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WO2018159808A1 - Antibody-drug conjugate of anti-il-7r antibody, and pharmaceutical composition for treating cancer or inflammation and containing antibody-drug conjugate of anti-il-7r antibody and cytotoxic drug - Google Patents

Antibody-drug conjugate of anti-il-7r antibody, and pharmaceutical composition for treating cancer or inflammation and containing antibody-drug conjugate of anti-il-7r antibody and cytotoxic drug Download PDF

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WO2018159808A1
WO2018159808A1 PCT/JP2018/007985 JP2018007985W WO2018159808A1 WO 2018159808 A1 WO2018159808 A1 WO 2018159808A1 JP 2018007985 W JP2018007985 W JP 2018007985W WO 2018159808 A1 WO2018159808 A1 WO 2018159808A1
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antibody
cancer
pharmaceutical composition
steroid
cells
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French (fr)
Japanese (ja)
Inventor
正浩 安永
保広 松村
眞鍋　史乃
厚至 辻
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National Cancer Center Japan
RIN Institute Inc
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National Cancer Center Japan
RIN Institute Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/475—Quinolines; Isoquinolines having an indole ring, e.g. yohimbine, reserpine, strychnine, vinblastine
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    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
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    • A—HUMAN NECESSITIES
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    • A61P35/00—Antineoplastic agents
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons

Definitions

  • the present invention relates to an antibody drug conjugate of an anti-IL-7R antibody (anti-IL-7 receptor antibody) and an antibody of an anti-IL-7R antibody and a cytotoxic agent for use in treating cancer or inflammation. It relates to a pharmaceutical composition comprising a drug conjugate.
  • SN-38 a topoisomerase 1 inhibitor
  • CPT-11 was developed as a compound that improves the blood translocation of SK-38.
  • CPT-11 is a prodrug of SN-38, and the conversion efficiency to the active form is high in cancer tissues of solid tumors, but is low in blood and low in blood. Therefore, CPT-11 could not be used to treat leukemia.
  • IL-7 is known as an essential cytokine for T cell generation and homeostasis (Non-patent Document 1).
  • IL-7R ⁇ moves into the cell slowly and partly returns to the cell surface, but partly decomposes (Non-patent Document 1).
  • the half-life of IL-7R ⁇ is shortened from about 24 hours to 3 hours by stimulation with IL-7 (Non-patent Document 1).
  • IL-7R is responsible for cancer steroid resistance (Non-patent Document 2).
  • Non-Patent Document 2 suggests that suppressing downstream signals of IL-7R is effective in treating steroid resistance.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody drug conjugate of an anti-IL-7R antibody and an antibody drug conjugate of an anti-IL-7R antibody and a cytotoxic agent for use in treating cancer or inflammation.
  • IL-7R is associated with steroid resistance and that IL-7R knockdown suppresses cell proliferation of cancer cell lines.
  • IL-7R knockdown dramatically improves the steroid sensitivity of cancer cells (especially lymphomas and leukemias), thereby increasing the cell killing effect of steroids, and steroid resistance
  • the expression of IL-7R is improved by obtaining.
  • the inventors further found that activation of NF ⁇ B occurs in steroid resistance without STAT or BCL2 activation of downstream signals of IL-7R.
  • the inventors further have improved expression of IL-7R after chemotherapy treatment with steroidal anti-inflammatory drugs, and IL-7R in cell lines that have been treated with steroidal anti-inflammatory drugs to gain steroid resistance.
  • anti-IL-7R antibodies accumulate in lysosomes when taken up by cells, and accumulate in tumor tissues and lymph nodes one week after administration.
  • the effect of the antibody blocking the binding between IL-7R and IL-7 is weak, and on the other hand, the ADC of the anti-IL-7R antibody shows a remarkable effect, And found to suppress lymphadenopathy.
  • the effect of ADC on tumors was also shown with a radioisotope-labeled anti-IL-7R antibody.
  • the present inventors have found that the symptoms of inflammatory diseases (for example, rheumatoid arthritis and ulcerative colitis) are reduced through a decrease in IL-7R positive cells of the immune system. The present invention is based on these findings.
  • a pharmaceutical composition comprising an antibody drug conjugate of an anti-IL-7R antibody or an antigen-binding fragment thereof and a cytotoxic agent, for use in treating cancer or inflammation.
  • the pharmaceutical composition according to the above (1) for use in treating inflammation.
  • the pharmaceutical composition according to (1) or (2) above wherein the inflammation is steroid-treated or steroid-resistant inflammation.
  • the pharmaceutical composition according to (2) or (3) above, wherein the inflammation is ulcerative colitis or rheumatoid arthritis.
  • a pharmaceutical composition comprising an antibody drug conjugate of an anti-IL-7R antibody or an antigen-binding fragment thereof and a cytotoxic agent, which is used for treating a disease or condition after steroid treatment or steroid resistance
  • Pharmaceutical composition for (11) A pharmaceutical composition comprising an antibody drug conjugate of an anti-IL-7R antibody or antigen-binding fragment thereof and a cytotoxic agent, which is used in combination with a steroid.
  • FIG. 1A shows the results of examining the expression level of IL-7R in various cancer cell lines by flow cytometry.
  • FIG. 1B is a graph showing that IL-7R expression level was actually decreased in various cancer cell lines by IL-7R knockdown assay.
  • FIG. 1C is a graph showing the influence on the growth of a cancer cell line in which IL-7R is knocked down.
  • FIG. 1D is a graph showing the influence of subcutaneously transplanted IL-7R on the growth of a cancer cell line knocked down in a nude mouse subcutaneous transplantation model.
  • FIG. 1E is a photomicrograph showing the viability of a cancer cell line in which IL-7R has been knocked down.
  • FIG. 1A shows the results of examining the expression level of IL-7R in various cancer cell lines by flow cytometry.
  • FIG. 1B is a graph showing that IL-7R expression level was actually decreased in various cancer cell lines by IL-7R knockdown assay.
  • FIG. 2A is a graph showing that IC50 of various cancer cell lines against dexamethasone (a kind of steroidal anti-inflammatory drug) is decreased by IL-7R knockdown.
  • FIG. 2B shows the effect of IL-7R knockdown on the resistance to dexamethasone in vitro by obtaining a dexamethasone resistant strain.
  • FIG. 2C shows that the expression of IL-7R is improved in the dexamethasone resistant strain.
  • FIG. 3 shows the results of staining various cancer cell lines and their IL-7R knockdown lines with anti-phosphorylated p65 / NF ⁇ B antibodies.
  • FIG. 4A is a diagram showing changes in gene expression before and after chemotherapy including steroids in B-cell acute leukemia.
  • FIG. 4A is a diagram showing changes in gene expression before and after chemotherapy including steroids in B-cell acute leukemia.
  • FIG. 4B is a diagram showing changes in gene expression before and after acquisition of steroid resistance by steroid treatment in B-cell acute leukemia.
  • FIG. 4C shows changes in gene expression before and after chemotherapy including steroids in T-cell acute leukemia.
  • FIG. 5A shows the results of confirming co-localization with lysosome by contacting Alexa647-labeled anti-IL-7R antibody with a cancer cell line in vitro.
  • FIG. 5B shows the results of administering a fluorescently labeled anti-IL-7R antibody to a tumor subcutaneous transplantation model and examining the accumulation of anti-IL-7R antibody in various tissues one week later.
  • FIG. 5C shows that an anti-IL-7R antibody having the ability to neutralize binding between IL-7R and IL-7 is effective against RAG2 ⁇ / ⁇ cells that survive in an IL-7-dependent manner, It shows that it has little effect on the cancer cell line CYG82.
  • FIG. 5D shows that an antibody drug conjugate (ADC) of an anti-IL-7R antibody and a cytotoxic agent exerts a killing effect on the cancer cell line CYG82.
  • FIG. 6A is a graph showing the effect of an antibody drug conjugate of an anti-IL-7R antibody and a cytotoxic agent on tumor volume increase in a subcutaneous transplantation model of a cancer cell line.
  • FIG. 6B is a graph showing the effect of an antibody drug conjugate of an anti-IL-7R antibody and a cytotoxic agent on tumor volume increase in a steroid-resistant cancer cell line subcutaneous transplantation model.
  • FIG. 7A shows the effect of IL-7R knockdown on lymphadenopathy in a subcutaneous transplantation model of cancer cell line CYG82.
  • FIG. 7B is a graph showing the effect of ADC of anti-IL-7R and SN-38 on intraperitoneal lymphadenopathy occurring through regional lymphadenopathy in a subcutaneous transplantation model of cancer cell line CYG82.
  • FIG. 8A is a graph showing the effect of a radioisotope-labeled ( 90 Y-labeled) anti-IL-7R antibody on the T-cell lymphoma cell line Sup-T1.
  • FIG. 8B shows the effect of radioisotope-labeled ( 90 Y-labeled) anti-IL-7R antibody on lung cancer cell line H2009.
  • FIG. 9 is a diagram showing the effect of anti-IL-7R on the immune system of ADC.
  • FIG. 10 shows that IL-7R expression is increased in rheumatoid arthritis and ulcerative colitis.
  • FIG. 11A shows the effect of ADC of anti-IL-7R antibody and monomethyl auristatin E (MMAE) on rheumatoid arthritis model.
  • FIG. 11B shows the effect of anti-IL-7R antibody and SN-38 ADC on rheumatoid arthritis model.
  • FIG. 11C shows the result of preparing a tissue section of a mouse forefoot lesion of a rheumatoid arthritis model and staining with hematoxylin and eosin.
  • FIG. 12A is a graph showing the effect of ADC of anti-IL-7R antibody and MMAE on ulcerative colitis model.
  • FIG. 12B is a graph showing the effect of ADC of anti-IL-7R antibody and SN-38 on ulcerative colitis model.
  • FIG. 13 shows that the colonic morphology of the ulcerative colitis model is improved with an anti-IL-7R antibody and MMAE ADC or an anti-IL-7R antibody and SN-38 ADC.
  • FIG. 14 is a graph showing the effect of ADC of anti-IL-7R antibody and MMAE or ADC of anti-IL-7R antibody and SN-38 on the histopathological image of a colon specimen removed from an ulcerative colitis model.
  • subject means a mammal, and may be a human in particular.
  • treatment is used to mean treatment (therapeutic treatment) and prevention (preventive treatment).
  • treatment means treatment, cure, prevention or amelioration of a disease or disorder or a reduction in the rate of progression of a disease or disorder.
  • prevention means reducing the likelihood of the onset of a disease or condition or delaying the onset of a disease or condition.
  • disease means a symptom for which treatment is beneficial.
  • therapeutically effective amount means the amount of a drug effective for treating (preventing or treating) a disease or condition.
  • a therapeutically effective amount of a drug reduces the rate of worsening of a symptom of a disease or condition, stops the worsening of the symptom, improves the symptom, cures the symptom, or develops or develops the symptom Can be suppressed.
  • resistant means not showing efficacy for treatment (eg, showing no significant effectiveness).
  • sensitivity means showing efficacy for treatment. Decreasing resistance has the same meaning as increasing sensitivity, and increasing resistance has the same meaning as decreasing sensitivity.
  • steroid resistance can be defined as no complete remission in the case of nephrotic syndrome, even if prednisolone is administered daily for more than 4 weeks.
  • other inflammatory / autoimmune diseases or malignant diseases such as leukemia / malignant lymphoma
  • other inflammatory / autoimmune diseases or malignant diseases such as leukemia / malignant lymphoma
  • other inflammatory / autoimmune diseases or malignant diseases such as leukemia / malignant lymphoma
  • other inflammatory / autoimmune diseases or malignant diseases such as leukemia / malignant lymphoma
  • other inflammatory / autoimmune diseases or malignant diseases such as leukemia / malignant lymphoma
  • steroids such as prednisolone, methylprednisolone
  • IL-7R means interleukin 7 receptor.
  • JAK1 and JAK3 are activated and receptor autophosphorylation occurs.
  • STAT binds to the phosphorylated receptor and is phosphorylated by JAK to dimerize. It is thought that transcriptional activation of genes is caused by dimerized STAT.
  • IL-7R has a high internalization activity of the molecule itself, and is internalized from the membrane and decomposed or recycled again to be exposed on the membrane.
  • blocking means neutralizing the binding of two proteins. Blocking includes partial dissociation in addition to complete dissociation. For example, it can be said that blocking occurs when the binding of two proteins is reduced by 50% or more.
  • steroid means steroidal anti-inflammatory drugs (SAIDs).
  • SAIDs steroidal anti-inflammatory drugs
  • glucocorticoids or their derivatives are mainly active ingredients.
  • GR ⁇ glucocorticoid receptor
  • GR ⁇ dissociates from hsp90, forms a dimer, moves into the nucleus, and regulates transcription.
  • Synthetic steroids include cortisols such as hydrocortisone and hydrocortisone succinate; prednisolones such as prednisolone, methylprednisolone and methylprednisolone succinate; triamcinolones such as triamcinolone and triamcinolone acetonide; dexamethasone and betamethasone. Both are derivatives of glucocorticoid and function in the cell by the same mechanism as glucocorticoid.
  • antibody drug conjugate means a substance in which a monoclonal antibody or an antigen-binding fragment thereof (hereinafter sometimes simply referred to as “antibody or the like”) is linked to a drug.
  • ADC a monoclonal antibody or the like and a drug can be linked via an appropriate linker.
  • the ADC binds to a membrane component on the cell membrane (for example, a transmembrane protein such as a receptor), is taken into the cell by endocytosis or internalization, is separated from the antibody or the like, and is released into the cell.
  • the linker By introducing a cleavable linker between the antibody and the drug in the cell, the linker can be cleaved in the cell, for example, in the endosome, and the drug can be released from the antibody and released into the cytoplasm. is there.
  • a cytotoxic agent is used as a drug, it is possible to kill cells to which the drug has been delivered.
  • a chemotherapeutic agent, a radioisotope, and a toxin can be used as the cytotoxic agent.
  • IL-7R expression is improved in many tumor cells.
  • tumor growth was greatly suppressed by reducing the expression of improved IL-7R.
  • IL-7R ADCs showed a strong killing ability.
  • antibodies capable of blocking the binding between IL-7R and IL-7 had little effect on cell survival. Therefore, as long as the anti-IL-7R antibody used in the ADC of the present invention binds to IL-7R on the cell membrane, the ability to block the binding between IL-7R and IL-7 is not questioned. It may be an antibody that can block the binding to -7, or an antibody that does not block the binding between IL-7R and IL-7.
  • an antibody drug conjugate of an anti-IL-7R antibody or antigen-binding fragment thereof and a cytotoxic agent is provided.
  • an antibody that recognizes IL-7R expressed on the cell membrane can be used as the anti-IL-7R antibody.
  • the antibody or antigen-binding fragment thereof and the cytotoxic agent in the antibody drug conjugate are linked via a linker.
  • cytotoxic agents include chemotherapeutic agents (for example, anticancer agents such as commercially available anticancer agents such as auristatin (auristatin E, auristatin F phenylenediamine (AFP), monomethyl auristatin E, monomethyl auristatin).
  • Statins F and their derivatives maytansinoids DM1 and DM4 and their derivatives), camptothecin (SN-38, topotecan and exotecan and their derivatives), DNA minor groove binders (enzyin, lexitropsin, duocarmycin) And their derivatives), taxanes (paclitaxel and docetaxel and their derivatives), polyketides (discodermolide and their derivatives), anthraquinones (mitoxantrone and their derivatives), benzodiazepines (pyrrolobenzodiazepine, indolinobenzo) Azepine, and oxazolidinobenzodiazepines and their derivatives), vinca alkaloids (vincristine, vinblastine, vindesine, and vinorelbine and their derivatives), doxorubicins (doxorubicin, morpholino-doxorubicin, and cyanomorpholino-doxorubicin and their derivatives), Cardiac glycosides (digit
  • the linker can be appropriately selected and synthesized by those skilled in the art in the production of ADC.
  • the linker can be a cleavable linker.
  • cleavable linkers include peptide linkers such as valine-citrulline (Val-Cit) and phenylalanine-lysine (Phe-lys) linkers, and hydrazone linkers that cleave in a pH-dependent manner.
  • Cleaveable linkers also include linkers that contain carbamate or ester bonds, which can be enzymatically degraded intracellularly. These linkers may be used in combination.
  • the bond between the antibody and the linker can be linked to, for example, a sulfhydryl group of the antibody via a maleimide group.
  • the linker may contain a polyethylene glycol block as necessary.
  • the present invention also provides a pharmaceutical composition for use in treating cancer, comprising an antibody drug conjugate of an anti-IL-7R antibody or antigen-binding fragment thereof and a cytotoxic agent.
