抗体を取得した。 [Example 3]
(1) Preparation of human chimeric antibody Total RNA was isolated from the established anti-CD44v9 antibody-producing hybridoma strain, and SMARTer ™ RACE cDNA. CDNA was synthesized using Amplification Kit (Clontech). From the obtained cDNA, cDNA encoding heavy chain and light chain variable regions was amplified by the polymerase chain reaction (PCR) method and subcloned into a cloning vector. The nucleotide sequence of the obtained cDNA was analyzed, and the amino acid sequences (sequences) of the variable regions of the heavy and light chains of each antibody were determined by a conventional method (described in <http://www.bioinf.org.uk/abs/>). The amino acid sequences (SEQ ID NOs: 11-40) of No. 1-10) and Complementarity determining region (CDR) were determined. CDR sequence-specific primers were synthesized and used to amplify CDRs from the extracted plasmid DNA and incorporated into a human chimeric antibody heavy chain expression vector or a human chimeric antibody light chain expression vector. The obtained plasmid was ligated using Ligase to prepare a human chimeric antibody production plasmid. Using FreeStyle ™ MAX Reagent (Invitrogen), a human chimeric antibody-producing plasmid is introduced into FreeStyle 293F cells (Invitrogen), and cultured after 96 hours of swirling culture (FreeStyle ™ 293 medium, 37 ° C, 8% CO 2 , 135 rpm) The supernatant was obtained. The culture supernatant was purified using Protein G Sepharose (GE Healthcare Bioscience) and then dialyzed with PBS (3 hours or more x 4 times) to obtain a human chimeric CD44v9 antibody.
（２）モノクローナル抗体の抗体依存性細胞障害活性の検討
　ベノジェクトＩＩ真空採血管（テルモ＃ＶＰ−Ｈ１００Ｋ）に採血したヒト末梢血６ｍＬを、リンフォセパールＩ（ＩＢＬ＃２３０１０）４ｍＬに対し静かに重層し、１８００ｒｐｍで３０分遠心して分離した単核球の層を回収した。ＰＢＳで倍量に希釈し、２００ｇで１０分間遠心した。ペレットにＰＢＳを加え、２００ｇで１０分間遠心する洗浄し、適切な細胞密度になるよう培地で懸濁した細胞をエフェクター細胞とした。一方、標的細胞としてヒト乳癌細胞株（ＭＤＡ−ＭＢ４６８）、ヒト正常乳腺由来細胞株（Ｈｓ６１７．Ｍｇ）およびヒト正常乳皮膚由来細胞株（Ｈｓ７１４．Ｓｋ）を２×１０５ｃｅｌｌｓ／ｍＬとなるように培地で調整した。丸底９６ｗｅｌｌ　ｐｌａｔｅに予めＡＣＳＣ０１０ヒトキメラ抗体５０μＬずつ分注し、標的細胞液を５０μＬ加えた。４℃で１５分インキュベート後、１５００ｒｐｍで３分遠心して、上清を捨てた。エフェクター細胞液１００μＬを加え、１２００ｒｐｍで１分遠心して３７℃で４時間インキュベートした。インキュベート後、１２００ｒｐｍで３分遠心し、上清５０μＬを平底９６ｗｅｌｌ　ｐｌａｔｅに移した。Ｓｕｂｓｔｒａｔｅ　ｍｉｘ（Ｃｙｔｏｔｏｘ９６　Ｎｏｎ−Ｒａｄｉｏａｃｔｉｖｅ　ｃｙｔｏｔｏｘｉｃｉｔｙ　ａｓｓａｙ（Ｐｒｏｍｅｇａ　＃Ｇ１７８０））を５０μＬ／ｗｅｌｌで加え、３０分静置した。Ｓｔｏｐ　ｓｏｌｕｔｉｏｎを５０μｌ／ｗｅｌｌ加え、Ｉｎｆｉｎｉｔｅ　Ｍ２００（ＴＥＣＡＮ）を用いて４９０ｎｍにおける吸光度を測定し、次の式で細胞障害率を算出した。細胞障害率（％）＝（ｓａｍｐｌｅ　ｒｅｌｅａｓｅ　−　ｓｐｏｎｔａｎｅｏｕｓ　ｒｅｌｅａｓｅ）／（ｔｏｔａｌ　ｒｅｌｅａｓｅ　−　ｓｐｏｎｔａｎｅｏｕｓ　ｒｅｌｅａｓｅ）×１００。図５に示すようにＡＣＳＣ０１０ヒトキメラ抗体のヒト乳癌細胞株（ＭＤＡ−ＭＢ４６８）に対するＡＤＣＣ活性が検出された。また、ヒト正常乳腺由来細胞株（Ｈｓ６１７．Ｍｇ）およびヒト正常乳皮膚由来細胞株（Ｈｓ７１４．Ｓｋ）は認められず、癌細胞特異的なＡＤＣＣ活性が示された。 (2) Examination of antibody-dependent cytotoxic activity of monoclonal antibody 6 mL of human peripheral blood collected in a Venoject II vacuum blood collection tube (Terumo # VP-H100K) was gently overlaid on 4 mL of Lymphosepar I (IBL # 23010) The separated mononuclear cell layer was recovered by centrifugation at 1800 rpm for 30 minutes. Dilute to volume with PBS and centrifuge at 200g for 10 minutes. PBS was added to the pellet, washed by centrifugation at 200 g for 10 minutes, and cells suspended in a medium so as to have an appropriate cell density were used as effector cells. On the other hand, human breast cancer cell line (MDA-MB468), human normal mammary gland-derived cell line (Hs617.Mg), and human normal breast skin-derived cell line (Hs714.Sk) are set to 2 × 10 5 cells / mL as target cells. Adjusted with medium. 50 μL of ACSC010 human chimeric antibody was dispensed in advance into a round bottom 96-well plate, and 50 μL of the target cell solution was added. After 15 minutes of incubation at 4 ° C., the mixture was centrifuged at 1500 rpm for 3 minutes, and the supernatant was discarded. 100 μL of the effector cell solution was added, centrifuged at 1200 rpm for 1 minute, and incubated at 37 ° C. for 4 hours. After incubation, the mixture was centrifuged at 1200 rpm for 3 minutes, and 50 μL of the supernatant was transferred to a flat-bottom 96-well plate. Substrate mix (Cytotox 96 Non-Radioactive Cytotoxicity Assay (Promega # G1780)) was added at 50 μL / well and allowed to stand for 30 minutes. Stop solution was added at 50 μl / well, the absorbance at 490 nm was measured using Infinite M200 (TECAN), and the cytotoxic rate was calculated by the following formula. Cell damage rate (%) = (sample release−spontaneous release) / (total release−spontaneous release) × 100. As shown in FIG. 5, the ADCC activity of the ACSC010 human chimeric antibody against the human breast cancer cell line (MDA-MB468) was detected. In addition, a human normal mammary gland-derived cell line (Hs617.Mg) and a human normal milk skin-derived cell line (Hs714.Sk) were not found, indicating cancer cell-specific ADCC activity.
