WO2023219147A1 - Novel anti-ccr8 antibodies for detecting ccr8 - Google Patents

Novel anti-ccr8 antibodies for detecting ccr8 Download PDF

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WO2023219147A1
WO2023219147A1 PCT/JP2023/017821 JP2023017821W WO2023219147A1 WO 2023219147 A1 WO2023219147 A1 WO 2023219147A1 JP 2023017821 W JP2023017821 W JP 2023017821W WO 2023219147 A1 WO2023219147 A1 WO 2023219147A1
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antibody
ccr8
seq
amino acid
acid sequence
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PCT/JP2023/017821
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French (fr)
Japanese (ja)
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哲也 吉田
舞 吉川
盛男 柳樂
道也 平田
里美 東雲
あずみ 上山
礼美 松本
秀和 田中
永也 大倉
志文 坂口
尚 和田
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塩野義製薬株式会社
国立大学法人大阪大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention relates to a novel anti-CCR8 antibody useful for detecting CCR8.
  • Non-Patent Document 1 Powerful negative regulatory mechanisms, including immunosuppression mediated by regulatory T cells (Treg cells) within the tumor microenvironment, are a major obstacle to tumor treatment.
  • CD4-positive Treg cells infiltrating tumors can strongly inhibit anti-tumor immune responses and can be a major obstacle to effective cancer treatment.
  • Tumor immunosuppression mediated by CD4-positive FoxP3-positive Treg cells has been well documented in animal tumor models, and systemic Treg cell removal, including within the tumor, results in an antitumor effect; It has been reported that no effect is observed in removing intratumoral infiltrating Treg cells (Non-Patent Document 2).
  • Non-patent Documents 3 to 8 In humans, an increased ratio of CD4+CD25+ Treg cells (cell population containing Treg cells) in the total CD4+ T cell population has been detected in tumors of various cancer patients, including lung, breast, and ovarian tumors. It has been reported that there is a negative correlation between the abundance ratio and patient survival rate (Non-patent Documents 3 to 8).
  • CCR8 also called CY6, CKR-L1, or TER1 is a G protein-coupled seven-transmembrane CC chemokine receptor protein expressed in the thymus and spleen, and its gene is located on 3p21 in human chromosomes. exist.
  • Human CCR8 consists of 355 amino acids (Non-Patent Document 9).
  • CCL1 is known as an endogenous ligand for CCR8 (Non-Patent Document 10).
  • Human CCR8 cDNA is Genbank ACC No.
  • the mouse CCR8 cDNA is composed of the base sequence shown in NM_005201, and the mouse CCR8 cDNA is shown in Genbank ACC No. It is composed of the base sequence shown in NM_007720.
  • CCR8 is also specifically expressed in tumor-infiltrating Treg cells, and when breast cancer cells were transplanted into CCR8-deficient mice and wild-type mice, the growth and metastasis of breast cancer in CCR8-deficient mice was significantly higher than in wild-type mice. It has been shown that this was suppressed (Patent Document 1 and Non-Patent Document 11).
  • Non-Patent Documents 12 and 13 also describe that CCR8 is involved in the pathology of cancer. Furthermore, it has been disclosed that administration of anti-CCR8 antibodies to cancer model animals showed antitumor effects (Patent Documents 2 to 12).
  • Patent Documents 1, 13, and 14 describe that CCR8 is useful as a cancer diagnostic marker, but do not specifically describe anti-CCR8 antibodies useful for CCR8 detection. Further, Patent Document 15 discloses anti-CCR8 antibodies useful for CCR8 detection, but all antibodies have neutralizing activity, and anti-CCR8 antibodies that do not have neutralizing activity useful for CCR8 detection Antibodies are not specifically listed. Furthermore, Patent Document 15 does not describe that anti-CCR8 antibodies are useful for cancer diagnosis.
  • An object of the present invention is to provide an anti-CCR8 antibody useful for detecting CCR8. Another object of the present invention is to provide a diagnostic agent containing the anti-CCR8 antibody.
  • the present inventors discovered a novel anti-CCR8 antibody that specifically binds to CCR8 and does not have neutralizing activity useful for CCR8 detection. Furthermore, it has been found that the novel CCR8 antibody of the present invention does not compete with anti-CCR8 antibodies having neutralizing activity in binding to CCR8. Furthermore, it has been found that the novel CCR8 antibody of the present invention detects CCR8 expressed in tumor-infiltrating Treg cells and is useful for cancer diagnosis and companion diagnosis.
  • the present invention relates to the following.
  • CDR1 consisting of the amino acid sequence of SEQ ID NO: 2
  • a light chain variable region comprising CDR2 consisting of the amino acid sequence of SEQ ID NO: 3 and CDR3 consisting of the amino acid sequence of SEQ ID NO: 4
  • CDR1 consisting of the amino acid sequence of SEQ ID NO: 5
  • the antibody or antibody fragment thereof according to (1) which comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 8 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 9.
  • the antibody or antibody fragment thereof according to (1) further having a light chain constant region having the amino acid sequence of SEQ ID NO: 10 and a heavy chain constant region having the amino acid sequence of SEQ ID NO: 11.
  • the method described in (4) for cancer diagnosis. (5-2) The method described in (4) for assisting cancer diagnosis.
  • (5-3) The method described in (4) for detecting intratumoral infiltrating Treg cells.
  • the method described in (4) for detecting CCR8-expressing tumor cells The method described in (4) for detecting CCR8-expressing tumor cells. (6) The method according to (4), wherein CCR8 contained in a sample collected from a subject to whom a medicine containing an anti-CCR8 antibody has been administered is detected. (7) The method according to (6), wherein the anti-CCR8 antibody contained in the anti-CCR8 antibody-containing medicine has neutralizing activity. (8) The anti-CCR8 antibody contained in the medicine containing the anti-CCR8 antibody contains an antibody comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 8 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 9. The method according to (6), wherein there is no conflict in binding to.
  • the anti-CCR8 antibody contained in the medicine containing an anti-CCR8 antibody is a monoclonal antibody comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 12 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13, or The method according to (6), which is a fragment thereof.
  • the method according to (6), wherein the subject is a cancer patient.
  • a CCR8 detection kit comprising a composition containing the antibody or antibody fragment thereof according to any one of (1) to (3).
  • (12-2) The kit according to (11) for assisting cancer diagnosis.
  • (12-3) The kit described in (11) for detecting intratumoral infiltrating Treg cells.
  • the kit described in (11) for detecting CCR8-expressing tumor cells (13) The kit according to (11), which detects CCR8 contained in a sample collected from a subject who has been administered a medicine containing an anti-CCR8 antibody. (14) The kit according to (13), wherein the anti-CCR8 antibody contained in the anti-CCR8 antibody-containing medicine has neutralizing activity. (15) The anti-CCR8 antibody contained in the medicine containing the anti-CCR8 antibody contains an antibody comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 8 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 9. The kit according to (13), which does not compete in binding to.
  • the anti-CCR8 antibody contained in the anti-CCR8 antibody-containing medicine is a monoclonal antibody comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 12 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13;
  • (18-3) Use of the antibody or antibody fragment thereof according to any one of (1) to (3) for producing a cancer diagnostic agent.
  • (19-3) Use of the antibody or antibody fragment thereof according to any one of (1) to (3) for producing a diagnostic agent for determining the suitability of administration of a medicine containing an anti-CCR8 antibody.
  • the anti-CCR8 antibody contained in the anti-CCR8 antibody-containing medicine is a monoclonal antibody comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 12 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13, or The diagnostic agent according to (19), which is a fragment thereof.
  • the anti-CCR8 antibody contained in the anti-CCR8 antibody-containing medicine is a monoclonal antibody comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 12 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13; The method according to (22), which is a fragment thereof.
  • the anti-CCR8 antibody contained in the anti-CCR8 antibody-containing medicine is used to infiltrate into tumor-infiltrating Treg cells. How to measure the amount of binding.
  • An expression vector comprising the polynucleotide described in (26).
  • novel anti-CCR8 antibody of the present invention specifically binds to CCR8, it can be used to detect CCR8 in biological samples. Furthermore, the novel anti-CCR8 antibody of the present invention detects CCR8 expressed in tumor-infiltrating Treg cells, and is therefore very useful for cancer diagnosis and companion diagnosis.
  • the competitive inhibitory activity of rat hybridoma supernatant against L263G8 antibody was evaluated by flow cytometry. If the antibody has competitive inhibitory activity against the L263G8 antibody, a peak shift will be observed compared to the negative control (Cont).
  • the X axis shows the detected fluorescence intensity, and the Y axis shows the number of detected cells.
  • the neutralizing activity of the rat hybridoma supernatant against the binding between CCL1 and CCR8 was evaluated by measuring intracellular calcium influx activity. When the antibody exhibits neutralizing activity, intracellular calcium influx activity is suppressed, and therefore the increase in fluorescence level over time is also suppressed.
  • the binding of the 3-3F antibody, L263G8 antibody, and 433H antibody to human CCR8-expressing Ramos cells was evaluated by flow cytometry.
  • the X axis shows the detected fluorescence intensity, and the Y axis shows the number of detected cells.
  • the X axis shows the detected fluorescence intensity, and the Y axis shows the number of detected cells.
  • the binding of the 3-3F antibody to cells expressing various chemokine receptors was evaluated by flow cytometry.
  • the X axis shows the detected fluorescence intensity
  • the Y axis shows the number of detected cells.
  • “3-3F” shows the peak of the 3-3F antibody
  • “Cont” shows the peak of the negative control.
  • the binding of the 3-3F antibody to intratumoral infiltrating Treg cells was evaluated by flow cytometry.
  • the X axis shows the detected fluorescence intensity
  • the Y axis shows the number of detected cells.
  • the binding of 3-3F antibody, fluorescently labeled 3-3F antibody, and L263G8 antibody to intratumoral infiltrating Treg cells was evaluated by flow cytometry.
  • the X axis shows the fluorescence intensity detected by FoxP3
  • the Y axis shows the fluorescence intensity detected by CCR8 by various anti-CCR8 antibodies.
  • the correlation between 3-3F antibody signal (Y axis) and CCR8 mRNA signal (X axis) is shown by flow cytometry analysis of intratumoral infiltrating Treg cells. Showing the correlation between the binding signal (X axis) to Treg cells of a neutralizing antibody (OP-1 antibody) against human CCR8 (OP-1 antibody) administered to human CCR8 knock-in mice and the 3-3F antibody signal (Y axis) in intratumoral infiltrating Treg cells. .
  • r indicates a correlation coefficient, and a straight line indicates linear regression.
  • antibody production techniques known in the art can be used. Examples include the method described in Immunochemistry in Practice (Blackwell Scientific Publications). Additionally, genetic engineering techniques known in the art are available. Examples include the methods described in Molecular Cloning, A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press (2012), Current Protocols Essential Laboratory Techniques, Current Protocols (2012).
  • the amino acid sequence of human CCR8 is shown in UniProtKB/Swiss-Prot:P51685 (SEQ ID NO: 1).
  • the extramembrane domain of human CCR8 corresponds to the N-terminal region consisting of amino acids 1 to 35, the loop 1 region consisting of amino acids 94 to 107, the loop 2 region consisting of amino acids 172 to 202, and the loop 3 region consisting of amino acids 264 to 280. do.
  • Position 17 in the amino acid sequence of human CCR8 is tyrosine, and position 17 is deleted in the amino acid sequence of mouse CCR8.
  • a hybridoma producing the anti-CCR8 antibody of the present invention can be produced using human CCR8 protein, a gene encoding the full-length human CCR8, human CCR8-expressing cells, etc. as an immunogen.
  • a hybridoma producing an anti-CCR8 antibody can be obtained by fusing mouse myeloma cells with spleen cells of a mouse immunized with DNA using the gene as an antigen. I can do it.
  • Examples of the basic medium for culturing hybridomas include D-MEM medium, RPMI1640 medium, IMDM medium, and ASF104 medium. Alternatively, various inorganic or organic substances can be contained.
  • the monoclonal antibody or antibody fragment thereof of the present invention includes a monoclonal antibody or a fragment of the antibody having the CDR or heavy chain variable region/light chain variable region described herein.
  • the antibody or antibody fragment may be derived from any class (e.g. IgG, IgE, IgM, IgD or IgA, preferably IgG) or subclass of immunoglobulin molecules, e.g. mouse, rat, shark, rabbit, It may be obtained from any species including pigs, hamsters, camels, llamas, goats or humans.
  • the antibody or antibody fragment is preferably a humanized monoclonal antibody or an antibody fragment of a humanized monoclonal antibody.
  • epitope refers to the region of an antigen that is bound by an antibody targeting that antigen, and when the antigen is a protein, includes specific amino acids that are in direct contact with the antibody.
  • the present invention includes not only monoclonal antibodies or antibody fragments thereof that recognize the completely same epitope as the monoclonal antibody or antibody fragment thereof of the present invention, but also monoclonal antibodies or antibody fragments thereof that recognize partially the same epitope. .
  • the CDR sequence of the monoclonal antibody of the present invention preferably has the following sequence.
  • Light chain CDR1 SEQ ID NO: 2.
  • Light chain CDR2 SEQ ID NO: 3.
  • Light chain CDR3 SEQ ID NO: 4.
  • Heavy chain CDR1 SEQ ID NO: 5.
  • Heavy chain CDR2 SEQ ID NO: 6.
  • Heavy chain CDR3 SEQ ID NO: 7.
  • antibody fragment of a monoclonal antibody refers to a fragment that is part of the monoclonal antibody of the present invention and that specifically binds to human CCR8 and selectively inhibits human CCR8 like the monoclonal antibody. means.
  • the "anti-CCR8 antibody” of the present invention includes “antibody fragment of a monoclonal antibody.”
  • Fab fragment of antigen binding
  • Fab' fragment of antigen binding
  • F(ab') 2 single chain antibody
  • scFv single chain Fv
  • Examples include disulfide stabilized antibodies (disulfide stabilized Fv; hereinafter referred to as dsFv), dimerized V region fragments (hereinafter referred to as diabody), peptides containing CDRs, etc. Pytic Patents, Vol. 6, No. 5, pp. 441-456, 1996).
  • Fab is obtained by decomposing the peptide part above the two disulfide bonds (SS bonds) that bridge the two H chains in the hinge region of IgG with the enzyme papain. It is an antibody fragment with a molecular weight of approximately 50,000 and antigen-binding activity, consisting of one half and the entire L chain.
  • Fab used in the present invention can be obtained by treating the monoclonal antibody of the present invention with papain.
  • Fab can also be produced by inserting DNA encoding the Fab of the monoclonal antibody of the present invention into a cellular expression vector, and expressing the vector by introducing the vector into cells.
  • Fab' is an antibody fragment with a molecular weight of approximately 50,000 and having antigen-binding activity obtained by cleaving the SS bond between the hinges of F(ab') 2 .
  • Fab' used in the present invention can be obtained by treating F(ab') 2 of the monoclonal antibody of the present invention with a reducing agent dithiothreitol.
  • Fab' can also be produced by inserting the DNA encoding Fab' of the monoclonal antibody of the present invention into a cellular expression vector, and expressing the vector by introducing it into E. coli, yeast, or animal cells. .
  • F(ab') 2 is obtained by decomposing the lower part of the two SS bonds in the hinge region of IgG with the enzyme pepsin, and is composed of two Fab' regions linked at the hinge region, and has a molecular weight of approximately It is an antibody fragment with an antigen binding activity of 100,000.
  • F(ab') 2 used in the present invention can be obtained by treating the monoclonal antibody of the present invention with pepsin.
  • F(ab') 2 of the monoclonal antibody of the present invention can also be expressed by inserting the DNA encoding it into an expression vector for cells and introducing the vector into E. coli, yeast, or animal cells. ) 2 can be manufactured.
  • scFv is a VH-P-VL or VL-P-VH polypeptide in which one VH and one VL are linked using an appropriate peptide linker (hereinafter referred to as P), and is capable of antigen binding. It is an active antibody fragment.
  • the VH and VL contained in the scFv used in the present invention may be those of the monoclonal antibody of the present invention.
  • the scFv used in the present invention is produced by constructing an scFv expression vector using cDNA encoding the VH and VL of the monoclonal antibody of the present invention, and expressing it by introducing it into E. coli, yeast, or animal cells. be able to.
  • dsFv refers to a polypeptide in which one amino acid residue in each of VH and VL is replaced with a cysteine residue, which are linked via an SS bond.
  • Amino acid residues to be substituted for cysteine residues can be selected based on the prediction of the three-dimensional structure of the antibody according to the method described by Reiter et al. (Protein Engineering, 7, 697, (1994)).
  • the VH or VL contained in the dsFv used in the present invention may be one of the monoclonal antibody of the present invention.
  • the dsFv used in the present invention is constructed by inserting the cDNA encoding the VH and VL of the monoclonal antibody of the present invention into an appropriate expression vector, and then transducing the expression vector into Escherichia coli, yeast, or an animal. It can be produced by introducing it into cells and expressing it.
  • Diabody is an antibody fragment in which scFv with the same or different antigen-binding specificity forms a dimer, and has bivalent antigen-binding activity for the same antigen or two types of specific antigen-binding activity for different antigens. It is.
  • a bivalent diabody that specifically reacts with the monoclonal antibody of the present invention encodes an scFv having a peptide linker of 3 to 10 residues using cDNA encoding the VH and VL of the monoclonal antibody of the present invention. It can be produced by constructing DNA, inserting the DNA into a cell expression vector, and introducing the expression vector into E. coli, yeast, or animal cells to express the diabody.
  • a peptide containing a CDR is composed of at least one region of a VH or VL CDR. Multiple CDRs can be linked directly or via a suitable peptide linker.
  • the CDR-containing peptide used in the present invention is obtained by constructing a CDR-encoding DNA using cDNA encoding the VH and VL of the monoclonal antibody of the present invention, and inserting the DNA into an expression vector for animal cells. It can be produced by introducing the vector into E. coli, yeast, or animal cells and expressing it.
  • Peptides containing CDRs can also be produced by chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method).
  • the monoclonal antibody of the present invention or its antibody fragment is characterized by binding to human CCR8. In particular, those that bind specifically are preferred.
  • Specific binding can be characterized by an equilibrium dissociation constant of at least about 1 ⁇ 10 ⁇ 6 M or less (eg, a lower Kd indicates tighter binding).
  • the Kd value is preferably 1 ⁇ 10 ⁇ 7 M or less, more preferably 1 ⁇ 10 ⁇ 8 M or less, and still more preferably 1 ⁇ 10 ⁇ 9 M or less.
  • the invention includes monoclonal antibodies or antibody fragments thereof that compete with the monoclonal antibodies or antibody fragments thereof described herein for binding to human CCR8.
  • “Competing monoclonal antibody or antibody fragment thereof” refers to the monoclonal antibody of the present invention or antibody fragment thereof (preferably a light chain variable region having the amino acid sequence of SEQ ID NO: 12 and a heavy chain having the amino acid sequence of SEQ ID NO: 13). It refers to an antibody or an antibody fragment thereof that inhibits the specific binding of a monoclonal antibody containing a variable region (or a fragment thereof) to human CCR8. It is determined whether a monoclonal antibody or antibody fragment thereof competes with a monoclonal antibody or antibody fragment thereof of the invention.
  • the monoclonal antibody of the present invention Among the antibodies that were confirmed to bind to the antigen (human CCR8) in the presence of the isotype control antibody, those antibodies whose binding signal was significantly reduced in the presence of the monoclonal antibody of the present invention or its antibody fragment were selected as the monoclonal antibody of the present invention. It can be identified as an antibody that competes with the antibody or antibody fragment thereof. The reduction in binding signal is preferably 50%, more preferably 70%. Further, the Ki value of an antibody that competes with the monoclonal antibody of the present invention or its antibody fragment for binding of the monoclonal antibody of the present invention or its antibody fragment to an antigen is preferably 1 ⁇ 10 ⁇ 7 M or less, more preferably It is 1 ⁇ 10 ⁇ 8 M or less, more preferably 1 ⁇ 10 ⁇ 9 M or less.
  • the monoclonal antibody of the present invention or its antibody fragment is characterized in that it hardly inhibits the binding between CCR8 and CCL1, that is, it has almost no neutralizing activity.
  • the ability to inhibit the binding between CCR8 and CCL1 for example, in the case of human CCL1, using human CCR8-expressing 293 cells, Ca 2+ influx upon addition of human CCL1 was measured, and the signal when human CCL1 was not added was determined to be 100% inhibition rate, human The IC50 value can be determined by calculating the IC50 value, assuming that the signal obtained when CCL1 is added and when the antibody is not added is regarded as an inhibition rate of 0%. For example, it can be measured by the method described in Example 3 below. Human CCL1 was obtained from UniProtKB/Swiss-Prot No. It has an amino acid sequence such as P22362.
  • the monoclonal antibody of the present invention can be produced by a conventional method in the art using the CDRs or heavy chain variable region/light chain variable region described herein.
  • Monoclonal antibodies of the invention also include chimeric, humanized, fully human, antibody small molecule conjugates (ADCs) and bispecific antibodies. Since humanized monoclonal antibodies have reduced antigenicity in the human body, they are useful when administered to humans for therapeutic purposes.
  • a humanized monoclonal antibody is one in which the complementarity determining regions (CDRs) of a non-human mammal, such as a mouse antibody, are grafted onto the framework regions (FRs) of a human antibody. Therefore, the FRs of the humanized monoclonal antibody are of human origin.
  • a suitable FR is Kabat E. A. You can make a selection by referring to the literature by et al.
  • FRs are selected that allow CDRs to form a good antigen-binding site.
  • amino acids in the FRs of the antibody variable region may be substituted so that the CDRs of the reconstituted humanized monoclonal antibody form a suitable antigen-binding site (Sato. et al, Cancer Res. (1993) ), 53, 851).
  • the proportion of FR amino acids to be replaced is 0-15%, preferably 0-5% of the total FR region.
  • the humanized monoclonal antibody of the present invention preferably comprises: CDR1 consisting of the amino acid sequence of SEQ ID NO: 2, A light chain variable region comprising CDR2 consisting of the amino acid sequence of SEQ ID NO: 3 and CDR3 consisting of the amino acid sequence of SEQ ID NO: 4, and CDR1 consisting of the amino acid sequence of SEQ ID NO: 5, It contains a heavy chain variable region including CDR2 consisting of the amino acid sequence of SEQ ID NO: 6 and CDR3 consisting of the amino acid sequence of SEQ ID NO: 7. More preferably, It includes a light chain variable region having the amino acid sequence of SEQ ID NO: 8 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 9.
  • sequence of the constant region of the monoclonal antibody of the present invention is not particularly limited, but examples include a light chain constant region having the amino acid sequence of SEQ ID NO: 10 and a heavy chain constant region having the amino acid sequence of SEQ ID NO: 11.
  • the constant region of a human antibody is used.
  • Preferred constant regions of human antibodies include C ⁇ as a heavy chain, such as C ⁇ 1, C ⁇ 2, C ⁇ 3, and C ⁇ 4, and C ⁇ and C ⁇ as light chains. Additionally, the C region of human antibodies may be modified to improve the stability of the antibody or its production.
  • the human antibody used in humanization may be of any isotype such as IgG, IgM, IgA, IgE, IgD, etc., but it is preferable to use IgG in the present invention.
  • the monoclonal antibody of the present invention can be produced by a general production method (for example, see WO95/14041, WO96/02576, etc.).
  • the antibody gene is inserted into an expression vector so that it is expressed under the control of an expression control region, such as an enhancer/promoter.
  • a host cell can then be transformed with this expression vector to express the antibody.
  • host cells for the above transformants include vertebrate cells such as COS cells and CHO cells, prokaryotic cells, and yeast.
  • the transformant can be cultured according to methods well known to those skilled in the art, and the monoclonal antibody of the present invention is produced within or outside the transformant cells.
  • the culture medium used for the culture can be appropriately selected from various commonly used media depending on the host cell employed. For example, in the case of COS cells, RPMI-1640 medium, Dulbecco's modified Eagle's minimum essential medium (DMEM), etc.
  • DMEM Dulbecco's modified Eagle's minimum essential medium
  • a culture medium to which a serum component such as fetal bovine serum (FBS) is added, if necessary, can be used.
  • FBS fetal bovine serum
  • the culture temperature for culturing the transformant may be any temperature as long as it does not significantly reduce the intracellular protein synthesis ability, but it is preferably cultured at 32 to 42°C, most preferably at 37°C. It is preferable. Furthermore, if necessary, the culture can be carried out in air containing 1 to 10% (v/v) carbon dioxide gas.
  • the fraction containing the monoclonal antibody of the present invention produced intracellularly or extracellularly in the transformant as described above can be separated and separated by various known separation methods that utilize the physical and chemical properties of the protein.
  • Can be purified include, for example, treatment with a normal protein precipitant, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC). ), various chromatography methods, dialysis methods, and combinations thereof can be employed.
  • a normal protein precipitant ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • the monoclonal antibody of the present invention or its antibody fragment may be further modified with various molecules such as polyethylene glycol (PEG), radioactive substances, and toxins. Methods known in this field can be used to modify antibodies.
  • PEG polyethylene glycol
  • the monoclonal antibody of the present invention may also be fused with other proteins to its N-terminus or C-terminus (Clinical Cancer Research, 2004, 10, 1274-1281). Proteins to be fused can be appropriately selected by those skilled in the art.
  • the monoclonal antibodies of the present invention include antibodies in which N-glycoside-linked sugar chains are bound to the Fc region of the antibody. Note that fucose does not need to be bound to N-acetylglucosamine at the reducing end of the N-glycoside-linked sugar chain.
  • An example of an antibody in which an N-glycoside-linked sugar chain is bound to the Fc region of the antibody and fucose is not bound to N-acetylglucosamine at the reducing end of the N-glycoside-linked sugar chain is, for example, an ⁇ 1,6-fucosyltransferase gene. Examples include antibodies produced using CHO cells deficient in CHO cells (WO 2005/035586, WO 02/31140).
  • the antibody of the present invention in which an N-glycoside-linked sugar chain is bound to the Fc region of the antibody and fucose is not bound to N-acetylglucosamine at the reducing end of the N-glycoside-linked sugar chain, has high ADCC activity.
  • the monoclonal antibody or antibody fragment thereof of the present invention has ADCC (Antibody-dependent cell mediated cytotoxicity) activity against cells expressing CCR8 in order to remove Treg cells or macrophage cells. You may do so.
  • ADCC activity means that in vivo, an antibody that binds to a cell surface antigen such as a target cell activates the effector cell through the binding between the Fc region of the antibody and the Fc receptor present on the surface of the effector cell. Refers to activity that damages cells, etc. Effector cells include natural killer cells, activated macrophages, and the like.
  • an antibody bound to a cell surface antigen (CCR8) such as a Treg cell or a macrophage cell is activated through the binding between the Fc region of the antibody and an Fc receptor present on the surface of an effector cell. It also includes cases where effector cells are activated, Treg cells or macrophage cells are injured, and as a result, tumor cells and the like are injured.
  • CCR8 cell surface antigen
  • the monoclonal antibody of the present invention or its antibody fragment does not compete with CCR8-neutralizing antibodies for binding to CCR8.