  • Many cancer cells are positive for IL-7R. Accordingly, many cancers can be treated by the medicament of the present invention.
  • the cancer may be an IL-7R-positive cancer or a cancer that has been confirmed to be IL-7R-positive. it can.
  • IL-7R positive cancers include leukemia, lymphoma, lung cancer, pancreatic cancer, head and neck cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, stomach cancer, colon cancer, uterine cancer, ovary Cancer, skin cancer, thyroid cancer, thymic cancer, kidney cancer, testicular cancer, penile cancer, liver cancer, biliary tract cancer, brain tumor, bone soft tissue tumor, retroperitoneal tumor, angio-lymphatic sarcoma , And these metastatic cancers (eg, metastatic solid tumors). Many cancers, such as leukemia, lymphoma, advanced cancer, and metastatic cancer, are positive for IL-7R. Whether IL-7R is positive or negative can be easily determined using techniques such as immunostaining and FACS using an anti-IL-7R antibody.
  • the pharmaceutical composition of the present invention can be used for treating IL-7R-positive cancer having resistance to a therapy that blocks the binding between IL-7R and IL-7.
  • IL-7R is involved in imparting steroid resistance.
  • IL-7R is positive in steroid resistant cells.
  • a pharmaceutical composition for use in treating a steroid resistant disease or condition comprising an antibody drug conjugate of an anti-IL-7R antibody or antigen-binding fragment thereof and a cytotoxic agent.
  • a pharmaceutical composition comprising a gate is provided.
  • a pharmaceutical composition for use in reducing steroid resistance in a disease or condition comprising an antibody drug conjugate of an anti-IL-7R antibody or antigen-binding fragment thereof and a cytotoxic agent.
  • a pharmaceutical composition is provided.
  • the steroid resistant disease or condition is cancer or inflammation. Furthermore, according to the present invention, it was also revealed that expression of IL-7R is improved after steroid treatment. Particularly after steroid treatment, IL-7R positive cells are more likely to survive, increasing the effectiveness of the conjugates of the invention. Even if IL-7R negative cells survive (for example, in patients after steroid treatment), a therapeutic effect can be expected. This is because the effect of killing IL-7R negative cells in the vicinity of IL-7R positive cells can be expected by the bystander effect of PDC.
  • a pharmaceutical composition for use in treating a disease or condition such as cancer or inflammation in a subject who has received steroid therapy wherein the anti-IL-7R antibody or antigen-binding property thereof is used.
  • Pharmaceutical compositions comprising antibody drug conjugates of fragments and cytotoxic agents are provided.
  • a pharmaceutical composition for use in treating a steroid resistant disease or condition, wherein the anti-IL-7R antibody or antigen-binding fragment thereof and cytotoxicity are used in combination with a steroid.
  • Pharmaceutical compositions comprising an antibody drug conjugate with an agent are provided.
  • a pharmaceutical composition for use in reducing steroid resistance of a disease or condition or increasing steroid sensitivity of the disease or condition
  • a pharmaceutical composition comprising an antibody drug conjugate of a sex fragment and a cytotoxic agent is provided.
  • the subject can be a subject with a steroid resistant disease or condition.
  • an antibody drug conjugate of an anti-IL-7R antibody or an antigen-binding fragment thereof and a cytotoxic agent can suppress lymphadenopathy in cancer subjects. Therefore, according to the present invention, a pharmaceutical composition for use in suppressing lymphadenopathy in a cancer subject, comprising an anti-IL-7R antibody or an antigen-binding fragment thereof and an antibody of a cytotoxic agent A pharmaceutical composition comprising a drug conjugate is provided.
  • an antibody drug conjugate of an anti-IL-7R antibody and a cytotoxic agent showed a symptom reducing effect.
  • IL-7R positive cells are strongly involved in the onset or maintenance of inflammatory diseases, and killing IL-7R positive cells in an inflammatory state is useful for alleviating inflammation. It means that there is.
  • a pharmaceutical composition for use in treating an inflammatory disease comprising an antibody drug conjugate of an anti-IL-7R antibody or antigen-binding fragment thereof and a cytotoxic agent A pharmaceutical composition is provided.
  • inflammatory diseases include collagen disease, autoimmune disease, type I diabetes, multiple sclerosis, systemic lupus erythematosus, allergic disease, rejection after organ transplantation, and graft versus host Diseases are also included. Inflammatory diseases also include inflammatory bowel diseases such as Crohn's disease.
  • autoimmune diseases and collagen diseases include Sjogren's syndrome, scleroderma, dermatomyositis, polyarteritis nodosa, mixed connective tissue disease, Corgan syndrome, polymyalgia rheumatica, adult Still's disease, giant cells Arteritis, antiphospholipid antibody syndrome, Guillain-Barre syndrome, myasthenia gravis, autoimmune hepatitis, autoimmune pancreatitis, primary biliary cholangitis, Takayasu arteritis, Goodpasture syndrome, rapidly progressive glomeruli Nephritis, autoimmune hemolytic anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura, Graves' disease, Hashimoto's disease, primary hypothyroidism, idiopathic Addison's disease, pemphigus vulgaris, pustular psoriasis Psoriasis vulgaris, pemphigoid, gestational herpe
  • lymphocytes are IL-7R positive in many inflammations. Specifically, many inflammatory diseases caused by these cells because they are positive in acquired immune system lymphocytes, T lymphocytes and B lymphocytes, and also in innate immune system lymphocytes. Can be the subject of treatment with the pharmaceutical composition of the present invention.
  • the inflammatory disease can be a disease or condition that is resistant to steroids or resistant to treatment that blocks the binding of IL-7R to IL-7.
  • a method of treating cancer or an inflammatory disease in a subject in need thereof comprising the step of administering to the subject an antibody drug conjugate of an anti-IL-7R antibody or antigen-binding fragment thereof and a cytotoxic agent.
  • a method is provided that includes administering a gate. The conjugate can be administered in a therapeutically effective amount.
  • the subject can be a subject that is resistant to steroid treatment or has received steroid treatment.
  • a method of treating cancer or an inflammatory disease in a subject in need thereof comprising the step of administering an antibody drug conjugate of an anti-IL-7R antibody or antigen-binding fragment thereof to a cytotoxic agent.
  • a method is provided comprising administering a gate and administering a steroidal anti-inflammatory drug to the subject.
  • the subject can be a subject that is resistant to steroid treatment or has received steroid treatment.
  • a method includes administering a conjugate.
  • a method of reducing steroid resistance of a disease or condition in a subject in need thereof, the subject comprising an antibody drug of an anti-IL-7R antibody or antigen-binding fragment thereof and a cytotoxic agent includes administering a conjugate.
  • a medicament for use in treating cancer or an inflammatory disease comprising an antibody drug conjugate of an anti-IL-7R antibody or antigen-binding fragment thereof and a cytotoxic agent.
  • the use of anti-IL-7R antibodies is provided.
  • in the manufacture of a medicament for use in treating cancer or an inflammatory disease comprising an antibody drug conjugate of an anti-IL-7R antibody or antigen-binding fragment thereof and a cytotoxic agent.
  • Use of an anti-IL-7R antibody or antigen-binding fragment thereof and a cytotoxic agent is provided.
  • an antibody drug conjugate of an anti-IL-7R antibody or antigen-binding fragment thereof and a cytotoxic agent in the manufacture of a medicament for use in treating cancer or an inflammatory disease.
  • One aspect of the invention provides the use of an antibody drug conjugate of an anti-IL-7R antibody or antigen-binding fragment thereof and a cytotoxic agent in a method for treating cancer or an inflammatory disease. Is done.
  • IL-7R expression may be suppressed in IL-7R-expressing cells.
  • a pharmaceutical composition for use in treating cancer or inflammatory disease comprising an IL-7R expression inhibitor.
  • the present invention also provides a pharmaceutical composition for use in treating a steroid resistant disease or condition, comprising an IL-7R expression inhibitor.
  • the present invention further provides a pharmaceutical composition for use in reducing steroid resistance in a disease or condition, comprising an IL-7R expression inhibitor.
  • the present invention further provides a pharmaceutical composition for use in treating a steroid resistant disease or condition, comprising an IL-7R expression inhibitor used in combination with a steroid.
  • the present invention further provides a pharmaceutical composition for use in suppressing lymphadenopathy in a cancer subject, comprising an IL-7R expression inhibitor.
  • IL-7R expression inhibitors include siRNA, shRNA and antisense oligos against IL-7R, which can be used in the present invention.
  • examples of the IL-7R expression inhibitor include an expression vector containing a DNA encoding siRNA or shRNA.
  • Example 1 Survival activity of IL-7 receptor and tumor cells
  • IL-7 receptor IL-7 receptor
  • IL-7R IL-7 receptor
  • IL-7R expression was measured by flow cytometry using an anti-human IL-7R antibody (eBioRDR5, eBioscience; R34-R34, TONB0) and an anti-mouse IL-7R antibody (A7R34 or SB / 199, eBioscience).
  • IL-7R antibody (A7R34) was prepared as in References 2 and 3 below and used in this example. The result was as shown in FIG. 1A.
  • IL-7R was positive in human T cell malignant lymphoma cell line SupT1, human B cell lymphocytic leukemia cell line NALM6, and mouse lymphocytic leukemia cell line CYG82.
  • lung cancer lymph node metastasis derived cell line H2009 lung cancer alveolar metastasis derived cell line H358, lung cancer pleural effusion cell line LC-2 / ad, pancreatic cancer liver metastasis derived cell line PK-45H, pharyngeal cancer IL-7R was positive in the cancerous pleural effusion cell line Detroite 562, prostate cancer bone metastasis-derived cell line PC3, and bladder cancer cell line TCCsup with bone metastasis (the histogram on the right side of each figure shows IL- 7R expression, left histogram is negative control).
  • IL-7R was positive in all cell lines of lymphoid malignancies and metastatic solid tumors examined. Expression was confirmed in metastatic solid tumors
  • IL-7R is expressed by expressing short hairpin RNA (shRNA, Sigma) incorporated into a lentiviral vector.
  • a gene knockdown (KD) cell line (IL-7R-KD) was generated.
  • a control cell (CTR) for comparison was prepared by introducing shRNA (Sigma) of the GFP gene into SupT1, NALM6, and CYG82, and non-specific shRNA (Sigma) into H2009 and PK-45H.
  • CDNA was synthesized from the total RNA of the cells using an extraction kit (QIAGEN) and a synthesis kit (Thermo Fisher Scientific or Takara).
  • Quantitative RT-PCR was performed. That is, ABI7500Fast (Thermo Fisher Scientific) was used in a total of 20 ⁇ l of the reaction mixture of 9 ⁇ l of cDNA dilution, 1 ⁇ l TaqMan primers / probe mixture (Thermo Fisher Scientific) and 10 ⁇ l TaqMan Fast Universal PCR Master Mix (Thermo Fisher Scientific). ). The expression of each gene was standardized by the comparative CT method using GAPDH for human genes and ACTb for mouse genes.
  • Example 2 Physiological function of IL-7R In this example, further physiological function of IL-7R was examined.
  • cytotoxic effect (IC50; 50% cytotoxic concentration) of dexamethasone in each cell in vitro was examined. confirmed. 1000 to 3000 cells were seeded at 100 ⁇ L / well in a 96-well plate, and dexamethasone was added to each well at a concentration of 0.1 ⁇ M to 1 mM.
  • WST-8 (Dojindo, Cell-Counting-Kit-8) was added at 10 ⁇ L / well, and after culturing for 3 hours, absorbance at 450 nm was measured with a microplate reader (Molecular Devices). IC50 was calculated by calculating the cell viability at each concentration. The result was as shown in FIG. 2A.
  • IL-7R-KD cells As shown in FIG. 2A, in SupT1, NALM6, and CYG82, the IC50 of steroid (dexamethasone) was decreased and the steroid sensitivity was increased in IL-7R-KD cells. . In H2009 and PK45H, there was no difference between IL-7R-KD cells and CTR cells. Thus, it is clear that IL-7R is greatly involved in steroid sensitivity in lymphoid malignant tumors, whereas in metastatic solid tumors H2009 and PK45H, steroid sensitivity is It became clear that IL-7R was hardly involved.
  • a steroid resistant strain was obtained, and an IL-7R knockdown strain and a control strain (CTR) with a specific shRNA were prepared to confirm the effect of IL-7R knockdown in the presence of dexamethasone. did. Since a CYG82 steroid resistant strain can be easily obtained from the CYG82 parental strain, the growth of cells in a state where a high concentration of steroid was added was observed. 10 6 cells were seeded at 3 ml / well in a 6- well plate, 10 nM or 100 nM dexamethasone was added, and the number of cells after 1 week and 2 weeks was counted. The result was as shown in FIG. 2B.
  • cDNA is synthesized from total RNA of cells using an extraction kit (QIAGEN) and a synthesis kit (Thermo Fisher Fisher or Takara), quantitative PCR is performed, and the expression of IL-7R gene and CD19 gene is performed. Examined. The result was as shown in FIG. 2C.
  • the steroid resistant strain of CYG82 cells obtained above shows a high IC50 against dexamethasone. Then, when the expression level of IL-7R was compared between the non-resistant parent strain and the resistant strain, it was revealed that the expression of IL-7R was significantly improved in the resistant strain. On the other hand, regarding the expression level of CD19, no significant difference was observed between the parent strain and the resistant strain.
  • Example 3 Relationship between IL-7R Signal Activation and Steroid Resistance
  • the relationship between IL-7R signal activation and steroid resistance was examined.
  • JAK / STAT was measured by fixing cells with 4% paraformaldehyde, treating with methanol, and detecting phosphorylated STAT5 using an anti-phospho-STAT5 antibody (eBioscience). Measurement was performed using a flow cytometry detection device, Guava easyCyte 10HT (Merck Millipore) or Aria flow cytometer (BD Biosciences). Dead cells were stained with Propidium iodide (Thermo Fisher Scientific) and excluded during measurement.
  • FlowJo program (Tree Star) was used for data analysis.
  • the BCL2 signal was subjected to total RNA extraction and cDNA synthesis from each cell, and the expression level of BCL2 was examined by quantitative PCR.
  • For NF ⁇ , cells were fixed with 4% paraformaldehyde and reacted with anti-phospho-p65 / NF-k ⁇ antibody (Abcam).
  • As the secondary antibody Alexa-488 / 555 / 647-labeled anti-rabbit IgG antibody (Thermo Fisher Scientific) was used. Nuclei were stained with DAPI (Thermo Fisher Scientific).
  • NFkB was decreased by IL-7R knockdown in all cells, but also decreased in IL-7R-KD of H2009 and PK-45H cells. Since these cells had no relationship with steroid resistance as shown in FIG. 2A, NFkB seemed to be more likely to be more strongly involved in cell proliferation and survival than steroid resistance.
  • Example 4 Acquisition of steroid resistance and change in gene expression
  • the relationship between IL-7R gene expression and steroid resistance in clinical samples was examined.
  • GSE39339 and GSE32962 were obtained from Gene® Expression® Omnibus® (GEO), and clinical data of E-MEXP-3916 was also acquired from Array® Express.
  • the data was subjected to statistical analysis after being standardized by RMA processing using analysis software R (open source freeware).
  • RMA analysis software