（３）モノクローナル抗体のインターナリゼーション活性の検討
　ヒト腫瘍細胞株を用いて抗ＣＤ４４ｖ９モノクローナル抗体のインターナリゼーション活性をＦＣＭ解析により測定した。ヒト前立腺癌癌細胞株ＰＣ３から（７）の方法によって濃縮したＣＤ４４ｖ９高発現細胞群を０．１％ＢＳＡ−ＰＢＳで懸濁し、Ｕ底９６ｗｅｌｌ　ｐｌａｔｅに１ｘ１０５ｃｅｌｌｓ／ｗｅｌｌになるように５０μＬずつ分注した。この細胞浮遊液に２０μｇ／ｍＬに調製した各モノクローナル抗体５０μＬを加え、４℃で４５分反応させた。０．１％ＢＳＡ−ＰＢＳ１５０μＬを加え、５００ｇで３分間、４℃で遠心する洗浄を３回行い、モノクローナル抗体を結合させた細胞ペレットを得た。この細胞ペレットに０．１％ＢＳＡ−ＰＢＳ１００μＬを加え、３７℃（インターナリゼーションが誘導される温度）または４℃（インターナリゼーションが誘導されない温度）で、それぞれ１時間インキュベートした。０．１％ＢＳＡ−ＰＢＳ１５０μＬを加え、５００ｇで３分間、４℃で遠心する洗浄を３回行った。２００倍希釈したＰＥ−標識抗マウス抗体を５０μＬ加え、遮光状態で４℃で３０分間反応させた。０．１％ＢＳＡ−ＰＢＳ１５０μＬを加え、５００ｇで３分間、４℃で遠心する洗浄を３回行い、ＰＢＳ５００μＬに懸濁し、ＦＣＭチューブに移した。死細胞を除くため測定直前にＰＩ１００μＬを添加し、ＦＡＣＳＣａｌｉｂｕｒ（Ｂｅｃｔｏｎ　Ｄｉｃｋｉｎｓｏｎ）にて測定した。図６に示すように、４℃でインキュベートした場合に比べ、３７℃でインキュベートした場合においてモノクローナル抗体の結合による蛍光強度の減少が認められた。これらの結果から、ＡＣＳＣ００２モノクローナル抗体、ＡＣＳＣ００５モノクローナル抗体、ＡＣＳＣ００７モノクローナル抗体、ＡＣＳＣ００９モノクローナル抗体、ＡＣＳＣ０１０モノクローナル抗体が抗原に結合することによって、細胞表面上の抗原−抗体複合体が減少、即ちインターナリゼーションが生じたことが示された。 (3) Examination of Internalization Activity of Monoclonal Antibody The internalization activity of anti-CD44v9 monoclonal antibody was measured by FCM analysis using a human tumor cell line. A group of high expression cells of CD44v9 concentrated from the human prostate cancer cell line PC3 by the method of (7) is suspended in 0.1% BSA-PBS, and 50 μL is dispensed on a U-bottom 96-well plate so that 1 × 10 5 cells / well. Noted. To this cell suspension, 50 μL of each monoclonal antibody prepared to 20 μg / mL was added and reacted at 4 ° C. for 45 minutes. 150 μL of 0.1% BSA-PBS was added, and washing was performed by centrifuging at 500 g for 3 minutes at 4 ° C. three times to obtain a cell pellet bound with a monoclonal antibody. To this cell pellet, 100 μL of 0.1% BSA-PBS was added and incubated at 37 ° C. (temperature at which internalization was induced) or 4 ° C. (temperature at which internalization was not induced), respectively, for 1 hour. Washing by adding 150 μL of 0.1% BSA-PBS and centrifuging at 500 g for 3 minutes at 4 ° C. was performed three times. 50 μL of a 200-fold diluted PE-labeled anti-mouse antibody was added and allowed to react at 4 ° C. for 30 minutes in the dark. 150 μL of 0.1% BSA-PBS was added, washing was performed by centrifuging at 500 g for 3 minutes at 4 ° C. three times, suspended in 500 μL of PBS, and transferred to an FCM tube. In order to remove dead cells, 100 μL of PI was added immediately before measurement, and measurement was performed with FACSCalibur (Becton Dickinson). As shown in FIG. 6, a decrease in fluorescence intensity due to the binding of the monoclonal antibody was observed when incubated at 37 ° C., compared to when incubated at 4 ° C. From these results, the binding of the ACSC002 monoclonal antibody, the ACSC005 monoclonal antibody, the ACSC007 monoclonal antibody, the ACSC009 monoclonal antibody, the ACSC010 monoclonal antibody to the antigen reduces the antigen-antibody complex on the cell surface, ie, internalization occurs. It was shown that
（４）モノクローナル抗体の活性酸素種（ＲＯＳ）上昇作用の効果の検討
　ヒト大腸癌細胞株ＨＣＴ−１１６を用いて抗ＣＤ４４ｖ９モノクローナル抗体のＲＯＳ上昇作用を測定した。培養を行った大腸癌細胞株ＨＣＴ−１１６に１０ｕｇの抗ＣＤ４４ｖ９モノクローナル抗体を添加し、１８時間培養を行った。５００μＭの過酸化水素（Ｈ２Ｏ２）を添加し、細胞にストレスを与えた後、細胞内のＲＯＳを測定した。ＲＯＳは、ＤＣＦ（２‘、７’−Ｄｉｃｈｌｏｒｏｄｉｈｙｄｒｏｆｌｕｏｒｅｓｃｅｉｎ）のシグナル（緑色）により検出し、顕微鏡下で観察を行うことで評価を行った。コントロールとしては、ｓｉＲＮＡによりＣＤ４４遺伝子をノックダウンして同様のＲＯＳの評価を行い、強いＲＯＳの上昇が検出された。図７に示すように、ＡＣＳＣ００２モノクローナル抗体、ＡＣＳＣ００５モノクローナル抗体、ＡＣＳＣ０１０モノクローナル抗体の添加によって、活性酸素腫（ＲＯＳ）の上昇作用が確認された。特に、ＡＣＳＣ００２モノクローナル抗体とＡＣＳＣ０１０モノクローナル抗体でより強いＲＯＳ上昇作用が確認された。 (4) Examination of the effect of the reactive oxygen species (ROS) increasing action of the monoclonal antibody The ROS increasing action of the anti-CD44v9 monoclonal antibody was measured using the human colon cancer cell line HCT-116. 10 ug of anti-CD44v9 monoclonal antibody was added to the cultured colon cancer cell line HCT-116 and cultured for 18 hours. After adding 500 μM hydrogen peroxide (H 2 O 2 ) and applying stress to the cells, intracellular ROS was measured. ROS was detected by DCF (2 ′, 7′-Dichlorodihydrofluorescein) signal (green) and evaluated by observation under a microscope. As a control, the CD44 gene was knocked down with siRNA and the same ROS was evaluated, and a strong rise in ROS was detected. As shown in FIG. 7, the action of raising active oxygenoma (ROS) was confirmed by the addition of the ACSC002 monoclonal antibody, the ACSC005 monoclonal antibody, and the ACSC010 monoclonal antibody. In particular, a stronger ROS increasing action was confirmed with the ACSC002 monoclonal antibody and the ACSC010 monoclonal antibody.
（５）前立腺細胞株ＰＣ−３におけるＣＤ４４ｖ９高発現群、ＣＤ４４ｖ９低発現群の分取およびＳｐｈｅｒｅ形成能評価
　（Ａ）ヒト前立腺癌細胞株（ＰＣ−３）から、本発明の抗体とはエピトープが異なる抗ＣＤ４４ｖ９抗体を用いてＣＤ４４ｖ９高発現群とＣＤ４４ｖ９低発現群を分取した。細胞はそれぞれ、２．０×１０５を回収し、２０日間培養した後、ＦＡＣＳによってＣＤ４４ｖ９の発現量の確認を実施した。さらに、培養を実施したそれぞれの細胞群から５．０×１０６を用いて、ＣＤ４４ｖ９高発現群とＣＤ４４ｖ９低発現群を分取した。それぞれ１．０×１０６の細胞を回収し、１３日間培養した後、ＦＡＣＳによってＣＤ４４ｖ９の発現量の確認を実施した。その結果、図８（Ａ）に示すように、各群において、ＣＤ４４ｖ９高発現細胞とＣＤ４４ｖ９低発現細胞が維持されており、ＣＤ４４ｖ９高発現群（ＰＣ３−ＣＤ４４ｖ９Ｈｉｇｈ）およびＣＤ４４ｖ９低発現群（ＰＣ３−ＣＤ４４ｖ９Ｌｏｗ）の分取が確認された。
　（Ｂ）ｉｎ　ｖｉｔｒｏ評価系を用いて、（７）−（Ａ）で得られた細胞群（ＰＣ３−ＣＤ４４ｖ９ＨｉｇｈおよびＰＣ３−ＤＣ４４ｖ９−Ｌｏｗ）の癌肝細胞の特性のひとつであるＳｐｈｅｒｅ形成能の評価を行った。ヒト前立腺癌細胞株（ＰＣ３−ＣＤ４４ｖ９ＨｉｇｈおよびＰＣ３−ＣＤ４４ｖ９Ｌｏｗ）を培養後、トリプシン処理により回収し、血清を含まない培地に懸濁後、１０回~２０回のピペッティングを行いｓｉｎｇｌｅ　ｃｅｌｌの懸濁液とした。２４ウェルのＵｌｔｒａ　Ｌｏｗ　ａｔｔａｃｈｍｅｎｔ　Ｐｌａｔｅ（Ｃｏｓｔａｒ＃３４７３）に１２５０細胞／ｗｅｌｌ、２５００細胞／ｗｅｌｌ、５０００細胞／ｗｅｌｌになるように細胞を播種し、ｂＦＧＦ（最終濃度１０ｎｇ／ｍＬ）、ＥＧＦ（最終濃度２０ｎｇ／ｍＬ）を添加し、培養を行った。３~５日後にＳｐｈｅｒｅ（Ｃｏｌｏｎｙ）の形成数を顕微鏡下でカウントすることにより評価を行った。図８（Ｂ）に示すように、ＰＣ３−ＣＤ４４ｖ９ＨｉｇｈはＰＣ３−ＣＤ４４ｖ９Ｌｏｗに比べてＳｐｈｅｒｅ形成能力が顕著に高く、癌幹細胞の特性を保有していることが示された。この結果からＰＣ３−ＣＤ４４ｖ９Ｈｉｇｈにおいて癌幹細胞が濃縮されていることを確認された。 (5) Sorting of CD44v9 high expression group and CD44v9 low expression group and evaluation of Sphere forming ability in prostate cell line PC-3 (A) From human prostate cancer cell line (PC-3), the antibody of the present invention has an epitope. Different anti-CD44v9 antibodies were used to separate a CD44v9 high expression group and a CD44v9 low expression group. Each cell was collected at 2.0 × 10 5 and cultured for 20 days, and then the expression level of CD44v9 was confirmed by FACS. Furthermore, the CD44v9 high expression group and the CD44v9 low expression group were separated from each of the cultured cell groups using 5.0 × 10 6 . Each 1.0 × 10 6 cells were collected and cultured for 13 days, and then the expression level of CD44v9 was confirmed by FACS. As a result, as shown in FIG. 8 (A), CD44v9 high-expressing cells and CD44v9 low-expressing cells were maintained in each group, and the CD44v9 high-expressing group (PC3-CD44v9High) and CD44v9 low-expressing group (PC3-CD44v9Low). ) Was confirmed.