  • a CCR8 neutralizing antibody means an antibody having neutralizing activity against CCR8. Whether or not it has neutralizing activity against CCR8 can be determined, for example, by measuring whether the physiological effect of any CCR8 ligand (eg, CCL1) on CCR8 is suppressed. Examples include, but are not limited to, measuring the binding of CCL1 to CCR8, the migration of CCR8-expressing cells by CCL1, an increase in intracellular Ca 2+ , or changes in the expression of genes sensitive to CCL1 stimulation. This can be mentioned. For example, it can be measured by the method described in Example 3 below.
  • the neutralizing activity of the "CCR8 neutralizing antibody” against the binding between CCR8 and CCL1 can be measured by adding a diluted antibody solution diluted with a medium to human CCR8-expressing 293 cells that have been pre-incorporated with a Ca 2+ indicator. can.
  • the affinity between CCR8 and CCR8 ligand is strongest for CCL1, and antibodies having high inhibitory activity against CCL1 are useful.
  • the "CCR8 neutralizing antibody” preferably has an IC50 value of neutralizing activity against the binding of CCR8 and CCL1 of 10 nM or less.
  • the neutralizing activity preferably has an IC50 value of 5 nM or less, further preferably 2 nM or less, particularly preferably 1 nM or less, and most preferably 0.5 nM or less.
  • the "CCR8 neutralizing antibody” used as the anti-CCR8 antibody contained in the anti-CCR8 antibody-containing medicine preferably has ADCC activity.
  • Examples of "neutralizing antibodies for CCR8” include antibodies described in Examples of Patent Documents 4 to 9 and 15, anti-CCR8 antibodies with product number L263G8 from BioLegend, and anti-CCR8 antibodies with product number 191704 from R&D.
  • an anti-CCR8 antibody that does not compete in binding to CCR8 with an antibody comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 8 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 9 is preferable; Most preferred is an anti-CCR8 antibody comprising a light chain variable region having the sequence and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13.
  • the monoclonal antibody or antibody fragment thereof of the present invention is preferably an antibody or antibody fragment thereof that strongly recognizes human CCR8.
  • an antibody or an antibody fragment thereof that strongly recognizes human CCR8 it is possible to select an antibody or an antibody fragment thereof based on the strength of binding activity and binding selectivity for CCR8 as an index, thereby creating an antibody that more strongly recognizes human CCR8. or an antibody fragment thereof can be selected.
  • the monoclonal antibody of the present invention or its antibody fragment does not compete with the neutralizing antibody for CCR8 in binding to CCR8, detection of CCR8 is possible even in a sample to which a neutralizing antibody for CCR8 has already bound. Furthermore, depending on the CCR8-neutralizing antibody bound to the sample, the CCR8-binding activity of the monoclonal antibody of the present invention or its antibody fragment may be increased.
  • the monoclonal antibody or antibody fragment thereof of the present invention can detect CCR8 contained in a sample collected from a subject, such as a cancer patient, who has been administered a medicine containing an anti-CCR8 antibody, and It is also possible to measure the amount of anti-CCR8 antibody contained in a medicament containing a CCR8 antibody that binds to intratumoral infiltrating Treg cells.
  • the anti-CCR8 antibody contained in the anti-CCR8 antibody-containing medicine the above-mentioned "CCR8 neutralizing antibody" is preferable, the anti-CCR8 antibodies described in the Examples of Patent Documents 4 to 9 are more preferable, and the anti-CCR8 antibodies described in the Examples of Patent Documents 4 to 9 are more preferable.
  • anti-CCR8 antibodies described in the Examples, which do not compete in binding to CCR8 with antibodies comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 8 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 9.
  • Anti-CCR8 antibodies are particularly preferred, and anti-CCR8 antibodies comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 12 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13 are most preferred.
  • the monoclonal antibody of the present invention or its antibody fragment specifically binds to CCR8, it can be used to detect CCR8 in samples collected from humans.
  • samples collected from humans include, but are not limited to, blood, plasma, serum, urine, organs, tissues, bone marrow, lymph nodes, and the like.
  • a labeled detection antibody When the monoclonal antibody of the present invention or an antibody fragment thereof is used as an antibody for detecting CCR8, a labeled detection antibody may be used when detecting CCR8, or a labeled secondary antibody for the detection antibody may be used. Good too.
  • substances used for labeling include fluorescent substances, microparticles, enzymes, biotin, radiolabels, magnetic particles, and the like, with fluorescent substances being preferred.
  • the fluorescent substance is not particularly limited as long as it does not impair the antigen binding property of the antibody or its antibody fragment, and includes, for example, rhodamine, coumarin, oxazine, carbopyronine, cyanine, pyromecene, naphthalene, biphenyl, Examples include anthracene, phenanthrene, pyrene, carbazole, Cy, EvoBlue, fluorescein, and derivatives thereof.
  • microparticles are not particularly limited as long as they do not impair the antigen binding properties of the antibody or antibody fragment thereof, and include, for example, metal colloid particles such as colloidal gold, colored particles such as colored latex particles, and the like.
  • the enzyme is not particularly limited as long as it does not impair the antigen binding property of the antibody or its antibody fragment, and examples include horseradish peroxidase (HRP), alkaline phosphatase, peroxidase, ⁇ -D-galactosidase, microperoxidase, and the like.
  • HRP horseradish peroxidase
  • alkaline phosphatase alkaline phosphatase
  • peroxidase peroxidase
  • ⁇ -D-galactosidase ⁇ -D-galactosidase
  • microperoxidase and the like.
  • radioactive labels examples include, but are not limited to, 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I, and the like.
  • a tag can be added to the antibody of the present invention or an antibody fragment thereof.
  • tags include FLAG, HA (hemagglutinin), GST (glutathione-S-transferase), Myc, polyhistidine (6 ⁇ His tag, etc.), and the like. These tags can be used for immobilization, purification, detection, etc. of antibodies.
  • a tagged antibody or antibody fragment is produced by adding a polynucleotide encoding a tag to a polynucleotide encoding the antibody of the present invention or an antibody fragment thereof, and then in vivo in animal cells or the like or in a cell-free manner. It can be produced by introducing it into an in vitro protein expression system such as a translation system.
  • the detection method of CCR8 of the present invention includes fluorescence immunoassay (FIA), enzyme immunoassay (ELISA or EIA), radioimmunoassay (RIA), chemiluminescence immunoassay, surface plasmon resonance, immunochromatography, etc.
  • FAA fluorescence immunoassay
  • ELISA enzyme immunoassay
  • RIA radioimmunoassay
  • chemiluminescence immunoassay chemiluminescence immunoassay
  • surface plasmon resonance immunochromatography
  • Flow cytometry, immunohistological staining, or ELISA is preferred, with flow cytometry being particularly preferred.
  • the method for detecting CCR8 of the present invention is a flow cytometry method, for example, it is a method of simultaneously measuring the expression of CCR8 and a marker specific to Treg cells, which is expressed in Treg cells contained in a sample such as intratumoral infiltrating cells. CCR8 can be detected. Furthermore, Treg cells expressing CCR8 can also be fractionated from a sample using a similar method. Examples of markers specific to Treg cells include FoxP3, CD25, and TIGIT, with FoxP3 being preferred.
  • the X axis is the detected fluorescence intensity of the Treg cell-specific marker
  • the Y axis is the detected fluorescence intensity of CCR8.
  • a plot showing Treg cells expressing Treg cells appears in the upper right area. Note that when plotting, the X-axis and Y-axis may be set in the opposite manner to the above.
  • the cell surface antigen to be gated before measuring the expression of markers specific to CCR8 and Treg cells can be selected as appropriate depending on the purpose of measurement. , for example, CD3, CD4, CD45, etc. may be selected.
  • the method for detecting CCR8 of the present invention is an immunohistological staining method
  • it can be carried out by known methods, including steps such as preparation of tissue sections such as paraffin sections, immunoreaction, washing, and color development.
  • the CCR8 detection method of the present invention is an ELISA method, it is carried out, for example, as follows. First, one antibody (capture antibody) is immobilized on the well surface of an ELISA plate. Next, after blocking is performed to prevent nonspecific adsorption to the well surface, a sample is added, and CCR8 in the sample is brought into contact with the antibody to form a complex. After removing proteins that did not bind to the antibody by washing, the other labeled antibody (detection antibody) is added to the well, brought into contact with CCR8 to form a complex, and detection and quantification are performed using the label. Quantification can be performed by a known method, such as quantifying a signal detected based on the label used using a calibration curve prepared using a standard sample of CCR8.
  • the monoclonal antibody of the present invention or its antibody fragment can be used as a diagnostic agent for cancer.
  • cancer for example, breast cancer, endometrial cancer, cervical cancer, ovarian cancer, prostate cancer, lung cancer, stomach cancer, pancreatic cancer, head and neck cancer, esophageal cancer, bladder cancer, melanoma, colorectal cancer, kidney cancer, non-Hodgkin lymphoma, urothelial cancer.
  • It can be used as a diagnostic agent for cancer, sarcoma, blood cell cancer (leukemia, lymphoma, etc.), cholangiocarcinoma, gallbladder cancer, thyroid cancer, testicular cancer, thymus cancer, liver cancer, etc., preferably breast cancer, ovarian cancer, lung cancer, etc.
  • gastric cancer pancreatic cancer, head and neck cancer, esophageal cancer, melanoma, colon cancer, renal cancer, and urothelial cancer.
  • cancer refers not only to epithelial malignant tumors such as ovarian cancer and gastric cancer, but also to non-epithelial malignant tumors including hematopoietic cancers such as chronic lymphocytic leukemia and Hodgkin's lymphoma.
  • cancer refers not only to epithelial malignant tumors such as ovarian cancer and gastric cancer, but also to non-epithelial malignant tumors including hematopoietic cancers such as chronic lymphocytic leukemia and Hodgkin's lymphoma.
  • cancer refers not only to epithelial malignant tumors such as ovarian cancer and gastric cancer, but also to non-epithelial malignant tumors including hematopoietic cancers such as chronic lymphocytic leukemia and Hodgkin's lymphoma.
  • cancer refers not only to epithelial malignant tumors such as ovarian cancer and gastric cancer, but also to non-epithelial mal
  • the monoclonal antibody of the present invention or its antibody fragment can also be used as a diagnostic agent to determine the suitability of administering a drug containing an anti-CCR8 antibody.
  • the anti-CCR8 antibody of the "medicine containing an anti-CCR8 antibody” the above-mentioned "CCR8 neutralizing antibody” is preferable, the anti-CCR8 antibodies described in the Examples of Patent Documents 4 to 9 are more preferable, and the anti-CCR8 antibodies described in the Examples of Patent Documents 4 to 9 are more preferable.
  • anti-CCR8 antibodies described in the Examples, which do not compete in binding to CCR8 with antibodies comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 8 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 9.
  • Anti-CCR8 antibodies are particularly preferred, and anti-CCR8 antibodies comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 12 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13 are most preferred.
  • “Pharmaceuticals containing anti-CCR8 antibodies” are useful as anticancer agents, such as breast cancer, endometrial cancer, cervical cancer, ovarian cancer, prostate cancer, lung cancer, stomach cancer, pancreatic cancer, head and neck cancer, esophageal cancer, and bladder cancer. , melanoma, colorectal cancer, kidney cancer, non-Hodgkin's lymphoma, urothelial cancer, sarcoma, blood cell cancer (leukemia, lymphoma, etc.), bile duct cancer, gallbladder cancer, thyroid cancer, testicular cancer, thymus cancer, liver cancer, etc.
  • it is very useful as a medicine for treating and/or preventing breast cancer, ovarian cancer, lung cancer, gastric cancer, pancreatic cancer, head and neck cancer, esophageal cancer, melanoma, colon cancer, kidney cancer, and urothelial cancer.
  • a kit containing the monoclonal antibody of the present invention can be used as a CCR8 detection kit.
  • the kit contains the monoclonal antibody of the present invention or an antibody fragment thereof, and further includes a gating antibody, a labeled secondary antibody, constituent reagents (diluting solution, washing solution, reaction stop solution, etc.), a fluorescence detection reagent, a CCR8 standard product, and usage. It may also include instructions, etc.
  • the pH buffer component contained in the constituent reagents is not particularly limited, but includes, for example, acids such as phosphoric acid, boric acid, acetic acid, citric acid, formic acid, and cacodylic acid, or salts thereof; amino acids such as glycine; trishydroxyaminomethane; (Tris), HEPES, MES, and other good buffers.
  • the surfactant contained in the constituent reagents is not particularly limited, but nonionic surfactants are preferred, and any of the ester ether type, ester type, and ether type can be used. More specifically, Tween20, Tween40, Tween80, Triton-X100, polyoxyethylene (60) sorbitan monostearate, polyoxyethylene (65) sorbitan tristearate, polyoxyethylene (80) sorbitan monooleate, polyoxy Examples include ethylene alkylphenyl ether, Nonidet P-40, CHAPS, and the like. These surfactants may be used alone or in combination of two or more.
  • the present invention includes polynucleotides encoding the light chain variable region or heavy chain variable region of the monoclonal antibody of the present invention.
  • the invention further includes expression vectors containing the polynucleotides.
  • the polynucleotide is not particularly limited as long as it encodes the light chain variable region or heavy chain variable region of the monoclonal antibody of the present invention, and may be a polymer consisting of multiple nucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). It is. It may contain non-natural bases.
  • the polynucleotide of the present invention can be used to produce antibodies by genetic engineering techniques. It can also be used as a probe to screen for antibodies having functions equivalent to the monoclonal antibodies of the present invention.
  • a polynucleotide encoding the monoclonal antibody of the present invention, or a portion thereof as a probe hybridize with the polynucleotide under stringent conditions by a technique such as hybridization or gene amplification technique (e.g. PCR), Moreover, DNA encoding an antibody having an activity equivalent to that of the monoclonal antibody of the present invention can be obtained. Such DNA is also included in the polynucleotide of the present invention.
  • Hybridization techniques (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58, Cold Spring Harbor Lab. press, 1989) are well known to those skilled in the art.
  • Examples of hybridization conditions include low stringency conditions. Low stringency conditions include, for example, 42°C, 0.1x SSC, 0.1% SDS in washing after hybridization, preferably 50°C, 0.1x SSC, 0.1%. This is a condition of SDS. More preferable hybridization conditions include highly stringent conditions. High stringency conditions are, for example, 65° C., 5 ⁇ SSC, and 0.1% SDS. Under these conditions, it can be expected that polynucleotides with higher homology can be obtained more efficiently as the temperature is raised. However, multiple factors such as temperature and salt concentration can be considered as factors that affect the stringency of hybridization, and those skilled in the art will be able to achieve similar stringency by appropriately selecting these factors. .
  • Antibodies that are functionally equivalent to the monoclonal antibodies of the present invention and encoded by polynucleotides obtained by these hybridization techniques or gene amplification techniques usually have high homology with these antibodies in their amino acid sequences.
  • the monoclonal antibodies of the present invention also include antibodies that are functionally equivalent to the monoclonal antibodies of the present invention and have high homology with the amino acid sequence of the antibodies.
  • High homology generally refers to at least 75% identity, preferably 85% or more identity, and more preferably 95% or more identity at the amino acid level.
  • the monoclonal antibody of the present invention or an antibody fragment thereof may have an effect of removing intratumoral infiltrating Treg cells or a tumor cell. Whether or not the monoclonal antibody of the present invention has an effect of removing tumor-infiltrating Treg cells can be determined, for example, by the method described in Examples of Patent Document 2.
  • the monoclonal antibody or antibody fragment thereof of the present invention is also useful in pharmaceutical compositions.
  • Pharmaceutical compositions containing the monoclonal antibodies of the present invention or antibody fragments thereof can be administered orally or parenterally, systemically or locally.
  • parenteral administration for example, intravenous injection such as drip, intramuscular injection, intraperitoneal injection, subcutaneous injection, intranasal administration, inhalation, etc. can be selected.
  • a pharmaceutical composition containing the monoclonal antibody or antibody fragment thereof of the present invention is very useful as a medicament for the treatment and/or prevention of CCR8-related diseases.
  • it is very useful as a medicament for treating and/or preventing cancer in which CCR8-expressing Treg cells infiltrate into tumors.
  • cancer for example, breast cancer, endometrial cancer, cervical cancer, ovarian cancer, prostate cancer, lung cancer, stomach cancer, pancreatic cancer, head and neck cancer, esophageal cancer, bladder cancer, melanoma, colorectal cancer, kidney cancer, non-Hodgkin lymphoma, urothelial cancer.
  • Cancer sarcoma, blood cell cancer (leukemia, lymphoma, etc.), bile duct cancer, gallbladder cancer, thyroid cancer, testicular cancer, thymus cancer, liver cancer, etc., preferably breast cancer, ovarian cancer, lung cancer, stomach cancer, pancreatic cancer, head and neck cancer. It is very useful as a medicine for treating and/or preventing cancer, esophageal cancer, melanoma, colon cancer, renal cancer, and urothelial cancer.
  • Example 1 Preparation of rat hybridoma producing anti-human CCR8 antibody (1) Preparation of hybridoma Human CCR8-expressing Rat-1 cells, which are the immunogen, were prepared using an expression vector in which the human CCR8 gene was cloned into pQCXIP (Clontech). The cells were transfected, followed by drug selection with puromycin (1 ⁇ g/ml) for one month. Rats were immunized once with human CCR8-expressing Rat-1 cells, and about two weeks later, lymph nodes were collected and hybridomas were produced by a conventional method.
  • DAPI 4,6-diamidino-2-phenylindole
  • the results are shown in Table 1. No. 30, No. 3.No. 12, No. The 37 hybridoma supernatants showed the strongest binding to human CCR8. In addition, binding to parent cells was No. 3 has the lowest coupling, and the S/N ratio (signal to noise ratio) is No. 3 was the highest. Next, competitive inhibition against anti-human CCR8 antibody (L263G8, BioLegend) was evaluated. The above four hybridoma supernatants or the hybridoma supernatant (No.
  • DAPI was added at a concentration of 100 ng/ml, and the fluorescence value (average value) bound to the cells was quantified by flow cytometry.
  • the inhibition rate (%) was calculated from the average fluorescence value of each test antibody, with the average fluorescence value when GIT medium was added as a negative control being 100% and the average fluorescence value of the positive control being 0%.
  • the results are shown in Table 1 and FIG. No.
  • the hybridoma supernatant of No. 2 exhibited competitive inhibitory activity against the L263G8 antibody, whereas the four hybridoma supernatants obtained in (1) above showed almost no competitive inhibitory activity against the L263G8 antibody.
  • GIT medium was used as a negative control.
  • hybridoma producing an anti-CCR8 antibody with neutralizing activity the supernatant of a hybridoma (No. 2) producing the 2-7B antibody described in the Examples of WO2020/138489 was used.
  • human CCL1 BioLegend
  • HITACHI F-7000 spectrofluorometer
  • No. 1 is the best hybridoma that produces anti-CCR8 antibodies without neutralizing activity. Three clones were selected.
  • Example 2 Determination of antibody sequence No. Regarding the 3-3F antibody produced by the three clones, the amino acid sequences of the light chain variable region and heavy chain variable region of the antibody were determined from hybridoma cells according to a conventional method (Table 3).
  • Example 3 Evaluation of neutralizing activity of purified antibody
  • the established hybridoma was cultured in a serum-free medium, and the culture supernatant was purified by Protein G affinity and gel filtration to obtain a purified antibody.
  • the neutralizing activity of this purified 3-3F antibody was measured by the method described below.
  • L263G8 antibody which is a commercially available anti-human CCR8 antibody, and 2C7 antibody, 10A11 antibody, and 2-7B antibody described in the Examples of WO2020/138489 were used. Using.
  • a diluted antibody solution diluted with a medium was added to human CCR8-expressing 293 cells into which a Ca 2+ indicator had been incorporated in advance, and Ca 2+ influx due to the addition of 200 nM human CCL1 (manufactured by Biolegend) was measured by FLIPR.
  • the inhibition rate was calculated by setting the signal when human CCL1 was not added as an inhibition rate of 100%, and the signal when human CCL1 was added and the antibody was not added as an inhibition rate of 0%, and the antibody concentration showing a 50% inhibition rate was defined as IC50. Evaluations were performed at least three times and IC50 was expressed as Average ⁇ SD. As a result, no neutralizing activity was observed in the purified 3-3F antibody (Table 4).
  • Example 4 Binding analysis to CCR8 (1) Evaluation of binding activity to CCR8 Binding of 3-3F antibody to human CCR8-expressing Ramos cells and its parent strain (Ramos cells), and OP-1 antibody (neutralizing antibody to human CCR8) In the presence of the humanized 10A11 antibody (light chain variable region: IGKV4-1 N53Q+G29R (SEQ ID NO: 12)/heavy chain variable region: IGHV3-15 T94R (SEQ ID NO: 13)) described in the Examples of WO2020/138489. The binding of the 3-3F antibody was evaluated. In this example, a non-fluorescently labeled 3-3F antibody and a fluorescently labeled Alexa647 antibody were used.
  • APC fluorescently labeled anti-rat IgG antibody (manufactured by Jackson ImmunoResearch Laboratories) was used as a secondary antibody to detect the non-fluorescently labeled substance.
  • APC fluorescently labeled L263G8 antibody and BV421 fluorescently labeled 433H antibody (manufactured by BD Biosciences), which are commercially available anti-human CCR8 antibodies, were used.
  • Rat IgG2a antibody (unlabeled, 100-fold diluted) was used as the isotype antibody. Thereafter, after washing with culture medium (10% FBS), APC fluorescently labeled anti-rat IgG antibody was added and reacted at 4°C for 30 minutes.
  • APC fluorescently labeled L263G8 antibody (100-fold dilution) was added and reacted at 4°C for 30 minutes.
  • APC-mouse IgG2a antibody (100-fold dilution) was used as the isotype antibody.
  • C BV421 fluorescently labeled 433H antibody (100 times diluted) was added and reacted at 4° C. for 30 minutes.
  • BV421-mouse IgG2a antibody (100 times diluted) was used as the isotype antibody.
  • the cells were washed with culture solution (10% FBS), Cellstain-DAPI solution (10,000-fold dilution, manufactured by Dojindo Laboratories) was added, and the cells were measured by flow cytometry.
  • eBioscience Fixable Viability Dye eFluor 780 solution (10,000-fold dilution, manufactured by Thermo Fisher Scientific) was added, reacted at 4°C for 10 minutes, and then washed with culture medium (10% FBS). , resuspended in culture medium (10% FBS) and measured by flow cytometry.
  • Human CCR8 cDNA (SEQ ID NO: 17) was cloned into the EcoRI/BamHI site of the pQCXIP vector to create a pQCXIP-hCCR8 expression vector. 5 ⁇ g of this expression vector was transformed into HEK293T cells (TAKARA) using lipofectamine 3000, and after 2 days drug selection was performed using 2 ⁇ g/ml of puromycin. Human CCR8 expressed on the cell surface of surviving drug-resistant HEK293T cells cultured in the presence of drugs for 3 weeks at 37°C and 5% CO was detected using a flow cytometer using a PE-labeled anti-CCR8 antibody (BioLegend).
  • Example 5 Evaluation of binding of 3-3F antibody to human tumor-infiltrating cells
  • the binding of 3-3F antibody to human tumor-infiltrating cells was evaluated using ovarian cancer patient specimens.
  • a non-fluorescently labeled 3-3F antibody and a fluorescently labeled Alexa647 antibody were used.
  • Alexa647 fluorescently labeled anti-rat IgG antibody (manufactured by Jackson ImmunoResearch Laboratories) was used as a secondary antibody to detect the unlabeled fluorescent substance.
  • an APC fluorescent label of L263G8 antibody which is a commercially available anti-human CCR8 antibody, was used as a positive control.
  • Intratumoral infiltrating cells extracted from ovarian cancer were seeded into a 96-well plate at a rate of 1.5 x 10 5 cells, and Zombie NIR Fixable Viability Kit (manufactured by Biolegend) was added and reacted at room temperature for 15 minutes, followed by Example 4.
  • OP-1 antibody which is a neutralizing antibody against human CCR8 used in , was added at various concentrations and allowed to react at 4°C for 60 minutes.
  • the positive rate of the FoxP3-positive CCR8-positive fraction was 46.6%.
  • the reactivity of each antibody in the presence of OP-1 antibody was also confirmed, no decrease in binding was observed for 3-3F antibody in the presence of OP-1 antibody.
  • the L236G8 antibody used as a comparison control a decrease in binding was observed in the presence of the OP-1 antibody, and the positivity rate of the FoxP3-positive CCR8-positive fraction was reduced to about half.
  • Example 6 Confirmation of CCR8-specific binding of 3-3F antibody to intratumoral infiltrating Treg cells Simultaneous detection of signal by 3-3F antibody and CCR8 mRNA expression on flow cytometry using PrimeFlow RNA assay technology The CCR8 specificity of the 3-3F antibody was confirmed.
  • Tumor tissues derived from ovarian cancer patients were dispersed using Tumor Dissociation Kit, human (Miltenyi Biotec) and cryopreserved. Frozen tumor dispersed cells were thawed and suspended in Stain Buffer (BD Bioscience) supplemented with Human TruStain FcX (BioLegend) and Zombie NIR Fixable Viability Kit (BioLegend). , and left at 4° C.
  • Example 7 Evaluation of binding of 3-3F antibody to mouse tumor-infiltrating cells
  • the binding of 3-3F antibody to mouse tumor-infiltrating cells was evaluated using a mouse kidney cancer lung metastasis model.
  • a BB515 fluorescently labeled 3-3F antibody was used.
  • a portion of this mouse model was prepared using the neutralizing antibody against human CCR8 used in Example 4.
  • OP-1 antibody which is an anti-human IgG Fc antibody
  • the BV421 fluorescent label of M1310G05 antibody which is an anti-human IgG Fc antibody
  • a BV421 fluorescent label of 433H antibody which is a commercially available anti-human CCR8 antibody, was used, and samples separated from the same sample were stained and measured. Renca, a mouse kidney cancer cell cultured in a knock-in mouse (female, 21 weeks old) in which CCR8 of Balb/C mouse described in Example 10 of WO2020/138489 was replaced with human CCR8 gene, was cultured at 4 ⁇ 10.
  • mice Five were injected into the tail vein. Twelve days later, OP-1 antibody was injected through the tail vein. The next day, the lungs to which tumor cells had metastasized were collected from the 0.05 mg/kg administration group (8 mice), the 0.5 mg/kg administration group (6 mice), and the untreated mice (9 mice) under inhalation anesthesia. Single cells were dispersed and purified from lung tissue using a Tumor Dissociation Kit, mouse (Miltenyi Biotec), and BD Pharma Lyse (BD Biosciences), and stained for flow cytometry.
  • Treg cells eFluor 780, CD8 negative, and TCR ⁇ , CD4, CD25, Foxp3 positive fraction for sample 1, eFluor 780, CD8 negative, and CD45, TCR fraction for sample 2) were analyzed using MACSQuantAnalyzer (Miltenyi Biotec).
  • the monoclonal antibody or antibody fragment thereof of the present invention can be used to detect CCR8 in a biological sample. Furthermore, a pharmaceutical composition containing the monoclonal antibody of the present invention or an antibody fragment thereof is very useful as a medicament for treating or preventing CCR8-related diseases.

Abstract

Provided are novel anti-CCR8 antibodies. Said antibodies detect CCR8 expressed in tumor invasive Treg cells or the like and are useful for cancer diagnosis or companion diagnostics.

Description

ＣＣＲ８検出用新規抗ＣＣＲ８抗体Novel anti-CCR8 antibody for CCR8 detection
　本発明は、ＣＣＲ８の検出に有用な新規抗ＣＣＲ８抗体に関する。 The present invention relates to a novel anti-CCR8 antibody useful for detecting CCR8.