  • SPSS Statistics Version 20 SPSS Statistics Version 20 (IBM) was used, and P ⁇ 0.05 or less was used as a criterion.
  • FIG. 4A shows the analysis result of GSE39339
  • FIG. 4B shows the analysis result of GSE32962
  • FIG. 4C shows the analysis result of E-MEXP-3916.
  • lymphocyte markers TdT, CD19, and CD22 significantly decreased after chemotherapy including steroid (dexamethasone) for human acute lymphoblastic leukemia.
  • steroid dexamethasone
  • IL-7R tended to increase after treatment.
  • Chemotherapy included anthracycline, vincristine, and L-asparakinase in addition to dexamethasone.
  • a steroid (prednisolone) sensitive and resistant strain was established from cells derived from a patient with human acute B lymphoblastic leukemia and subjected to DNA microarray analysis. There was no change in TdT, CD19, and CD22 between the two groups, but IL-7R was significantly higher in the steroid (prednisolone) resistant strain.
  • DNA microarray analysis was performed in a non-treated group and a treated group containing steroid (dexamethasone) in a patient with human acute T lymphocytic leukemia.
  • IL-7R expression was significantly higher in the treatment group.
  • Chemotherapy included daunorubicin, vincristine, and L-asparakinase in addition to dexamethasone.
  • Example 5 Characteristics of anti-IL-7R antibody
  • the kinetics when an anti-IL-7R antibody was brought into contact with cells were examined.
  • Alexa647 (Thermo Fisher Scientific) was labeled with an anti-IL-7R antibody (A7R34). Alexa647-labeled anti-IL-7R antibody (A7R34) was administered to CYG62 cells under in vitro culture conditions, and intracellular uptake after 10 and 30 minutes was observed. Lysosomes were visualized with a lyso tracker (Thermo Fisher Fisher Scientific). The result was as shown in FIG. 5A.
  • the anti-IL-7R antibody was taken up into cells within 30 minutes after administration and localized in lysosomes.
  • the kinetics of anti-IL-7R antibody in vivo was confirmed. Specifically, after the CYG82 parent strain was formed into a tumor after subcutaneous implantation of female nude mice, Alexa647-labeled anti-IL-7R antibody (A7R34) or control antibody was administered from the tail vein of the mouse. One week after administration, the tumor, lymph node, spleen, bone, and liver were removed, and accumulation of anti-IL-7R antibody (A7R34) in each tissue was confirmed. The result was as shown in FIG. 5B.
  • the anti-IL-7R antibody (A7R34) accumulated in the tumor and the diseased lymph node, whereas the control antibody did not accumulate in these tissues.
  • CYG82 cells and IL-7-dependent RAG2 ⁇ / ⁇ cells were added to each 96-well plate at 2000 or 4000 cells / 100 ⁇ L / well and cultured.
  • IL-7R antibody A7R34 was added in a dilution series of 10 ⁇ 4 to 10 2 ⁇ g / ml, and 72 hours later, WST-8 (Dojindo, Cell Counting Kit-8) was added at 10 ⁇ L / well and cultured for 3 hours. Later, the absorbance at 450 nm was measured with a microplate reader (Molecular Devices). The cell viability was calculated and the cell killing effect due to the neutralizing activity of IL-7 signal of the antibody was measured. The result was as shown in FIG. 5C.
  • the anti-IL-7R antibody (A7R34) showed a cell-killing effect due to neutralizing activity in IL-7-dependent RAG2 ⁇ / ⁇ cells, but almost no effect in CYG82 cells. I could't. This revealed that CYG82 cells do not require IL-7. In CYG82 cells, cell proliferation and survival are maintained by autoactivation of IL-7R, and it is considered that the influence of the neutralizing activity of IL-7R antibody (A7R34) is avoided.
  • the linker and drug use maleimide for binding to the antibody.
  • Mal-PEG 12 -OSu was purchased from Quanta Biodesign or IRIS Biotech GMBH and used to increase polarity.
  • a carbamate bond was introduced into the linker site as a cleavage site for releasing the drug inside the cell.
  • SN-38 was dissolved in dimethyl sulfoxide at a concentration of 10 mM and stored at ⁇ 80 ° C.
  • the ADC was prepared by reducing the SS bond in the antibody and reacting the maleimide group of the linker with the drug. It was possible to obtain an ADC in which 4 to 6 drugs were bound per antibody. The result was as shown in FIG. 5D.
  • A7R34-ADC (SN-38) was 100 times more potent than CPT-11 and showed a cell killing effect similar to free SN-38. This indicates that A7R34-ADC is effective against CYG82 that survives independent of IL-7. This indicates that IL-7R constitutively internalizes even in the absence of IL-7 signal.
  • the anti-IL-7R antibody (A7R34)
  • the neutralizing activity between IL-7 and IL-7R did not significantly affect the cell viability and tumor volume suppression effects.
  • a remarkable antitumor effect was exhibited by the ADC of the anti-IL-7R antibody and the cytotoxic agent.
  • Example 6 Anti-tumor effect in leukemia model
  • the anti-tumor effect by ADC of anti-IL-7R antibody and drug was confirmed in leukemia model.
  • a mouse having a tumor volume of about 50 mm 3 after subcutaneous implantation of female nude mice (5 mice per group) with the CYG82 parent strain was used as a leukemia model mouse.
  • A7R34-ADC (SN-38) prepared in Example 5 was used as the ADC.
  • A7R34-ADC (SN-38) was administered as an ADC amount of 50 mg / kg on days 0, 4 and 8 from the tail vein of the mouse, and 0.6 mg / kg as an SN-38 equivalent amount.
  • Tumor volume was measured at 2-day intervals and measured until day 26 after administration.
  • 50 mg / kg of A7R34-ADC [0.6 mg / kg in terms of SN-38] was transferred to the mouse via the tail vein. Administered on day 8.
  • ADC of anti-IL-7R antibody and drug showed the most excellent inhibitory effect on tumor volume increase. On the other hand, no significant effect was observed with the anti-IL-7R antibody alone. Further, as shown in FIG. 6B, the ADC of the anti-IL-7R antibody and the drug showed a strong antitumor effect even against a steroid-resistant lymphocytic leukemia model.
  • Example 7 Lymph node enlargement suppression CYG82 cells in a leukemia model were transplanted into the front sole of female nude mice (5 mice per group), and the average volume of lymph nodes was measured on the 16th day after transplantation. The result was as shown in FIG. 7A. As shown in FIG. 7A, the enlargement of axillary lymph nodes was markedly suppressed in IL-7R knockdown mice.
  • A7R34-ADC (SN-38) was administered on days 0, 4 and 8 from the mouse tail vein.
  • physiological saline, A7R34 antibody, isotype antibody ADC (SN-38), dexamethasone, or CPT-11 was administered.
  • the volume of the lymph nodes was measured. The result was as shown in FIG. 7B.
  • A7R34-ADC (SN-38) showed a strong lymph node enlargement suppressing effect. Almost no therapeutic effect was observed with A7R34 monotherapy.
  • Example 8 Radioisotope-labeled anti-IL-7R antibody
  • ADC radioisotope-labeled anti-IL-7R antibody
  • the preparation of 90 Y-labeled antibody was performed according to Reference Document 10 below.
  • 1M acetate buffer (pH 6.0) and 90 YCl 3 were mixed at 1: 1 and allowed to stand at room temperature for 5 minutes.
  • a commercially available anti-IL-7R antibody (clone 40131, R & D) was added and allowed to stand at room temperature for 30 minutes.
  • purification was performed using a Sephadex G50 (GE Healthcare) column (swelled with 0.1 M acetate buffer (pH 6.0)). Thereafter, the dose was adjusted to 20 ⁇ g in terms of unlabeled anti-human IL-7R antibody.
  • 90 Y-labeled anti-IL-7R antibody was administered to the tail vein of nude mice transplanted subcutaneously with either SupT1 or H2009 to confirm the drug efficacy.
  • a single dose of 90 ⁇ g of 90 Y-labeled anti-IL-7R antibody was administered. The results were as shown in FIGS. 8A and B.
  • FIGS. 8A and 8B it was revealed that at 90 ⁇ Ci, 90 Y-labeled anti-IL-7R antibody exhibits an anti-tumor effect. This revealed that the anti-IL-7R antibody can be used for radioimmunotherapy.
  • Example 9 Effect of ADC on immune response
  • the effect of ADC on tumor was confirmed.
  • mice Healthy female BALB / c mice receive saline, 50 mg / kg A7R34-ADC (SN-38) [0.6 mg / kg in SN-38 equivalent], 10 mg / kg dexamethasone, or 40 mg / kg dexamethasone. Each was administered from the tail vein of mice.
  • IL-7R positive T cells and B cells were reduced to 1/10 or less in any of the thymus, bone marrow, and spleen.
  • T cells including IL-7R positive T cells in the thymus were generally decreased, but conversely, IL-7R positive B cells in the bone marrow increased more than twice.
  • the ratio of IL-7R positive T cells is increased, and physiologically IL-7R is involved in steroid resistance of T cells and B cells. It was shown that.
  • A7R34-ADC is considered effective in controlling steroid-resistant IL-7R positive cells.
  • Example 10 Increase in IL-7R positive cells in immune diseases
  • IL-7R gene expression in clinical samples was examined in the same manner as in Example 4.
  • Raw data of GSE55235 and GSE14580 were obtained from Gene Expression Omnibus (GEO). Data was analyzed using analysis software Qlucore Omics Explorer (Filgen), and P ⁇ 0.05 or less was used as a criterion for judgment. The result was as shown in FIG.
  • IL-7R gene expression tended to be higher in normal rheumatoid arthritis (RA) patients than in healthy subjects.
  • RA normal rheumatoid arthritis
  • U ulcerative colitis
  • IL-7R gene expression tended to be higher than that in healthy subjects, and in particular, IL-7R gene expression was enhanced in patients who did not succeed in both anti-TNF.
  • Example 11 Therapeutic effect of rheumatoid arthritis model using anti-IL-7R antibody
  • the anti-IL-7R antibody-drug conjugate was administered to a rheumatoid arthritis model, and the effect was examined.
  • A7R34-ADC As a conjugate of the anti-IL-7R antibody and the drug, A7R34-ADC (MMAE) in which A7R34 monoclonal antibody and monomethyl auristatin E (MMAE) are linked by Val-Cit bond (valine-citrulline dipeptide) was used. The Val-Cit bond and MMAE were linked by p-aminobenzylcarbamate (PABC). MMAE is known to have stronger cytotoxic activity than SN-38.
  • A7R34-ADC (MMAE) was prepared as described in References 6 to 9 as in Example 5. The chemical structural formula of A7R34-ADC (MMAE) was as follows.
  • a rheumatoid arthritis model was prepared by the method described in Reference 7 below. That is, 2 mg of anti-collagen 4 antibody (Chondrex) was intraperitoneally administered to female DBA / 1J mice (day 0). On day 3, 50 ⁇ g of LPS (Chondrex) was administered intraperitoneally. On the 5th day, 50 mg / kg of A7R34-ADC (MMAE) [MMAE equivalent 1 mg / kg] or 50 mg / kg of A7R34-ADC (SN-38) [SN-38 equivalent] The dose was 0.6 mg / kg].
  • MMAE A7R34-ADC
  • SN-38 A7R34-ADC
  • physiological saline 1 mg / kg MMAE, 50 mg / kg isotype control antibody ADC (MMAE) [1 mg / kg in MMAE equivalent amount], 10 mg / kg dexamethasone, or 50 mg / kg anti-TNF preparation etanercept was administered instead.
  • MMAE isotype control antibody ADC
  • SN-38 physiological saline as a control
  • 50 mg / kg anti-IL-7R antibody (A7R34) 50 mg / Kg anti-CD19 antibody ADC
  • SN-38 10 mg / kg dexamethasone was used.
  • the severity of arthritis was evaluated by measuring the arthritis score of each individual according to the instructions (the higher the score is, the more severe it is). The results were as shown in FIGS. 11A-C.
  • the arthritis score was determined by observing three joints: fingers, back and wrists of each limb. Specifically, the arthritis score of each limb was determined based on the following criteria. Evaluation criteria score of arthritis score of each limb 0: When there is no inflammation in any joint, Score 1: If inflammation is observed in one of the joints, Score 2: If inflammation is observed in any two of the joints, Score 3: If inflammation is observed in all joints, Score 4: All joints are inflamed and the entire limb is swollen red. Based on the above scoring, the arthritis score was measured for each limb (4 points maximum for each limb), and the limbs were summed (16 points maximum) to determine the arthritis score of the individual.
  • A7R34-ADC remarkably suppressed the exacerbation of the arthritic coefficient in the rheumatoid arthritis model, like dexamethasone.
  • the dexamethasone-administered group there was an individual (an example of poor control of inflammation) in which inflammation in the forelimbs was marked compared to the hindlimbs.
  • the A7R34-ADC (MMAE) administration group both the forelimbs and hindlimbs showed stable and strong anti-inflammatory effects. This anti-inflammatory effect was superior to anti-TNF therapy with etanercept.
  • FIG. 11B Similar results were obtained with A7R34-ADC (SN-38).
  • anti-IL-7R antibody A7R34 antibody alone was observed to suppress the deterioration of arthritis coefficient in a rheumatoid arthritis model, but the effect was limited (see FIG. 11B).
  • FIG. 11C a tissue section of a mouse forefoot lesion on day 9 after drug administration was prepared and observed with hematoxylin and eosin staining (HE staining). As shown in FIG. 11C, it can be seen that inflammation was significantly improved in the ADC administration group.
  • Example 12 Treatment effect of ulcerative colitis using anti-IL-7R antibody
  • the effect of anti-IL-7R antibody on ulcerative colitis was examined.
  • the ulcerative colitis model was prepared as shown in Reference Material 11 below. That is, female BALB / c mice were allowed to drink 5% dextran sulfate (DSS; MP Biomedicals). From the second day after drinking DSS, A7R34-ADC (MMAE) or A7R34-ADC (SN-38) was intraperitoneally administered in the same manner and dose as in the rheumatoid arthritis model.
  • DSS dextran sulfate
  • MMAE isotype control antibody ADC
  • dexamethasone dexamethasone
  • anti-TNF preparation etanercept was administered instead of ADC.
  • A7R34-ADC physiological saline, anti-IL-7R antibody (A7R34), anti-CD19 antibody ADC (SN-38), and dexamethasone were used as controls as in the rheumatoid arthritis model.
  • the large intestine on the 8th day after drinking was removed and the total length was measured. Furthermore, a tissue specimen was prepared and a pathological image was confirmed by HE staining.
  • the severity of ulcerative colitis was observed in terms of weight loss (the greater the weight loss, the greater the severity), the entire length of the large intestine (the more severe the length of the large intestine), and histological changes.
  • the results of changes in body weight were as shown in FIGS. 12A and B. Moreover, the result of the histological change of the entire length of the large intestine was as shown in FIG.
  • A7R34-ADC significantly suppressed weight loss after the fifth day.
  • This inhibitory effect was significantly higher than the suppression of weight loss in the etanercept administration group.
  • MMAE and control antibody ADC showed almost no inhibitory effect on weight loss.
  • A7R34-ADC also remarkably suppressed weight loss in model mice.
  • FIG. 13 in the negative control for ulcerative colitis, the total length was significantly reduced compared to the DSS-untreated group, but in the A7R34-ADC (MMAE) administration group, the total length of the large intestine was DSS. The laparotomy was at the same level as the untreated group. In the A7R34-ADC (SN-38) administration group, the total length tended to be long.
  • IL-7R is expressed on acquired immune system lymphocytes, T cells and B cells, and in particular on innate immune system lymphocytes. Since the inflammation could be sedated by the ADC against IL-7R, the ADC is effective against the sedation of inflammation via the acquired immune system lymphocytes, T cells and B cells, and in particular the innate immune system lymphocytes. It was suggested to be effective. Therefore, according to the present invention, against all inflammation via innate immune system lymphocytes, diseases caused by enhancement of acquired immune system (for example, autoimmune diseases, graft-versus-host diseases, etc.) and other diseases or conditions described above It has been shown that antibody drug conjugates of anti-IL-7R antibodies and cytotoxic agents are effective.