(B) Evaluation of Sphere forming ability which is one of the characteristics of cancer hepatocytes of the cell groups (PC3-CD44v9High and PC3-DC44v9-Low) obtained in (7)-(A) using an in vitro evaluation system. Went. Human prostate cancer cell lines (PC3-CD44v9High and PC3-CD44v9Low) are cultured and then collected by trypsin treatment, suspended in a serum-free medium, and then pipetted 10 to 20 times to obtain a single cell suspension. It was. 24-well Ultra Low attachment Plate (Costar # 3473) was seeded at 1250 cells / well, 2500 cells / well, 5000 cells / well, bFGF (final concentration 10 ng / mL), EGF (final concentration 20 ng) / ML) was added and culture was performed. Evaluation was performed 3 to 5 days later by counting the number of Sphere (Colony) formed under a microscope. As shown in FIG. 8B, PC3-CD44v9High has significantly higher Sphere-forming ability than PC3-CD44v9Low, indicating that it possesses the characteristics of cancer stem cells. From this result, it was confirmed that cancer stem cells were concentrated in PC3-CD44v9High.
（６）モノクローナル抗体によるＳｐｈｅｒｅ形成能抑制効果の評価
　ｉｎｖｉｔｒｏ評価系用いて、モノクローナル抗体のＳｐｈｅｒｅ形成能の抑制効果の評価を行った。（７）で確立したヒト前立腺癌細胞株ＰＣ３−ＣＤ４４ｖ９Ｈｉｇｈを培養後、トリプシン処理により回収し、血清を含まない培地に懸濁後、１０回~２０回のピペッティングを行いｓｉｎｇｌｅ　ｃｅｌｌの懸濁液とした。２４ウェルのＵｌｔｒａ　Ｌｏｗ　ａｔｔａｃｈｍｅｎｔ　Ｐｌａｔｅ（Ｃｏｓｔａｒ＃３４７３）に１２５０細胞／ｗｅｌｌになるように細胞を播種し、ｂＦＧＦ（最終濃度１０ｎｇ／ｍＬ）、ＥＧＦ（最終濃度２０ｎｇ／ｍＬ）、およびモノクローナル抗体を添加した。培養開始から７日後にＳｐｈｅｒｅ（Ｃｏｌｏｎｙ）の形成数を顕微鏡下でカウントすることにより評価を行った。図９に示すように、ＡＣＳＣ００２モノクローナル抗体、ＡＣＳＣ００５モノクローナル抗体、ＡＣＳＣ００９モノクローナル抗体、ＡＣＳＣ０１０モノクローナル抗体の添加により、コントロールであるマウスＩｇＧ抗体と比較して２０％以上のＳｐｈｅｒｅ形成抑制効果が示された。特に、ＡＣＳＣ００２抗体では、より高いＳｐｈｅｒｅ形成抑制効果が示された。 (6) Evaluation of Sphere formation ability inhibitory effect by monoclonal antibody The inhibition effect of the Sphere formation ability of the monoclonal antibody was evaluated using an in vitro evaluation system. After culturing the human prostate cancer cell line PC3-CD44v9High established in (7), it is recovered by trypsin treatment, suspended in a medium not containing serum, and then pipetting 10 to 20 times to obtain a single cell suspension. It was. Cells were seeded in a 24-well Ultra Low attachment plate (Costar # 3473) to 1250 cells / well, and bFGF (final concentration 10 ng / mL), EGF (final concentration 20 ng / mL), and monoclonal antibody were added. . Seven days after the start of the culture, the number of Sphere (Colony) formed was evaluated by counting under a microscope. As shown in FIG. 9, the addition of the ACSC002 monoclonal antibody, the ACSC005 monoclonal antibody, the ACSC009 monoclonal antibody, and the ACSC010 monoclonal antibody showed a Sphere formation inhibitory effect of 20% or more compared to the mouse IgG antibody as a control. In particular, the ACSC002 antibody showed a higher Sphere formation inhibitory effect.
（７）モノクローナル抗体による転移巣形成の抑制効果の評価
　微量細胞のマウスへの尾静注による転移巣形成能（ｉｎ　ｖｉｖｏ）評価系を用いて、抗ＣＤ４４ｖ９抗体の転移巣形成能抑制効果の評価を行った。（７）の方法を用いて確立したヒト膵癌細胞株（ＡｓＰＣ−１）のＣＤ４４ｖ９高発現細胞群１．０×１０５を２００ｕＬのＰＢＳに懸濁し、マウス（ｂａｌｂ／ｃ、ｎｕ／ｎｕ、♀、７週令）に尾静脈注射により投与した。モノクローナル抗体０．２５ｍｇ、モノクローナル抗体１．００ｍｇまたはＰＢＳ（コントロール）は週２回の頻度で腹腔内投与した。移植後４２日後に解剖を行い、肺組織の観察により、抗体による肺転移巣形成への影響を評価した。図１０（Ａ）に示すように、コントロールであるＰＢＳと比べ、抗体濃度依存的に解剖した肺表面への生着コロニー数の減少が確認され、ＡＣＳＣ００２モノクローナル抗体による転移巣形成抑制効果が示された。 (7) Evaluation of inhibitory effect of metastatic focus formation by monoclonal antibody Evaluation of anti-CD44v9 antibody suppressive effect of metastatic focus formation using an in vivo evaluation system by injecting a small amount of cells into mice. Went. The human pancreatic cancer cell line (AsPC-1) CD44v9 high-expressing cell group 1.0 × 10 5 established using the method of (7) is suspended in 200 uL of PBS, and the mouse (balb / c, nu / nu, ♀) , 7 weeks of age). Monoclonal antibody 0.25 mg, monoclonal antibody 1.00 mg or PBS (control) was intraperitoneally administered twice a week. Dissection was performed 42 days after transplantation, and the effect of antibodies on the formation of lung metastases was evaluated by observation of lung tissue. As shown in FIG. 10 (A), a decrease in the number of colonies engrafted on the dissected lung surface in an antibody concentration-dependent manner was confirmed as compared with PBS as a control, and the effect of inhibiting the formation of metastasis by the ACSC002 monoclonal antibody was shown. It was.
　さらにＡＣＳＣ００２モノクローナル抗体の転移巣形成抑制効果について、特許文献１に記載のＬＧａｄｈ０１−０１モノクローナル抗体との比較を行った。図１０（Ｂ）に示すように、ＬＧａｄｈ０１−０１モノクローナル抗体に比べて、ＡＣＳＣ００２モノクローナル抗体において、有意に高い転移巣形成抑制効果が確認された。 Further, the effect of the ACSC002 monoclonal antibody on the formation of metastatic lesions was compared with the LGadh01-01 monoclonal antibody described in Patent Document 1. As shown in FIG. 10 (B), a significantly higher metastasis formation inhibitory effect was confirmed in the ACSC002 monoclonal antibody as compared with the LGadh01-01 monoclonal antibody.
　本発明は、癌幹細胞の機能を標的とした治療または診断のための医薬等として特に有用である。 The present invention is particularly useful as a pharmaceutical or the like for treatment or diagnosis targeting the function of cancer stem cells.