　腫瘍微小環境内の制御性Ｔ細胞（Ｔｒｅｇ細胞）が媒介する免疫抑制をはじめとする強力な負の調節機構は、腫瘍の治療にとって大きな障害である（非特許文献１）。
　例えば、腫瘍に浸潤するＣＤ４陽性Ｔｒｅｇ細胞は、抗腫瘍免疫応答を強力に阻害している可能性があり、効果的な癌治療の大きな障害となりうる。
　ＣＤ４陽性ＦｏｘＰ３陽性Ｔｒｅｇ細胞が媒介する腫瘍免疫抑制は、動物腫瘍モデルで十分に立証されており、腫瘍内も含めた全身性のＴｒｅｇ細胞除去により抗腫瘍効果が得られるが、一方で５０％程度の腫瘍内浸潤Ｔｒｅｇ細胞除去では効果が認められないことが報告されている（非特許文献２）。 Powerful negative regulatory mechanisms, including immunosuppression mediated by regulatory T cells (Treg cells) within the tumor microenvironment, are a major obstacle to tumor treatment (Non-Patent Document 1).
For example, CD4-positive Treg cells infiltrating tumors can strongly inhibit anti-tumor immune responses and can be a major obstacle to effective cancer treatment.
Tumor immunosuppression mediated by CD4-positive FoxP3-positive Treg cells has been well documented in animal tumor models, and systemic Treg cell removal, including within the tumor, results in an antitumor effect; It has been reported that no effect is observed in removing intratumoral infiltrating Treg cells (Non-Patent Document 2).
　ヒトにおいて全ＣＤ４陽性Ｔ細胞集団中のＣＤ４陽性ＣＤ２５陽性Ｔｒｅｇ細胞比（Ｔｒｅｇ細胞を含む細胞集団）の増大が、肺、乳房及び卵巣腫瘍をはじめとする、種々の癌患者の腫瘍内で検出され、存在比と患者生存率に負の相関があることが報告されている（非特許文献３～８）。 In humans, an increased ratio of CD4+CD25+ Treg cells (cell population containing Treg cells) in the total CD4+ T cell population has been detected in tumors of various cancer patients, including lung, breast, and ovarian tumors. It has been reported that there is a negative correlation between the abundance ratio and patient survival rate (Non-patent Documents 3 to 8).
　ＣＣＲ８はＣＹ６、ＣＫＲ－Ｌ１又はＴＥＲ１とも呼ばれていた胸腺や脾臓等で発現しているＧ蛋白共役型７回膜貫通型のＣＣケモカイン受容体タンパク質であり、その遺伝子はヒトクロモゾームでは３ｐ２１に存在する。ヒトＣＣＲ８は３５５アミノ酸からなる（非特許文献９）。ＣＣＲ８に対する内因性リガンドとしてはＣＣＬ１が知られている（非特許文献１０）。ヒトＣＣＲ８　ｃＤＮＡはＧｅｎｂａｎｋ　ＡＣＣ　Ｎｏ．ＮＭ＿００５２０１に示される塩基配列で構成され、マウスＣＣＲ８　ｃＤＮＡはＧｅｎｂａｎｋ　ＡＣＣ　Ｎｏ．ＮＭ＿００７７２０に示される塩基配列で構成される。 CCR8, also called CY6, CKR-L1, or TER1, is a G protein-coupled seven-transmembrane CC chemokine receptor protein expressed in the thymus and spleen, and its gene is located on 3p21 in human chromosomes. exist. Human CCR8 consists of 355 amino acids (Non-Patent Document 9). CCL1 is known as an endogenous ligand for CCR8 (Non-Patent Document 10). Human CCR8 cDNA is Genbank ACC No. The mouse CCR8 cDNA is composed of the base sequence shown in NM_005201, and the mouse CCR8 cDNA is shown in Genbank ACC No. It is composed of the base sequence shown in NM_007720.
　ＣＣＲ８は、腫瘍内浸潤Ｔｒｅｇ細胞にも特異的に発現しており、ＣＣＲ８欠損マウスと野生型マウスに乳癌細胞を移植したところ、ＣＣＲ８欠損マウスにおける乳癌の増殖と転移が野生型マウスと比較して抑制されていたことが示されている（特許文献１及び非特許文献１１）。非特許文献１２～１３にも、ＣＣＲ８が癌の病態に関与していることが記載されている。さらに、抗ＣＣＲ８抗体を癌モデル動物に投与することにより抗腫瘍効果を示したことが開示されている（特許文献２～１２）。 CCR8 is also specifically expressed in tumor-infiltrating Treg cells, and when breast cancer cells were transplanted into CCR8-deficient mice and wild-type mice, the growth and metastasis of breast cancer in CCR8-deficient mice was significantly higher than in wild-type mice. It has been shown that this was suppressed (Patent Document 1 and Non-Patent Document 11). Non-Patent Documents 12 and 13 also describe that CCR8 is involved in the pathology of cancer. Furthermore, it has been disclosed that administration of anti-CCR8 antibodies to cancer model animals showed antitumor effects (Patent Documents 2 to 12).
　特許文献１、１３及び１４には、ＣＣＲ８が癌診断マーカーとして有用であることが記載されているが、ＣＣＲ８検出に有用な抗ＣＣＲ８抗体は具体的に記載されていない。また、特許文献１５には、ＣＣＲ８検出に有用な抗ＣＣＲ８抗体が開示されているが、いずれの抗体も中和活性を有しており、ＣＣＲ８検出に有用な中和活性を有さない抗ＣＣＲ８抗体は具体的に記載されていない。さらに、特許文献１５には、抗ＣＣＲ８抗体が癌診断に有用であることも記載されていない。 Patent Documents 1, 13, and 14 describe that CCR8 is useful as a cancer diagnostic marker, but do not specifically describe anti-CCR8 antibodies useful for CCR8 detection. Further, Patent Document 15 discloses anti-CCR8 antibodies useful for CCR8 detection, but all antibodies have neutralizing activity, and anti-CCR8 antibodies that do not have neutralizing activity useful for CCR8 detection Antibodies are not specifically listed. Furthermore, Patent Document 15 does not describe that anti-CCR8 antibodies are useful for cancer diagnosis.
米国特許第１００８７２５９号明細書US Patent No. 10087259 国際公開第２０１８／１８１４２５号International Publication No. 2018/181425 国際公開第２０１８／１１２０３２号International Publication No. 2018/112032 国際公開第２０２０／１３８４８９号International Publication No. 2020/138489 国際公開第２０２１／１４２００２号International Publication No. 2021/142002 国際公開第２０２１／１５２１８６号International Publication No. 2021/152186 国際公開第２０２１／１６３０６４号International Publication No. 2021/163064 国際公開第２０２１／１７８７４９号International Publication No. 2021/178749 国際公開第２０２１／１９４９４２号International Publication No. 2021/194942 国際公開第２０２１／２６０２０８号International Publication No. 2021/260208 国際公開第２０２１／２６０２０９号International Publication No. 2021/260209 国際公開第２０２１／２６０２１０号International Publication No. 2021/260210 国際公開第０３／０９６０２０号International Publication No. 03/096020 国際公開第２０１７／１９８６３１号International Publication No. 2017/198631 国際公開第２００７／０４４７５６号International Publication No. 2007/044756
　本発明の目的は、ＣＣＲ８の検出に有用な抗ＣＣＲ８抗体を提供することである。さらには、該抗ＣＣＲ８抗体を含有する診断薬を提供することである。 An object of the present invention is to provide an anti-CCR8 antibody useful for detecting CCR8. Another object of the present invention is to provide a diagnostic agent containing the anti-CCR8 antibody.
　本発明者らは、鋭意研究の結果、ＣＣＲ８に特異的に結合し、ＣＣＲ８検出に有用な中和活性を有さない新規抗ＣＣＲ８抗体を見出した。また、本発明の新規ＣＣＲ８抗体は、ＣＣＲ８に対する結合が中和活性を有する抗ＣＣＲ８抗体とは競合しないことを見出した。さらに、本発明の新規ＣＣＲ８抗体は、腫瘍内浸潤Ｔｒｅｇ細胞等に発現するＣＣＲ８を検出し、癌診断やコンパニオン診断に有用であることを見出した。 As a result of extensive research, the present inventors discovered a novel anti-CCR8 antibody that specifically binds to CCR8 and does not have neutralizing activity useful for CCR8 detection. Furthermore, it has been found that the novel CCR8 antibody of the present invention does not compete with anti-CCR8 antibodies having neutralizing activity in binding to CCR8. Furthermore, it has been found that the novel CCR8 antibody of the present invention detects CCR8 expressed in tumor-infiltrating Treg cells and is useful for cancer diagnosis and companion diagnosis.
　本発明は、以下に関する。
（１）配列番号２のアミノ酸配列からなるＣＤＲ１、
配列番号３のアミノ酸配列からなるＣＤＲ２及び
配列番号４のアミノ酸配列からなるＣＤＲ３を含む軽鎖可変領域並びに
配列番号５のアミノ酸配列からなるＣＤＲ１、
配列番号６のアミノ酸配列からなるＣＤＲ２及び
配列番号７のアミノ酸配列からなるＣＤＲ３を含む重鎖可変領域
を含む、ＣＣＲ８に結合するモノクローナル抗体又はその抗体断片。
（２）配列番号８のアミノ酸配列を有する軽鎖可変領域、及び、配列番号９のアミノ酸配列を有する重鎖可変領域を含む、（１）記載の抗体又はその抗体断片。
（３）さらに、配列番号１０のアミノ酸配列を有する軽鎖定常領域、及び、配列番号１１のアミノ酸配列を有する重鎖定常領域を有する、（１）記載の抗体又はその抗体断片。
（４）（１）～（３）のいずれかに記載の抗体又はその抗体断片を含有する組成物を使用する、ＣＣＲ８の検出方法。
（５）癌診断のための、（４）記載の方法。
（５－２）癌診断を補助するための、（４）記載の方法。
（５－３）腫瘍内浸潤Ｔｒｅｇ細胞を検出するための、（４）記載の方法。
（５－４）ＣＣＲ８発現腫瘍細胞を検出するための、（４）記載の方法。
（６）抗ＣＣＲ８抗体を含有する医薬を投与した被験者から採取した試料に含まれるＣＣＲ８を検出する、（４）記載の方法。
（７）抗ＣＣＲ８抗体を含有する医薬に含まれる抗ＣＣＲ８抗体が中和活性を有する、（６）記載の方法。
（８）抗ＣＣＲ８抗体を含有する医薬に含まれる抗ＣＣＲ８抗体が、配列番号８のアミノ酸配列を有する軽鎖可変領域、及び、配列番号９のアミノ酸配列を有する重鎖可変領域を含む抗体とＣＣＲ８に対する結合が競合しない、（６）記載の方法。
（９）抗ＣＣＲ８抗体を含有する医薬に含まれる抗ＣＣＲ８抗体が、配列番号１２のアミノ酸配列を有する軽鎖可変領域、及び、配列番号１３のアミノ酸配列を有する重鎖可変領域を含むモノクローナル抗体又はその断片である、（６）記載の方法。
（１０）被験者が癌の患者である、（６）記載の方法。
（１１）（１）～（３）のいずれかに記載の抗体又はその抗体断片を含有する組成物を含む、ＣＣＲ８の検出キット。
（１２）癌診断のための、（１１）記載のキット。
（１２－２）癌診断を補助するための、（１１）記載のキット。
（１２－３）腫瘍内浸潤Ｔｒｅｇ細胞を検出するための、（１１）記載のキット。
（１２－４）ＣＣＲ８発現腫瘍細胞を検出するための、（１１）記載のキット。
（１３）抗ＣＣＲ８抗体を含有する医薬を投与した被験者から採取した試料に含まれるＣＣＲ８を検出する、（１１）記載のキット。
（１４）抗ＣＣＲ８抗体を含有する医薬に含まれる抗ＣＣＲ８抗体が中和活性を有する、（１３）記載のキット。
（１５）抗ＣＣＲ８抗体を含有する医薬に含まれる抗ＣＣＲ８抗体が、配列番号８のアミノ酸配列を有する軽鎖可変領域、及び、配列番号９のアミノ酸配列を有する重鎖可変領域を含む抗体とＣＣＲ８に対する結合が競合しない、（１３）記載のキット。
（１６）抗ＣＣＲ８抗体を含有する医薬に含まれる抗ＣＣＲ８抗体が、配列番号１２のアミノ酸配列を有する軽鎖可変領域、及び、配列番号１３のアミノ酸配列を有する重鎖可変領域を含むモノクローナル抗体又はその断片である、（１３）記載のキット。
（１７）被験者が癌の患者である、（１３）記載のキット。
（１８）（１）～（３）のいずれかに記載の抗体又はその抗体断片を含有する、癌の診断薬。
（１８－２）癌診断のための、（１）～（３）のいずれかに記載の抗体又はその抗体断片。
（１８－３）癌の診断薬を製造するための、（１）～（３）のいずれかに記載の抗体又はその抗体断片の使用。
（１９）（１）～（３）のいずれかに記載の抗体又はその抗体断片を含有する、抗ＣＣＲ８抗体を含有する医薬の投与の適否を判断するための診断薬。
（１９－２）抗ＣＣＲ８抗体を含有する医薬の投与の適否を判断するための、（１）～（３）のいずれかに記載の抗体又はその抗体断片。
（１９－３）、抗ＣＣＲ８抗体を含有する医薬の投与の適否を判断する診断薬を製造するための、（１）～（３）のいずれかに記載の抗体又はその抗体断片の使用。
（２０）抗ＣＣＲ８抗体を含有する医薬が抗癌剤である、（１９）記載の診断薬。
（２１）抗ＣＣＲ８抗体を含有する医薬に含まれる抗ＣＣＲ８抗体が、配列番号１２のアミノ酸配列を有する軽鎖可変領域、及び、配列番号１３のアミノ酸配列を有する重鎖可変領域を含むモノクローナル抗体又はその断片である、（１９）記載の診断薬。
（２２）（１）～（３）のいずれかに記載の抗体又はその抗体断片を含有する組成物を使用する、抗ＣＣＲ８抗体を含有する医薬の投与の適否を判断する方法。
（２３）抗ＣＣＲ８抗体を含有する医薬が抗癌剤である、（２２）記載の方法。
（２４）抗ＣＣＲ８抗体を含有する医薬に含まれる抗ＣＣＲ８抗体が、配列番号１２のアミノ酸配列を有する軽鎖可変領域、及び、配列番号１３のアミノ酸配列を有する重鎖可変領域を含むモノクローナル抗体又はその断片である、（２２）記載の方法。
（２５）（１）～（３）のいずれかに記載の抗体又はその抗体断片を含有する組成物を使用する、抗ＣＣＲ８抗体を含有する医薬に含まれる抗ＣＣＲ８抗体の腫瘍内浸潤Ｔｒｅｇ細胞への結合量を測定する方法。
（２６）（１）又は（２）記載の抗体の軽鎖可変領域又は重鎖可変領域をコードするポリヌクレオチド。
（２７）（２６）記載のポリヌクレオチドを含む発現ベクター。 The present invention relates to the following.
(1) CDR1 consisting of the amino acid sequence of SEQ ID NO: 2,
A light chain variable region comprising CDR2 consisting of the amino acid sequence of SEQ ID NO: 3 and CDR3 consisting of the amino acid sequence of SEQ ID NO: 4, and CDR1 consisting of the amino acid sequence of SEQ ID NO: 5,
A monoclonal antibody or antibody fragment thereof that binds to CCR8 and includes a heavy chain variable region including CDR2 consisting of the amino acid sequence of SEQ ID NO: 6 and CDR3 consisting of the amino acid sequence of SEQ ID NO: 7.
(2) The antibody or antibody fragment thereof according to (1), which comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 8 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 9.
(3) The antibody or antibody fragment thereof according to (1), further having a light chain constant region having the amino acid sequence of SEQ ID NO: 10 and a heavy chain constant region having the amino acid sequence of SEQ ID NO: 11.
(4) A method for detecting CCR8 using a composition containing the antibody or antibody fragment thereof according to any one of (1) to (3).
(5) The method described in (4) for cancer diagnosis.
(5-2) The method described in (4) for assisting cancer diagnosis.
(5-3) The method described in (4) for detecting intratumoral infiltrating Treg cells.
(5-4) The method described in (4) for detecting CCR8-expressing tumor cells.
(6) The method according to (4), wherein CCR8 contained in a sample collected from a subject to whom a medicine containing an anti-CCR8 antibody has been administered is detected.
(7) The method according to (6), wherein the anti-CCR8 antibody contained in the anti-CCR8 antibody-containing medicine has neutralizing activity.
(8) The anti-CCR8 antibody contained in the medicine containing the anti-CCR8 antibody contains an antibody comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 8 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 9. The method according to (6), wherein there is no conflict in binding to.
(9) The anti-CCR8 antibody contained in the medicine containing an anti-CCR8 antibody is a monoclonal antibody comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 12 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13, or The method according to (6), which is a fragment thereof.
(10) The method according to (6), wherein the subject is a cancer patient.
(11) A CCR8 detection kit comprising a composition containing the antibody or antibody fragment thereof according to any one of (1) to (3).
(12) The kit according to (11) for cancer diagnosis.
(12-2) The kit according to (11) for assisting cancer diagnosis.
(12-3) The kit described in (11) for detecting intratumoral infiltrating Treg cells.
(12-4) The kit described in (11) for detecting CCR8-expressing tumor cells.
(13) The kit according to (11), which detects CCR8 contained in a sample collected from a subject who has been administered a medicine containing an anti-CCR8 antibody.
(14) The kit according to (13), wherein the anti-CCR8 antibody contained in the anti-CCR8 antibody-containing medicine has neutralizing activity.
(15) The anti-CCR8 antibody contained in the medicine containing the anti-CCR8 antibody contains an antibody comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 8 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 9. The kit according to (13), which does not compete in binding to.
(16) The anti-CCR8 antibody contained in the anti-CCR8 antibody-containing medicine is a monoclonal antibody comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 12 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13; The kit according to (13), which is a fragment thereof.
(17) The kit according to (13), wherein the subject is a cancer patient.
(18) A cancer diagnostic agent containing the antibody or antibody fragment thereof according to any one of (1) to (3).
(18-2) The antibody or antibody fragment thereof according to any one of (1) to (3) for cancer diagnosis.
(18-3) Use of the antibody or antibody fragment thereof according to any one of (1) to (3) for producing a cancer diagnostic agent.
(19) A diagnostic agent containing the antibody or antibody fragment thereof according to any one of (1) to (3), for determining the appropriateness of administration of a medicine containing an anti-CCR8 antibody.
(19-2) The antibody or antibody fragment thereof according to any one of (1) to (3) for determining suitability of administration of a medicine containing an anti-CCR8 antibody.
(19-3) Use of the antibody or antibody fragment thereof according to any one of (1) to (3) for producing a diagnostic agent for determining the suitability of administration of a medicine containing an anti-CCR8 antibody.
(20) The diagnostic agent according to (19), wherein the medicine containing the anti-CCR8 antibody is an anticancer agent.
(21) The anti-CCR8 antibody contained in the anti-CCR8 antibody-containing medicine is a monoclonal antibody comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 12 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13, or The diagnostic agent according to (19), which is a fragment thereof.
(22) A method for determining the suitability of administration of a medicament containing an anti-CCR8 antibody, using a composition containing the antibody or antibody fragment thereof according to any one of (1) to (3).
(23) The method according to (22), wherein the medicine containing the anti-CCR8 antibody is an anticancer agent.
(24) The anti-CCR8 antibody contained in the anti-CCR8 antibody-containing medicine is a monoclonal antibody comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 12 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13; The method according to (22), which is a fragment thereof.
(25) Using a composition containing the antibody or antibody fragment thereof according to any one of (1) to (3), the anti-CCR8 antibody contained in the anti-CCR8 antibody-containing medicine is used to infiltrate into tumor-infiltrating Treg cells. How to measure the amount of binding.
(26) A polynucleotide encoding the light chain variable region or heavy chain variable region of the antibody according to (1) or (2).
(27) An expression vector comprising the polynucleotide described in (26).
　本発明の新規抗ＣＣＲ８抗体は、ＣＣＲ８に特異的に結合するので、生体試料においてＣＣＲ８を検出するために使用することができる。さらに、本発明の新規抗ＣＣＲ８抗体は、腫瘍内浸潤Ｔｒｅｇ細胞に発現するＣＣＲ８を検出するので、癌診断やコンパニオン診断に非常に有用である。 Since the novel anti-CCR8 antibody of the present invention specifically binds to CCR8, it can be used to detect CCR8 in biological samples. Furthermore, the novel anti-CCR8 antibody of the present invention detects CCR8 expressed in tumor-infiltrating Treg cells, and is therefore very useful for cancer diagnosis and companion diagnosis.
ラットハイブリドーマ上清のＬ２６３Ｇ８抗体に対する競合阻害活性をフローサイトメトリー法により評価した。抗体にＬ２６３Ｇ８抗体に対する競合阻害活性がある場合は、陰性コントロール（Ｃｏｎｔ）と比較してピークのシフトが認められる。Ｘ軸は検出蛍光強度、Ｙ軸は検出細胞数を示す。The competitive inhibitory activity of rat hybridoma supernatant against L263G8 antibody was evaluated by flow cytometry. If the antibody has competitive inhibitory activity against the L263G8 antibody, a peak shift will be observed compared to the negative control (Cont). The X axis shows the detected fluorescence intensity, and the Y axis shows the number of detected cells. ラットハイブリドーマ上清による、ＣＣＬ１とＣＣＲ８の結合に対する中和活性を細胞内カルシウム流入活性測定により評価した。抗体が中和活性を示す場合は、細胞内カルシウム流入活性が抑制されるため、蛍光量の経時的上昇も抑制される。The neutralizing activity of the rat hybridoma supernatant against the binding between CCL1 and CCR8 was evaluated by measuring intracellular calcium influx activity. When the antibody exhibits neutralizing activity, intracellular calcium influx activity is suppressed, and therefore the increase in fluorescence level over time is also suppressed. ヒトＣＣＲ８発現Ｒａｍｏｓ細胞における３－３Ｆ抗体、Ｌ２６３Ｇ８抗体及び４３３Ｈ抗体の結合をフローサイトメトリー法により評価した。Ｘ軸は検出蛍光強度、Ｙ軸は検出細胞数を示す。The binding of the 3-3F antibody, L263G8 antibody, and 433H antibody to human CCR8-expressing Ramos cells was evaluated by flow cytometry. The X axis shows the detected fluorescence intensity, and the Y axis shows the number of detected cells. ヒトＣＣＲ８発現Ｒａｍｏｓ細胞における、ヒトＣＣＲ８に対する中和抗体であるＯＰ－１抗体存在下での、３－３Ｆ抗体、Ｌ２６３Ｇ８抗体及び４３３Ｈ抗体の結合をフローサイトメトリー法により評価した。Ｘ軸は検出蛍光強度、Ｙ軸は検出細胞数を示す。The binding of 3-3F antibody, L263G8 antibody, and 433H antibody to human CCR8-expressing Ramos cells in the presence of OP-1 antibody, which is a neutralizing antibody against human CCR8, was evaluated by flow cytometry. The X axis shows the detected fluorescence intensity, and the Y axis shows the number of detected cells. 各種ケモカイン受容体発現細胞に対する３－３Ｆ抗体の結合をフローサイトメトリー法により評価した。Ｘ軸は検出蛍光強度、Ｙ軸は検出細胞数を示し、各グラフで「３－３Ｆ」は３－３Ｆ抗体のピーク、「Ｃｏｎｔ」は陰性コントロールのピークを示す。The binding of the 3-3F antibody to cells expressing various chemokine receptors was evaluated by flow cytometry. The X axis shows the detected fluorescence intensity, and the Y axis shows the number of detected cells. In each graph, "3-3F" shows the peak of the 3-3F antibody, and "Cont" shows the peak of the negative control. 腫瘍内浸潤Ｔｒｅｇ細胞に対する３－３Ｆ抗体の結合をフローサイトメトリー法により評価した。Ｘ軸は検出蛍光強度、Ｙ軸は検出細胞数を示す。The binding of the 3-3F antibody to intratumoral infiltrating Treg cells was evaluated by flow cytometry. The X axis shows the detected fluorescence intensity, and the Y axis shows the number of detected cells. 腫瘍内浸潤Ｔｒｅｇ細胞に対する３－３Ｆ抗体、蛍光標識３－３Ｆ抗体及びＬ２６３Ｇ８抗体の結合をフローサイトメトリー法により評価した。Ｘ軸はＦｏｘＰ３検出蛍光強度、Ｙ軸は各種抗ＣＣＲ８抗体によるＣＣＲ８検出蛍光強度を示す。The binding of 3-3F antibody, fluorescently labeled 3-3F antibody, and L263G8 antibody to intratumoral infiltrating Treg cells was evaluated by flow cytometry. The X axis shows the fluorescence intensity detected by FoxP3, and the Y axis shows the fluorescence intensity detected by CCR8 by various anti-CCR8 antibodies. 腫瘍内浸潤Ｔｒｅｇ細胞のフローサイトメトリー解析による、３－３Ｆ抗体シグナル（Ｙ軸）とＣＣＲ８ ｍＲＮＡシグナル（Ｘ軸）の相関を示す。The correlation between 3-3F antibody signal (Y axis) and CCR8 mRNA signal (X axis) is shown by flow cytometry analysis of intratumoral infiltrating Treg cells. 腫瘍内浸潤Ｔｒｅｇ細胞における、ヒトＣＣＲ８ノックインマウスに投与したヒトＣＣＲ８に対する中和抗体（ＯＰ－１抗体）のＴｒｅｇ細胞に対する結合シグナル（Ｘ軸）と３－３Ｆ抗体シグナル（Ｙ軸）の相関を示す。図中のｒは相関係数、直線は線形回帰を示す。Showing the correlation between the binding signal (X axis) to Treg cells of a neutralizing antibody (OP-1 antibody) against human CCR8 (OP-1 antibody) administered to human CCR8 knock-in mice and the 3-3F antibody signal (Y axis) in intratumoral infiltrating Treg cells. . In the figure, r indicates a correlation coefficient, and a straight line indicates linear regression. 腫瘍内浸潤Ｔｒｅｇ細胞における、ヒトＣＣＲ８ノックインマウスに投与したヒトＣＣＲ８に対する中和抗体（ＯＰ－１抗体）のＴｒｅｇ細胞に対する結合シグナル（Ｘ軸）と４３３Ｈ抗体シグナル（Ｙ軸）の相関を示す。図中のｒは相関係数、直線は線形回帰を示す。The correlation between the binding signal (X axis) to Treg cells of a neutralizing antibody (OP-1 antibody) against human CCR8 (OP-1 antibody) administered to human CCR8 knockin mice and the 433H antibody signal (Y axis) in tumor-infiltrating Treg cells is shown. In the figure, r indicates a correlation coefficient, and a straight line indicates linear regression.
　本明細書において使用される用語は、特に言及する場合を除いて、当該分野で通常用いられる意味で用いられる。 The terms used herein have the meanings commonly used in the field, unless otherwise specified.