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Abstract

The present invention provides: an antibody-drug conjugate of an anti-IL-7R antibody; and a pharmaceutical composition for treating cancer or inflammation and containing an antibody-drug conjugate of an anti-IL-7R antibody and a cytotoxic drug. The present invention provides: an antibody-drug conjugate of an anti-IL-7R antibody and a cytotoxic drug; and a pharmaceutical composition for use in treatment of cancer or inflammation, and containing an antibody-drug conjugate of an anti-IL-7R antibody and a cytotoxic drug.

Description

抗ＩＬ－７Ｒ抗体の抗体薬物コンジュゲートと、がんまたは炎症を処置することに用いるための、抗ＩＬ－７Ｒ抗体と細胞傷害剤との抗体薬物コンジュゲートを含む医薬組成物Pharmaceutical composition comprising an antibody drug conjugate of an anti-IL-7R antibody and an antibody drug conjugate of an anti-IL-7R antibody and a cytotoxic agent for use in treating cancer or inflammation

　本発明は、抗ＩＬ－７Ｒ抗体（抗ＩＬ－７受容体抗体）の抗体薬物コンジュゲートと、がんまたは炎症を処置することに用いるための、抗ＩＬ－７Ｒ抗体と細胞傷害剤との抗体薬物コンジュゲートを含む医薬組成物に関する。 The present invention relates to an antibody drug conjugate of an anti-IL-7R antibody (anti-IL-7 receptor antibody) and an antibody of an anti-IL-7R antibody and a cytotoxic agent for use in treating cancer or inflammation. It relates to a pharmaceutical composition comprising a drug conjugate.

　トポイソメラーゼ1阻害剤であるＳＮ－３８は、その阻害効果から抗がん作用が期待されているが難溶性であり、血中移行性の点で改善の余地があった。ＳＫ－３８の血中移行性を改善する化合物として開発されたのがＣＰＴ－１１である。ＣＰＴ－１１は、ＳＮ－３８のプロドラッグであり、活性体への変換効率は固形腫瘍のがん組織では高いが、血中では低く、血中での活性が低かった。従って、白血病の治療にはＣＰＴ－１１を利用することはできなかった。 SN-38, a topoisomerase 1 inhibitor, is expected to have an anticancer effect due to its inhibitory effect, but is poorly soluble and has room for improvement in terms of its ability to migrate into the blood. CPT-11 was developed as a compound that improves the blood translocation of SK-38. CPT-11 is a prodrug of SN-38, and the conversion efficiency to the active form is high in cancer tissues of solid tumors, but is low in blood and low in blood. Therefore, CPT-11 could not be used to treat leukemia.