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配列番号：１１　ＡＣＳＣ００２軽鎖相補性決定領域１（ＣＤＲＬ１）
配列番号：１２　ＡＣＳＣ００２軽鎖相補性決定領域２（ＣＤＲＬ２）
配列番号：１３　ＡＣＳＣ００２軽鎖相補性決定領域３（ＣＤＲＬ３）
配列番号：１４　ＡＣＳＣ００２重鎖相補性決定領域１（ＣＤＲＨ１）
配列番号：１５　ＡＣＳＣ００２重鎖相補性決定領域２（ＣＤＲＨ２）
配列番号：１６　ＡＣＳＣ００２重鎖相補性決定領域３（ＣＤＲＨ３）
配列番号：１７　ＡＣＳＣ００５軽鎖相補性決定領域１（ＣＤＲＬ１）
配列番号：１８　ＡＣＳＣ００５軽鎖相補性決定領域２（ＣＤＲＬ２）
配列番号：１９　ＡＣＳＣ００５軽鎖相補性決定領域３（ＣＤＲＬ３）
配列番号：２０　ＡＣＳＣ００５重鎖相補性決定領域１（ＣＤＲＨ１）
配列番号：２１　ＡＣＳＣ００５重鎖相補性決定領域２（ＣＤＲＨ２）
配列番号：２２　ＡＣＳＣ００５重鎖相補性決定領域３（ＣＤＲＨ３）
配列番号：２３　ＡＣＳＣ００７軽鎖相補性決定領域１（ＣＤＲＬ１）
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配列番号：３０　ＡＣＳＣ００９軽鎖相補性決定領域２（ＣＤＲＬ２）
配列番号：３１　ＡＣＳＣ００９軽鎖相補性決定領域３（ＣＤＲＬ３）
配列番号：３２　ＡＣＳＣ００９重鎖相補性決定領域１（ＣＤＲＨ１）
配列番号：３３　ＡＣＳＣ００９重鎖相補性決定領域２（ＣＤＲＨ２）
配列番号：３４　ＡＣＳＣ００９重鎖相補性決定領域３（ＣＤＲＨ３）
配列番号：３５　ＡＣＳＣ０１０軽鎖相補性決定領域１（ＣＤＲＬ１）
配列番号：３６　ＡＣＳＣ０１０軽鎖相補性決定領域２（ＣＤＲＬ２）
配列番号：３７　ＡＣＳＣ０１０軽鎖相補性決定領域３（ＣＤＲＬ３）
配列番号：３８　ＡＣＳＣ０１０重鎖相補性決定領域１（ＣＤＲＨ１）
配列番号：３９　ＡＣＳＣ０１０重鎖相補性決定領域２（ＣＤＲＨ２）
配列番号：４０　ＡＣＳＣ０１０重鎖相補性決定領域３（ＣＤＲＨ３）
SEQ ID NO: 1 ACSC002 light chain variable region SEQ ID NO: 2 ACSC002 heavy chain variable region SEQ ID NO: 3 ACSC005 light chain variable region SEQ ID NO: 4 ACSC005 heavy chain variable region SEQ ID NO: 5 ACSC007 light chain variable region SEQ ID NO: 6 ACSC007 Heavy chain variable region SEQ ID NO: 7 ACSC009 light chain variable region SEQ ID NO: 8 ACSC009 heavy chain variable region SEQ ID NO: 9 ACSC010 light chain variable region SEQ ID NO: 10 ACSC010 heavy chain variable region SEQ ID NO: 11 ACSC002 light chain complementarity determination Region 1 (CDRL1)
SEQ ID NO: 12 ACSC002 light chain complementarity determining region 2 (CDRL2)
SEQ ID NO: 13 ACSC002 light chain complementarity determining region 3 (CDRL3)
SEQ ID NO: 14 ACSC002 heavy chain complementarity determining region 1 (CDRH1)
SEQ ID NO: 15 ACSC002 heavy chain complementarity determining region 2 (CDRH2)
SEQ ID NO: 16 ACSC002 heavy chain complementarity determining region 3 (CDRH3)
SEQ ID NO: 17 ACSC005 light chain complementarity determining region 1 (CDRL1)
SEQ ID NO: 18 ACSC005 light chain complementarity determining region 2 (CDRL2)
SEQ ID NO: 19 ACSC005 light chain complementarity determining region 3 (CDRL3)
SEQ ID NO: 20 ACSC005 heavy chain complementarity determining region 1 (CDRH1)
SEQ ID NO: 21 ACSC005 heavy chain complementarity determining region 2 (CDRH2)
SEQ ID NO: 22 ACSC005 heavy chain complementarity determining region 3 (CDRH3)
SEQ ID NO: 23 ACSC007 light chain complementarity determining region 1 (CDRL1)
SEQ ID NO: 24 ACSC007 light chain complementarity determining region 2 (CDRL2)
SEQ ID NO: 25 ACSC007 light chain complementarity determining region 3 (CDRL3)
SEQ ID NO: 26 ACSC007 heavy chain complementarity determining region 1 (CDRH1)
SEQ ID NO: 27 ACSC007 heavy chain complementarity determining region 2 (CDRH2)
SEQ ID NO: 28 ACSC007 heavy chain complementarity determining region 3 (CDRH3)
SEQ ID NO: 29 ACSC009 light chain complementarity determining region 1 (CDRL1)
SEQ ID NO: 30 ACSC009 light chain complementarity determining region 2 (CDRL2)
SEQ ID NO: 31 ACSC009 light chain complementarity determining region 3 (CDRL3)
SEQ ID NO: 32 ACSC009 heavy chain complementarity determining region 1 (CDRH1)
SEQ ID NO: 33 ACSC009 heavy chain complementarity determining region 2 (CDRH2)
SEQ ID NO: 34 ACSC009 heavy chain complementarity determining region 3 (CDRH3)
SEQ ID NO: 35 ACSC010 light chain complementarity determining region 1 (CDRL1)
SEQ ID NO: 36 ACSC010 light chain complementarity determining region 2 (CDRL2)
SEQ ID NO: 37 ACSC010 light chain complementarity determining region 3 (CDRL3)
SEQ ID NO: 38 ACSC010 heavy chain complementarity determining region 1 (CDRH1)
SEQ ID NO: 39 ACSC010 heavy chain complementarity determining region 2 (CDRH2)
SEQ ID NO: 40 ACSC010 heavy chain complementarity determining region 3 (CDRH3)

Claims (36)

  1. 　ＣＤ４４ｖ９を発現する細胞のＣＤ４４ｖ９の細胞外領域に特異的に結合する抗体またはその抗原結合断片であって、
    　（１）軽鎖相補性決定領域１（ＣＤＲＬ１）、軽鎖相補性決定領域２（ＣＤＲＬ２）、および軽鎖相補性決定領域３（ＣＤＲＬ３）として、それぞれ、配列番号１１、配列番号１２、および配列番号１３のアミノ酸配列、または配列番号１１、配列番号１２、および配列番号１３のアミノ酸配列に１個もしくは数個のアミノ酸残基の変異をそれぞれ有するアミノ酸配列を含み、
    　重鎖相補性決定領域１（ＣＤＲＨ１）、重鎖相補性決定領域２（ＣＤＲＨ２）、および重鎖相補性決定領域３（ＣＤＲＨ３）として、それぞれ、配列番号１４、配列番号１５、および配列番号１６のアミノ酸配列、もしくは配列番号１４、配列番号１５、および配列番号１６のアミノ酸配列に１個もしくは数個のアミノ酸残基の変異をそれぞれ有するアミノ酸配列を含む、抗体またはその抗原結合断片、
    　（２）ＣＤＲＬ１、ＣＤＲＬ２、およびＣＤＲＬ３として、それぞれ、配列番号１７、配列番号１８、および配列番号１９のアミノ酸配列、または配列番号１７、配列番号１８、および配列番号１９のアミノ酸配列に１個もしくは数個のアミノ酸残基の変異をそれぞれ有するアミノ酸配列を含み、
    　ＣＤＲＨ１、ＣＤＲＨ２、およびＣＤＲＨ３として、それぞれ、配列番号２０、配列番号２１、および配列番号２２のアミノ酸配列、もしくは配列番号２０、配列番号２１、および配列番号２２のアミノ酸配列に１個もしくは数個のアミノ酸残基の変異をそれぞれ有するアミノ酸配列を含む、抗体またはその抗原結合断片、
    　（３）ＣＤＲＬ１、ＣＤＲＬ２、およびＣＤＲＬ３として、それぞれ、配列番号２３、配列番号２４、および配列番号２５のアミノ酸配列、または配列番号２３、配列番号２４、および配列番号２５のアミノ酸配列に１個もしくは数個のアミノ酸残基の変異をそれぞれ有するアミノ酸配列を含み、
    　ＣＤＲＨ１、ＣＤＲＨ２、およびＣＤＲＨ３として、それぞれ、配列番号２６、配列番号２７、および配列番号２８のアミノ酸配列、もしくは配列番号２６、配列番号２７、および配列番号２８のアミノ酸配列に１個もしくは数個のアミノ酸残基の変異をそれぞれ有するアミノ酸配列を含む、抗体またはその抗原結合断片、
    　（４）ＣＤＲＬ１、ＣＤＲＬ２、およびＣＤＲＬ３として、それぞれ、配列番号２９、配列番号３０、および配列番号３１のアミノ酸配列、または配列番号２９、配列番号３０、および配列番号３１のアミノ酸配列に１個もしくは数個のアミノ酸残基の変異をそれぞれ有するアミノ酸配列を含み、
    　ＣＤＲＨ１、ＣＤＲＨ２、およびＣＤＲＨ３として、それぞれ、配列番号３２、配列番号３３、および配列番号３４のアミノ酸配列、もしくは配列番号３２、配列番号３３、および配列番号３４のアミノ酸配列に１個もしくは数個のアミノ酸残基の変異をそれぞれ有するアミノ酸配列を含む、抗体またはその抗原結合断片、または
    　（５）ＣＤＲＬ１、ＣＤＲＬ２、およびＣＤＲＬ３として、それぞれ、配列番号３５、配列番号３６、および配列番号３７のアミノ酸配列、または配列番号３５、配列番号３６、および配列番号３７のアミノ酸配列に１個もしくは数個のアミノ酸残基の変異をそれぞれ有するアミノ酸配列を含み、
    　ＣＤＲＨ１、ＣＤＲＨ２、およびＣＤＲＨ３として、それぞれ、配列番号３８、配列番号３９、および配列番号４０のアミノ酸配列、もしくは配列番号３８、配列番号３９、および配列番号４０のアミノ酸配列に１個もしくは数個のアミノ酸残基の変異をそれぞれ有するアミノ酸配列を含む、抗体またはその抗原結合断片。     An antibody or antigen-binding fragment thereof that specifically binds to an extracellular region of CD44v9 of a cell expressing CD44v9,
    (1) as light chain complementarity determining region 1 (CDRL1), light chain complementarity determining region 2 (CDRL2), and light chain complementarity determining region 3 (CDRL3), respectively, SEQ ID NO: 11, SEQ ID NO: 12, and sequence An amino acid sequence of No. 13, or an amino acid sequence having a mutation of one or several amino acid residues in the amino acid sequences of SEQ ID No. 11, SEQ ID No. 12 and SEQ ID No. 13, respectively;