　本発明においては、当該分野で公知の抗体作製手法が利用可能である。例えば、Immunochemistry in Practice (Blackwell Scientific Publications)に記載された方法等が挙げられる。
　また、当該分野で公知の遺伝子操作的手法が利用可能である。例えば、Molecular Cloning, A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press (2012), Current Protocols Essential Laboratory Techniques, Current Protocols (2012)に記載された方法等が挙げられる。 In the present invention, antibody production techniques known in the art can be used. Examples include the method described in Immunochemistry in Practice (Blackwell Scientific Publications).
Additionally, genetic engineering techniques known in the art are available. Examples include the methods described in Molecular Cloning, A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press (2012), Current Protocols Essential Laboratory Techniques, Current Protocols (2012).
　ヒトＣＣＲ８のアミノ酸配列は、ＵｎｉＰｒｏｔＫＢ／Ｓｗｉｓｓ－Ｐｒｏｔ：Ｐ５１６８５に示されている（配列番号１）。ヒトＣＣＲ８の膜外ドメインは、１～３５位アミノ酸からなるＮ末領域、９４～１０７位アミノ酸からなるｌｏｏｐ１領域、１７２～２０２位アミノ酸からなるｌｏｏｐ２領域、２６４～２８０位アミノ酸からなるｌｏｏｐ３領域が該当する。ヒトＣＣＲ８のアミノ酸配列における１７位はチロシンであり、マウスＣＣＲ８のアミノ酸配列においては、１７位は欠損している。 The amino acid sequence of human CCR8 is shown in UniProtKB/Swiss-Prot:P51685 (SEQ ID NO: 1). The extramembrane domain of human CCR8 corresponds to the N-terminal region consisting of amino acids 1 to 35, the loop 1 region consisting of amino acids 94 to 107, the loop 2 region consisting of amino acids 172 to 202, and the loop 3 region consisting of amino acids 264 to 280. do. Position 17 in the amino acid sequence of human CCR8 is tyrosine, and position 17 is deleted in the amino acid sequence of mouse CCR8.
　本発明の抗ＣＣＲ８抗体を産生するハイブリドーマは、ヒトＣＣＲ８タンパク質、ヒトＣＣＲ８の全長をコードする遺伝子、ヒトＣＣＲ８発現細胞等を免疫原として作製することができる。ヒトＣＣＲ８の全長をコードする遺伝子を免疫原とする場合は、例えば、該遺伝子を抗原としてＤＮＡ免疫したマウスの脾臓細胞とマウスミエローマ細胞を融合させることにより、抗ＣＣＲ８抗体を産生するハイブリドーマを得ることができる。 A hybridoma producing the anti-CCR8 antibody of the present invention can be produced using human CCR8 protein, a gene encoding the full-length human CCR8, human CCR8-expressing cells, etc. as an immunogen. When using a gene encoding the full length of human CCR8 as an immunogen, for example, a hybridoma producing an anti-CCR8 antibody can be obtained by fusing mouse myeloma cells with spleen cells of a mouse immunized with DNA using the gene as an antigen. I can do it.
　ハイブリドーマを培養するための基本培地としては、例えば、Ｄ－ＭＥＭ培地、ＲＰＭＩ１６４０培地、ＩＭＤＭ培地、ＡＳＦ１０４培地等が挙げられ、該基本培地には、目的に応じて、例えば血清、ホルモン、サイトカイン及び／または種々無機あるいは有機物質等を含有させることができる。 Examples of the basic medium for culturing hybridomas include D-MEM medium, RPMI1640 medium, IMDM medium, and ASF104 medium. Alternatively, various inorganic or organic substances can be contained.
　本発明のモノクローナル抗体又はその抗体断片の一つの態様としては、本明細書記載のＣＤＲ又は重鎖可変領域／軽鎖可変領域を有するモノクローナル抗体又は該抗体の断片が挙げられる。該抗体又は抗体断片は、免疫グロブリン分子の任意のクラス（例えば、ＩｇＧ、ＩｇＥ、ＩｇＭ、ＩｇＤ若しくはＩｇＡ。好ましくは、ＩｇＧ。）又はサブクラス由来であり得、例えば、マウス、ラット、サメ、ウサギ、ブタ、ハムスター、ラクダ、ラマ、ヤギ又はヒトを含む任意の種から取得されてもよい。該抗体又は抗体断片として、好ましくは、ヒト化モノクローナル抗体又はヒト化モノクローナル抗体の抗体断片である。 One embodiment of the monoclonal antibody or antibody fragment thereof of the present invention includes a monoclonal antibody or a fragment of the antibody having the CDR or heavy chain variable region/light chain variable region described herein. The antibody or antibody fragment may be derived from any class (e.g. IgG, IgE, IgM, IgD or IgA, preferably IgG) or subclass of immunoglobulin molecules, e.g. mouse, rat, shark, rabbit, It may be obtained from any species including pigs, hamsters, camels, llamas, goats or humans. The antibody or antibody fragment is preferably a humanized monoclonal antibody or an antibody fragment of a humanized monoclonal antibody.
　本明細書中、エピトープは、その抗原を標的とする抗体によって結合される抗原の領域を意味し、抗原がタンパク質である場合、抗体に直接接触する特定のアミノ酸を含む。本発明には、本発明のモノクローナル抗体又はその抗体断片と完全に同じエピトープを認識するモノクローナル抗体又はその抗体断片のみならず、部分的に同じエピトープを認識するモノクローナル抗体又はその抗体断片も包含される。 As used herein, epitope refers to the region of an antigen that is bound by an antibody targeting that antigen, and when the antigen is a protein, includes specific amino acids that are in direct contact with the antibody. The present invention includes not only monoclonal antibodies or antibody fragments thereof that recognize the completely same epitope as the monoclonal antibody or antibody fragment thereof of the present invention, but also monoclonal antibodies or antibody fragments thereof that recognize partially the same epitope. .
　本発明のモノクローナル抗体のＣＤＲ配列は、好ましくは以下の配列を有している。
（１）軽鎖ＣＤＲ１：配列番号２。
（２）軽鎖ＣＤＲ２：配列番号３。
（３）軽鎖ＣＤＲ３：配列番号４。
（４）重鎖ＣＤＲ１：配列番号５。
（５）重鎖ＣＤＲ２：配列番号６。
（６）重鎖ＣＤＲ３：配列番号７。 The CDR sequence of the monoclonal antibody of the present invention preferably has the following sequence.
(1) Light chain CDR1: SEQ ID NO: 2.
(2) Light chain CDR2: SEQ ID NO: 3.
(3) Light chain CDR3: SEQ ID NO: 4.
(4) Heavy chain CDR1: SEQ ID NO: 5.
(5) Heavy chain CDR2: SEQ ID NO: 6.
(6) Heavy chain CDR3: SEQ ID NO: 7.
　本発明において「モノクローナル抗体の抗体断片」とは、本発明のモノクローナル抗体の一部であって、当該モノクローナル抗体と同様にヒトＣＣＲ８に特異的に結合し、ヒトＣＣＲ８を選択的に阻害する断片を意味する。
　本発明の「抗ＣＣＲ８抗体」には、「モノクローナル抗体の抗体断片」を含む。 In the present invention, the term "antibody fragment of a monoclonal antibody" refers to a fragment that is part of the monoclonal antibody of the present invention and that specifically binds to human CCR8 and selectively inhibits human CCR8 like the monoclonal antibody. means.
The "anti-CCR8 antibody" of the present invention includes "antibody fragment of a monoclonal antibody."
　具体的には、ヒトＣＣＲ８に対して特異的に結合するＦａｂ（ｆｒａｇｍｅｎｔ　ｏｆ　ａｎｔｉｇｅｎ　ｂｉｎｄｉｎｇ）、Ｆａｂ’、Ｆ（ａｂ’）２、一本鎖抗体（ｓｉｎｇｌｅ　ｃｈａｉｎ　Ｆｖ；以下、ｓｃＦｖと表記する）、ジスルフィド安定化抗体（ｄｉｓｕｌｆｉｄｅ　ｓｔａｂｉｌｉｚｅｄ　Ｆｖ；　以下、ｄｓＦｖと表記する）、２量化体Ｖ領域断片（以下、Ｄｉａｂｏｄｙと表記する）、ＣＤＲを含むペプチド等を挙げることができる（エキスパート・オピニオン・オン・テラピューティック・パテンツ、第６巻、第５号、第４４１～４５６頁、１９９６年）。 Specifically, Fab (fragment of antigen binding), Fab', F(ab') 2 , single chain antibody (single chain Fv; hereinafter referred to as scFv) that specifically binds to human CCR8, Examples include disulfide stabilized antibodies (disulfide stabilized Fv; hereinafter referred to as dsFv), dimerized V region fragments (hereinafter referred to as diabody), peptides containing CDRs, etc. Pytic Patents, Vol. 6, No. 5, pp. 441-456, 1996).
　Ｆａｂは、ＩｇＧのヒンジ領域で２本のＨ鎖を架橋している２つのジスルフィド結合（Ｓ－Ｓ結合）の上部のペプチド部分を酵素パパインで分解して得られる、Ｈ鎖のＮ末端側約半分とＬ鎖全体で構成される、分子量約５万の抗原結合活性を有する抗体断片である。本発明で使用されるＦａｂは、本発明のモノクローナル抗体をパパイン処理して得ることができる。また、本発明のモノクローナル抗体のＦａｂをコードするＤＮＡを細胞用発現ベクターに挿入し、該ベクターを細胞へ導入することにより発現させることによってもＦａｂを製造することができる。 Fab is obtained by decomposing the peptide part above the two disulfide bonds (SS bonds) that bridge the two H chains in the hinge region of IgG with the enzyme papain. It is an antibody fragment with a molecular weight of approximately 50,000 and antigen-binding activity, consisting of one half and the entire L chain. Fab used in the present invention can be obtained by treating the monoclonal antibody of the present invention with papain. Furthermore, Fab can also be produced by inserting DNA encoding the Fab of the monoclonal antibody of the present invention into a cellular expression vector, and expressing the vector by introducing the vector into cells.
　Ｆａｂ’は、Ｆ（ａｂ’）２のヒンジ間のＳ－Ｓ結合を切断した分子量約５万の抗原結合活性を有する抗体断片である。本発明で使用されるＦａｂ’は、本発明のモノクローナル抗体のＦ（ａｂ’）２を還元剤ジチオスレイトール処理して得ることができる。また、本発明のモノクローナル抗体のＦａｂ’をコードするＤＮＡを細胞用発現ベクターに挿入し、該ベクターを大腸菌、酵母又は動物細胞へ導入することにより発現させることによってもＦａｂ’を製造することができる。 Fab' is an antibody fragment with a molecular weight of approximately 50,000 and having antigen-binding activity obtained by cleaving the SS bond between the hinges of F(ab') 2 . Fab' used in the present invention can be obtained by treating F(ab') 2 of the monoclonal antibody of the present invention with a reducing agent dithiothreitol. Fab' can also be produced by inserting the DNA encoding Fab' of the monoclonal antibody of the present invention into a cellular expression vector, and expressing the vector by introducing it into E. coli, yeast, or animal cells. .
　Ｆ（ａｂ’）２は、ＩｇＧのヒンジ領域の２個のＳ－Ｓ結合の下部を酵素ペプシンで分解して得られる、２つのＦａｂ’領域がヒンジ部分で結合して構成される、分子量約１０万の抗原結合活性を有する抗体断片である。本発明で使用されるＦ（ａｂ’）２は、本発明のモノクローナル抗体をペプシン処理して得ることができる。また、本発明のモノクローナル抗体のＦ（ａｂ’）２をコードするＤＮＡを細胞用発現ベクターに挿入し、該ベクターを大腸菌、酵母又は動物細胞へ導入することにより発現させることによってもＦ（ａｂ’）２を製造することができる。 F(ab') 2 is obtained by decomposing the lower part of the two SS bonds in the hinge region of IgG with the enzyme pepsin, and is composed of two Fab' regions linked at the hinge region, and has a molecular weight of approximately It is an antibody fragment with an antigen binding activity of 100,000. F(ab') 2 used in the present invention can be obtained by treating the monoclonal antibody of the present invention with pepsin. F(ab') 2 of the monoclonal antibody of the present invention can also be expressed by inserting the DNA encoding it into an expression vector for cells and introducing the vector into E. coli, yeast, or animal cells. ) 2 can be manufactured.
　ｓｃＦｖは、一本のＶＨと一本のＶＬとを適当なペプチドリンカー（以下、Ｐと表記する）を用いて連結した、ＶＨ－Ｐ－ＶＬ又はＶＬ－Ｐ－ＶＨポリペプチドであり、抗原結合活性を有する抗体断片である。本発明で使用されるｓｃＦｖに含まれるＶＨ及びＶＬは、本発明のモノクローナル抗体のものであればよい。本発明で使用されるｓｃＦｖは、本発明のモノクローナル抗体のＶＨ及びＶＬをコードするｃＤＮＡを用いて、ｓｃＦｖ発現ベクターを構築し、大腸菌、酵母又は動物細胞へ導入することにより発現させることによって製造することができる。 scFv is a VH-P-VL or VL-P-VH polypeptide in which one VH and one VL are linked using an appropriate peptide linker (hereinafter referred to as P), and is capable of antigen binding. It is an active antibody fragment. The VH and VL contained in the scFv used in the present invention may be those of the monoclonal antibody of the present invention. The scFv used in the present invention is produced by constructing an scFv expression vector using cDNA encoding the VH and VL of the monoclonal antibody of the present invention, and expressing it by introducing it into E. coli, yeast, or animal cells. be able to.
　ｄｓＦｖは、ＶＨ及びＶＬ中のそれぞれ１アミノ酸残基をシステイン残基に置換したポリペプチドをＳ－Ｓ結合を介して結合させたものをいう。システイン残基に置換するアミノ酸残基はＲｅｉｔｅｒらにより示された方法（Protein Engineering, 7, 697, (1994)）に従って、抗体の立体構造予測に基づいて選択することができる。本発明で使用されるｄｓＦｖに含まれるＶＨ又はＶＬは、本発明のモノクローナル抗体のものであればよい。本発明で使用されるｄｓＦｖは、本発明のモノクローナル抗体のＶＨ及びＶＬをコードするｃＤＮＡを用いて、適当な発現ベクターに挿入してｄｓＦｖ発現ベクターを構築し、該発現ベクターを大腸菌、酵母又は動物細胞へ導入し、発現させることによって製造することができる。 dsFv refers to a polypeptide in which one amino acid residue in each of VH and VL is replaced with a cysteine residue, which are linked via an SS bond. Amino acid residues to be substituted for cysteine residues can be selected based on the prediction of the three-dimensional structure of the antibody according to the method described by Reiter et al. (Protein Engineering, 7, 697, (1994)). The VH or VL contained in the dsFv used in the present invention may be one of the monoclonal antibody of the present invention. The dsFv used in the present invention is constructed by inserting the cDNA encoding the VH and VL of the monoclonal antibody of the present invention into an appropriate expression vector, and then transducing the expression vector into Escherichia coli, yeast, or an animal. It can be produced by introducing it into cells and expressing it.
　Ｄｉａｂｏｄｙは、抗原結合特異性が同じ又は異なるｓｃＦｖが２量体を形成した抗体断片であり、同じ抗原に対する２価の抗原結合活性又は異なる抗原に対する２種類の特異的な抗原結合活性を有する抗体断片である。例えば、本発明のモノクローナル抗体に特異的に反応する２価のＤｉａｂｏｄｙは、本発明のモノクローナル抗体のＶＨ及びＶＬをコードするｃＤＮＡを用いて、３～１０残基のペプチドリンカーを有するｓｃＦｖをコードするＤＮＡを構築し、該ＤＮＡを細胞用発現ベクターに挿入し、該発現ベクターを大腸菌、酵母又は動物細胞へ導入することによりＤｉａｂｏｄｙを発現させることによって製造することができる。 Diabody is an antibody fragment in which scFv with the same or different antigen-binding specificity forms a dimer, and has bivalent antigen-binding activity for the same antigen or two types of specific antigen-binding activity for different antigens. It is. For example, a bivalent diabody that specifically reacts with the monoclonal antibody of the present invention encodes an scFv having a peptide linker of 3 to 10 residues using cDNA encoding the VH and VL of the monoclonal antibody of the present invention. It can be produced by constructing DNA, inserting the DNA into a cell expression vector, and introducing the expression vector into E. coli, yeast, or animal cells to express the diabody.
　ＣＤＲを含むペプチドは、ＶＨ又はＶＬのＣＤＲの少なくとも１領域以上を含んで構成される。複数のＣＤＲは、直接又は適当なペプチドリンカーを介して結合させることができる。本発明で使用されるＣＤＲを含むペプチドは、本発明のモノクローナル抗体のＶＨ及びＶＬをコードするｃＤＮＡを用いて、ＣＤＲをコードするＤＮＡを構築し、該ＤＮＡを動物細胞用発現ベクターに挿入し、該ベクターを大腸菌、酵母又は動物細胞へ導入することにより発現させることにより、製造することができる。また、ＣＤＲを含むペプチドは、Ｆｍｏｃ法（フルオレニルメチルオキシカルボニル法）、ｔＢｏｃ法（ｔ－ブチルオキシカルボニル法）等の化学合成法によって製造することもできる。 A peptide containing a CDR is composed of at least one region of a VH or VL CDR. Multiple CDRs can be linked directly or via a suitable peptide linker. The CDR-containing peptide used in the present invention is obtained by constructing a CDR-encoding DNA using cDNA encoding the VH and VL of the monoclonal antibody of the present invention, and inserting the DNA into an expression vector for animal cells. It can be produced by introducing the vector into E. coli, yeast, or animal cells and expressing it. Peptides containing CDRs can also be produced by chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method).
　本発明のモノクローナル抗体又はその抗体断片は、ヒトＣＣＲ８に結合することが特徴である。特に、特異的に結合するものが好ましい。 The monoclonal antibody of the present invention or its antibody fragment is characterized by binding to human CCR8. In particular, those that bind specifically are preferred.
　特異的結合は、少なくとも約１×１０－６Ｍ又はそれ未満の平衡解離定数（例えば、Ｋｄが小さいほど、より緊密な結合を表す）を特徴とすることができる。Ｋｄ値は、好ましくは１×１０－７Ｍ以下、より好ましくは１×１０－８Ｍ以下、さらに好ましくは１×１０－９Ｍ以下である。２つの分子が特異的に結合するかどうかを測定する方法は、当該技術分野で周知であり、競合ＥＬＩＳＡ法、表面プラズモン共鳴及び同様のものが挙げられる。 Specific binding can be characterized by an equilibrium dissociation constant of at least about 1×10 −6 M or less (eg, a lower Kd indicates tighter binding). The Kd value is preferably 1×10 −7 M or less, more preferably 1×10 −8 M or less, and still more preferably 1×10 −9 M or less. Methods of determining whether two molecules specifically bind are well known in the art and include competitive ELISA methods, surface plasmon resonance, and the like.
　本発明は、ヒトＣＣＲ８への結合に関して、本明細書中に記載されているモノクローナル抗体又はその抗体断片と競合するモノクローナル抗体又はその抗体断片を含む。
　「競合するモノクローナル抗体又はその抗体断片」は、本発明のモノクローナル抗体又はその抗体断片（好ましくは、配列番号１２のアミノ酸配列を有する軽鎖可変領域、及び、配列番号１３のアミノ酸配列を有する重鎖可変領域を含むモノクローナル抗体又はその断片）の、ヒトＣＣＲ８への特異的結合を阻害する抗体又はその抗体断片を意味する。あるモノクローナル抗体又はその抗体断片が本発明のモノクローナル抗体又はその抗体断片と競合するかどうかを決定する。Ｉｓｏｔｙｐｅ　ｃｏｎｔｒｏｌ抗体の共存下、抗原（ヒトＣＣＲ８）との結合が確認できた抗体のうち、本発明のモノクローナル抗体又はその抗体断片の共存下では結合シグナルが顕著に低下する抗体を、本発明のモノクローナル抗体又はその抗体断片と競合する抗体として同定することができる。該結合シグナルの低下は、好ましくは５０％、より好ましくは７０％である。また、本発明のモノクローナル抗体又はその抗体断片と競合する抗体の、該本発明のモノクローナル抗体又はその抗体断片と抗原との結合に対するＫｉ値は、好ましくは１×１０－７Ｍ以下、より好ましくは１×１０－８Ｍ以下、さらに好ましくは１×１０－９Ｍ以下である。 The invention includes monoclonal antibodies or antibody fragments thereof that compete with the monoclonal antibodies or antibody fragments thereof described herein for binding to human CCR8.
"Competing monoclonal antibody or antibody fragment thereof" refers to the monoclonal antibody of the present invention or antibody fragment thereof (preferably a light chain variable region having the amino acid sequence of SEQ ID NO: 12 and a heavy chain having the amino acid sequence of SEQ ID NO: 13). It refers to an antibody or an antibody fragment thereof that inhibits the specific binding of a monoclonal antibody containing a variable region (or a fragment thereof) to human CCR8. It is determined whether a monoclonal antibody or antibody fragment thereof competes with a monoclonal antibody or antibody fragment thereof of the invention. Among the antibodies that were confirmed to bind to the antigen (human CCR8) in the presence of the isotype control antibody, those antibodies whose binding signal was significantly reduced in the presence of the monoclonal antibody of the present invention or its antibody fragment were selected as the monoclonal antibody of the present invention. It can be identified as an antibody that competes with the antibody or antibody fragment thereof. The reduction in binding signal is preferably 50%, more preferably 70%. Further, the Ki value of an antibody that competes with the monoclonal antibody of the present invention or its antibody fragment for binding of the monoclonal antibody of the present invention or its antibody fragment to an antigen is preferably 1×10 −7 M or less, more preferably It is 1×10 −8 M or less, more preferably 1×10 −9 M or less.
　本発明のモノクローナル抗体又はその抗体断片は、ＣＣＲ８とＣＣＬ１との結合をほとんど阻害しない、つまり中和活性をほとんど有さないことが特徴である。
　ＣＣＲ８とＣＣＬ１の結合阻害能については、例えば、ヒトＣＣＬ１の場合、ヒトＣＣＲ８発現２９３細胞を用い、ヒトＣＣＬ１添加によるＣａ２＋流入を測定し、ヒトＣＣＬ１非添加時のシグナルを阻害率１００％、ヒトＣＣＬ１添加及び抗体非添加時のシグナルを阻害率０％として、ＩＣ５０値を計算することにより決定することができる。例えば、後述の実施例３記載の方法等にて測定することができる。
　ヒトＣＣＬ１は、ＵｎｉＰｒｏｔＫＢ／Ｓｗｉｓｓ－Ｐｒｏｔ　Ｎｏ．Ｐ２２３６２などに示されるアミノ酸配列を有する。 The monoclonal antibody of the present invention or its antibody fragment is characterized in that it hardly inhibits the binding between CCR8 and CCL1, that is, it has almost no neutralizing activity.
Regarding the ability to inhibit the binding between CCR8 and CCL1, for example, in the case of human CCL1, using human CCR8-expressing 293 cells, Ca 2+ influx upon addition of human CCL1 was measured, and the signal when human CCL1 was not added was determined to be 100% inhibition rate, human The IC50 value can be determined by calculating the IC50 value, assuming that the signal obtained when CCL1 is added and when the antibody is not added is regarded as an inhibition rate of 0%. For example, it can be measured by the method described in Example 3 below.
Human CCL1 was obtained from UniProtKB/Swiss-Prot No. It has an amino acid sequence such as P22362.
　本発明のモノクローナル抗体は、本明細書記載のＣＤＲ又は重鎖可変領域／軽鎖可変領域を用いて当該技術分野の定法によって作製することができる。 The monoclonal antibody of the present invention can be produced by a conventional method in the art using the CDRs or heavy chain variable region/light chain variable region described herein.
　本発明のモノクローナル抗体は、キメラ、ヒト化、完全ヒト、抗体低分子複合体（ＡＤＣ）及び二重特異的抗体も含む。
　ヒト化モノクローナル抗体はヒト体内における抗原性が低下しているため、治療目的等でヒトに投与する場合に有用である。ヒト化モノクローナル抗体は、ヒト以外の哺乳動物、例えばマウス抗体の相補性決定領域（ＣＤＲ；　ｃｏｍｐｌｅｍｅｎｔａｒｉｔｙ　ｄｅｔｅｒｍｉｎｉｎｇ　ｒｅｇｉｏｎ）をヒト抗体のフレームワーク領域（ＦＲ；　ｆｒａｍｅｗｏｒｋ　ｒｅｇｉｏｎ）へ移植したものである。従って、ヒト化モノクローナル抗体のＦＲは、ヒト由来のものである。適当なＦＲは、Ｋａｂａｔ　Ｅ．Ａ．らの文献を参照すれば選択できる。この場合のＦＲとしては、ＣＤＲが良好な抗原結合部位を形成することができるものが選択される。必要に応じ、再構成したヒト化モノクローナル抗体のＣＤＲが適切な抗原結合部位を形成するように、抗体の可変領域のＦＲのアミノ酸を置換してもよい（Sato. et al, Cancer Res. (1993), 53, 851）。置換されるＦＲのアミノ酸の割合は、全ＦＲ領域の０～１５％、好ましくは０～５％である。 Monoclonal antibodies of the invention also include chimeric, humanized, fully human, antibody small molecule conjugates (ADCs) and bispecific antibodies.
Since humanized monoclonal antibodies have reduced antigenicity in the human body, they are useful when administered to humans for therapeutic purposes. A humanized monoclonal antibody is one in which the complementarity determining regions (CDRs) of a non-human mammal, such as a mouse antibody, are grafted onto the framework regions (FRs) of a human antibody. Therefore, the FRs of the humanized monoclonal antibody are of human origin. A suitable FR is Kabat E. A. You can make a selection by referring to the literature by et al. In this case, FRs are selected that allow CDRs to form a good antigen-binding site. If necessary, amino acids in the FRs of the antibody variable region may be substituted so that the CDRs of the reconstituted humanized monoclonal antibody form a suitable antigen-binding site (Sato. et al, Cancer Res. (1993) ), 53, 851). The proportion of FR amino acids to be replaced is 0-15%, preferably 0-5% of the total FR region.
　本発明のヒト化モノクローナル抗体は、好ましくは、
配列番号２のアミノ酸配列からなるＣＤＲ１、
配列番号３のアミノ酸配列からなるＣＤＲ２及び
配列番号４のアミノ酸配列からなるＣＤＲ３を含む軽鎖可変領域並びに
配列番号５のアミノ酸配列からなるＣＤＲ１、
配列番号６のアミノ酸配列からなるＣＤＲ２及び
配列番号７のアミノ酸配列からなるＣＤＲ３を含む重鎖可変領域
を含む。
　より好ましくは、
配列番号８のアミノ酸配列を有する軽鎖可変領域、及び、配列番号９のアミノ酸配列を有する重鎖可変領域を含む。 The humanized monoclonal antibody of the present invention preferably comprises:
CDR1 consisting of the amino acid sequence of SEQ ID NO: 2,
A light chain variable region comprising CDR2 consisting of the amino acid sequence of SEQ ID NO: 3 and CDR3 consisting of the amino acid sequence of SEQ ID NO: 4, and CDR1 consisting of the amino acid sequence of SEQ ID NO: 5,
It contains a heavy chain variable region including CDR2 consisting of the amino acid sequence of SEQ ID NO: 6 and CDR3 consisting of the amino acid sequence of SEQ ID NO: 7.
More preferably,
It includes a light chain variable region having the amino acid sequence of SEQ ID NO: 8 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 9.