　ＩＬ－７は、Ｔ細胞の発生と恒常性維持において必須のサイトカインとして知られている（非特許文献１）。また、ＩＬ－７Ｒαは、ゆっくりと細胞内移行し、一部は細胞表面に戻るが一部は分解される（非特許文献１）。ＩＬ－７による刺激によってＩＬ－７Ｒαの半減期は約２４時間から３時間に短縮される（非特許文献１）。また、ＩＬ－７Ｒががんのステロイド耐性の原因になっていることが明らかとなっている（非特許文献２）。非特許文献２では、ＩＬ－７Ｒの下流シグナルを抑制することがステロイド耐性の処置に有効であることが示唆されている。 IL-7 is known as an essential cytokine for T cell generation and homeostasis (Non-patent Document 1). In addition, IL-7Rα moves into the cell slowly and partly returns to the cell surface, but partly decomposes (Non-patent Document 1). The half-life of IL-7Rα is shortened from about 24 hours to 3 hours by stimulation with IL-7 (Non-patent Document 1). It has also been clarified that IL-7R is responsible for cancer steroid resistance (Non-patent Document 2). Non-Patent Document 2 suggests that suppressing downstream signals of IL-7R is effective in treating steroid resistance.

Li et al., 2016, PLOS Medicine, 13(12): e1002200Li et al., 2016, PLOS Medicine, 13 (12): e1002200 Henriques et al., 2010, Blood, 115(16): 3269-3277Henriques et al., 2010, Blood, 115 (16) :( 3269-3277

　本発明は、抗ＩＬ－７Ｒ抗体の抗体薬物コンジュゲートと、がんまたは炎症を処置することに用いるための、抗ＩＬ－７Ｒ抗体と細胞傷害剤との抗体薬物コンジュゲートを含む医薬組成物を提供する。 The present invention relates to a pharmaceutical composition comprising an antibody drug conjugate of an anti-IL-7R antibody and an antibody drug conjugate of an anti-IL-7R antibody and a cytotoxic agent for use in treating cancer or inflammation. provide.

　本発明者らは、ＩＬ－７Ｒがステロイド耐性と関連すること、およびＩＬ－７Ｒのノックダウンががん細胞株の細胞増殖を抑えることを見出した。本発明者らはまた、ＩＬ－７Ｒのノックダウンががん細胞（特にリンパ腫および白血病）のステロイド感受性を劇的に向上させること、これによりステロイドによる細胞殺傷作用を高めることができること、およびステロイド耐性を獲得するとＩＬ－７Ｒの発現が向上することを見出した。発明者らはさらに、ステロイド耐性にはＩＬ－７Ｒの下流シグナルのＳＴＡＴやＢＣＬ２の活性化は見られず、ＮＦκＢの活性化が生じていることを見出した。本発明者らはさらにまた、ステロイド系抗炎症剤を含む化学療法処置後にＩＬ－７Ｒの発現が向上すること、またステロイド系抗炎症薬で処置してステロイド耐性を得た細胞株においてＩＬ－７Ｒの発現が向上していることを見出した。本発明者らはさらにまた、抗ＩＬ－７Ｒ抗体が細胞に取り込まれるとライソゾームに集積すること、投与１週間後に腫瘍組織およびリンパ節に集積していることを見出した。一方で、がんの細胞生存機能に対しては、ＩＬ－７ＲとＩＬ－７との結合を遮断する抗体の効果は弱く、他方で抗ＩＬ－７Ｒ抗体のＡＤＣが顕著な効果を示すこと、およびリンパ節腫大を抑制することを見出した。腫瘍に対するＡＤＣの効果は放射性同位体標識抗ＩＬ－７Ｒ抗体でも同様に示されることを見出した。本発明者らはさらにまた、免疫系のＩＬ－７Ｒ陽性細胞の減少を通じて炎症疾患（例えば、関節リウマチや潰瘍性大腸炎）の症状を軽減することを見出した。本発明はこれらの知見に基づくものである。 The present inventors have found that IL-7R is associated with steroid resistance and that IL-7R knockdown suppresses cell proliferation of cancer cell lines. We have also seen that IL-7R knockdown dramatically improves the steroid sensitivity of cancer cells (especially lymphomas and leukemias), thereby increasing the cell killing effect of steroids, and steroid resistance It was found that the expression of IL-7R is improved by obtaining. The inventors further found that activation of NFκB occurs in steroid resistance without STAT or BCL2 activation of downstream signals of IL-7R. The inventors further have improved expression of IL-7R after chemotherapy treatment with steroidal anti-inflammatory drugs, and IL-7R in cell lines that have been treated with steroidal anti-inflammatory drugs to gain steroid resistance. It has been found that the expression of is improved. Furthermore, the present inventors have found that anti-IL-7R antibodies accumulate in lysosomes when taken up by cells, and accumulate in tumor tissues and lymph nodes one week after administration. On the other hand, on the cell survival function of cancer, the effect of the antibody blocking the binding between IL-7R and IL-7 is weak, and on the other hand, the ADC of the anti-IL-7R antibody shows a remarkable effect, And found to suppress lymphadenopathy. It was found that the effect of ADC on tumors was also shown with a radioisotope-labeled anti-IL-7R antibody. Furthermore, the present inventors have found that the symptoms of inflammatory diseases (for example, rheumatoid arthritis and ulcerative colitis) are reduced through a decrease in IL-7R positive cells of the immune system. The present invention is based on these findings.

　すなわち、本発明によれば、以下の発明が提供される。
（１）抗ＩＬ－７Ｒ抗体またはその抗原結合性断片と細胞傷害剤との抗体薬物コンジュゲートを含む医薬組成物であって、がんまたは炎症を処置することに用いるための医薬組成物。
（２）炎症を処置することに用いるための、上記（１）に記載の医薬組成物。
（３）炎症が、ステロイド治療後のまたはステロイド抵抗性の炎症である、上記（１）または（２）に記載の医薬組成物。
（４）炎症が、潰瘍性大腸炎または関節リウマチである、上記（２）または（３）に記載の医薬組成物。
（５）がんを処置することに用いるための上記（１）に記載の医薬組成物。
（６）がんが、白血病、リンパ腫、または転移性のがんである、上記（１）または（５）に記載の医薬組成物。
（７）がんが、ステロイド治療後のまたはステロイド耐性のがんである、上記（１）、（５）または（６）に記載の医薬組成物。
（８）がんが、転移性のがんである、上記（１）および（５）～（７）のいずれか一項に記載の医薬組成物。
（９）がんが、ＩＬ－７ＲとＩＬ－７との結合を中和する治療に対して抵抗性である、上記（１）および（５）～（８）のいずれか一項に記載の医薬組成物。
（１０）抗ＩＬ－７Ｒ抗体またはその抗原結合性断片と細胞傷害剤との抗体薬物コンジュゲートを含む医薬組成物であって、ステロイド治療後の若しくはステロイド耐性の疾患または状態を処置することに用いるための医薬組成物。
（１１）抗ＩＬ－７Ｒ抗体またはその抗原結合性断片と細胞傷害剤との抗体薬物コンジュゲートを含む医薬組成物であって、ステロイドと併用される、医薬組成物。
（１２）がんを処置することに用いるための、上記（１１）に記載の医薬組成物。
（１３）細胞傷害剤が、放射性同位体、化学療法剤、または毒素である、上記（１）～（１２）のいずれか一項に記載の医薬組成物。
（１４）抗ＩＬ－７Ｒ抗体またはその抗原結合性断片とモノメチルオーリスタチンＥとの抗体薬物コンジュゲート。 That is, according to the present invention, the following inventions are provided.
(1) A pharmaceutical composition comprising an antibody drug conjugate of an anti-IL-7R antibody or an antigen-binding fragment thereof and a cytotoxic agent, for use in treating cancer or inflammation.
(2) The pharmaceutical composition according to the above (1) for use in treating inflammation.
(3) The pharmaceutical composition according to (1) or (2) above, wherein the inflammation is steroid-treated or steroid-resistant inflammation.
(4) The pharmaceutical composition according to (2) or (3) above, wherein the inflammation is ulcerative colitis or rheumatoid arthritis.
(5) The pharmaceutical composition according to the above (1) for use in treating cancer.
(6) The pharmaceutical composition according to (1) or (5) above, wherein the cancer is leukemia, lymphoma, or metastatic cancer.
(7) The pharmaceutical composition according to the above (1), (5) or (6), wherein the cancer is after steroid treatment or steroid-resistant cancer.
(8) The pharmaceutical composition according to any one of (1) and (5) to (7) above, wherein the cancer is metastatic cancer.
(9) The cancer according to any one of (1) and (5) to (8), wherein the cancer is resistant to a treatment that neutralizes binding between IL-7R and IL-7. Pharmaceutical composition.
(10) A pharmaceutical composition comprising an antibody drug conjugate of an anti-IL-7R antibody or an antigen-binding fragment thereof and a cytotoxic agent, which is used for treating a disease or condition after steroid treatment or steroid resistance Pharmaceutical composition for
(11) A pharmaceutical composition comprising an antibody drug conjugate of an anti-IL-7R antibody or antigen-binding fragment thereof and a cytotoxic agent, which is used in combination with a steroid.
(12) The pharmaceutical composition according to the above (11) for use in treating cancer.
(13) The pharmaceutical composition according to any one of (1) to (12) above, wherein the cytotoxic agent is a radioisotope, a chemotherapeutic agent, or a toxin.
(14) An antibody drug conjugate of an anti-IL-7R antibody or an antigen-binding fragment thereof and monomethyl auristatin E.