    As heavy chain complementarity determining region 1 (CDRH1), heavy chain complementarity determining region 2 (CDRH2), and heavy chain complementarity determining region 3 (CDRH3), SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively. An antibody or an antigen-binding fragment thereof comprising an amino acid sequence or an amino acid sequence each having a mutation of one or several amino acid residues in the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16,
    (2) As CDRL1, CDRL2, and CDRL3, one or a number in the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, or the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively An amino acid sequence each having a mutation of one amino acid residue,
    CDRH1, CDRH2, and CDRH3 are one or several amino acids in the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, or the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively. An antibody or antigen-binding fragment thereof comprising an amino acid sequence each having a mutation of a residue,
    (3) As CDRL1, CDRL2, and CDRL3, one or more amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25, or amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25, respectively An amino acid sequence each having a mutation of one amino acid residue,
    CDRH1, CDRH2, and CDRH3 are one or several amino acids in the amino acid sequences of SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, or the amino acid sequences of SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, respectively. An antibody or antigen-binding fragment thereof comprising an amino acid sequence each having a mutation of a residue,
    (4) As CDRL1, CDRL2, and CDRL3, one or more amino acid sequences of SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, or SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, respectively. An amino acid sequence each having a mutation of one amino acid residue,
    CDRH1, CDRH2, and CDRH3 are one or several amino acids in the amino acid sequences of SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34, or the amino acid sequences of SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34, respectively. An antibody or antigen-binding fragment thereof comprising an amino acid sequence each having a mutation of residues, or (5) the amino acid sequences of SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37, respectively, as CDRL1, CDRL2, and CDRL3, or Comprising amino acid sequences each having a mutation of one or several amino acid residues in the amino acid sequences of SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37;
    CDRH1, CDRH2, and CDRH3 are one or several amino acids in the amino acid sequences of SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, or the amino acid sequences of SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively. An antibody or antigen-binding fragment thereof comprising an amino acid sequence each having a residue mutation.
  2. 　ＣＤ４４ｖ９を発現する細胞のＣＤ４４ｖ９の細胞外領域に特異的に結合する抗体またはその抗原結合断片であって、
    　（１）ＲＳ−Ｘ１−Ｋ−Ｘ２−ＬＬＨ−Ｘ３−ＮＧＮ−Ｘ４−ＹＬＹ（配列番号４１）のアミノ酸配列を含む軽鎖相補性決定領域１（ＣＤＲＬ１）、
    　（２）Ｒ−Ｘ５−Ｓ−Ｘ６−ＬＡＳ（配列番号４２）のアミノ酸配列を含む軽鎖相補性決定領域２（ＣＤＲＬ２）、
    　（３）Ｘ７−ＱＨＬＥＹＰ−Ｘ８−Ｔ（配列番号４３）のアミノ酸配列を含む軽鎖相補性決定領域３（ＣＤＲＬ３）、
    　（４）ＧＦＮ−Ｘ９−ＫＤＴＹ−Ｘ１０−Ｈ（配列番号４４）のアミノ酸配列を含む重鎖相補性決定領域１（ＣＤＲＨ１）、
    　（５）ＲＩＤＰＡＮ−Ｘ１１−Ｘ１２−Ｘ１３−Ｘ１４−Ｙ−Ｘ１５−ＰＫＦＱ−Ｘ１６（配列番号４５）のアミノ酸配列を含む重鎖相補性決定領域２（ＣＤＲＨ２）、および
    　（６）Ｒ−Ｘｓｅｑ−Ｆ−Ｘ１７−Ｘ１８のアミノ酸配列を含む重鎖相補性決定領域３（ＣＤＲＨ３）
    （但し、Ｘ１−Ｘ１８は、任意の天然に存在するアミノ酸残基であり、Ｘｓｅｑは、以下からなる群：
    　ＨＬＧＬＰ（配列番号４６）
    　ＱＬＧＬＰ（配列番号４７）
    　ＤＶＳＳＭＩＰＴＧ（配列番号４８）
    　ＳＧＶＧ（配列番号４９）、および
    　ＤＱＴＲＡＴＧ（配列番号５０）
    から選択されるアミノ酸配列である）
    を含む、抗体またはその抗原結合断片。     An antibody or antigen-binding fragment thereof that specifically binds to an extracellular region of CD44v9 of a cell expressing CD44v9,
    (1) Light chain complementarity determining region 1 (CDRL1) comprising the amino acid sequence of RS-X 1 -KX 2 -LLH-X 3 -NGN-X 4 -YLY (SEQ ID NO: 41),
    (2) a light chain complementarity determining region 2 (CDRL2) comprising the amino acid sequence of R-X 5 -SX 6 -LAS (SEQ ID NO: 42),
    (3) a light chain complementarity determining region 3 (CDRL3) comprising the amino acid sequence of X 7 -QHLEYP-X 8 -T (SEQ ID NO: 43),
    (4) heavy chain complementarity determining region 1 (CDRH1) comprising the amino acid sequence of GFN-X 9 -KDTY-X 10 -H (SEQ ID NO: 44),
    (5) RIDPAN-X 11 -X 12 -X 13 -X 14 -Y-X 15 -PKFQ-X 16 ( SEQ ID NO: 45) heavy chain complementarity determining region 2 comprising an amino acid sequence of (CDRH2), and (6 ) Heavy chain complementarity determining region 3 (CDRH3) comprising the amino acid sequence of R-X seq -F-X 17 -X 18
    (Where X 1 -X 18 are any naturally occurring amino acid residue and X seq is a group consisting of:
    HLGLP (SEQ ID NO: 46)
    QLGLP (SEQ ID NO: 47)
    DVSSMIPTG (SEQ ID NO: 48)
    SGVG (SEQ ID NO: 49), and DQTRATG (SEQ ID NO: 50)
    Is an amino acid sequence selected from
    Or an antigen-binding fragment thereof.
  3. 　Ｘ１が、ＳまたはＮであり、
    　Ｘ２が、ＳまたはＴであり、
    　Ｘ３が、ＳまたはＴであり、
    　Ｘ４が、ＴまたはＩであり、
    　Ｘ５が、ＶまたはＭであり、
    　Ｘ６が、ＮまたはＳであり、
    　Ｘ７が、ＬまたはＭであり、
    　Ｘ８が、Ｌ、ＦまたはＹであり、
    　Ｘ９が、ＩまたはＶであり、
    　Ｘ１０が、ＭまたはＩであり、
    　Ｘ１１が、ＧまたはＤであり、
    　Ｘ１２が、ＮまたはＳであり、
    　Ｘ１３が、ＴまたはＣであり、
    　Ｘ１４が、Ｑ、Ｅ、またはＫであり、
    　Ｘ１５が、ＤまたはＮであり、
    　Ｘ１６が、ＤまたはＧであり、
    　Ｘ１７が、ＡまたはＧであり、
    　Ｘ１８が、ＹまたはＤである、
    　請求項２に記載の抗体またはその抗原結合断片。     X 1 is S or N;
    X 2 is S or T;
    X 3 is S or T;
    X 4 is T or I;
    X 5 is V or M;
    X 6 is N or S;
    X 7 is L or M;
    X 8 is L, F or Y,
    X 9 is I or V;
    X 10 is M or I;
    X 11 is G or D;
    X 12 is N or S;
    X 13 is T or C;
    X 14 is Q, E, or K;
    X 15 is D or N;
    X 16 is D or G;
    X 17 is A or G;
    X 18 is Y or D,
    The antibody or antigen-binding fragment thereof according to claim 2.