　本発明のモノクローナル抗体の定常領域の配列は特に限定されないが、配列番号１０のアミノ酸配列を有する軽鎖定常領域、及び、配列番号１１のアミノ酸配列を有する重鎖定常領域が例示される。 The sequence of the constant region of the monoclonal antibody of the present invention is not particularly limited, but examples include a light chain constant region having the amino acid sequence of SEQ ID NO: 10 and a heavy chain constant region having the amino acid sequence of SEQ ID NO: 11.
　本発明のモノクローナル抗体がヒト化モノクローナル抗体の場合、ヒト抗体の定常領域が使用される。好ましいヒト抗体の定常領域としては、重鎖としてはＣγが挙げられ、例えば、Ｃγ１，Ｃγ２，Ｃγ３，Ｃγ４を、軽鎖としてはＣκ、Ｃλを使用することができる。また、抗体又はその産生の安定性を改善するために、ヒト抗体のＣ領域を修飾してもよい。ヒト化の際に用いられるヒト抗体は、ＩｇＧ、ＩｇＭ、ＩｇＡ、ＩｇＥ、ＩｇＤ等いかなるアイソタイプのヒト抗体でもよいが、本発明においてはＩｇＧを用いることが好ましい。 When the monoclonal antibody of the present invention is a humanized monoclonal antibody, the constant region of a human antibody is used. Preferred constant regions of human antibodies include Cγ as a heavy chain, such as Cγ1, Cγ2, Cγ3, and Cγ4, and Cκ and Cλ as light chains. Additionally, the C region of human antibodies may be modified to improve the stability of the antibody or its production. The human antibody used in humanization may be of any isotype such as IgG, IgM, IgA, IgE, IgD, etc., but it is preferable to use IgG in the present invention.
　本発明のモノクローナル抗体は一般的な製造方法によって作製することができる（例えば、ＷＯ９５／１４０４１号公報、ＷＯ９６／０２５７６号公報等参照）。本発明で使用される抗体を製造するには、抗体遺伝子を発現制御領域、例えば、エンハンサー／プロモーターの制御のもとで発現するよう発現ベクターに組み込む。次に、この発現ベクターにより宿主細胞を形質転換し、抗体を発現させることができる。 The monoclonal antibody of the present invention can be produced by a general production method (for example, see WO95/14041, WO96/02576, etc.). To produce the antibody used in the present invention, the antibody gene is inserted into an expression vector so that it is expressed under the control of an expression control region, such as an enhancer/promoter. A host cell can then be transformed with this expression vector to express the antibody.
　上記の形質転換体の宿主細胞としては、例えば、ＣＯＳ細胞、ＣＨＯ細胞等の脊椎動物細胞、原核細胞、酵母等が挙げられる。形質転換体は、当業者に周知の方法に従って培養することができ、該培養により、形質転換体細胞内又は細胞外に本発明のモノクローナル抗体が産生される。該培養に用いられる培地としては、採用した宿主細胞に応じて慣用される各種のものを適宜選択でき、例えば、ＣＯＳ細胞の場合、ＲＰＭＩ－１６４０培地やダルベッコ修正イーグル最小必須培地（ＤＭＥＭ）等の培地に、必要に応じウシ胎児血清（ＦＢＳ）等の血清成分を添加したものを使用できる。該形質転換体の培養の際の培養温度は、細胞内のタンパク質合成能を著しく低下せしめない温度であればいずれでもよいが、好適には３２～４２℃、最も好適には３７℃で培養することが好ましい。また必要に応じて、１～１０％（ｖ／ｖ）の炭酸ガスを含む空気中で培養することができる。 Examples of host cells for the above transformants include vertebrate cells such as COS cells and CHO cells, prokaryotic cells, and yeast. The transformant can be cultured according to methods well known to those skilled in the art, and the monoclonal antibody of the present invention is produced within or outside the transformant cells. The culture medium used for the culture can be appropriately selected from various commonly used media depending on the host cell employed. For example, in the case of COS cells, RPMI-1640 medium, Dulbecco's modified Eagle's minimum essential medium (DMEM), etc. A culture medium to which a serum component such as fetal bovine serum (FBS) is added, if necessary, can be used. The culture temperature for culturing the transformant may be any temperature as long as it does not significantly reduce the intracellular protein synthesis ability, but it is preferably cultured at 32 to 42°C, most preferably at 37°C. It is preferable. Furthermore, if necessary, the culture can be carried out in air containing 1 to 10% (v/v) carbon dioxide gas.
　上記により形質転換体の細胞内又は細胞外に生産される本発明のモノクローナル抗体を含む画分は、該タンパク質の物理的性質や化学的性質等を利用した各種公知の分離操作法により、分離・精製することができる。かかる方法としては、具体的には例えば通常のタンパク質沈澱剤による処理、限外濾過、分子ふるいクロマトグラフィー（ゲル濾過）、吸着クロマトグラフィー、イオン交換クロマトグラフィー、アフィニティークロマトグラフィー、高速液体クロマトグラフィー（ＨＰＬＣ）等の各種クロマトグラフィー、透析法、及びこれらの組み合わせ等を採用できる。該方法により、容易に高収率、高純度で本発明のモノクローナル抗体を製造できる。 The fraction containing the monoclonal antibody of the present invention produced intracellularly or extracellularly in the transformant as described above can be separated and separated by various known separation methods that utilize the physical and chemical properties of the protein. Can be purified. Specifically, such methods include, for example, treatment with a normal protein precipitant, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC). ), various chromatography methods, dialysis methods, and combinations thereof can be employed. By this method, the monoclonal antibody of the present invention can be easily produced with high yield and high purity.
　本発明のモノクローナル抗体又はその抗体断片は、さらに、ポリエチレングリコール（ＰＥＧ）、放射性物質、トキシン等の各種分子により修飾されていてもよい。抗体の修飾方法はこの分野で公知の方法を利用することができる。 The monoclonal antibody of the present invention or its antibody fragment may be further modified with various molecules such as polyethylene glycol (PEG), radioactive substances, and toxins. Methods known in this field can be used to modify antibodies.
　また本発明のモノクローナル抗体は、そのＮ末端あるいはＣ末端に他のタンパク質を融合してもよい（Clinical Cancer Research, 2004, 10, 1274-1281）。融合するタンパク質は当業者が適宜選択することができる。 The monoclonal antibody of the present invention may also be fused with other proteins to its N-terminus or C-terminus (Clinical Cancer Research, 2004, 10, 1274-1281). Proteins to be fused can be appropriately selected by those skilled in the art.
　本発明のモノクローナル抗体には、抗体のＦｃ領域にＮ－グリコシド結合糖鎖が結合している抗体が含まれる。なお、該Ｎ－グリコシド結合糖鎖の還元末端のＮ－アセチルグルコサミンにフコースが結合していなくてもよい。抗体のＦｃ領域にＮ－グリコシド結合糖鎖が結合し、該Ｎ－グリコシド結合糖鎖の還元末端のＮ－アセチルグルコサミンにフコースが結合していない抗体としては、例えばα１，６－フコース転移酵素遺伝子が欠損したＣＨＯ細胞（国際公開第２００５／０３５５８６号、国際公開第０２／３１１４０号）を用いて作製される抗体が挙げられる。抗体のＦｃ領域にＮ－グリコシド結合糖鎖が結合し、該Ｎ－グリコシド結合糖鎖の還元末端のＮ－アセチルグルコサミンにフコースが結合していない本発明の抗体は、高いＡＤＣＣ活性を有する。 The monoclonal antibodies of the present invention include antibodies in which N-glycoside-linked sugar chains are bound to the Fc region of the antibody. Note that fucose does not need to be bound to N-acetylglucosamine at the reducing end of the N-glycoside-linked sugar chain. An example of an antibody in which an N-glycoside-linked sugar chain is bound to the Fc region of the antibody and fucose is not bound to N-acetylglucosamine at the reducing end of the N-glycoside-linked sugar chain is, for example, an α1,6-fucosyltransferase gene. Examples include antibodies produced using CHO cells deficient in CHO cells (WO 2005/035586, WO 02/31140). The antibody of the present invention, in which an N-glycoside-linked sugar chain is bound to the Fc region of the antibody and fucose is not bound to N-acetylglucosamine at the reducing end of the N-glycoside-linked sugar chain, has high ADCC activity.
　本発明のモノクローナル抗体又はその抗体断片は、Ｔｒｅｇ細胞又はマクロファージ細胞を除去させるため、ＣＣＲ８を発現する細胞に対してＡＤＣＣ（Ａｎｔｉｂｏｄｙ－ｄｅｐｅｎｄｅｎｔ　ｃｅｌｌ　ｍｅｄｉａｔｅｄ　ｃｙｔｏｔｏｘｉｃｉｔｙ；抗体依存性細胞介在性細胞傷害）活性を有していてもよい。ＡＤＣＣ活性とは、生体内で、標的細胞等の細胞表面抗原などに結合した抗体が、抗体のＦｃ領域とエフェクター細胞表面上に存在するＦｃレセプターとの結合を介してエフェクター細胞を活性化し、標的細胞等を傷害する活性を意味する。エフェクター細胞としては、ナチュラルキラー細胞、活性化されたマクロファージ等があげられる。なお、本発明においては、生体内で、Ｔｒｅｇ細胞又はマクロファージ細胞等の細胞表面抗原（ＣＣＲ８）に結合した抗体が、抗体のＦｃ領域とエフェクター細胞表面上に存在するＦｃレセプターとの結合を介してエフェクター細胞を活性化し、Ｔｒｅｇ細胞又はマクロファージ細胞等を傷害し、結果として腫瘍細胞等を傷害する場合も含む。 The monoclonal antibody or antibody fragment thereof of the present invention has ADCC (Antibody-dependent cell mediated cytotoxicity) activity against cells expressing CCR8 in order to remove Treg cells or macrophage cells. You may do so. ADCC activity means that in vivo, an antibody that binds to a cell surface antigen such as a target cell activates the effector cell through the binding between the Fc region of the antibody and the Fc receptor present on the surface of the effector cell. Refers to activity that damages cells, etc. Effector cells include natural killer cells, activated macrophages, and the like. In addition, in the present invention, in vivo, an antibody bound to a cell surface antigen (CCR8) such as a Treg cell or a macrophage cell is activated through the binding between the Fc region of the antibody and an Fc receptor present on the surface of an effector cell. It also includes cases where effector cells are activated, Treg cells or macrophage cells are injured, and as a result, tumor cells and the like are injured.
　本発明のモノクローナル抗体又はその抗体断片は、ＣＣＲ８に対する結合がＣＣＲ８の中和抗体と競合しない。ＣＣＲ８の中和抗体とは、ＣＣＲ８に対する中和活性を有する抗体を意味する。ＣＣＲ８に対する中和活性を有するか否かは、例えば、ＣＣＲ８リガンドのいずれか（例えば、ＣＣＬ１）のＣＣＲ８に対する生理作用を抑制するか否かを測定することにより判別できる。例としては、これらに限定されるものではないが、ＣＣＬ１のＣＣＲ８への結合、あるいはＣＣＬ１によるＣＣＲ８発現細胞の遊走又は細胞内Ｃａ２＋増加又はＣＣＬ１刺激に感受性のある遺伝子の発現変動などを測定することが挙げられる。例えば、後述の実施例３記載の方法等にて測定することができる。
　「ＣＣＲ８の中和抗体」のＣＣＲ８とＣＣＬ１の結合に対する中和活性は、あらかじめＣａ２＋指示薬を取り込ませたヒトＣＣＲ８発現２９３細胞に対し、培地で希釈した抗体希釈液を添加して測定することができる。ＣＣＲ８とＣＣＲ８リガンドの親和性は、ＣＣＬ１が最も強く、ＣＣＬ１に対して高い阻害活性を有する抗体が有用である。
　「ＣＣＲ８の中和抗体」は、ＣＣＲ８とＣＣＬ１の結合に対する中和活性のＩＣ５０値が１０ｎＭ以下であることが好ましい。中和活性は、より好ましくは、ＩＣ５０値が５ｎＭ以下、さらに好ましくは２ｎＭ以下、特に好ましくは１ｎＭ以下、最も好ましくは０．５ｎＭ以下である。
　抗ＣＣＲ８抗体を含有する医薬に含まれる抗ＣＣＲ８抗体として用いる「ＣＣＲ８の中和抗体」は、ＡＤＣＣ活性を有するのが好ましい。 
　「ＣＣＲ８の中和抗体」としては、特許文献４～９及び１５の実施例に記載された抗体、ＢｉｏＬｅｇｅｎｄ社の品番Ｌ２６３Ｇ８の抗ＣＣＲ８抗体、Ｒ＆Ｄ社の品番１９１７０４の抗ＣＣＲ８抗体等が例示されるが、配列番号８のアミノ酸配列を有する軽鎖可変領域、及び、配列番号９のアミノ酸配列を有する重鎖可変領域を含む抗体とＣＣＲ８に対する結合が競合しない抗ＣＣＲ８抗体が好ましく、配列番号１２のアミノ酸配列を有する軽鎖可変領域、及び、配列番号１３のアミノ酸配列を有する重鎖可変領域を含む抗ＣＣＲ８抗体が最も好ましい。 The monoclonal antibody of the present invention or its antibody fragment does not compete with CCR8-neutralizing antibodies for binding to CCR8. A CCR8 neutralizing antibody means an antibody having neutralizing activity against CCR8. Whether or not it has neutralizing activity against CCR8 can be determined, for example, by measuring whether the physiological effect of any CCR8 ligand (eg, CCL1) on CCR8 is suppressed. Examples include, but are not limited to, measuring the binding of CCL1 to CCR8, the migration of CCR8-expressing cells by CCL1, an increase in intracellular Ca 2+ , or changes in the expression of genes sensitive to CCL1 stimulation. This can be mentioned. For example, it can be measured by the method described in Example 3 below.
The neutralizing activity of the "CCR8 neutralizing antibody" against the binding between CCR8 and CCL1 can be measured by adding a diluted antibody solution diluted with a medium to human CCR8-expressing 293 cells that have been pre-incorporated with a Ca 2+ indicator. can. The affinity between CCR8 and CCR8 ligand is strongest for CCL1, and antibodies having high inhibitory activity against CCL1 are useful.
The "CCR8 neutralizing antibody" preferably has an IC50 value of neutralizing activity against the binding of CCR8 and CCL1 of 10 nM or less. The neutralizing activity preferably has an IC50 value of 5 nM or less, further preferably 2 nM or less, particularly preferably 1 nM or less, and most preferably 0.5 nM or less.
The "CCR8 neutralizing antibody" used as the anti-CCR8 antibody contained in the anti-CCR8 antibody-containing medicine preferably has ADCC activity.
Examples of "neutralizing antibodies for CCR8" include antibodies described in Examples of Patent Documents 4 to 9 and 15, anti-CCR8 antibodies with product number L263G8 from BioLegend, and anti-CCR8 antibodies with product number 191704 from R&D. However, an anti-CCR8 antibody that does not compete in binding to CCR8 with an antibody comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 8 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 9 is preferable; Most preferred is an anti-CCR8 antibody comprising a light chain variable region having the sequence and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13.
　本発明のモノクローナル抗体又はその抗体断片は、ヒトＣＣＲ８を強く認識する抗体又はその抗体断片が好ましい。ヒトＣＣＲ８を強く認識する抗体又はその抗体断片を選択する際に、ＣＣＲ８に対する結合活性の強度や結合選択性を指標にして抗体又はその抗体断片を選択することで、より強くヒトＣＣＲ８を認識する抗体又はその抗体断片を選択することができる。 The monoclonal antibody or antibody fragment thereof of the present invention is preferably an antibody or antibody fragment thereof that strongly recognizes human CCR8. When selecting an antibody or an antibody fragment thereof that strongly recognizes human CCR8, it is possible to select an antibody or an antibody fragment thereof based on the strength of binding activity and binding selectivity for CCR8 as an index, thereby creating an antibody that more strongly recognizes human CCR8. or an antibody fragment thereof can be selected.
　本発明のモノクローナル抗体又はその抗体断片は、ＣＣＲ８に対する結合がＣＣＲ８の中和抗体と競合しないため、ＣＣＲ８の中和抗体がすでに結合した試料であっても、ＣＣＲ８の検出が可能である。さらに、試料に結合させたＣＣＲ８の中和抗体によっては、本発明のモノクローナル抗体又はその抗体断片のＣＣＲ８に対する結合活性をむしろ増加させうる。
　したがって、本発明のモノクローナル抗体又はその抗体断片は、抗ＣＣＲ８抗体を含有する医薬を投与した、癌の患者等の被験者から採取した試料に含まれるＣＣＲ８を検出することが可能であり、さらに、抗ＣＣＲ８抗体を含有する医薬に含まれる抗ＣＣＲ８抗体の腫瘍内浸潤Ｔｒｅｇ細胞への結合量を測定することもできる。抗ＣＣＲ８抗体を含有する医薬に含まれる抗ＣＣＲ８抗体としては、上記「ＣＣＲ８の中和抗体」が好ましく、特許文献４～９の実施例に記載された抗ＣＣＲ８抗体がより好ましく、特許文献４の実施例に記載された抗ＣＣＲ８抗体がさらに好ましく、配列番号８のアミノ酸配列を有する軽鎖可変領域、及び、配列番号９のアミノ酸配列を有する重鎖可変領域を含む抗体とＣＣＲ８に対する結合が競合しない抗ＣＣＲ８抗体が特に好ましく、配列番号１２のアミノ酸配列を有する軽鎖可変領域、及び、配列番号１３のアミノ酸配列を有する重鎖可変領域を含む抗ＣＣＲ８抗体が最も好ましい。 Since the monoclonal antibody of the present invention or its antibody fragment does not compete with the neutralizing antibody for CCR8 in binding to CCR8, detection of CCR8 is possible even in a sample to which a neutralizing antibody for CCR8 has already bound. Furthermore, depending on the CCR8-neutralizing antibody bound to the sample, the CCR8-binding activity of the monoclonal antibody of the present invention or its antibody fragment may be increased.
Therefore, the monoclonal antibody or antibody fragment thereof of the present invention can detect CCR8 contained in a sample collected from a subject, such as a cancer patient, who has been administered a medicine containing an anti-CCR8 antibody, and It is also possible to measure the amount of anti-CCR8 antibody contained in a medicament containing a CCR8 antibody that binds to intratumoral infiltrating Treg cells. As the anti-CCR8 antibody contained in the anti-CCR8 antibody-containing medicine, the above-mentioned "CCR8 neutralizing antibody" is preferable, the anti-CCR8 antibodies described in the Examples of Patent Documents 4 to 9 are more preferable, and the anti-CCR8 antibodies described in the Examples of Patent Documents 4 to 9 are more preferable. More preferred are the anti-CCR8 antibodies described in the Examples, which do not compete in binding to CCR8 with antibodies comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 8 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 9. Anti-CCR8 antibodies are particularly preferred, and anti-CCR8 antibodies comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 12 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13 are most preferred.
　本発明のモノクローナル抗体又はその抗体断片は、ＣＣＲ８に特異的に結合するため、ヒトから採取された試料においてＣＣＲ８を検出するために使用することができる。試料としては、特に限定されないが、例えば、血液、血漿、血清、尿、臓器、組織、骨髄、リンパ節等が挙げられる。 Since the monoclonal antibody of the present invention or its antibody fragment specifically binds to CCR8, it can be used to detect CCR8 in samples collected from humans. Examples of the sample include, but are not limited to, blood, plasma, serum, urine, organs, tissues, bone marrow, lymph nodes, and the like.
　本発明のモノクローナル抗体又はその抗体断片がＣＣＲ８検出用抗体として用いられる場合、ＣＣＲ８を検出する際に、標識された検出用抗体を用いてもよいし、検出用抗体に対する標識二次抗体を用いてもよい。標識に用いる物質としては、蛍光物質、微小粒子、酵素、ビオチン、放射性標識体、磁性粒子等が例示されるが、蛍光物質が好ましい。 When the monoclonal antibody of the present invention or an antibody fragment thereof is used as an antibody for detecting CCR8, a labeled detection antibody may be used when detecting CCR8, or a labeled secondary antibody for the detection antibody may be used. Good too. Examples of substances used for labeling include fluorescent substances, microparticles, enzymes, biotin, radiolabels, magnetic particles, and the like, with fluorescent substances being preferred.
　蛍光物質としては、抗体又はその抗体断片の抗原結合性を損なわない限りにおいて特に限定されず、例えば、ローダミン系、クマリン系、オキサジン系、カルボピロニン系、シアニン系、ピロメセン系、ナフタレン系、ビフェニル系、アントラセン系、フェナントレン系、ピレン系、カルバゾール系、Ｃｙ系、ＥｖｏＢｌｕｅ系、フルオレセイン系又はこれらの誘導体等が挙げられる。 The fluorescent substance is not particularly limited as long as it does not impair the antigen binding property of the antibody or its antibody fragment, and includes, for example, rhodamine, coumarin, oxazine, carbopyronine, cyanine, pyromecene, naphthalene, biphenyl, Examples include anthracene, phenanthrene, pyrene, carbazole, Cy, EvoBlue, fluorescein, and derivatives thereof.
　微小粒子としては、抗体又はその抗体断片の抗原結合性を損なわない限りにおいて特に限定されず、例えば、金コロイドのような金属コロイド粒子、着色ラテックス粒子のような着色粒子等が挙げられる。 The microparticles are not particularly limited as long as they do not impair the antigen binding properties of the antibody or antibody fragment thereof, and include, for example, metal colloid particles such as colloidal gold, colored particles such as colored latex particles, and the like.
　酵素としては、抗体又はその抗体断片の抗原結合性を損なわない限りにおいて特に限定されず、ホースラディッシュ・ペルオキシダーゼ（ＨＲＰ）、アルカリホスファターゼ、ペルオキシダーゼ、β－Ｄ－ガラクトシダーゼ、マイクロペルオキシダーゼ等が挙げられる。 The enzyme is not particularly limited as long as it does not impair the antigen binding property of the antibody or its antibody fragment, and examples include horseradish peroxidase (HRP), alkaline phosphatase, peroxidase, β-D-galactosidase, microperoxidase, and the like.
　放射性標識体としては、特に限定されないが、例えば、３Ｈ、１４Ｃ、１５Ｎ、３５Ｓ、９０Ｙ、９９Ｔｃ、１１１Ｉｎ、１２５Ｉ、１３１Ｉ等が挙げられる。 Examples of radioactive labels include, but are not limited to, 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I, and the like.
　本発明の抗体又はその抗体断片は、タグを付加することができる。このようなタグとしては、例えば、ＦＬＡＧ、ＨＡ（ヘマグルチニン）、ＧＳＴ（グルタチオン－Ｓ－トランスフェラーゼ）、Ｍｙｃ、ポリヒスチジン（６×Ｈｉｓタグ等）等が挙げられる。これらのタグは抗体の固相化、精製、検出等に用いることができる。タグ化された抗体又は抗体断片は、例えば、本発明の抗体又はその抗体断片をコードするポリヌクレオチドに、タグをコードするポリヌクレオチドを付加したものを作製し、動物細胞等のｉｎ　ｖｉｖｏ又は無細胞翻訳系等のｉｎ　ｖｉｔｒｏのタンパク質発現システムに導入することにより製造することができる。 A tag can be added to the antibody of the present invention or an antibody fragment thereof. Examples of such tags include FLAG, HA (hemagglutinin), GST (glutathione-S-transferase), Myc, polyhistidine (6×His tag, etc.), and the like. These tags can be used for immobilization, purification, detection, etc. of antibodies. A tagged antibody or antibody fragment is produced by adding a polynucleotide encoding a tag to a polynucleotide encoding the antibody of the present invention or an antibody fragment thereof, and then in vivo in animal cells or the like or in a cell-free manner. It can be produced by introducing it into an in vitro protein expression system such as a translation system.
　本発明のＣＣＲ８の検出方法は、蛍光免疫測定法（ＦＩＡ）、酵素免疫測定法（ＥＬＩＳＡまたはＥＩＡ）、放射免疫測定法（ＲＩＡ）、化学発光免疫測定法、表面プラズモン共鳴法、イムノクロマトグラフィー法等の免疫測定法により測定され、フローサイトメトリー法、免疫組織染色法又はＥＬＩＳＡ法による測定が好ましく、フローサイトメトリー法が特に好ましい。 The detection method of CCR8 of the present invention includes fluorescence immunoassay (FIA), enzyme immunoassay (ELISA or EIA), radioimmunoassay (RIA), chemiluminescence immunoassay, surface plasmon resonance, immunochromatography, etc. Flow cytometry, immunohistological staining, or ELISA is preferred, with flow cytometry being particularly preferred.
　本発明のＣＣＲ８の検出方法がフローサイトメトリー法の場合、例えば、ＣＣＲ８及びＴｒｅｇ細胞に特異的なマーカーの発現を同時に測定する方法で、腫瘍内浸潤細胞等の試料中に含まれるＴｒｅｇ細胞において発現したＣＣＲ８を検出することができる。さらに、同様の方法で、試料よりＣＣＲ８を発現するＴｒｅｇ細胞を分画することもできる。Ｔｒｅｇ細胞に特異的なマーカーとしては、ＦｏｘＰ３、ＣＤ２５、ＴＩＧＩＴなどが例示されるが、ＦｏｘＰ３が好ましい。
　フローサイトメトリー法によりＣＣＲ８及びＴｒｅｇ細胞に特異的なマーカーの発現を同時に測定した場合、Ｘ軸をＴｒｅｇ細胞に特異的なマーカーの検出蛍光強度、Ｙ軸をＣＣＲ８の検出蛍光強度とした場合、ＣＣＲ８を発現するＴｒｅｇ細胞を示すプロットは右上の領域に現れる。なお、プロットの際に、Ｘ軸とＹ軸は上記と逆に設定してもよい。 When the method for detecting CCR8 of the present invention is a flow cytometry method, for example, it is a method of simultaneously measuring the expression of CCR8 and a marker specific to Treg cells, which is expressed in Treg cells contained in a sample such as intratumoral infiltrating cells. CCR8 can be detected. Furthermore, Treg cells expressing CCR8 can also be fractionated from a sample using a similar method. Examples of markers specific to Treg cells include FoxP3, CD25, and TIGIT, with FoxP3 being preferred.
When the expression of CCR8 and Treg cell-specific markers is simultaneously measured by flow cytometry, the X axis is the detected fluorescence intensity of the Treg cell-specific marker, and the Y axis is the detected fluorescence intensity of CCR8. A plot showing Treg cells expressing Treg cells appears in the upper right area. Note that when plotting, the X-axis and Y-axis may be set in the opposite manner to the above.
　本発明のＣＣＲ８の検出方法がフローサイトメトリー法の場合、ＣＣＲ８及びＴｒｅｇ細胞に特異的なマーカーの発現を測定する前にゲートする細胞の表面抗原としては、測定の目的により適宜選択可能であるが、例えば、ＣＤ３、ＣＤ４、ＣＤ４５等が選択されうる。 When the method for detecting CCR8 of the present invention is flow cytometry, the cell surface antigen to be gated before measuring the expression of markers specific to CCR8 and Treg cells can be selected as appropriate depending on the purpose of measurement. , for example, CD3, CD4, CD45, etc. may be selected.
