WO2023219147A1 - Nouveaux anticorps anti-ccr8 pour la détection de ccr8 - Google Patents

Nouveaux anticorps anti-ccr8 pour la détection de 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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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.

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Abstract

L'invention concerne de nouveaux anticorps anti-CCR8. Lesdits anticorps détectent CCR8 exprimé dans des cellules Treg invasives tumorales ou similaires et sont utiles pour le diagnostic du cancer ou le diagnostic compagnon.
PCT/JP2023/017821 2022-05-13 2023-05-12 Nouveaux anticorps anti-ccr8 pour la détection de ccr8 WO2023219147A1 (fr)

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WO2024165468A1 (fr) 2023-02-06 2024-08-15 Bayer Aktiengesellschaft Combinaison d'anticorps anti-ccr8 avec des inhibiteurs de dgk dans le traitement du cancer

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WO2024165468A1 (fr) 2023-02-06 2024-08-15 Bayer Aktiengesellschaft Combinaison d'anticorps anti-ccr8 avec des inhibiteurs de dgk dans le traitement du cancer

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