図１Ａは、様々ながん細胞株におけるＩＬ－７Ｒの発現量をフローサイトメトリーにより調べた結果である。FIG. 1A shows the results of examining the expression level of IL-7R in various cancer cell lines by flow cytometry. 図１Ｂは、ＩＬ－７Ｒノックダウンアッセイで実際に各種がん細胞株においてＩＬ－７Ｒの発現量が低下したことを示す図である。FIG. 1B is a graph showing that IL-7R expression level was actually decreased in various cancer cell lines by IL-7R knockdown assay. 図１Ｃは、ＩＬ－７Ｒがノックダウンされたがん細胞株の増殖に対する影響を示す図である。FIG. 1C is a graph showing the influence on the growth of a cancer cell line in which IL-7R is knocked down. 図１Ｄは、ヌードマウスの皮下移植モデルにおいて、皮下移植されたＩＬ－７Ｒがノックダウンされたがん細胞株の増殖に対する影響を示す図である。FIG. 1D is a graph showing the influence of subcutaneously transplanted IL-7R on the growth of a cancer cell line knocked down in a nude mouse subcutaneous transplantation model. 図１Ｅは、ＩＬ－７Ｒがノックダウンされたがん細胞株の生存能を示す顕微鏡写真である。FIG. 1E is a photomicrograph showing the viability of a cancer cell line in which IL-7R has been knocked down. 図２Ａは、デキサメタゾン（ステロイド系抗炎症薬の一種）に対する各種がん細胞株のＩＣ５０がＩＬ－７Ｒのノックダウンにより低下することを示す図である。FIG. 2A is a graph showing that IC50 of various cancer cell lines against dexamethasone (a kind of steroidal anti-inflammatory drug) is decreased by IL-7R knockdown. 図２Ｂは、デキサメタゾン耐性株を得て、インビトロでデキサメタゾンに対する耐性にＩＬ－７Ｒのノックダウンが及ぼす影響を示す図である。FIG. 2B shows the effect of IL-7R knockdown on the resistance to dexamethasone in vitro by obtaining a dexamethasone resistant strain. 図２Ｃは、デキサメタゾン耐性株においてＩＬ－７Ｒの発現が向上していることを示す図である。FIG. 2C shows that the expression of IL-7R is improved in the dexamethasone resistant strain. 図３は、各種がん細胞株およびそのＩＬ－７Ｒノックダウン株を抗リン酸化ｐ６５／ＮＦκＢ抗体で染色した結果を示す図である。FIG. 3 shows the results of staining various cancer cell lines and their IL-7R knockdown lines with anti-phosphorylated p65 / NFκB antibodies. 図４Ａは、Ｂ細胞急性白血病において、ステロイドを含む化学療法の前後での遺伝子発現の変化を示す図である。FIG. 4A is a diagram showing changes in gene expression before and after chemotherapy including steroids in B-cell acute leukemia. 図４Ｂは、Ｂ細胞急性白血病において、ステロイド治療によるステロイド耐性の獲得前後における遺伝子発現の変化を示す図である。FIG. 4B is a diagram showing changes in gene expression before and after acquisition of steroid resistance by steroid treatment in B-cell acute leukemia. 図４Ｃは、Ｔ細胞急性白血病において、ステロイドを含む化学療法の前後での遺伝子発現の変化を示す図である。FIG. 4C shows changes in gene expression before and after chemotherapy including steroids in T-cell acute leukemia. 図５Ａは、インビトロでがん細胞株にAlexa647標識抗ＩＬ－７Ｒ抗体を接触させ、ライソゾームとの共局在を確認した結果を示す。FIG. 5A shows the results of confirming co-localization with lysosome by contacting Alexa647-labeled anti-IL-7R antibody with a cancer cell line in vitro. 図５Ｂは、腫瘍の皮下移植モデルに蛍光標識抗ＩＬ－７Ｒ抗体を投与し、１週間後に各種組織における抗ＩＬ－７Ｒ抗体の蓄積を調べた結果を示す。FIG. 5B shows the results of administering a fluorescently labeled anti-IL-7R antibody to a tumor subcutaneous transplantation model and examining the accumulation of anti-IL-7R antibody in various tissues one week later. 図５Ｃは、ＩＬ－７ＲとＩＬ－７との結合中和能を有する抗ＩＬ－７Ｒ抗体がＩＬ－７依存性に生存するＲＡＧ２－／－細胞に対しては効果が奏される一方で、がん細胞株ＣＹＧ８２に対してはほとんど効果を奏さないことを示す。FIG. 5C shows that an anti-IL-7R antibody having the ability to neutralize binding between IL-7R and IL-7 is effective against RAG2 − / − cells that survive in an IL-7-dependent manner, It shows that it has little effect on the cancer cell line CYG82. 図５Ｄは、抗ＩＬ－７Ｒ抗体と細胞傷害剤との抗体薬物コンジュゲート（ＡＤＣ）が、がん細胞株ＣＹＧ８２に対して殺傷作用を奏することを示す。FIG. 5D shows that an antibody drug conjugate (ADC) of an anti-IL-7R antibody and a cytotoxic agent exerts a killing effect on the cancer cell line CYG82. 図６Ａは、がん細胞株の皮下移植モデルにおける、抗ＩＬ－７Ｒ抗体と細胞傷害剤との抗体薬物コンジュゲートの腫瘍体積増加に対する効果を示す図である。FIG. 6A is a graph showing the effect of an antibody drug conjugate of an anti-IL-7R antibody and a cytotoxic agent on tumor volume increase in a subcutaneous transplantation model of a cancer cell line. 図６Ｂは、ステロイド耐性になったがん細胞株の皮下移植モデルにおける、抗ＩＬ－７Ｒ抗体と細胞傷害剤との抗体薬物コンジュゲートの腫瘍体積増加に対する効果を示す図である。FIG. 6B is a graph showing the effect of an antibody drug conjugate of an anti-IL-7R antibody and a cytotoxic agent on tumor volume increase in a steroid-resistant cancer cell line subcutaneous transplantation model. 図７Ａは、がん細胞株ＣＹＧ８２の皮下移植モデルにおけるリンパ節腫大に対するＩＬ－７Ｒノックダウンの影響を示す図である。FIG. 7A shows the effect of IL-7R knockdown on lymphadenopathy in a subcutaneous transplantation model of cancer cell line CYG82. 図７Ｂは、がん細胞株ＣＹＧ８２の皮下移植モデルで所属リンパ節腫大を経て生じる腹腔内リンパ節腫大に対する抗ＩＬ－７ＲとＳＮ－３８とのＡＤＣの効果を示す図である。FIG. 7B is a graph showing the effect of ADC of anti-IL-7R and SN-38 on intraperitoneal lymphadenopathy occurring through regional lymphadenopathy in a subcutaneous transplantation model of cancer cell line CYG82. 図８Ａは、Ｔ細胞リンパ腫細胞株Ｓｕｐ－Ｔ１に対する放射線同位体標識（90Ｙ標識）抗ＩＬ－７Ｒ抗体の効果を示す図である。FIG. 8A is a graph showing the effect of a radioisotope-labeled ( 90 Y-labeled) anti-IL-7R antibody on the T-cell lymphoma cell line Sup-T1. 図８Ｂは、肺がん細胞株Ｈ２００９に対する放射線同位体標識（90Ｙ標識）抗ＩＬ－７Ｒ抗体の効果を示す図である。FIG. 8B shows the effect of radioisotope-labeled ( 90 Y-labeled) anti-IL-7R antibody on lung cancer cell line H2009. 図９は、抗ＩＬ－７ＲのＡＤＣの免疫系に与える影響を示す図である。FIG. 9 is a diagram showing the effect of anti-IL-7R on the immune system of ADC. 図１０は、慢性関節リウマチおよび潰瘍性大腸炎でＩＬ－７Ｒの発現が向上していることを示す。FIG. 10 shows that IL-7R expression is increased in rheumatoid arthritis and ulcerative colitis. 図１１Ａは、関節リウマチモデルに対する抗ＩＬ－７Ｒ抗体とモノメチルオーリスタチンＥ（ＭＭＡＥ）のＡＤＣの影響を示す。FIG. 11A shows the effect of ADC of anti-IL-7R antibody and monomethyl auristatin E (MMAE) on rheumatoid arthritis model. 図１１Ｂは、関節リウマチモデルに対する抗ＩＬ－７Ｒ抗体とＳＮ－３８のＡＤＣの影響を示す。FIG. 11B shows the effect of anti-IL-7R antibody and SN-38 ADC on rheumatoid arthritis model. 図１１Ｃは、関節リウマチモデルのマウス前足病変部の組織切片を作製し、ヘマトキシリン・エオシン染色した結果を示す。FIG. 11C shows the result of preparing a tissue section of a mouse forefoot lesion of a rheumatoid arthritis model and staining with hematoxylin and eosin. 図１２Ａは、潰瘍性大腸炎モデルに対する抗ＩＬ－７Ｒ抗体とＭＭＡＥとのＡＤＣの効果を示す図である。FIG. 12A is a graph showing the effect of ADC of anti-IL-7R antibody and MMAE on ulcerative colitis model. 図１２Ｂは、潰瘍性大腸炎モデルに対する抗ＩＬ－７Ｒ抗体とＳＮ－３８とのＡＤＣの効果を示す図である。FIG. 12B is a graph showing the effect of ADC of anti-IL-7R antibody and SN-38 on ulcerative colitis model. 図１３は、潰瘍性大腸炎モデルの大腸の組織形態が抗ＩＬ－７Ｒ抗体とＭＭＡＥとのＡＤＣまたは抗ＩＬ－７Ｒ抗体とＳＮ－３８とのＡＤＣとで改善することを示す。FIG. 13 shows that the colonic morphology of the ulcerative colitis model is improved with an anti-IL-7R antibody and MMAE ADC or an anti-IL-7R antibody and SN-38 ADC. 図１４は、潰瘍性大腸炎モデルから摘出した大腸標本の病理組織像において抗ＩＬ－７Ｒ抗体とＭＭＡＥとのＡＤＣまたは抗ＩＬ－７Ｒ抗体とＳＮ－３８とのＡＤＣの効果を示す図である。FIG. 14 is a graph showing the effect of ADC of anti-IL-7R antibody and MMAE or ADC of anti-IL-7R antibody and SN-38 on the histopathological image of a colon specimen removed from an ulcerative colitis model.

発明の具体的説明Detailed description of the invention

　本発明では、「対象」とは、哺乳動物を意味し、特にヒトであり得る。 In the present invention, “subject” means a mammal, and may be a human in particular.

　本明細書では、「処置」とは、治療（治療的処置）と予防（予防的処置）とを含む意味で用いられる。本明細書では、「治療」とは、疾患若しくは障害の治療、治癒、防止若しくは、寛解の改善、または、疾患若しくは障害の進行速度の低減を意味する。本明細書では、「予防」とは、疾患もしくは病態の発症の可能性を低下させる、または疾患もしくは病態の発症を遅らせることを意味する。 In this specification, “treatment” is used to mean treatment (therapeutic treatment) and prevention (preventive treatment). As used herein, “treatment” means treatment, cure, prevention or amelioration of a disease or disorder or a reduction in the rate of progression of a disease or disorder. As used herein, “prevention” means reducing the likelihood of the onset of a disease or condition or delaying the onset of a disease or condition.