　本発明のＣＣＲ８の検出方法が免疫組織染色法の場合、パラフィン切片等の組織切片の作成、免疫反応、洗浄、発色等の各工程を含め、公知の方法で行うことができる。 When the method for detecting CCR8 of the present invention is an immunohistological staining method, it can be carried out by known methods, including steps such as preparation of tissue sections such as paraffin sections, immunoreaction, washing, and color development.
　本発明のＣＣＲ８の検出方法がＥＬＩＳＡ法の場合、例えば、次のように行う。まず、一方の抗体（捕捉抗体）をＥＬＩＳＡ用プレートのウェル表面に固相化する。次いでウェル表面への非特異的な吸着を防ぐためにブロッキングを行った後、試料を添加し、試料中のＣＣＲ８を該抗体と接触させて複合体を形成させる。該抗体と結合しなかったタンパク質を洗浄により除去した後、標識した他方の抗体（検出抗体）をウェルに加え、ＣＣＲ８に接触させて複合体を形成させ、該標識により検出・定量を行う。定量は、用いた標識に基づき検出したシグナルを、ＣＣＲ８の標準試料を使用して作成した検量線を利用して数値化すること等、公知の方法により行うことができる。 When the CCR8 detection method of the present invention is an ELISA method, it is carried out, for example, as follows. First, one antibody (capture antibody) is immobilized on the well surface of an ELISA plate. Next, after blocking is performed to prevent nonspecific adsorption to the well surface, a sample is added, and CCR8 in the sample is brought into contact with the antibody to form a complex. After removing proteins that did not bind to the antibody by washing, the other labeled antibody (detection antibody) is added to the well, brought into contact with CCR8 to form a complex, and detection and quantification are performed using the label. Quantification can be performed by a known method, such as quantifying a signal detected based on the label used using a calibration curve prepared using a standard sample of CCR8.
　本発明のモノクローナル抗体又はその抗体断片は、癌の診断薬として使用することができる。例えば、乳癌、子宮体癌、子宮頸癌、卵巣癌、前立腺癌、肺癌、胃癌、膵臓癌、頭頚部癌、食道癌、膀胱癌、メラノーマ、大腸癌、腎癌、非ホジキンリンパ腫、尿路上皮癌、肉腫、血球癌（白血病、リンパ腫等）、胆管癌、胆のう癌、甲状腺癌、精巣癌、胸腺癌、肝臓癌等の診断薬として使用することができ、好ましくは乳癌、卵巣癌、肺癌、胃癌、膵臓癌、頭頚部癌、食道癌、メラノーマ、大腸癌、腎癌、尿路上皮癌の診断薬として使用することができる。 The monoclonal antibody of the present invention or its antibody fragment can be used as a diagnostic agent for cancer. For example, breast cancer, endometrial cancer, cervical cancer, ovarian cancer, prostate cancer, lung cancer, stomach cancer, pancreatic cancer, head and neck cancer, esophageal cancer, bladder cancer, melanoma, colorectal cancer, kidney cancer, non-Hodgkin lymphoma, urothelial cancer. It can be used as a diagnostic agent for cancer, sarcoma, blood cell cancer (leukemia, lymphoma, etc.), cholangiocarcinoma, gallbladder cancer, thyroid cancer, testicular cancer, thymus cancer, liver cancer, etc., preferably breast cancer, ovarian cancer, lung cancer, etc. It can be used as a diagnostic agent for gastric cancer, pancreatic cancer, head and neck cancer, esophageal cancer, melanoma, colon cancer, renal cancer, and urothelial cancer.
　なお、本明細書記載の「癌」は、卵巣癌、胃癌等の上皮性の悪性腫瘍のみならず、慢性リンパ性白血病やホジキンリンパ腫等の造血器がんを含む非上皮性の悪性腫瘍も意味するものとし、本明細書において、「がん（ｃａｎｃｅｒ）」、「癌（ｃａｒｃｉｎｏｍａ）」、「腫瘍（ｔｕｍｏｒ）」、「新生物（ｎｅｏｐｌａｓｍ）」等の用語は互いに区別されず、相互に交換可能である。 Note that "cancer" as used herein refers not only to epithelial malignant tumors such as ovarian cancer and gastric cancer, but also to non-epithelial malignant tumors including hematopoietic cancers such as chronic lymphocytic leukemia and Hodgkin's lymphoma. In this specification, terms such as "cancer," "carcinoma," "tumor," and "neoplasm" are not distinguished from each other and are used interchangeably. It is possible.
　本発明のモノクローナル抗体又はその抗体断片は、抗ＣＣＲ８抗体を含有する医薬の投与の適否を判断するための診断薬としても使用することができる。「抗ＣＣＲ８抗体を含有する医薬」の抗ＣＣＲ８抗体としては、上記「ＣＣＲ８の中和抗体」が好ましく、特許文献４～９の実施例に記載された抗ＣＣＲ８抗体がより好ましく、特許文献４の実施例に記載された抗ＣＣＲ８抗体がさらに好ましく、配列番号８のアミノ酸配列を有する軽鎖可変領域、及び、配列番号９のアミノ酸配列を有する重鎖可変領域を含む抗体とＣＣＲ８に対する結合が競合しない抗ＣＣＲ８抗体が特に好ましく、配列番号１２のアミノ酸配列を有する軽鎖可変領域、及び、配列番号１３のアミノ酸配列を有する重鎖可変領域を含む抗ＣＣＲ８抗体が最も好ましい。
　「抗ＣＣＲ８抗体を含有する医薬」は抗癌剤として有用であり、例えば、乳癌、子宮体癌、子宮頸癌、卵巣癌、前立腺癌、肺癌、胃癌、膵臓癌、頭頚部癌、食道癌、膀胱癌、メラノーマ、大腸癌、腎癌、非ホジキンリンパ腫、尿路上皮癌、肉腫、血球癌（白血病、リンパ腫等）、胆管癌、胆のう癌、甲状腺癌、精巣癌、胸腺癌、肝臓癌等の癌、好ましくは乳癌、卵巣癌、肺癌、胃癌、膵臓癌、頭頚部癌、食道癌、メラノーマ、大腸癌、腎癌、尿路上皮癌を治療及び／又は予防するための医薬として非常に有用である。 The monoclonal antibody of the present invention or its antibody fragment can also be used as a diagnostic agent to determine the suitability of administering a drug containing an anti-CCR8 antibody. As the anti-CCR8 antibody of the "medicine containing an anti-CCR8 antibody", the above-mentioned "CCR8 neutralizing antibody" is preferable, the anti-CCR8 antibodies described in the Examples of Patent Documents 4 to 9 are more preferable, and the anti-CCR8 antibodies described in the Examples of Patent Documents 4 to 9 are more preferable. More preferred are the anti-CCR8 antibodies described in the Examples, which do not compete in binding to CCR8 with antibodies comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 8 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 9. Anti-CCR8 antibodies are particularly preferred, and anti-CCR8 antibodies comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 12 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13 are most preferred.
"Pharmaceuticals containing anti-CCR8 antibodies" are useful as anticancer agents, such as breast cancer, endometrial cancer, cervical cancer, ovarian cancer, prostate cancer, lung cancer, stomach cancer, pancreatic cancer, head and neck cancer, esophageal cancer, and bladder cancer. , melanoma, colorectal cancer, kidney cancer, non-Hodgkin's lymphoma, urothelial cancer, sarcoma, blood cell cancer (leukemia, lymphoma, etc.), bile duct cancer, gallbladder cancer, thyroid cancer, testicular cancer, thymus cancer, liver cancer, etc. Preferably, it is very useful as a medicine for treating and/or preventing breast cancer, ovarian cancer, lung cancer, gastric cancer, pancreatic cancer, head and neck cancer, esophageal cancer, melanoma, colon cancer, kidney cancer, and urothelial cancer.
　本発明のモノクローナル抗体を含有するキットは、ＣＣＲ８検出用キットとして使用することができる。該キットは、本発明のモノクローナル抗体又はその抗体断片を含み、さらに、ゲート用抗体、標識二次抗体、構成試薬（希釈液、洗浄液、反応停止液等）、蛍光検出試薬、ＣＣＲ８標準品、使用説明書等を含んでいてもよい。 A kit containing the monoclonal antibody of the present invention can be used as a CCR8 detection kit. The kit contains the monoclonal antibody of the present invention or an antibody fragment thereof, and further includes a gating antibody, a labeled secondary antibody, constituent reagents (diluting solution, washing solution, reaction stop solution, etc.), a fluorescence detection reagent, a CCR8 standard product, and usage. It may also include instructions, etc.
　構成試薬に含まれるｐＨ緩衝成分としては、特に限定されないが、例えば、リン酸、ホウ酸、酢酸、クエン酸、ギ酸、カコジル酸等の酸又はこれらの塩；グリシン等のアミノ酸；トリスヒドロキシアミノメタン（Ｔｒｉｓ）、ＨＥＰＥＳ、ＭＥＳ等のグッドバッファー等が挙げられる。 The pH buffer component contained in the constituent reagents is not particularly limited, but includes, for example, acids such as phosphoric acid, boric acid, acetic acid, citric acid, formic acid, and cacodylic acid, or salts thereof; amino acids such as glycine; trishydroxyaminomethane; (Tris), HEPES, MES, and other good buffers.
　構成試薬に含まれる界面活性剤としては、特に限定されないが、非イオン性界面活性剤が好ましく、エステルエーテル型、エステル型、エーテル型のいずれも用いることができる。より具体的にはＴｗｅｅｎ２０、Ｔｗｅｅｎ４０、Ｔｗｅｅｎ８０、Ｔｒｉｔｏｎ－Ｘ１００、ポリオキシエチレン（６０）ソルビタンモノステアレート、ポリオキシエチレン（６５）ソルビタントリステアレート、ポリオキシエチレン（８０）ソルビタンモノオレエート、ポリオキシエチレンアルキルフェニルエーテル、ノニデットＰ－４０、ＣＨＡＰＳ等が挙げられる。これらの界面活性剤は、いずれかを単独で用いても良いし、２種以上を組み合わせても良い。 The surfactant contained in the constituent reagents is not particularly limited, but nonionic surfactants are preferred, and any of the ester ether type, ester type, and ether type can be used. More specifically, Tween20, Tween40, Tween80, Triton-X100, polyoxyethylene (60) sorbitan monostearate, polyoxyethylene (65) sorbitan tristearate, polyoxyethylene (80) sorbitan monooleate, polyoxy Examples include ethylene alkylphenyl ether, Nonidet P-40, CHAPS, and the like. These surfactants may be used alone or in combination of two or more.
　本発明は、本発明のモノクローナル抗体の軽鎖可変領域又は重鎖可変領域をコードするポリヌクレオチドを包含する。本発明は、さらに、該ポリヌクレオチドを含む発現ベクターを包含する。 The present invention includes polynucleotides encoding the light chain variable region or heavy chain variable region of the monoclonal antibody of the present invention. The invention further includes expression vectors containing the polynucleotides.
　該ポリヌクレオチドは、本発明のモノクローナル抗体の軽鎖可変領域又は重鎖可変領域をコードする限り、特に限定されず、複数のデオキシリボ核酸（ＤＮＡ）又はリボ核酸（ＲＮＡ）等のヌクレオチドからなる重合体である。天然以外の塩基を含んでいてよい。本発明のポリヌクレオチドは、抗体を遺伝子工学的な手法により製造するために使用することができる。また本発明のモノクローナル抗体と同等な機能を有する抗体をスクリーニングするために、プローブとして用いることもできる。即ち本発明のモノクローナル抗体をコードするポリヌクレオチド、又はその一部をプローブとして用い、ハイブリダイゼーション、遺伝子増幅技術（例えばＰＣＲ）等の技術により、該ポリヌクレオチドとストリンジェントな条件下でハイブリダイズし、かつ本発明のモノクローナル抗体と同等の活性を有する抗体をコードするＤＮＡを得ることができる。このようなＤＮＡも本発明のポリヌクレオチドに含まれる。 The polynucleotide is not particularly limited as long as it encodes the light chain variable region or heavy chain variable region of the monoclonal antibody of the present invention, and may be a polymer consisting of multiple nucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). It is. It may contain non-natural bases. The polynucleotide of the present invention can be used to produce antibodies by genetic engineering techniques. It can also be used as a probe to screen for antibodies having functions equivalent to the monoclonal antibodies of the present invention. That is, using a polynucleotide encoding the monoclonal antibody of the present invention, or a portion thereof as a probe, hybridize with the polynucleotide under stringent conditions by a technique such as hybridization or gene amplification technique (e.g. PCR), Moreover, DNA encoding an antibody having an activity equivalent to that of the monoclonal antibody of the present invention can be obtained. Such DNA is also included in the polynucleotide of the present invention.
　ハイブリダイゼーション技術（Sambrook,J et al., Molecular Cloning 2nd ed., 9.47-9.58, Cold Spring Harbor Lab. press, 1989）は当業者によく知られた技術である。ハイブリダイゼーションの条件としては、例えば、低ストリンジェントな条件が挙げられる。低ストリンジェントな条件とは、ハイブリダイゼーション後の洗浄において、例えば４２℃、０．１×ＳＳＣ、０．１％ＳＤＳの条件であり、好ましくは５０℃、０．１×ＳＳＣ　、０．１％ＳＤＳの条件である。より好ましいハイブリダイゼーションの条件としては、高ストリンジェントな条件が挙げられる。高ストリンジェントな条件とは、例えば６５℃、５×ＳＳＣ及び０．１％ＳＤＳの条件である。これらの条件において、温度を上げる程に高い相同性を有するポリヌクレオチドが効率的に得られることが期待できる。但し、ハイブリダイゼーションのストリンジェンシーに影響する要素としては温度や塩濃度等複数の要素が考えられ、当業者であればこれら要素を適宜選択することで同様のストリンジェンシーを実現することが可能である。 Hybridization techniques (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58, Cold Spring Harbor Lab. press, 1989) are well known to those skilled in the art. Examples of hybridization conditions include low stringency conditions. Low stringency conditions include, for example, 42°C, 0.1x SSC, 0.1% SDS in washing after hybridization, preferably 50°C, 0.1x SSC, 0.1%. This is a condition of SDS. More preferable hybridization conditions include highly stringent conditions. High stringency conditions are, for example, 65° C., 5×SSC, and 0.1% SDS. Under these conditions, it can be expected that polynucleotides with higher homology can be obtained more efficiently as the temperature is raised. However, multiple factors such as temperature and salt concentration can be considered as factors that affect the stringency of hybridization, and those skilled in the art will be able to achieve similar stringency by appropriately selecting these factors. .
　これらハイブリダイゼーション技術や遺伝子増幅技術により得られるポリヌクレオチドがコードする、本発明のモノクローナル抗体と機能的に同等な抗体は、通常、これら抗体とアミノ酸配列において高い相同性を有する。本発明のモノクローナル抗体には、本発明のモノクローナル抗体と機能的に同等であり、かつ該抗体のアミノ酸配列と高い相同性を有する抗体も含まれる。高い相同性とは、アミノ酸レベルにおいて、通常、少なくとも７５％以上の同一性、好ましくは８５％以上の同一性、さらに好ましくは９５％以上の同一性を指す。ポリペプチドの相同性を決定するには、文献（Wilbur, W. J. and Lipman, D. J. Proc. Natl. Acad. Sci. USA, (1983), 80, 726-730）記載のアルゴリズムに従えばよい。 Antibodies that are functionally equivalent to the monoclonal antibodies of the present invention and encoded by polynucleotides obtained by these hybridization techniques or gene amplification techniques usually have high homology with these antibodies in their amino acid sequences. The monoclonal antibodies of the present invention also include antibodies that are functionally equivalent to the monoclonal antibodies of the present invention and have high homology with the amino acid sequence of the antibodies. High homology generally refers to at least 75% identity, preferably 85% or more identity, and more preferably 95% or more identity at the amino acid level. To determine polypeptide homology, an algorithm described in the literature (Wilbur, W. J. and Lipman, D. J. Proc. Natl. Acad. Sci. USA, (1983), 80, 726-730) was used. Just follow.
　本発明のモノクローナル抗体又はその抗体断片は、腫瘍内浸潤Ｔｒｅｇ細胞除去作用や腫瘍細胞除去作用を有していてもよい。本発明のモノクローナル抗体が腫瘍内浸潤Ｔｒｅｇ細胞除去作用を有しているか否かは、例えば、特許文献２の実施例記載の方法にて測定することができる。 The monoclonal antibody of the present invention or an antibody fragment thereof may have an effect of removing intratumoral infiltrating Treg cells or a tumor cell. Whether or not the monoclonal antibody of the present invention has an effect of removing tumor-infiltrating Treg cells can be determined, for example, by the method described in Examples of Patent Document 2.
　本発明のモノクローナル抗体又はその抗体断片は、医薬組成物にも有用である。本発明のモノクローナル抗体又はその抗体断片を含有する医薬組成物は、経口的又は非経口的に全身あるいは局所的に投与することができる。非経口的投与としては、例えば、点滴などの静脈内注射、筋肉内注射、腹腔内注射、皮下注射、鼻腔内投与、吸入などを選択することができる。 The monoclonal antibody or antibody fragment thereof of the present invention is also useful in pharmaceutical compositions. Pharmaceutical compositions containing the monoclonal antibodies of the present invention or antibody fragments thereof can be administered orally or parenterally, systemically or locally. As parenteral administration, for example, intravenous injection such as drip, intramuscular injection, intraperitoneal injection, subcutaneous injection, intranasal administration, inhalation, etc. can be selected.
　本発明のモノクローナル抗体又はその抗体断片を含有する医薬組成物は、ＣＣＲ８関連疾患の治療及び／又は予防のための医薬として非常に有用である。特に、ＣＣＲ８発現Ｔｒｅｇ細胞の腫瘍内浸潤が生じている癌を治療及び／又は予防するための医薬として非常に有用である。例えば、乳癌、子宮体癌、子宮頸癌、卵巣癌、前立腺癌、肺癌、胃癌、膵臓癌、頭頚部癌、食道癌、膀胱癌、メラノーマ、大腸癌、腎癌、非ホジキンリンパ腫、尿路上皮癌、肉腫、血球癌（白血病、リンパ腫等）、胆管癌、胆のう癌、甲状腺癌、精巣癌、胸腺癌、肝臓癌等の癌、好ましくは乳癌、卵巣癌、肺癌、胃癌、膵臓癌、頭頚部癌、食道癌、メラノーマ、大腸癌、腎癌、尿路上皮癌を治療及び／又は予防するための医薬として非常に有用である。 A pharmaceutical composition containing the monoclonal antibody or antibody fragment thereof of the present invention is very useful as a medicament for the treatment and/or prevention of CCR8-related diseases. In particular, it is very useful as a medicament for treating and/or preventing cancer in which CCR8-expressing Treg cells infiltrate into tumors. For example, breast cancer, endometrial cancer, cervical cancer, ovarian cancer, prostate cancer, lung cancer, stomach cancer, pancreatic cancer, head and neck cancer, esophageal cancer, bladder cancer, melanoma, colorectal cancer, kidney cancer, non-Hodgkin lymphoma, urothelial cancer. Cancer, sarcoma, blood cell cancer (leukemia, lymphoma, etc.), bile duct cancer, gallbladder cancer, thyroid cancer, testicular cancer, thymus cancer, liver cancer, etc., preferably breast cancer, ovarian cancer, lung cancer, stomach cancer, pancreatic cancer, head and neck cancer. It is very useful as a medicine for treating and/or preventing cancer, esophageal cancer, melanoma, colon cancer, renal cancer, and urothelial cancer.
　以下に本発明の実施例を挙げて本発明をさらに詳しく説明するが、本発明はこれらにより限定されるものではない。 The present invention will be explained in more detail with reference to Examples below, but the present invention is not limited thereto.
実施例１：抗ヒトＣＣＲ８抗体産生ラットハイブリドーマの作製
（１）ハイブリドーマの作製
　免疫原であるヒトＣＣＲ８発現Ｒａｔ－１細胞は、ｐＱＣＸＩＰ（クロンテック社）に
ヒトＣＣＲ８遺伝子をクローニングした発現ベクターをＲａｔ－１細胞にトランスフェクションしたのちに、ピューロマイシン（１μｇ／ｍｌ）で一ヶ月間薬剤選択することにより作製した。
　ヒトＣＣＲ８発現Ｒａｔ－１細胞をラットに一回免疫し、約２週間後にリンパ節を回収して常法によりハイブリドーマを作製した。 Example 1: Preparation of rat hybridoma producing anti-human CCR8 antibody (1) Preparation of hybridoma Human CCR8-expressing Rat-1 cells, which are the immunogen, were prepared using an expression vector in which the human CCR8 gene was cloned into pQCXIP (Clontech). The cells were transfected, followed by drug selection with puromycin (1 μg/ml) for one month.
Rats were immunized once with human CCR8-expressing Rat-1 cells, and about two weeks later, lymph nodes were collected and hybridomas were produced by a conventional method.
（２）クローンの選抜
１）ＣＣＲ８結合活性評価
　上記（１）で得られた４種のハイブリドーマ（Ｎｏ．３、Ｎｏ．１２、Ｎｏ．３０、Ｎｏ．３７）の上清を用いて、ヒトＣＣＲ８発現２９３細胞及び、親細胞の２９３細胞に対する結合を評価した。二次抗体としてＡｌｅｘａ６４７標識抗ラットＩｇＧ抗体（サーモフィッシャーサイエンティフィック社製）を使用した。ハイブリドーマの上清とヒトＣＣＲ８発現２９３細胞又は親細胞の２９３細胞を混合し、４℃で２０分間反応させ、ＦＡＣＳバッファーで３回洗浄した。二次抗体を３００００倍希釈で添加し、４℃で２０分間反応させ、ＦＡＣＳバッファーで３回洗浄した。ＤＡＰＩ（４’，６－ジアミジノ－２－フェニルインドール）を１００ｎｇ／ｍｌとなるように添加し、フローサイトメトリーで細胞に結合した蛍光値（平均値）を定量した。
　その結果を表１に示す。Ｎｏ．３０、Ｎｏ．３、Ｎｏ．１２、Ｎｏ．３７のハイブリドーマ上清の順にヒトＣＣＲ８に対する結合が強かった。また、親細胞に対する結合はＮｏ．３が最も結合が低く、Ｓ／Ｎ比（シグナル対ノイズ比）はＮｏ．３がもっとも高かった。
　次に、抗ヒトＣＣＲ８抗体（Ｌ２６３Ｇ８、ＢｉｏＬｅｇｅｎｄ社）に対する競合阻害を評価した。上記４種のハイブリドーマ上清又は比較対照としてＷＯ２０２０／１３８４８９号公報の実施例に記載された２－７Ｂ抗体を産生するハイブリドーマ（Ｎｏ．２）上清をヒトＣＣＲ８発現２９３細胞に添加し、４℃で３０分間静置した。陽性コントロールとして、ハイブリドーマ上清の代わりにＧＩＴ培地で希釈した未標識抗ヒトＣＣＲ８抗体（Ｌ２６３Ｇ８、ＢｉｏＬｅｇｅｎｄ社）を２μｇ／ｍｌの濃度で使用した。ＰＥ標識抗ヒトＣＣＲ８抗体（Ｌ２６３Ｇ８、ＢｉｏＬｅｇｅｎｄ社）を０．２μｇ／ｍｌの濃度で添加し、３０分間４℃静置した。その後、ＦＡＣＳバッファーで３回洗浄した。ＤＡＰＩを１００ｎｇ／ｍｌとなるように添加し、フローサイトメトリーで細胞に結合した蛍光値（平均値）を定量した。陰性コントロールとしてＧＩＴ培地を添加した場合の蛍光平均値を１００％とし、陽性コントロールの蛍光平均値を０％として、各試験抗体の蛍光平均値から、阻害率（％）を算出した。
　その結果を表１及び図１に示す。Ｎｏ．２のハイブリドーマ上清はＬ２６３Ｇ８抗体に対する競合阻害活性を示したのに対し、上記（１）で得られた４種のハイブリドーマ上清はＬ２６３Ｇ８抗体に対する競合阻害活性をほとんど示さなかった。 (2) Clone selection 1) CCR8 binding activity evaluation Using the supernatants of the four hybridomas (No. 3, No. 12, No. 30, and No. 37) obtained in (1) above, human CCR8 Binding to expressing 293 cells and parental cells to 293 cells was evaluated. Alexa647-labeled anti-rat IgG antibody (manufactured by Thermo Fisher Scientific) was used as a secondary antibody. The hybridoma supernatant and human CCR8-expressing 293 cells or parental 293 cells were mixed, reacted at 4°C for 20 minutes, and washed three times with FACS buffer. A secondary antibody was added at a 30,000-fold dilution, reacted at 4°C for 20 minutes, and washed three times with FACS buffer. DAPI (4',6-diamidino-2-phenylindole) was added at a concentration of 100 ng/ml, and the fluorescence value (average value) bound to the cells was quantified by flow cytometry.
The results are shown in Table 1. No. 30, No. 3.No. 12, No. The 37 hybridoma supernatants showed the strongest binding to human CCR8. In addition, binding to parent cells was No. 3 has the lowest coupling, and the S/N ratio (signal to noise ratio) is No. 3 was the highest.
Next, competitive inhibition against anti-human CCR8 antibody (L263G8, BioLegend) was evaluated. The above four hybridoma supernatants or the hybridoma supernatant (No. 2) producing the 2-7B antibody described in the Examples of WO2020/138489 as a comparison were added to human CCR8-expressing 293 cells, and the mixture was incubated at 4°C. It was left standing for 30 minutes. As a positive control, an unlabeled anti-human CCR8 antibody (L263G8, BioLegend) diluted in GIT medium was used at a concentration of 2 μg/ml instead of the hybridoma supernatant. PE-labeled anti-human CCR8 antibody (L263G8, BioLegend) was added at a concentration of 0.2 μg/ml, and the mixture was allowed to stand at 4° C. for 30 minutes. Thereafter, it was washed three times with FACS buffer. DAPI was added at a concentration of 100 ng/ml, and the fluorescence value (average value) bound to the cells was quantified by flow cytometry. The inhibition rate (%) was calculated from the average fluorescence value of each test antibody, with the average fluorescence value when GIT medium was added as a negative control being 100% and the average fluorescence value of the positive control being 0%.