  4. 　（１）Ｘ１がＳ、Ｘ２がＳ、Ｘ３がＳ、Ｘ４がＴ、Ｘ５がＶ、Ｘ６がＮ、Ｘ７がＬ、Ｘ８がＬ、Ｘ９がＩ、Ｘ１０がＭ、Ｘ１１がＧ、Ｘ１２がＮ、Ｘ１３がＴ、Ｘ１４がＱ、Ｘ１５がＤ、Ｘ１６がＤ、Ｘ１７がＡ、Ｘ１８がＹ、およびＸｓｅｑがＨＬＧＬＰ（配列番号４６）であるか、
    　（２）Ｘ１がＳ、Ｘ２がＳ、Ｘ３がＳ、Ｘ４がＴ、Ｘ５がＭ、Ｘ６がＮ、Ｘ７がＭ、Ｘ８がＬ、Ｘ９がＩ、Ｘ１０がＭ、Ｘ１１がＤ、Ｘ１２がＮ、Ｘ１３がＴ、Ｘ１４がＥ、Ｘ１５がＤ、Ｘ１６がＧ、Ｘ１７がＡ、Ｘ１８がＹ、およびＸｓｅｑがＱＬＧＬＰ（配列番号４７）であるか、
    　（３）Ｘ１がＳ、Ｘ２がＳ、Ｘ３がＳ、Ｘ４がＩ、Ｘ５がＭ、Ｘ６がＮ、Ｘ７がＭ、Ｘ８がＦ、Ｘ９がＩ、Ｘ１０がＭ、Ｘ１１がＤ、Ｘ１２がＮ、Ｘ１３がＴ、Ｘ１４がＫ、Ｘ１５がＮ、Ｘ１６がＧ、Ｘ１７がＡ、Ｘ１８がＹ、およびＸｓｅｑがＤＶＳＳＭＩＰＴＧ（配列番号４８）であるか、
    　（４）Ｘ１がＮ、Ｘ２がＴ、Ｘ３がＴ、Ｘ４がＴ、Ｘ５がＭ、Ｘ６がＮ、Ｘ７がＭ、Ｘ８がＦ、Ｘ９がＩ、Ｘ１０がＭ、Ｘ１１がＧ、Ｘ１２がＮ、Ｘ１３がＣ、Ｘ１４がＫ、Ｘ１５がＤ、Ｘ１６がＧ、Ｘ１７がＧ、Ｘ１８がＹ、およびＸｓｅｑがＳＧＶＧ（配列番号４９）であるか、または
    　（５）Ｘ１がＳ、Ｘ２がＳ、Ｘ３がＳ、Ｘ４がＴ、Ｘ５がＶ、Ｘ６がＳ、Ｘ７がＭ、Ｘ８がＹ、Ｘ９がＶ、Ｘ１０がＩ、Ｘ１１がＧ、Ｘ１２がＳ、Ｘ１３がＴ、Ｘ１４がＫ、Ｘ１５がＤ、Ｘ１６がＧ、Ｘ１７がＡ、Ｘ１８がＤ、およびＸｓｅｑがＤＱＴＲＡＴＧ（配列番号５０）である、請求項２に記載の抗体またはその抗原結合断片。     (1) X 1 is S, X 2 is S, X 3 is S, X 4 is T, X 5 is V, X 6 is N, X 7 is L, X 8 is L, X 9 is I, X 10 Is M, X 11 is G, X 12 is N, X 13 is T, X 14 is Q, X 15 is D, X 16 is D, X 17 is A, X 18 is Y, and X seq is HLGLP (sequence Number 46)
    (2) X 1 is S, X 2 is S, X 3 is S, X 4 is T, X 5 is M, X 6 is N, X 7 is M, X 8 is L, X 9 is I, X 10 Is M, X 11 is D, X 12 is N, X 13 is T, X 14 is E, X 15 is D, X 16 is G, X 17 is A, X 18 is Y, and X seq is QLGLP (sequence Number 47)
    (3) X 1 is S, X 2 is S, X 3 is S, X 4 is I, X 5 is M, X 6 is N, X 7 is M, X 8 is F, X 9 is I, X 10 Is M, X 11 is D, X 12 is N, X 13 is T, X 14 is K, X 15 is N, X 16 is G, X 17 is A, X 18 is Y, and X seq is DVSS MIPTG (array Number 48),
    (4) X 1 is N, X 2 is T, X 3 is T, X 4 is T, X 5 is M, X 6 is N, X 7 is M, X 8 is F, X 9 is I, X 10 Is M, X 11 is G, X 12 is N, X 13 is C, X 14 is K, X 15 is D, X 16 is G, X 17 is G, X 18 is Y, and X seq is SGVG (array (5) X 1 is S, X 2 is S, X 3 is S, X 4 is T, X 5 is V, X 6 is S, X 7 is M, X 8 is Y , X 9 is V, X 10 is I, X 11 is G, X 12 is S, X 13 is T, X 14 is K, X 15 is D, X 16 is G, X 17 is A, X 18 is D And the antibody or antigen-binding fragment thereof according to claim 2, wherein X seq is DQTRATG (SEQ ID NO: 50).
  5. 　請求項１~４のいずれか一項に記載の抗体またはその抗原結合断片であって、
    （１）配列番号１のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含む軽鎖可変領域（ＬＨ）、および配列番号２のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含む重鎖可変領域（ＶＨ）、
    （２）配列番号３のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含むＬＨ、および配列番号４のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含むＶＨ、
    （３）配列番号５のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含むＬＨ、および配列番号６のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含むＶＨ、
    （４）配列番号７のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含むＬＨ、および配列番号８のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含むＶＨ、または
    （５）配列番号９のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含むＬＨ、および配列番号１０のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含むＶＨ
    を含有する、抗体もしくはその抗原結合断片。     The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4,
    (1) including a light chain variable region (LH) comprising an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 1, and an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 2. Heavy chain variable region (VH),
    (2) LH including an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 3, and VH including an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 4,
    (3) LH including an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 5, and VH including an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 6,
    (4) LH including an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 7 and VH including an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 8, or (5) LH including an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 9, and VH including an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 10
    Or an antigen-binding fragment thereof, comprising
  6. 　請求項１~５のいずれか一項に記載の抗体もしくはその抗原結合断片であって、
    （１）配列番号１のアミノ酸配列を含む軽鎖可変領域（ＶＬ）、および配列番号２のアミノ酸配列を含む重鎖可変領域（ＶＨ）、
    （２）配列番号３のアミノ酸配列を含むＶＬ、および配列番号４のアミノ酸配列を含むＶＨ、
    （３）配列番号５のアミノ酸配列を含むＶＬ、および配列番号６のアミノ酸配列を含むＶＨ、
    （４）配列番号７のアミノ酸配列を含むＶＬ、および配列番号８のアミノ酸配列を含むＶＨ、または
    （５）配列番号９のアミノ酸配列を含むＶＬ、および配列番号１０のアミノ酸配列を含むＶＨ
    を含有する、抗体もしくはその抗原結合断片。     The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5,
    (1) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 2,
    (2) VL comprising the amino acid sequence of SEQ ID NO: 3 and VH comprising the amino acid sequence of SEQ ID NO: 4,
    (3) a VL comprising the amino acid sequence of SEQ ID NO: 5, and a VH comprising the amino acid sequence of SEQ ID NO: 6,
    (4) VL including the amino acid sequence of SEQ ID NO: 7 and VH including the amino acid sequence of SEQ ID NO: 8, or (5) VL including the amino acid sequence of SEQ ID NO: 9 and VH including the amino acid sequence of SEQ ID NO: 10.
    Or an antigen-binding fragment thereof, comprising
  7. 　ＣＤ４４ｖ９を発現する細胞に対する細胞障害活性および／またはインターナリゼーション活性を有する、請求項１~６のいずれか一項に記載の抗体またはその抗原結合断片。 The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, which has cytotoxic activity and / or internalization activity against cells expressing CD44v9.
  8. 　ＣＤ４４ｖ９を発現する細胞のＣＤ４４ｖ９に特異的に結合し、ＣＤ４４ｖ９とアミノ酸トランスポーターｘＣＴとの相互作用を阻害する活性を有する、請求項１~６のいずれか一項に記載の抗体またはその抗原結合断片。 The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, which specifically binds to CD44v9 of a cell expressing CD44v9 and has an activity of inhibiting an interaction between CD44v9 and amino acid transporter xCT. .
