WO2015076425A1 - Nouvel anticorps monoclonal - Google Patents

Nouvel anticorps monoclonal Download PDF

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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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seq
amino acid
antibody
acid sequence
antigen
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PCT/JP2014/081674
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Japanese (ja)
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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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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

L'invention concerne un médicament destiné à la prévention ou au traitement de différentes tumeurs malignes jusque là incurables, telles que des tumeurs solides, lequel médicament comprenant comme principe actif de nouveaux anticorps qui sont aptes à se lier au CD44v9 humain et qui, plus précisément, induisent dans les cellules cancéreuses une cytotoxicité cellulaire fonction de l'anticorps. L'invention concerne également un anticorps humain CD44v9 et un médicament contenant cet anticorps et destiné à la prévention ou au traitement des tumeurs.
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WO2018181878A1 (fr) * 2017-03-30 2018-10-04 株式会社キャンサーステムテック Cellules souches cancéreuses
JPWO2018181878A1 (ja) * 2017-03-30 2020-04-09 株式会社キャンサーステムテック 癌幹細胞
JP2021519608A (ja) * 2018-02-22 2021-08-12 マルティチュード・インコーポレーテッド 治療抗体およびその使用
EP3755717A4 (fr) * 2018-02-22 2022-01-26 Multitude Inc. Anticorps thérapeutique et utilisations associées
WO2024040194A1 (fr) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditionnement pour l'ingénierie de cellules immunitaires in vivo
WO2024040195A1 (fr) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditionnement pour l'ingénierie de cellules immunitaires in vivo

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