The results are shown in Table 1 and FIG. No. The hybridoma supernatant of No. 2 exhibited competitive inhibitory activity against the L263G8 antibody, whereas the four hybridoma supernatants obtained in (1) above showed almost no competitive inhibitory activity against the L263G8 antibody.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
２）中和活性評価
　上記（１）で得られた４種のハイブリドーマ上清を用いて、ヒトＣＣＬ１によるヒトＣＣＲ８を介したヒトＣＣＲ８発現２９３細胞内カルシウム流入活性に対する阻害活性評価を実施した。ヒトＣＣＲ８発現２９３細胞をトリプシンで剥離し、常法に従いＦｌｕｏ３－ＡＭ（同人化学社）で３７℃、６０分間細胞を処理することで、細胞にＦｌｕｏ３を取り込ませた。その後ＤＭＥＭ／１０％ＦＢＳで２回洗浄した。６００μＬのＤＭＥＭ／１０％ＦＢＳに懸濁した上記ラベルされた細胞に各ハイブリドーマ上清３００μＬを添加し、３０分間室温で静置した。陰性コントロールはＧＩＴ培地を使用した。なお、比較対照とする中和活性を有する抗ＣＣＲ８抗体を産生するハイブリドーマとして、ＷＯ２０２０／１３８４８９号公報の実施例に記載された２－７Ｂ抗体を産生するハイブリドーマ（Ｎｏ．２）上清を用いた。その後ヒトＣＣＬ１（ＢｉｏＬｅｇｅｎｄ社）を終濃度５ｎＭになるように添加し、Ｆ－７０００形分光蛍光光度計（ＨＩＴＡＣＨＩ）を用いて励起波長：　５０８．０　ｎｍ、　蛍光波長：　５２７．０　ｎｍで、経時的に細胞内カルシウム濃度を５分間測定した。定量化方法は、ヒトＣＣＬ１を添加後３秒後の値をバックグラウンド値とし、カルシウムの蛍光値の最大値をＭａｘとし、以下の計算式で％阻害を算出した。
１００－（各ＩＴＭ抗体ｍａｘ－各ＩＴＭ抗体のＢＧ）／（ネガコンのＭａｘ－ネガコンのＢＧ）×１００　（％）
　その結果を表２及び図２に示す。なお、Ｎｏ．２及びＮｏ．３に対応する陰性コントロールは左列下のコントロールが対応し、Ｎｏ．３０、Ｎｏ．１２及びＮｏ．３７に対応する陰性コントロールは右列下のコントロールが対応している。Ｎｏ．３、Ｎｏ．３０、Ｎｏ．１２、Ｎｏ．３７のハイブリドーマ上清はいずれもヒトＣＣＬ１に対する中和活性をほとんど示さず、この順に中和活性が上昇した。Ｎｏ．３の上清がもっともヒトＣＣＬ１に対する中和活性が低かった。 2) Evaluation of neutralizing activity Using the four types of hybridoma supernatants obtained in (1) above, an evaluation of the inhibitory activity of human CCL1 against human CCR8-mediated calcium influx activity into human CCR8-expressed 293 cells was performed. Human CCR8-expressing 293 cells were detached with trypsin, and the cells were treated with Fluo3-AM (Dojin Kagakusha) for 60 minutes at 37°C to incorporate Fluo3 into the cells. Thereafter, it was washed twice with DMEM/10% FBS. 300 μL of each hybridoma supernatant was added to the labeled cells suspended in 600 μL of DMEM/10% FBS, and the cells were allowed to stand at room temperature for 30 minutes. GIT medium was used as a negative control. As a comparison control hybridoma producing an anti-CCR8 antibody with neutralizing activity, the supernatant of a hybridoma (No. 2) producing the 2-7B antibody described in the Examples of WO2020/138489 was used. . Thereafter, human CCL1 (BioLegend) was added to a final concentration of 5 nM, and the mixture was incubated at an excitation wavelength of 508.0 nm and a fluorescence wavelength of 527.0 nm using an F-7000 spectrofluorometer (HITACHI) over time. Intracellular calcium concentration was measured for 5 minutes. In the quantification method, the value 3 seconds after addition of human CCL1 was taken as the background value, the maximum value of the fluorescence value of calcium was taken as Max, and % inhibition was calculated using the following formula.
100 - (Max of each ITM antibody - BG of each ITM antibody) / (Max of negative control - BG of negative control) x 100 (%)
The results are shown in Table 2 and FIG. In addition, No. 2 and no. The negative control corresponding to No. 3 corresponds to the control at the bottom of the left column. 30, No. 12 and no. The negative control corresponding to No. 37 corresponds to the control at the bottom of the right column. No. 3.No. 30, No. 12, No. All 37 hybridoma supernatants showed almost no neutralizing activity against human CCL1, and the neutralizing activity increased in this order. No. The supernatant of No. 3 had the lowest neutralizing activity against human CCL1.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　上記（１）で得られた４種のハイブリドーマのうちで、Ｎｏ．３クローンはヒトＣＣＲ８の結合活性が２番目に強く、Ｓ／Ｎ比が最も高く、ヒトＣＣＬ１によるヒトＣＣＲ８を介した細胞内カルシウム流入に対する中和活性が最も低い抗体であった。以上より、中和活性を有さない抗ＣＣＲ８抗体を産生するハイブリドーマとして、もっとも優れているＮｏ．３クローンを選抜した。 Among the four hybridomas obtained in (1) above, No. The three clones were antibodies with the second strongest binding activity to human CCR8, the highest S/N ratio, and the lowest neutralizing activity against intracellular calcium influx via human CCR8 by human CCL1. From the above, No. 1 is the best hybridoma that produces anti-CCR8 antibodies without neutralizing activity. Three clones were selected.
実施例２：抗体配列の決定
　Ｎｏ．３クローンが産生する３－３Ｆ抗体について、ハイブリドーマ細胞より、常法に従って抗体の軽鎖可変領域及び重鎖可変領域のアミノ酸配列を決定した（表３）。 Example 2: Determination of antibody sequence No. Regarding the 3-3F antibody produced by the three clones, the amino acid sequences of the light chain variable region and heavy chain variable region of the antibody were determined from hybridoma cells according to a conventional method (Table 3).
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
実施例３：精製抗体の中和活性評価
　樹立したハイブリドーマを無血清培地で培養し、その培養上清をＰｒｏｔｅｉｎＧアフィニティー精製及びゲル濾過精製を行うことで、精製抗体を得た。この精製３－３Ｆ抗体について、下記の方法で中和活性を測定した。なお、比較対照とする中和活性を有する抗ＣＣＲ８抗体として、市販抗ヒトＣＣＲ８抗体であるＬ２６３Ｇ８抗体ならびに、ＷＯ２０２０／１３８４８９号公報の実施例に記載された２Ｃ７抗体、１０Ａ１１抗体及び２－７Ｂ抗体を用いた。
　あらかじめＣａ２＋指示薬を取り込ませたヒトＣＣＲ８発現２９３細胞に対して培地で希釈した抗体希釈液を添加し、ＦＬＩＰＲにより２００ｎＭのヒトＣＣＬ１（バイオレジェンド社製）添加によるＣａ２＋流入を測定した。ヒトＣＣＬ１非添加時のシグナルを阻害率１００％、ヒトＣＣＬ１添加及び抗体非添加時のシグナルを阻害率０％として阻害率を計算し、５０％の阻害率を示す抗体濃度をＩＣ５０とした。少なくとも３回評価を行い、ＩＣ５０をＡｖｅｒａｇｅ±ＳＤとして表記した。
　その結果、精製した３－３Ｆ抗体にも中和活性が認められなかった（表４）。 Example 3: Evaluation of neutralizing activity of purified antibody The established hybridoma was cultured in a serum-free medium, and the culture supernatant was purified by Protein G affinity and gel filtration to obtain a purified antibody. The neutralizing activity of this purified 3-3F antibody was measured by the method described below. In addition, as anti-CCR8 antibodies with neutralizing activity for comparison, L263G8 antibody, which is a commercially available anti-human CCR8 antibody, and 2C7 antibody, 10A11 antibody, and 2-7B antibody described in the Examples of WO2020/138489 were used. Using.
A diluted antibody solution diluted with a medium was added to human CCR8-expressing 293 cells into which a Ca 2+ indicator had been incorporated in advance, and Ca 2+ influx due to the addition of 200 nM human CCL1 (manufactured by Biolegend) was measured by FLIPR. The inhibition rate was calculated by setting the signal when human CCL1 was not added as an inhibition rate of 100%, and the signal when human CCL1 was added and the antibody was not added as an inhibition rate of 0%, and the antibody concentration showing a 50% inhibition rate was defined as IC50. Evaluations were performed at least three times and IC50 was expressed as Average±SD.
As a result, no neutralizing activity was observed in the purified 3-3F antibody (Table 4).
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
実施例４：ＣＣＲ８に対する結合解析
（１）ＣＣＲ８に対する結合活性評価
　ヒトＣＣＲ８発現Ｒａｍｏｓ細胞及びその親株（Ｒａｍｏｓ細胞）に対する３－３Ｆ抗体の結合及び、ヒトＣＣＲ８に対する中和抗体であるＯＰ－１抗体（ＷＯ２０２０／１３８４８９号公報の実施例に記載されたヒト化１０Ａ１１抗体（軽鎖可変領域：ＩＧＫＶ４－１　Ｎ５３Ｑ＋Ｇ２９Ｒ（配列番号１２）／重鎖可変領域：ＩＧＨＶ３－１５　Ｔ９４Ｒ（配列番号１３）））存在下の３－３Ｆ抗体の結合について評価した。
　本実施例では、３－３Ｆ抗体の蛍光非標識体およびＡｌｅｘａ６４７蛍光標識体を用いた。蛍光非標識体の検出には二次抗体としてＡＰＣ蛍光標識抗ラットＩｇＧ抗体（Ｊａｃｋｓｏｎ　ＩｍｍｕｎｏＲｅｓｅａｒｃｈ　Ｌａｂｏｒａｔｏｒｉｅｓ社製）を使用した。比較対照のため、市販抗ヒトＣＣＲ８抗体であるＬ２６３Ｇ８抗体のＡＰＣ蛍光標識体および４３３Ｈ抗体（ＢＤバイオサイエンス社製）のＢＶ４２１蛍光標識体を使用した。ヒトＣＣＲ８発現Ｒａｍｏｓ細胞およびＲａｍｏｓ細胞を６．０×１０５細胞ずつマイクロチューブへ分注し、（Ａ）３－３Ｆ抗体の蛍光非標識体を５μｇ／ｍＬを添加し、４℃で３０分間反応させた。アイソタイプ抗体としてはＲａｔ　ＩｇＧ２ａ抗体（未標識、　１００倍希釈）を使用した。その後、培養液（１０％ＦＢＳ）で洗浄後、ＡＰＣ蛍光標識抗ラットＩｇＧ抗体を加え、４℃で３０分間反応させた。（Ｂ）Ｌ２６３Ｇ８抗体のＡＰＣ蛍光標識体（１００倍希釈）を添加し、４℃で３０分間反応させた。アイソタイプ抗体としてはＡＰＣ－ｍｏｕｓｅ　ＩｇＧ２ａ抗体（１００倍希釈）を使用した。（Ｃ）４３３Ｈ抗体のＢＶ４２１蛍光標識体（１００倍希釈）を添加し、４℃で３０分間反応させたアイソタイプ抗体としてはＢＶ４２１－ｍｏｕｓｅ　ＩｇＧ２ａ抗体（１００倍希釈）を使用した。（Ａ）及び（Ｂ）の反応後、培養液（１０％ＦＢＳ）で洗浄して、Ｃｅｌｌｓｔａｉｎ－ＤＡＰＩ　ｓｏｌｕｔｉｏｎ（１００００倍希釈、同仁化学研究所社製）を加えて、フローサイトメトリーで測定した。（Ｃ）の反応後、ｅＢｉｏｓｃｉｅｎｃｅ　Ｆｉｘａｂｌｅ　Ｖｉａｂｉｌｉｔｙ　Ｄｙｅ　ｅＦｌｕｏｒ　７８０溶液（１００００倍希釈、Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社製）を加えて、４℃で１０分間反応させた後、培養液（１０％ＦＢＳ）で洗浄して、培養液（１０％ＦＢＳ）で再懸濁後、フローサイトメトリーで測定した。また、競合試験においては、マイクロチューブに分注したヒトＣＣＲ８発現Ｒａｍｏｓ細胞にＯＰ－１抗体を各種濃度（１，１０，１００μｇ／ｍＬ）で添加し、４℃で３０分間反応させ、培養液（１０％ＦＢＳ）で洗浄後に上記（Ａ）、（Ｂ）及び（Ｃ）の抗体反応を行った。
　その結果、ヒトＣＣＲ８発現Ｒａｍｏｓ細胞において３－３Ｆ抗体、Ｌ２６３Ｇ８抗体及び４３３Ｈ抗体の結合が認められた（図３）。また、ＯＰ－１抗体存在下での各抗体の反応性についても確認したところ、３－３Ｆ抗体については、ＯＰ－１抗体存在下で結合の減弱が認めらなかった。（図４） Example 4: Binding analysis to CCR8 (1) Evaluation of binding activity to CCR8 Binding of 3-3F antibody to human CCR8-expressing Ramos cells and its parent strain (Ramos cells), and OP-1 antibody (neutralizing antibody to human CCR8) In the presence of the humanized 10A11 antibody (light chain variable region: IGKV4-1 N53Q+G29R (SEQ ID NO: 12)/heavy chain variable region: IGHV3-15 T94R (SEQ ID NO: 13)) described in the Examples of WO2020/138489. The binding of the 3-3F antibody was evaluated.
In this example, a non-fluorescently labeled 3-3F antibody and a fluorescently labeled Alexa647 antibody were used. APC fluorescently labeled anti-rat IgG antibody (manufactured by Jackson ImmunoResearch Laboratories) was used as a secondary antibody to detect the non-fluorescently labeled substance. For comparison purposes, APC fluorescently labeled L263G8 antibody and BV421 fluorescently labeled 433H antibody (manufactured by BD Biosciences), which are commercially available anti-human CCR8 antibodies, were used. Dispense human CCR8-expressing Ramos cells and Ramos cells at 6.0 x 10 5 cells into microtubes, add 5 μg/mL of (A) non-fluorescent labeled 3-3F antibody, and react at 4°C for 30 minutes. I let it happen. Rat IgG2a antibody (unlabeled, 100-fold diluted) was used as the isotype antibody. Thereafter, after washing with culture medium (10% FBS), APC fluorescently labeled anti-rat IgG antibody was added and reacted at 4°C for 30 minutes. (B) APC fluorescently labeled L263G8 antibody (100-fold dilution) was added and reacted at 4°C for 30 minutes. APC-mouse IgG2a antibody (100-fold dilution) was used as the isotype antibody. (C) BV421 fluorescently labeled 433H antibody (100 times diluted) was added and reacted at 4° C. for 30 minutes. BV421-mouse IgG2a antibody (100 times diluted) was used as the isotype antibody. After the reactions in (A) and (B), the cells were washed with culture solution (10% FBS), Cellstain-DAPI solution (10,000-fold dilution, manufactured by Dojindo Laboratories) was added, and the cells were measured by flow cytometry. After the reaction in (C), eBioscience Fixable Viability Dye eFluor 780 solution (10,000-fold dilution, manufactured by Thermo Fisher Scientific) was added, reacted at 4°C for 10 minutes, and then washed with culture medium (10% FBS). , resuspended in culture medium (10% FBS) and measured by flow cytometry. In the competition test, OP-1 antibody was added at various concentrations (1, 10, 100 μg/mL) to human CCR8-expressing Ramos cells dispensed into microtubes, reacted at 4°C for 30 minutes, and culture medium ( After washing with 10% FBS), the antibody reactions in (A), (B), and (C) above were performed.
As a result, binding of the 3-3F antibody, L263G8 antibody, and 433H antibody was observed in human CCR8-expressing Ramos cells (FIG. 3). Furthermore, when the reactivity of each antibody in the presence of OP-1 antibody was confirmed, no decrease in binding was observed for 3-3F antibody in the presence of OP-1 antibody. (Figure 4)
（２）ケモカイン受容体における結合選択性評価
　表５の各ケモカイン受容体について、それぞれ対応するアクセッション番号のｃＤＮＡ配列を組み込んだ発現プラスミドを２又は６μｇ用いて、Ｌｉｐｏｆｅｃｔａｍｉｎｅ　ＬＴＸ又はＬｉｐｏｆｅｃｔａｍｉｎｅ　３０００（いずれもＴｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）により２９３ｃ１８細胞にトランスフェクションを行い、３７℃、５％ＣＯ２で７２時間培養して、ケモカイン受容体発現細胞を得た。
　ＣＣＲ３発現細胞については、配列番号１６に示すヒトＣＣＲ３のｃＤＮＡ配列を用い、上記と同様の方法で発現細胞を得た。
　なお、ヒトＣＣＲ８発現細胞については、以下の方法で安定発現細胞株を得た。ｐＱＣＸＩＰベクターのＥｃｏＲＩ／ＢａｍＨＩサイトにヒトＣＣＲ８のｃＤＮＡ（配列番号１７）をクローニングし、ｐＱＣＸＩＰ－ｈＣＣＲ８発現ベクターを作製した。この発現ベクター５μｇをｌｉｐｏｆｅｃｔａｍｉｎｅ３０００によりＨＥＫ２９３Ｔ細胞（ＴＡＫＡＲＡ）に形質転換し、２日後よりピューロマイシン２μｇ／ｍｌで薬剤選択した。薬剤存在下で３週間、３７℃、５％ＣＯ２で培養し、生き残った薬剤耐性ＨＥＫ２９３Ｔ細胞の細胞表面上に発現するヒトＣＣＲ８を、ＰＥ標識抗ＣＣＲ８抗体（ＢｉｏＬｅｇｅｎｄ）を用いてフローサイトメーターにより発現確認し、安定発現細胞ｈＣＣＲ８／ＨＥＫ２９３Ｔを樹立した。
　上記ケモカイン受容体発現細胞に死細胞染色試薬（Ｚｏｍｂｉｅ　ＮＩＲ　Ｆｉｘａｂｌｅ　Ｖｉａｂｉｌｉｔｙ　Ｄｙｅ、ＢｉｏＬｅｇｅｎｄ社）を２０００倍希釈で添加後、検出抗体としてＡＦ６４７標識３－３Ｆ抗体又はＡＦ６４７標識ｒａｔ　ＩｇＧ２ａ抗体（＃４０７５１２）を５００ｎｇ／ｍＬの濃度で添加し、４℃で３０分間静置した。その後Ｓｔａｉｎ　Ｂｕｆｆｅｒ（ＢＤ　Ｐｈａｒｍｉｎｇｅｎ）で２回洗浄した。Ｎｏｖｏｃｙｔｅ３０００を用いて各抗体の蛍光を測定し、それぞれの陰性コントロール抗体のヒストグラムとオーバーレイした。なお、各ケモカイン受容体発現細胞におけるケモカイン受容体の発現確認のため、上記と同様の方法（ただし、検出抗体として、各ケモカイン受容体の抗体（表６）又は対応する陰性コントロール抗体（表７）を４０倍希釈で添加）で解析したところ、各ケモカイン受容体発現細胞においてケモカイン受容体が正常に発現していることを確認した。
　その結果、ＣＣＲ８についてはＡＦ６４７標識３－３Ｆ抗体のピークが陰性コントロール抗体のピークからシフトしたが、その他のケモカイン受容体について両ピークが重なったことから、ＡＦ６４７標識３－３Ｆ抗体のＣＣＲ８以外のケモカイン受容体への結合は認められなかった（図５）。







(2) Evaluation of binding selectivity in chemokine receptors For each chemokine receptor in Table 5, 2 or 6 μg of an expression plasmid incorporating the cDNA sequence with the corresponding accession number was used to inject Lipofectamine LTX or Lipofectamine 3000 (both with Thermo (Fisher Scientific), 293c18 cells were transfected and cultured at 37° C. and 5% CO 2 for 72 hours to obtain chemokine receptor-expressing cells.
CCR3-expressing cells were obtained in the same manner as above using the human CCR3 cDNA sequence shown in SEQ ID NO: 16.
In addition, regarding human CCR8 expressing cells, a stable expressing cell line was obtained by the following method. Human CCR8 cDNA (SEQ ID NO: 17) was cloned into the EcoRI/BamHI site of the pQCXIP vector to create a pQCXIP-hCCR8 expression vector. 5 μg of this expression vector was transformed into HEK293T cells (TAKARA) using lipofectamine 3000, and after 2 days drug selection was performed using 2 μg/ml of puromycin. Human CCR8 expressed on the cell surface of surviving drug-resistant HEK293T cells cultured in the presence of drugs for 3 weeks at 37°C and 5% CO was detected using a flow cytometer using a PE-labeled anti-CCR8 antibody (BioLegend). Expression was confirmed and stable expressing cell hCCR8/HEK293T was established.
After adding a dead cell staining reagent (Zombie NIR Fixable Viability Dye, BioLegend) to the chemokine receptor-expressing cells at a 2000-fold dilution, 500 ng of AF647-labeled 3-3F antibody or AF647-labeled rat IgG2a antibody (#407512) was added as a detection antibody. /mL and left at 4°C for 30 minutes. Thereafter, it was washed twice with Stain Buffer (BD Pharmingen). Fluorescence of each antibody was measured using Novocyte 3000 and overlaid with the histogram of each negative control antibody. In addition, to confirm the expression of chemokine receptors in each chemokine receptor-expressing cell, the same method as above (however, as a detection antibody, the antibody for each chemokine receptor (Table 6) or the corresponding negative control antibody (Table 7)) (added at a 40-fold dilution), it was confirmed that chemokine receptors were normally expressed in each chemokine receptor-expressing cell.
As a result, for CCR8, the peak of AF647-labeled 3-3F antibody shifted from the peak of the negative control antibody, but for other chemokine receptors, both peaks overlapped. No binding to the receptor was observed (Figure 5).
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005




























Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006










Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
実施例５：ヒト腫瘍内浸潤細胞に対する３－３Ｆ抗体の結合評価
　ヒト腫瘍内浸潤細胞に対する３－３Ｆ抗体の結合について、卵巣癌患者検体を用いて評価した。本実施例では、３－３Ｆ抗体の蛍光非標識体およびＡｌｅｘａ６４７蛍光標識体を用いた。蛍光非標識体の検出には二次抗体としてＡｌｅｘａ６４７蛍光標識抗ラットＩｇＧ抗体（Ｊａｃｋｓｏｎ　ＩｍｍｕｎｏＲｅｓｅａｒｃｈ　Ｌａｂｏｒａｔｏｒｉｅｓ社製）を使用した。比較対照のため、陽性コントロールとして市販抗ヒトＣＣＲ８抗体であるＬ２６３Ｇ８抗体のＡＰＣ蛍光標識体を使用した。
　卵巣癌より抽出した腫瘍内浸潤細胞を１．５×１０５細胞ずつ９６ウェルプレートへ播種し、Ｚｏｍｂｉｅ　ＮＩＲ　Ｆｉｘａｂｌｅ　Ｖｉａｂｉｌｉｔｙ　Ｋｉｔ（Ｂｉｏｌｅｇｅｎｄ社製）を添加し室温で１５分反応させた後、実施例４で用いたヒトＣＣＲ８に対する中和抗体であるＯＰ－１抗体を各種濃度で添加し、４℃で６０分間反応させた。ＨＢＳＳ（２％ＦＢＳ＋１０ｍｍｏｌ／Ｌ　Ｈｅｐｅｓ）で洗浄後、Ｈｕｍａｎ　ＴｒｕＳｔａｉｎ　ＦｃＸ（ＢｉｏＬｅｇｅｎｄ社製）を添加し、室温で１５分反応させた後、各表面抗原（ＣＤ４５、ＣＤ４５ＲＡ、ＣＤ３、ＣＤ４、ＣＤ８、ＣＤ２５及びＣＣＲ８）に対する抗体を加えて４℃で３０分間反応させた。ＨＢＳＳで洗浄後、３－３Ｆ抗体の蛍光非標識体を反応させたウェルにＡｌｅｘａ６４７蛍光標識抗ラットＩｇＧ抗体を加え、４℃で３０分間反応させた。ＨＢＳＳで洗浄後、Ｆｏｘｐ３／Ｔｒａｎｓｃｒｉｐｔｉｏｎ　Ｆａｃｔｏｒ　Ｓｔａｉｎｉｎｇ　Ｂｕｆｆｅｒ　Ｓｅｔ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社製）及びＦｏｘＰ３に対する抗体を用いて、細胞内ＦｏｘＰ３染色を行った。同Ｂｕｆｆｅｒ　ＳｅｔのＰｅｒｍｅａｂｉｌｉｚａｔｉｏｎ　Ｂｕｆｆｅｒで洗浄後、フローサイトメトリーで測定した。ゲート方法は、リンパ球をゲート後、ＣＤ４５陽性細胞ゲート、ＣＤ３ゲート、ＣＤ４をゲートした後に、ＣＤ４５ＲＡとＦｏｘＰ３の２軸で展開し、ＣＤ４５ＲＡ陰性ＦｏｘＰ３陽性細胞（Ｔｒｅｇ細胞）を解析対象とした。
　その結果を図６に示す。蛍光標識二次抗体のみで染色した細胞を陰性対照とした際に、３－３Ｆ抗体において、Ｔｒｅｇ細胞中の６１．４％が陽性シグナルとして検出された。
　また、ＣＤ４をゲートするまでを前述したゲート方法と同じ手法で行い、ＣＤ４でゲートした細胞をＣＣＲ８とＦｏｘＰ３の２軸で展開し、蛍光標識体及び蛍光非標識体の染色性を比較した。
　その結果を図７に示す。ＦｏｘＰ３陽性ＣＣＲ８陽性分画の陽性率は、蛍光非標識体２μｇ／ｍＬの反応で５４．１％、蛍光標識体２μｇ／ｍＬ反応で４７．８％となり、蛍光標識体においても蛍光非標識体と同等の染色パターンを示した。また、比較対照として用いたＬ２３６Ｇ８抗体２μｇ／ｍＬの反応において、ＦｏｘＰ３陽性ＣＣＲ８陽性分画の陽性率は、４６．６％であった。ＯＰ－１抗体存在下での各抗体の反応性についても確認したところ、３－３Ｆ抗体については、ＯＰ－１抗体存在下で結合の減弱が認めらなかった。比較対照として用いたＬ２３６Ｇ８抗体については、ＯＰ－１抗体存在下で結合の減弱が認められ、ＦｏｘＰ３陽性ＣＣＲ８陽性分画の陽性率は、約半分に低下した。 Example 5: Evaluation of binding of 3-3F antibody to human tumor-infiltrating cells The binding of 3-3F antibody to human tumor-infiltrating cells was evaluated using ovarian cancer patient specimens. In this example, a non-fluorescently labeled 3-3F antibody and a fluorescently labeled Alexa647 antibody were used. Alexa647 fluorescently labeled anti-rat IgG antibody (manufactured by Jackson ImmunoResearch Laboratories) was used as a secondary antibody to detect the unlabeled fluorescent substance. For comparison, an APC fluorescent label of L263G8 antibody, which is a commercially available anti-human CCR8 antibody, was used as a positive control.
Intratumoral infiltrating cells extracted from ovarian cancer were seeded into a 96-well plate at a rate of 1.5 x 10 5 cells, and Zombie NIR Fixable Viability Kit (manufactured by Biolegend) was added and reacted at room temperature for 15 minutes, followed by Example 4. OP-1 antibody, which is a neutralizing antibody against human CCR8 used in , was added at various concentrations and allowed to react at 4°C for 60 minutes. After washing with HBSS (2% FBS + 10 mmol/L Hepes), Human TruStain FcX (manufactured by BioLegend) was added, and after reacting for 15 minutes at room temperature, each surface antigen (CD45, CD45RA, CD3, CD4, CD8, CD25 and CCR8) was added and allowed to react at 4°C for 30 minutes. After washing with HBSS, Alexa647 fluorescently labeled anti-rat IgG antibody was added to the wells in which the non-fluorescently labeled 3-3F antibody had been reacted, and the wells were allowed to react at 4°C for 30 minutes. After washing with HBSS, intracellular FoxP3 staining was performed using Foxp3/Transcription Factor Staining Buffer Set (manufactured by Thermo Fisher Scientific) and an antibody against FoxP3. After washing with the Permeabilization Buffer of the same Buffer Set, measurement was performed by flow cytometry. In the gating method, after gating lymphocytes, gating CD45-positive cells, CD3 gate, and gating CD4, two axes of CD45RA and FoxP3 were used, and CD45RA-negative FoxP3-positive cells (Treg cells) were targeted for analysis.