  9. （ｉ）細胞内活性酸素種の低下を抑制するか、または（ｉｉ）薬剤の細胞外排出を阻害する、請求項１~６のいずれか一項に記載の抗体またはその抗原結合断片。 The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, which suppresses (i) a decrease in intracellular reactive oxygen species, or (ii) inhibits extracellular discharge of a drug.
  10. 　ＣＤ４４ｖ９を発現する細胞のＣＤ４４ｖ９に特異的に結合し、癌幹細胞および／または癌細胞の腫瘍形成能および転移巣形成能を阻害する活性を有する、請求項１~６のいずれか一項に記載の抗体またはその抗原結合断片。 The cell according to any one of claims 1 to 6, which specifically binds to CD44v9 of a cell expressing CD44v9 and has an activity of inhibiting the ability of cancer stem cells and / or cancer cells to form tumors and metastasis. An antibody or antigen-binding fragment thereof.
  11. 　サブクラスがＩｇＧである、請求項１~１０のいずれか一項に記載の抗体またはその抗原結合断片。 The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the subclass is IgG.
  12. 　上記ＩｇＧが、ＩｇＧ２ｂまたはＩｇＧ１である、請求項１１に記載の抗体またはその抗原結合断片。 The antibody or antigen-binding fragment thereof according to claim 11, wherein the IgG is IgG 2b or IgG 1 .
  13. 　上記抗原結合断片が、Ｆａｂ、Ｆａｂ’、Ｆ（ａｂ’）２、ｓｃＦｖ、ｄｓＦｖ、ｄｓ−ｓｃＦｖ、それらのダイマー、ミニボディ（ｍｉｎｏｂｏｄｉｅｓ）、ダイアボディ（ｄｉａｂｏｄｉｅｓ）、およびマルチマー、または二重特異性抗体断片である、請求項１~１２のいずれか一項に記載の抗体またはその抗原結合断片。 The antigen-binding fragment is Fab, Fab ′, F (ab ′) 2 , scFv, dsFv, ds-scFv, their dimers, minibodies, diabodies, and multimers, or bispecific The antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, which is an antibody fragment.
  14. 　モノクローナル抗体である、請求項１~１３のいずれか一項に記載の抗体またはその抗原結合断片。 The antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, which is a monoclonal antibody.
  15. 　ラット、マウス、霊長類、またはヒト抗体である、請求項１~１４のいずれか一項に記載の抗体またはその抗原結合断片。 The antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, which is a rat, mouse, primate, or human antibody.
  16. 　キメラ抗体またはヒト化抗体である、請求項１~１４のいずれか一項に記載の抗体またはその抗原結合断片。 The antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, which is a chimeric antibody or a humanized antibody.
  17. 　請求項１~１６のいずれか一項に記載の抗体またはその抗原結合断片と少なくとも１つの細胞毒性薬、抗腫瘍剤または放射性同位体を含んでなるイムノコンジュゲート。 An immunoconjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 16 and at least one cytotoxic drug, antitumor agent or radioisotope.
  18. 　請求項１~１７のいずれか一項に記載の抗体またはその抗原結合断片をコードする単離された核酸分子。 An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 17.
  19. 　請求項１８に記載の核酸分子を含む発現ベクター。 An expression vector comprising the nucleic acid molecule according to claim 18.
  20. 　請求項１８に記載の核酸分子を含むかまたは該核酸分子を導入した、請求項１~１６のいずれか一項に記載の抗体を産生する、ハイブリドーマ細胞または遺伝子導入細胞。 A hybridoma cell or a gene-introduced cell that contains the nucleic acid molecule according to claim 18 or that has introduced the nucleic acid molecule and produces the antibody according to any one of claims 1 to 16.
  21. 　請求項２０に記載の細胞を培養し、得られる培養物からＣＤ４４ｖ９特異的に結合する抗体を採取することを含む、ＣＤ４４ｖ９に特異的に結合するモノクローナル抗体の製造方法。 A method for producing a monoclonal antibody that specifically binds to CD44v9, comprising culturing the cells of claim 20 and collecting an antibody that specifically binds to CD44v9 from the resulting culture.
  22. 　請求項２０に記載の細胞から抗ＣＤ４４ｖ９モノクローナル抗体をコードする遺伝子を単離し、該遺伝子を含む発現ベクターを構築し、該発現ベクターを宿主に導入して上記モノクローナル抗体を発現せしめ、得られる宿主、宿主の培養上清または宿主の分泌物からＣＤ４４ｖ９に特異的に結合するモノクローナル抗体を採取することを含む、ＣＤ４４ｖ９に特異的に結合するモノクローナル抗体の製造方法。 A host obtained by isolating a gene encoding an anti-CD44v9 monoclonal antibody from the cell of claim 20, constructing an expression vector containing the gene, introducing the expression vector into a host and expressing the monoclonal antibody, A method for producing a monoclonal antibody that specifically binds to CD44v9, comprising collecting a monoclonal antibody that specifically binds to CD44v9 from a culture supernatant of a host or a secretion of a host.
  23. 　上記宿主が、大腸菌、酵母細胞、昆虫細胞、哺乳動物細胞、植物細胞、または哺乳動物である、請求項２２に記載の製造方法。 The production method according to claim 22, wherein the host is Escherichia coli, yeast cells, insect cells, mammalian cells, plant cells, or mammals.
  24. 　請求項１~１７のいずれか一項に記載の抗体またはその抗原結合断片またはイムノコンジュゲートを含有する組成物であって、ＣＤ４４ｖ９を発現する細胞に適用して該細胞のアポトーシスを誘導するために使用する、組成物。 A composition containing the antibody or antigen-binding fragment thereof or immunoconjugate thereof according to any one of claims 1 to 17, which is applied to a cell expressing CD44v9 to induce apoptosis of the cell. The composition to use.
  25. 　請求項１~１７のいずれか一項に記載の抗体またはその抗原結合断片またはイムノコンジュゲートを含有する、腫瘍の予防または治療のための医薬。 A medicament for the prevention or treatment of a tumor, comprising the antibody according to any one of claims 1 to 17, an antigen-binding fragment thereof, or an immunoconjugate.
  26. 　請求項１~１７のいずれか一項に記載の抗体またはその抗原結合断片またはイムノコンジュゲートを含有する組成物であって、ＣＤ４４ｖ９を発現する細胞を検出するために使用する、組成物。 A composition containing the antibody or antigen-binding fragment thereof or immunoconjugate thereof according to any one of claims 1 to 17, which is used for detecting a cell expressing CD44v9.
  27. 　請求項２６の組成物と、生体から採取された試料とを反応させ、反応したシグナルを検出することを特徴とする、腫瘍の検出方法。 A method for detecting a tumor, comprising reacting the composition of claim 26 with a sample collected from a living body, and detecting a reacted signal.
  28. 　請求項２６の組成物を癌細胞と接触する工程を含む試験管内の腫瘍の免疫検出方法。 A method for immunodetection of a tumor in a test tube comprising a step of contacting the composition of claim 26 with cancer cells.
  29. 　請求項２６の組成物を個体に投与する工程および近赤外光イメージング、ＰＥＴ、ＭＲＩまたは超音波イメージングによって得られる検出映像を得る工程を含む生体内で腫瘍の映像化方法。 A method for imaging a tumor in vivo, comprising a step of administering the composition of claim 26 to an individual and a step of obtaining a detection image obtained by near-infrared light imaging, PET, MRI or ultrasonic imaging.
  30. 　請求項１~１７のいずれか一項に記載の抗体またはその抗原結合断片またはイムノコンジュゲートを含有する、腫瘍の診断のための医薬。 A medicament for diagnosis of a tumor, comprising the antibody according to any one of claims 1 to 17, an antigen-binding fragment thereof, or an immunoconjugate.