The results are shown in FIG. When cells stained only with a fluorescently labeled secondary antibody were used as a negative control, 61.4% of Treg cells were detected as positive signals with the 3-3F antibody.
In addition, the steps up to gating CD4 were performed using the same method as the gating method described above, and the cells gated by CD4 were developed on two axes of CCR8 and FoxP3, and the stainability of fluorescently labeled and non-fluorescently labeled cells was compared.
The results are shown in FIG. The positive rate of the FoxP3-positive CCR8-positive fraction was 54.1% in the reaction with 2 μg/mL of the unlabeled fluorescent substance and 47.8% in the reaction with 2 μg/mL of the fluorescent label; showed comparable staining patterns. Furthermore, in the reaction with 2 μg/mL of L236G8 antibody used as a comparison control, the positive rate of the FoxP3-positive CCR8-positive fraction was 46.6%. When the reactivity of each antibody in the presence of OP-1 antibody was also confirmed, no decrease in binding was observed for 3-3F antibody in the presence of OP-1 antibody. Regarding the L236G8 antibody used as a comparison control, a decrease in binding was observed in the presence of the OP-1 antibody, and the positivity rate of the FoxP3-positive CCR8-positive fraction was reduced to about half.
実施例６：３－３Ｆ抗体の腫瘍内浸潤Ｔｒｅｇ細胞中へのＣＣＲ８特異的結合の確認
　ＰｒｉｍｅＦｌｏｗ ＲＮＡアッセイ技術を用い、フローサイトメトリー上で、３－３Ｆ抗体によるシグナルとＣＣＲ８のｍＲＮＡ 発現を同時に検出し、３－３Ｆ抗体のＣＣＲ８特異性を確認した。
　卵巣癌患者由来腫瘍組織をＴｕｍｏｒ Ｄｉｓｓｏｃｉａｔｉｏｎ Ｋｉｔ, ｈｕｍａｎ（Ｍｉｌｔｅｎｙｉ Ｂｉｏｔｅｃ社）を用いて分散し、凍結保存した。凍結した腫瘍分散細胞を融解し、Ｈｕｍａｎ　ＴｒｕＳｔａｉｎ　ＦｃＸ（ＢｉｏＬｅｇｅｎｄ社）およびＺｏｍｂｉｅ ＮＩＲ Ｆｉｘａｂｌｅ Ｖｉａｂｉｌｉｔｙ Ｋｉｔ（Ｂｉｏｌｅｇｅｎｄ社）を添加したＳｔａｉｎ Ｂｕｆｆｅｒ（ＢＤ Ｂｉｏｓｃｉｅｎｃｅ社）に懸濁し、４℃に３０分間静置した。洗浄後、３－３Ｆ抗体（４μｇ／ｍＬ）を添加して４℃に３０分間静置した。洗浄後、ヒトＣＤ４５、ＣＤ３及びＣＤ８に対する蛍光標識抗体とＡｌｅｘａＦｌｏｕｒ４８８標識抗ラットＩｇＧ抗体を添加して４℃に３０分間静置した。洗浄後、ＰｒｉｍｅＦｌｏｗ ＲＮＡアッセイキット（サーモフィッシャー社）の試薬にて固定および膜透過処理後、ＰＥ標識抗ヒトＦｏｘｐ３抗体を添加して４℃に３０分間静置した。洗浄後、ＣＣＲ８ ｍＲＮＡに対するプローブを添加して４０℃に１２０分間静置した。洗浄後、シグナル増幅用プローブＰｒｅａｍｐｌｉｆｉｅｒおよびＡｍｐｌｉｆｉｅｒをそれぞれ４０℃ ９０分にてハイブリダイゼーション後、ＡｌｅｘａＦｌｏｕｒ６４７標識プローブを添加し、４０℃にて６０分間静置した。洗浄後、Ｎｏｖｏｃｙｔｅ Ｑｕａｎｔｅｏｎフローサイトメーターを用いて、Ｔｒｅｇ細胞（Ｚｏｍｂｉｅ ＮＩＲ陰性かつＣＤ４５・ＣＤ３・ＣＤ４・Ｆｏｘｐ３陽性画分）における３－３Ｆ抗体シグナルとＣＣＲ８ ｍＲＮＡシグナルの相関を解析した。
　その結果、３－３Ｆ抗体シグナルとＣＣＲ８ ｍＲＮＡシグナルが正に相関する結果が得られた（図８）。 Example 6: Confirmation of CCR8-specific binding of 3-3F antibody to intratumoral infiltrating Treg cells Simultaneous detection of signal by 3-3F antibody and CCR8 mRNA expression on flow cytometry using PrimeFlow RNA assay technology The CCR8 specificity of the 3-3F antibody was confirmed.
Tumor tissues derived from ovarian cancer patients were dispersed using Tumor Dissociation Kit, human (Miltenyi Biotec) and cryopreserved. Frozen tumor dispersed cells were thawed and suspended in Stain Buffer (BD Bioscience) supplemented with Human TruStain FcX (BioLegend) and Zombie NIR Fixable Viability Kit (BioLegend). , and left at 4° C. for 30 minutes. After washing, 3-3F antibody (4 μg/mL) was added and left at 4° C. for 30 minutes. After washing, fluorescently labeled antibodies against human CD45, CD3, and CD8 and AlexaFlour488-labeled anti-rat IgG antibody were added and left at 4°C for 30 minutes. After washing, fixation and membrane permeabilization with the reagents of PrimeFlow RNA Assay Kit (Thermo Fisher), PE-labeled anti-human Foxp3 antibody was added, and the mixture was allowed to stand at 4°C for 30 minutes. After washing, a probe for CCR8 mRNA was added and left at 40°C for 120 minutes. After washing, signal amplification probes Preamplifier and Amplifier were each hybridized at 40°C for 90 minutes, Alexa Flour647 labeled probe was added, and the mixture was left standing at 40°C for 60 minutes. After washing, the correlation between the 3-3F antibody signal and CCR8 mRNA signal in Treg cells (Zombie NIR negative and CD45/CD3/CD4/Foxp3 positive fraction) was analyzed using a Novocyte Quanteon flow cytometer.
As a result, a positive correlation was obtained between the 3-3F antibody signal and the CCR8 mRNA signal (FIG. 8).
実施例７：マウス腫瘍内浸潤細胞に対する３－３Ｆ抗体の結合評価
　マウス腫瘍内浸潤細胞に対する３－３Ｆ抗体の結合について、マウス腎臓癌の肺転移モデルを用いて評価した。本実施例では、３－３Ｆ抗体のＢＢ５１５蛍光標識体を用いた。ＣＣＲ８に結合した３－３Ｆとは異なる抗ＣＣＲ８抗体が３－３Ｆ抗体の結合に及ぼす影響を評価するために、本マウスモデルの一部には、実施例４で用いたヒトＣＣＲ８に対する中和抗体であるＯＰ－１抗体を投与しており、その際のヒトＣＣＲ８に結合したＯＰ－１抗体を検出する抗体として、抗ヒトＩｇＧ　Ｆｃ抗体であるＭ１３１０Ｇ０５抗体のＢＶ４２１蛍光標識体を使用した。比較対照として市販抗ヒトＣＣＲ８抗体である４３３Ｈ抗体のＢＶ４２１蛍光標識体を使用し、同一の試料から分けた試料について染色、測定を行った。
　ＷＯ２０２０／１３８４８９号公報の実施例１０に記載されたＢａｌｂ／ＣマウスのＣＣＲ８をヒト型のＣＣＲ８遺伝子で置換したノックインマウス（雌、２１週齢）に培養したマウス腎臓癌細胞、Ｒｅｎｃａを４×１０５個、尾静脈に注入した。その１２日後にＯＰ－１抗体を尾静脈から注入した。翌日、０．０５ｍｇ／ｋｇ投与群（８匹）、０．５ｍｇ／ｋｇ投与群（６匹）、および無処置マウス（９匹）より吸入麻酔下で腫瘍細胞が転移した肺を採取した。Ｔｕｍｏｒ　Ｄｉｓｓｏｃｉａｔｉｏｎ　Ｋｉｔ，ｍｏｕｓｅ（Ｍｉｌｔｅｎｙｉ Ｂｉｏｔｅｃ社）、およびＢＤ　Ｐｈａｒｍａ　Ｌｙｓｅ（ＢＤ　Ｂｉｏｓｃｉｅｎｃｅｓ社）を用い、肺組織より単細胞を分散、精製し、フローサイトメトリー用の染色を行った。染色は、まずｅＢｉｏｓｃｉｅｎｃｅ　Ｆｉｘａｂｌｅ　Ｖｉａｂｉｌｉｔｙ　Ｄｙｅ　ｅＦｌｕｏｒ　７８０（ｅＦｌｕｏｒ　７８０、Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）で死細胞を染色し、その後、Ｍｏｕｓｅ　ＢＤ　Ｆｃ　Ｂｌｏｃｋ（ＢＤ　Ｂｉｏｓｃｉｅｎｃｅｓ社）で処理し非特異的な抗体結合を阻害した。その後、３－３Ｆ抗体、Ｍ１３１０Ｇ０５抗体を含む各種の表面抗原（ＴＣＲβ、ＣＤ４、ＣＤ８、ＣＤ２５）に対する抗体を加えて４℃で３０分間反応させた（サンプル１）。同時にすべての試料から別のサンプルをとり４３３Ｈ抗体を含む各種表面抗原（ＣＤ４５、ＴＣＲβ、ＣＤ４、ＣＤ８、ＣＤ２５）に対する抗体で同様に反応させた（サンプル２）。洗浄後、Ｔｒａｎｓｃｒｉｐｔｉｏｎ　Ｆａｃｔｏｒ　Ｂｕｆｆｅｒ　Ｓｅｔ（ＢＤ　Ｂｉｏｓｃｉｅｎｃｅｓ社）を用い細胞を固定、及び膜透過処理した後、ふたたびＭｏｕｓｅ　ＢＤ　Ｆｃ　Ｂｌｏｃｋで処理した後にＰＥ標識抗ヒトＦｏｘｐ３抗体を添加し、４℃に５０分間（サンプル１）、もしくは一晩（サンプル２）静置し染色を行った。洗浄後、ＭＡＣＳＱｕａｎｔＡｎａｌｙｚｅｒ（Ｍｉｌｔｅｎｙｉ Ｂｉｏｔｅｃ社）を用いて、Ｔｒｅｇ細胞（サンプル１ではｅＦｌｕｏｒ　７８０、ＣＤ８陰性、かつＴＣＲβ、ＣＤ４、ＣＤ２５、Ｆｏｘｐ３陽性分画、サンプル２ではｅＦｌｕｏｒ　７８０、ＣＤ８陰性、かつＣＤ４５、ＴＣＲβ、ＣＤ４、ＣＤ２５、Ｆｏｘｐ３陽性分画）における３－３Ｆ抗体のシグナルとＭ１３１０Ｇ０５抗体シグナルの相関を解析した（図９）。各抗体の示す蛍光シグナルの指標として、Ｔｒｅｇ細胞中に占める陽性細胞の率とその陽性細胞集団の示すＭＦＩ（Ｍｅａｎ　Ｆｌｕｏｒｅｓｃｅｎｃｅ　Ｉｎｔｅｎｓｉｔｙ）を掛け合わせた値（％ｘＭＦＩ）を用いた。また、比較対照として４３３Ｈ抗体のシグナルとＭ１３１０Ｇ０５抗体シグナルの相関を解析した（図１０）。
　その結果、ＯＰ－１抗体が結合したＴｒｅｇ細胞に対する３－３Ｆ抗体の結合量は正に相関することが判明した（図９）。一方、市販の抗ＣＣＲ８抗体である４３３Ｈ抗体の結合量は、ＯＰ－１抗体の結合量が増大するにつれ減少する事が確認された（図１０）。 Example 7: Evaluation of binding of 3-3F antibody to mouse tumor-infiltrating cells The binding of 3-3F antibody to mouse tumor-infiltrating cells was evaluated using a mouse kidney cancer lung metastasis model. In this example, a BB515 fluorescently labeled 3-3F antibody was used. In order to evaluate the effect of an anti-CCR8 antibody different from 3-3F binding to CCR8 on the binding of the 3-3F antibody, a portion of this mouse model was prepared using the neutralizing antibody against human CCR8 used in Example 4. OP-1 antibody, which is an anti-human IgG Fc antibody, was administered, and the BV421 fluorescent label of M1310G05 antibody, which is an anti-human IgG Fc antibody, was used as an antibody to detect the OP-1 antibody bound to human CCR8. As a comparison, a BV421 fluorescent label of 433H antibody, which is a commercially available anti-human CCR8 antibody, was used, and samples separated from the same sample were stained and measured.
Renca, a mouse kidney cancer cell cultured in a knock-in mouse (female, 21 weeks old) in which CCR8 of Balb/C mouse described in Example 10 of WO2020/138489 was replaced with human CCR8 gene, was cultured at 4×10. Five were injected into the tail vein. Twelve days later, OP-1 antibody was injected through the tail vein. The next day, the lungs to which tumor cells had metastasized were collected from the 0.05 mg/kg administration group (8 mice), the 0.5 mg/kg administration group (6 mice), and the untreated mice (9 mice) under inhalation anesthesia. Single cells were dispersed and purified from lung tissue using a Tumor Dissociation Kit, mouse (Miltenyi Biotec), and BD Pharma Lyse (BD Biosciences), and stained for flow cytometry. For staining, dead cells were first stained with eBioscience Fixable Viability Dye eFluor 780 (eFluor 780, Thermo Fisher Scientific), and then Mouse BD Fc Block (BD Biosc to inhibit non-specific antibody binding. Thereafter, antibodies against various surface antigens (TCRβ, CD4, CD8, CD25), including 3-3F antibody and M1310G05 antibody, were added and reacted at 4°C for 30 minutes (Sample 1). At the same time, another sample was taken from all the samples and reacted in the same manner with antibodies against various surface antigens (CD45, TCRβ, CD4, CD8, CD25), including the 433H antibody (Sample 2). After washing, cells were fixed and permeabilized using Transcription Factor Buffer Set (BD Biosciences), treated again with Mouse BD Fc Block, PE-labeled anti-human Foxp3 antibody was added, and incubated at 4°C for 50 minutes ( Sample 1) or overnight (sample 2) was left standing and stained. After washing, Treg cells (eFluor 780, CD8 negative, and TCRβ, CD4, CD25, Foxp3 positive fraction for sample 1, eFluor 780, CD8 negative, and CD45, TCR fraction for sample 2) were analyzed using MACSQuantAnalyzer (Miltenyi Biotec). β , CD4, CD25, Foxp3 positive fraction) and the correlation between the 3-3F antibody signal and the M1310G05 antibody signal was analyzed (FIG. 9). As an index of the fluorescence signal exhibited by each antibody, the value obtained by multiplying the ratio of positive cells among Treg cells by the MFI (Mean Fluorescence Intensity) of the positive cell population (% x MFI) was used. Furthermore, as a comparison, the correlation between the 433H antibody signal and the M1310G05 antibody signal was analyzed (FIG. 10).
As a result, it was found that the binding amount of 3-3F antibody to Treg cells bound to OP-1 antibody was positively correlated (FIG. 9). On the other hand, it was confirmed that the binding amount of 433H antibody, which is a commercially available anti-CCR8 antibody, decreased as the binding amount of OP-1 antibody increased (FIG. 10).
　本発明のモノクローナル抗体又はその抗体断片は、生体試料においてＣＣＲ８を検出するために使用することができる。さらに、本発明のモノクローナル抗体又はその抗体断片を含む医薬組成物は、ＣＣＲ８関連疾患の治療又は予防のための医薬として非常に有用である。 The monoclonal antibody or antibody fragment thereof of the present invention can be used to detect CCR8 in a biological sample. Furthermore, a pharmaceutical composition containing the monoclonal antibody of the present invention or an antibody fragment thereof is very useful as a medicament for treating or preventing CCR8-related diseases.

Claims (27)

  1. 配列番号２のアミノ酸配列からなるＣＤＲ１、
    配列番号３のアミノ酸配列からなるＣＤＲ２及び
    配列番号４のアミノ酸配列からなるＣＤＲ３を含む軽鎖可変領域並びに
    配列番号５のアミノ酸配列からなるＣＤＲ１、
    配列番号６のアミノ酸配列からなるＣＤＲ２及び
    配列番号７のアミノ酸配列からなるＣＤＲ３を含む重鎖可変領域
    を含む、ＣＣＲ８に結合するモノクローナル抗体又はその抗体断片。     CDR1 consisting of the amino acid sequence of SEQ ID NO: 2,
    A light chain variable region comprising CDR2 consisting of the amino acid sequence of SEQ ID NO: 3 and CDR3 consisting of the amino acid sequence of SEQ ID NO: 4, and CDR1 consisting of the amino acid sequence of SEQ ID NO: 5,
    A monoclonal antibody or antibody fragment thereof that binds to CCR8 and includes a heavy chain variable region including CDR2 consisting of the amino acid sequence of SEQ ID NO: 6 and CDR3 consisting of the amino acid sequence of SEQ ID NO: 7.
  2. 配列番号８のアミノ酸配列を有する軽鎖可変領域、及び、配列番号９のアミノ酸配列を有する重鎖可変領域を含む、請求項１記載の抗体又はその抗体断片。 The antibody or antibody fragment thereof according to claim 1, comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 8 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 9.
  3. さらに、配列番号１０のアミノ酸配列を有する軽鎖定常領域、及び、配列番号１１のアミノ酸配列を有する重鎖定常領域を有する、請求項１記載の抗体又はその抗体断片。 The antibody or antibody fragment thereof according to claim 1, further comprising a light chain constant region having the amino acid sequence of SEQ ID NO: 10 and a heavy chain constant region having the amino acid sequence of SEQ ID NO: 11.
  4. 請求項１～３のいずれかに記載の抗体又はその抗体断片を含有する組成物を使用する、ＣＣＲ８の検出方法。 A method for detecting CCR8 using a composition containing the antibody or antibody fragment thereof according to any one of claims 1 to 3.
  5. 癌診断のための、請求項４記載の方法。 5. The method according to claim 4, for cancer diagnosis.
  6. 抗ＣＣＲ８抗体を含有する医薬を投与した被験者から採取した試料に含まれるＣＣＲ８を検出する、請求項４記載の方法。 5. The method according to claim 4, wherein CCR8 contained in a sample collected from a subject to whom a medicine containing an anti-CCR8 antibody has been administered is detected.
  7. 抗ＣＣＲ８抗体を含有する医薬に含まれる抗ＣＣＲ８抗体が中和活性を有する、請求項６記載の方法。 7. The method according to claim 6, wherein the anti-CCR8 antibody contained in the anti-CCR8 antibody-containing medicament has neutralizing activity.
  8. 抗ＣＣＲ８抗体を含有する医薬に含まれる抗ＣＣＲ８抗体が、配列番号８のアミノ酸配列を有する軽鎖可変領域、及び、配列番号９のアミノ酸配列を有する重鎖可変領域を含む抗体とＣＣＲ８に対する結合が競合しない、請求項６記載の方法。 The anti-CCR8 antibody contained in the anti-CCR8 antibody-containing medicine has the ability to bind to CCR8 with an antibody comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 8 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 9. 7. The method of claim 6, which is non-conflicting.
  9. 抗ＣＣＲ８抗体を含有する医薬に含まれる抗ＣＣＲ８抗体が、配列番号１２のアミノ酸配列を有する軽鎖可変領域、及び、配列番号１３のアミノ酸配列を有する重鎖可変領域を含むモノクローナル抗体又はその断片である、請求項６記載の方法。 The anti-CCR8 antibody contained in the anti-CCR8 antibody-containing medicine is a monoclonal antibody or a fragment thereof comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 12 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13. 7. The method of claim 6, wherein:
  10. 被験者が癌の患者である、請求項６記載の方法。 7. The method according to claim 6, wherein the subject is a cancer patient.
  11. 請求項１～３のいずれかに記載の抗体又はその抗体断片を含有する組成物を含む、ＣＣＲ８の検出キット。 A CCR8 detection kit comprising a composition containing the antibody or antibody fragment thereof according to any one of claims 1 to 3.
  12. 癌診断のための、請求項１１記載のキット。 The kit according to claim 11, for cancer diagnosis.
  13. 抗ＣＣＲ８抗体を含有する医薬を投与した被験者から採取した試料に含まれるＣＣＲ８を検出する、請求項１１記載のキット。 The kit according to claim 11, which detects CCR8 contained in a sample collected from a subject who has been administered a medicine containing an anti-CCR8 antibody.
  14. 抗ＣＣＲ８抗体を含有する医薬に含まれる抗ＣＣＲ８抗体が中和活性を有する、請求項１３記載のキット。 The kit according to claim 13, wherein the anti-CCR8 antibody contained in the anti-CCR8 antibody-containing medicament has neutralizing activity.
  15. 抗ＣＣＲ８抗体を含有する医薬に含まれる抗ＣＣＲ８抗体が、配列番号８のアミノ酸配列を有する軽鎖可変領域、及び、配列番号９のアミノ酸配列を有する重鎖可変領域を含む抗体とＣＣＲ８に対する結合が競合しない、請求項１３記載のキット。 The anti-CCR8 antibody contained in the anti-CCR8 antibody-containing medicine has the ability to bind to CCR8 with an antibody comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 8 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 9. 14. The kit of claim 13, which is non-competitive.
  16. 抗ＣＣＲ８抗体を含有する医薬に含まれる抗ＣＣＲ８抗体が、配列番号１２のアミノ酸配列を有する軽鎖可変領域、及び、配列番号１３のアミノ酸配列を有する重鎖可変領域を含むモノクローナル抗体又はその断片である、請求項１３記載のキット。 The anti-CCR8 antibody contained in the anti-CCR8 antibody-containing medicine is a monoclonal antibody or a fragment thereof comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 12 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13. 14. The kit of claim 13.
  17. 被験者が癌の患者である、請求項１３記載のキット。 The kit according to claim 13, wherein the subject is a cancer patient.
  18. 請求項１～３のいずれかに記載の抗体又はその抗体断片を含有する、癌の診断薬。 A diagnostic agent for cancer, comprising the antibody or antibody fragment thereof according to any one of claims 1 to 3.
  19. 請求項１～３のいずれかに記載の抗体又はその抗体断片を含有する、抗ＣＣＲ８抗体を含有する医薬の投与の適否を判断するための診断薬。 A diagnostic agent containing the antibody or antibody fragment thereof according to any one of claims 1 to 3, for determining the suitability of administration of a medicament containing an anti-CCR8 antibody.
  20. 抗ＣＣＲ８抗体を含有する医薬が抗癌剤である、請求項１９記載の診断薬。 The diagnostic agent according to claim 19, wherein the medicament containing the anti-CCR8 antibody is an anticancer agent.
  21. 抗ＣＣＲ８抗体を含有する医薬に含まれる抗ＣＣＲ８抗体が、配列番号１２のアミノ酸配列を有する軽鎖可変領域、及び、配列番号１３のアミノ酸配列を有する重鎖可変領域を含むモノクローナル抗体又はその断片である、請求項１９記載の診断薬。 The anti-CCR8 antibody contained in the anti-CCR8 antibody-containing medicine is a monoclonal antibody or a fragment thereof comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 12 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13. The diagnostic agent according to claim 19.
  22. 請求項１～３のいずれかに記載の抗体又はその抗体断片を含有する組成物を使用する、抗ＣＣＲ８抗体を含有する医薬の投与の適否を判断する方法。 A method for determining the suitability of administration of a medicament containing an anti-CCR8 antibody, using a composition containing the antibody or antibody fragment thereof according to any one of claims 1 to 3.
  23. 抗ＣＣＲ８抗体を含有する医薬が抗癌剤である、請求項２２記載の方法。 23. The method according to claim 22, wherein the medicament containing the anti-CCR8 antibody is an anticancer drug.
  24. 抗ＣＣＲ８抗体を含有する医薬に含まれる抗ＣＣＲ８抗体が、配列番号１２のアミノ酸配列を有する軽鎖可変領域、及び、配列番号１３のアミノ酸配列を有する重鎖可変領域を含むモノクローナル抗体又はその断片である、請求項２２記載の方法。 The anti-CCR8 antibody contained in the anti-CCR8 antibody-containing medicine is a monoclonal antibody or a fragment thereof comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 12 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13. 23. The method of claim 22, wherein:
  25. 請求項１～３のいずれかに記載の抗体又はその抗体断片を含有する組成物を使用する、抗ＣＣＲ８抗体を含有する医薬に含まれる抗ＣＣＲ８抗体の腫瘍内浸潤Ｔｒｅｇ細胞への結合量を測定する方法。 Using a composition containing the antibody or antibody fragment thereof according to any one of claims 1 to 3, measuring the amount of binding of an anti-CCR8 antibody contained in a medicament containing an anti-CCR8 antibody to intratumoral infiltrating Treg cells. how to.
  26. 請求項１又は２記載の抗体の軽鎖可変領域又は重鎖可変領域をコードするポリヌクレオチド。 A polynucleotide encoding the light chain variable region or heavy chain variable region of the antibody according to claim 1 or 2.
  27. 請求項２６記載のポリヌクレオチドを含む発現ベクター。 An expression vector comprising the polynucleotide according to claim 26.
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001523446A (en) * 1997-11-13 2001-11-27 シェーリング コーポレイション Th2 cell depletion, compositions, methods
WO2018181064A1 (en) * 2017-03-27 2018-10-04 国立大学法人北海道大学 Anti-pd-l1 antibody for detecting pd-l1
JP2020511932A (en) * 2016-06-20 2020-04-23 カイマブ・リミテッド Anti-PD-L1 and IL-2 cytokine
JP2020511937A (en) * 2016-12-07 2020-04-23 ジェネンテック， インコーポレイテッド Anti-TAU antibody and method of use
WO2020138489A1 (en) * 2018-12-27 2020-07-02 塩野義製薬株式会社 Novel anti-ccr8 antibody

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001523446A (en) * 1997-11-13 2001-11-27 シェーリング コーポレイション Th2 cell depletion, compositions, methods
JP2020511932A (en) * 2016-06-20 2020-04-23 カイマブ・リミテッド Anti-PD-L1 and IL-2 cytokine
JP2020511937A (en) * 2016-12-07 2020-04-23 ジェネンテック， インコーポレイテッド Anti-TAU antibody and method of use
WO2018181064A1 (en) * 2017-03-27 2018-10-04 国立大学法人北海道大学 Anti-pd-l1 antibody for detecting pd-l1
WO2020138489A1 (en) * 2018-12-27 2020-07-02 塩野義製薬株式会社 Novel anti-ccr8 antibody

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SUZUKI HIROYUKI, SAITO MASAKI, ASANO TEIZO, TANAKA TOMOHIRO, KITAMURA KAISHI, KUDO YUMA, KANEKO MIKA K., KATO YUKINARI: "C 8 Mab-3: An Anti-Mouse CCR8 Monoclonal Antibody for Immunocytochemistry", MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY, MARY ANN LIEBERT, INC. PUBLISHERS, US, vol. 41, no. 2, 1 April 2022 (2022-04-01), US , pages 110 - 114, XP093109349, ISSN: 2167-9436, DOI: 10.1089/mab.2022.0002 *

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