  31. 　上記腫瘍が、大腸癌、結腸直腸癌、肺癌、乳癌、脳腫瘍、黒色腫、腎細胞癌、膀胱癌、白血病、リンパ腫、Ｔ細胞リンパ腫、多発性骨髄腫、胃癌、膵臓癌、子宮頸癌、子宮内膜癌、卵巣癌、食道癌、肝臓癌、頭頸部扁平上皮癌、皮膚癌、尿路癌、前立腺癌、絨毛癌、咽頭癌、喉頭癌、舌癌、口腔癌、胆嚢癌、甲状腺癌、中皮腫、胸膜腫、男性胚腫、子宮内膜過形成、子宮内膜症、胚芽腫、線維肉腫、カポジ肉腫、血管腫、海綿状血管腫、血管芽腫、網膜芽腫、星状細胞腫、神経線維腫、稀突起謬腫、髄芽腫、神経芽腫、神経膠腫、横紋筋肉腫、謬芽腫、骨原性肉腫、平滑筋肉腫、甲状肉腫、およびウィルムス腫瘍からなる群から選択される少なくとも１つである、請求項２５または請求項３０に記載の医薬。 The above tumors are colorectal cancer, colorectal cancer, lung cancer, breast cancer, brain tumor, melanoma, renal cell carcinoma, bladder cancer, leukemia, lymphoma, T cell lymphoma, multiple myeloma, stomach cancer, pancreatic cancer, cervical cancer, uterus Endometrial cancer, ovarian cancer, esophageal cancer, liver cancer, squamous cell carcinoma of the head and neck, skin cancer, urinary tract cancer, prostate cancer, choriocarcinoma, pharyngeal cancer, laryngeal cancer, tongue cancer, oral cancer, gallbladder cancer, thyroid cancer, Mesothelioma, pleurioma, male embryo, endometrial hyperplasia, endometriosis, embryonal, fibrosarcoma, Kaposi's sarcoma, hemangioma, cavernous hemangioma, hemangioblastoma, retinoblastoma, astrocytes Group consisting of tumor, neurofibroma, oligodendroma, medulloblastoma, neuroblastoma, glioma, rhabdomyosarcoma, glioblastoma, osteogenic sarcoma, leiomyosarcoma, thyroid sarcoma, and Wilms tumor The medicament according to claim 25 or claim 30, which is at least one selected from the group consisting of:
  32. 　上記腫瘍が、大腸癌、結腸直腸癌、肺癌、乳癌、脳腫瘍、膀胱癌、リンパ腫、胃癌、膵臓癌、肝臓癌、または前立腺癌である、請求項２５または請求項３０に記載の医薬。 The medicament according to claim 25 or 30, wherein the tumor is colon cancer, colorectal cancer, lung cancer, breast cancer, brain tumor, bladder cancer, lymphoma, gastric cancer, pancreatic cancer, liver cancer, or prostate cancer.
  33. 　薬学的に許容し得る細胞増殖抑制剤と同時、個別、又は逐次投与するための、請求項３０~３２のいずれか一項に記載の医薬。 The medicament according to any one of claims 30 to 32, for simultaneous, separate or sequential administration with a pharmaceutically acceptable cell growth inhibitor.
  34. 　上記細胞増殖抑制剤が、アルキル化剤、代謝拮抗剤、分子標的薬、植物アルカロイド、抗癌性抗生物質、およびプラチナ製剤からなる群から選択される少なくとも１つである、請求項３３に記載の医薬。 34. The cytostatic agent according to claim 33, wherein the cytostatic agent is at least one selected from the group consisting of an alkylating agent, an antimetabolite, a molecular target drug, a plant alkaloid, an anticancer antibiotic, and a platinum preparation. Medicine.
  35. 　上記アルキル化剤が、イホスファミド、シクロフォスファミド、ダカルバシン、テモゾロミド、ニムスチン、ブスルファン、プロカルバジン、メルファラン、およびラニムスチンからなる群から選択され、
    　上記代謝拮抗剤が、エノシタビン、カペシタビン、カルモフール、クラドリピン、ゲムシタビン、シタラビン、シタラビンオクホスファート、テガフール、テガフールウラシル、ＴＳ−１、ドキシフルリジン、ネララビン、ヒドロキシカルバミド、フルオロウラシル、フルダラビン、ペメトレキセド、ペントスタチン、メルカプトプリン、およびメトトレキサートからなる群から選択され、
    　上記分子標的薬が、ゼヴァリン、イマチニブ、エルロチニブ、ゲフィチニブ、スニチニブ、セツキシマブ、ソラフェニブ、ダサチニブ、トラスツズマブ、トレチノイン、パニツムマブ、ベバシズマブ、ボルテゾミブ、およびリツキシマブからなる群から選択され、
    　植物アルカロイドが、イリノテカン、エトポシド、ソブゾキサン、ドセタキセル、ノギテカン、パクリタキセル、ビノレルビン、ビンクリスチン、ビンデシン、およびビンブラスチンからなる群から選択され、
    　上記抗癌性抗生物質が、アクチノマイシンＤ、アクラルビシン、アムルビシン、イダルビシン、エピルビシン、スマンクス、ダウノルビシン、ドキソルビシン、ピラルビシン、ブレオマイシン、ペプロマイシン、マイトマイシンＣ、ミトキサントロン、およびドキシルからなる群から選択され、
    　上記プラチナ製剤が、オキサリプラチン、シスプラチン、カルボプラチン、およびネダプラチンからなる群から選択される少なくとも１つである、請求項３３に記載の医薬。     The alkylating agent is selected from the group consisting of ifosfamide, cyclophosphamide, dacarbacin, temozolomide, nimustine, busulfan, procarbazine, melphalan, and ranimustine;
    The antimetabolite is enocitabine, capecitabine, carmofur, cladripine, gemcitabine, cytarabine, cytarabine ocphosate, tegafur, tegafur uracil, TS-1, doxyfluridine, nelarabine, hydroxycarbamide, fluorouracil, fludarabine, pemetrexed captostatin, pentostatin, pentostatin And selected from the group consisting of methotrexate,
    The molecular targeted drug is selected from the group consisting of Zevalin, Imatinib, Erlotinib, Gefitinib, Sunitinib, Cetuximab, Sorafenib, Dasatinib, Trastuzumab, Tretinoin, Panitumumab, Bevacizumab, Bortezomib, and Rituximab
    The plant alkaloid is selected from the group consisting of irinotecan, etoposide, sobuzoxan, docetaxel, nogitecan, paclitaxel, vinorelbine, vincristine, vindesine, and vinblastine;
    The anticancer antibiotic is selected from the group consisting of actinomycin D, aclarubicin, amrubicin, idarubicin, epirubicin, smux, daunorubicin, doxorubicin, pirarubicin, bleomycin, peomycin, mitomycin C, mitoxantrone, and doxil;
    The medicament according to claim 33, wherein the platinum preparation is at least one selected from the group consisting of oxaliplatin, cisplatin, carboplatin, and nedaplatin.
  36. 　以下の（１）~（６）からなる群から選択される相補性決定領域（ＣＤＲ）：
    　（１）配列番号１１、１７、２３、２９、３５のアミノ酸配列を含むＣＤＲＬ１、
    　（２）配列番号１２、１８、２４、３０、３６のアミノ酸配列を含むＣＤＲＬ２、
    　（３）配列番号１３、１９、２５、３１、３７のアミノ酸配列を含むＣＤＲＬ３、
    　（４）配列番号１４、２０、２６、３２、３８のアミノ酸配列を含むＣＤＲＨ１、
    　（５）配列番号１５、２１、２７、３３、３９のアミノ酸配列を含むＣＤＲＨ２、および
    　（６）配列番号１６、２２、２８、３４、４０のアミノ酸配列を含むＣＤＲＨ３。     Complementarity determining region (CDR) selected from the group consisting of the following (1) to (6):
    (1) CDRL1, comprising the amino acid sequences of SEQ ID NOs: 11, 17, 23, 29, and 35,
    (2) CDRL2, comprising the amino acid sequences of SEQ ID NOs: 12, 18, 24, 30, 36
    (3) CDRL3 comprising the amino acid sequences of SEQ ID NOs: 13, 19, 25, 31, 37
    (4) CDRH1, comprising the amino acid sequences of SEQ ID NOs: 14, 20, 26, 32, 38,
    (5) CDRH2 comprising the amino acid sequences of SEQ ID NOs: 15, 21, 27, 33, 39, and (6) CDRH3 comprising the amino acid sequences of SEQ ID NOs: 16, 22, 28, 34, 40.
PCT/JP2014/081674 2013-11-25 2014-11-25 New monoclonal antibody WO2015076425A1 (en)

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JP2021519608A (en) * 2018-02-22 2021-08-12 マルティチュード・インコーポレーテッド Therapeutic antibodies and their use
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Publication number Priority date Publication date Assignee Title
WO2017215638A1 (en) * 2016-06-15 2017-12-21 李翀 Marker and antibody of human endometrial cancer and use thereof
WO2018181878A1 (en) * 2017-03-30 2018-10-04 株式会社キャンサーステムテック Cancer stem cell
JPWO2018181878A1 (en) * 2017-03-30 2020-04-09 株式会社キャンサーステムテック Cancer stem cells
JP2021519608A (en) * 2018-02-22 2021-08-12 マルティチュード・インコーポレーテッド Therapeutic antibodies and their use
EP3755717A4 (en) * 2018-02-22 2022-01-26 Multitude Inc. Therapeutic antibody and uses thereof
WO2024040194A1 (en) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditioning for in vivo immune cell engineering
WO2024040195A1 (en) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditioning for in vivo immune cell engineering

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