　本明細書では、「疾患」とは、治療が有益な症状を意味する。 As used herein, “disease” means a symptom for which treatment is beneficial.

　本明細書では、「治療上有効量」とは、疾患や状態を処置（予防または治療）するために有効な薬剤の量を意味する。治療上有効量の薬剤は、疾患または状態の症状の悪化速度を低下させること、前記症状の悪化を止めること、前記症状を改善すること、前記症状を治癒すること、または前記症状の発症または発展を抑制することが可能である。 As used herein, “therapeutically effective amount” means the amount of a drug effective for treating (preventing or treating) a disease or condition. A therapeutically effective amount of a drug reduces the rate of worsening of a symptom of a disease or condition, stops the worsening of the symptom, improves the symptom, cures the symptom, or develops or develops the symptom Can be suppressed.

　本明細書では、「抵抗性」とは、治療に対して有効性を示さないこと（例えば、有意な有効性を示さないこと）を意味する。本明細書では、「感受性」とは、治療に対して有効性を示すことを意味する。抵抗性を低下させることは感受性を増大させることとは同じ意味をもち、抵抗性を増大させることは感受性を低下させることと同じ意味を持つ。本明細書では、例えば、ステロイド抵抗性とは、ネフローゼ症候群の場合にはプレドニゾロンを４週間以上連日投与しても、完全寛解しないものと定義され得る。本明細書ではまた、他の炎症・自己免疫疾患或いは白血病・悪性リンパ腫などの悪性疾患に関しては、プレドニゾロン、メチルプレドニゾロン、デキサメゾン、およびベタメタゾンなど各種の治療用ステロイドを投与しても症状が改善されないか、または症状が増悪するものとして扱われ得る。医師であれば、ステロイド治療の分野における知見に基づいて、抵抗性については、どのようなものであるかを理解し、把握することができ、また、適宜対象が抵抗性か否かを決定することができる。 As used herein, “resistant” means not showing efficacy for treatment (eg, showing no significant effectiveness). As used herein, “sensitivity” means showing efficacy for treatment. Decreasing resistance has the same meaning as increasing sensitivity, and increasing resistance has the same meaning as decreasing sensitivity. As used herein, for example, steroid resistance can be defined as no complete remission in the case of nephrotic syndrome, even if prednisolone is administered daily for more than 4 weeks. Also, in this specification, regarding other inflammatory / autoimmune diseases or malignant diseases such as leukemia / malignant lymphoma, is the symptom improved by administration of various therapeutic steroids such as prednisolone, methylprednisolone, dexamezone, and betamethasone? Or may be treated as exacerbating symptoms. Physicians can understand and understand what resistance is based on their knowledge in the field of steroid therapy, and determine whether the subject is resistant as appropriate be able to.

　本明細書では、「ＩＬ－７Ｒ」とは、インターロイキン７受容体を意味する。ＩＬ－７ＲにＩＬ－７（インターロイキン－７）が結合すると、ＪＡＫ１およびＪＡＫ３が活性化し、受容体の自己リン酸化が起こる。リン酸化された受容体には、ＳＴＡＴが結合し、ＪＡＫによりリン酸化を受けて二量体化する。二量体化したＳＴＡＴにより遺伝子の転写活性化が生じると考えられている。ＩＬ－７Ｒは、分子自体のインターナライズ活性が高く、膜上からインターナライズし、分解されるか再びリサイクリングして膜上に表出する。 As used herein, “IL-7R” means interleukin 7 receptor. When IL-7 (interleukin-7) binds to IL-7R, JAK1 and JAK3 are activated and receptor autophosphorylation occurs. STAT binds to the phosphorylated receptor and is phosphorylated by JAK to dimerize. It is thought that transcriptional activation of genes is caused by dimerized STAT. IL-7R has a high internalization activity of the molecule itself, and is internalized from the membrane and decomposed or recycled again to be exposed on the membrane.

　本明細書では、「遮断」とは、２つのタンパク質の結合を中和することを意味する。遮断には、完全な解離に加えて部分的な解離も含まれる。例えば、２つのタンパク質の結合を５０％以上低減させる場合にも遮断するということができる。 As used herein, “blocking” means neutralizing the binding of two proteins. Blocking includes partial dissociation in addition to complete dissociation. For example, it can be said that blocking occurs when the binding of two proteins is reduced by 50% or more.

　本明細書では、「ステロイド」とは、ステロイド系抗炎症薬（ＳＡＩＤｓ）を意味する。ステロイドでは、主に糖質コルチコイドまたはその誘導体が有効成分となっている。ステロイドは、細胞内でグルココルチコイド受容体（ＧＲα）と結合するとＧＲαは、ｈｓｐ９０から解離し、二量体を形成して核内に移行し、転写調節を行う。合成系ステロイドとしては、ヒドロコルチゾンおよびコハク酸ヒドロコルチゾンなどのコルチゾール；プレドニゾロン、メチルプレドニゾロンおよびコハク酸メチルプレドニゾロンなどのプレドニゾロン；トリアムシノロンおよびトリアムシノロンアセトニドなどのトリアムシノロン；デキサメタゾン、並びにベタメタゾンなどが挙げられる。いずれもグルココルチコイドの誘導体であり、グルココルチコイドと同様のメカニズムで細胞内において機能する。 As used herein, “steroid” means steroidal anti-inflammatory drugs (SAIDs). In steroids, glucocorticoids or their derivatives are mainly active ingredients. When a steroid binds to a glucocorticoid receptor (GRα) in a cell, GRα dissociates from hsp90, forms a dimer, moves into the nucleus, and regulates transcription. Synthetic steroids include cortisols such as hydrocortisone and hydrocortisone succinate; prednisolones such as prednisolone, methylprednisolone and methylprednisolone succinate; triamcinolones such as triamcinolone and triamcinolone acetonide; dexamethasone and betamethasone. Both are derivatives of glucocorticoid and function in the cell by the same mechanism as glucocorticoid.

　本明細書では、「抗体薬物コンジュゲート」（ＡＤＣ）とは、モノクローナル抗体またはその抗原結合性断片（以下、単に「抗体等」ということがある）と薬物とが連結した物質を意味する。ＡＤＣでは、モノクローナル抗体等と薬物とは適切なリンカーを介して連結させることができる。ＡＤＣは、細胞膜上の膜成分（例えば、受容体等の膜貫通タンパク質）に結合し、エンドサイトーシスやインターナライゼーションにより、細胞内に取り込まれ、抗体等と切り離されて細胞内に放出される。細胞内で抗体等と薬物との間に開裂性リンカーを導入しておくことで、細胞内、例えばエンドソーム内でリンカーを開裂させ薬物を抗体等から乖離させて細胞質内に放出させることが可能である。薬物として、細胞傷害剤を用いると薬物が送達された細胞を死滅させることが可能である。細胞傷害剤としては、化学療法剤、放射性同位体、および毒素を用いることができる。 In the present specification, “antibody drug conjugate” (ADC) means a substance in which a monoclonal antibody or an antigen-binding fragment thereof (hereinafter sometimes simply referred to as “antibody or the like”) is linked to a drug. In ADC, a monoclonal antibody or the like and a drug can be linked via an appropriate linker. The ADC binds to a membrane component on the cell membrane (for example, a transmembrane protein such as a receptor), is taken into the cell by endocytosis or internalization, is separated from the antibody or the like, and is released into the cell. By introducing a cleavable linker between the antibody and the drug in the cell, the linker can be cleaved in the cell, for example, in the endosome, and the drug can be released from the antibody and released into the cytoplasm. is there. When a cytotoxic agent is used as a drug, it is possible to kill cells to which the drug has been delivered. As the cytotoxic agent, a chemotherapeutic agent, a radioisotope, and a toxin can be used.

　本発明によれば、多くの腫瘍細胞でＩＬ－７Ｒの発現が向上していることを見出した。本発明によれば、向上したＩＬ－７Ｒの発現を減少させることにより、腫瘍の増殖が大きく抑制された。これらの細胞に対して、ＩＬ－７ＲのＡＤＣは強い殺傷能力を示した。一方で、ＩＬ－７ＲとＩＬ－７との結合を遮断できる抗体は、細胞生存にはほとんど効果を示さなかった。従って、本発明のＡＤＣにおいて用いられる抗ＩＬ－７Ｒ抗体は、細胞膜上のＩＬ－７Ｒに結合する限り、そのＩＬ－７ＲとＩＬ－７との結合の遮断能力は問われず、ＩＬ－７ＲとＩＬ－７との結合を遮断できる抗体であってもよいし、ＩＬ－７ＲとＩＬ－７との結合を遮断しない抗体であってもよい。 According to the present invention, it was found that IL-7R expression is improved in many tumor cells. According to the present invention, tumor growth was greatly suppressed by reducing the expression of improved IL-7R. For these cells, IL-7R ADCs showed a strong killing ability. On the other hand, antibodies capable of blocking the binding between IL-7R and IL-7 had little effect on cell survival. Therefore, as long as the anti-IL-7R antibody used in the ADC of the present invention binds to IL-7R on the cell membrane, the ability to block the binding between IL-7R and IL-7 is not questioned. It may be an antibody that can block the binding to -7, or an antibody that does not block the binding between IL-7R and IL-7.

　従って、本発明によれば、抗ＩＬ－７Ｒ抗体またはその抗原結合性断片と細胞傷害剤との抗体薬物コンジュゲートが提供される。本発明の抗体薬物コンジュゲートでは、抗ＩＬ－７Ｒ抗体としては、細胞膜上に発現するＩＬ－７Ｒを認識する抗体を用いることができる。また、本発明のある態様では、抗体薬物コンジュゲートにおいて抗体またはその抗原結合性断片と細胞傷害剤とは、リンカーを介して連結している。細胞傷害剤としては、化学療法剤（例えば、市販の抗がん剤などの抗がん剤、例えば、アウリスタチン（アウリスタチンＥ、アウリスタチンＦフェニレンジアミン（ＡＦＰ）、モノメチルアウリスタチンＥ、モノメチルアウリスタチンＦとそれらの誘導体）、メイタンシノイドＤＭ１およびＤＭ４とそれらの誘導体）、カンプトテシン（ＳＮ－３８、トポテカンおよびエキソテカンとそれらの誘導体）、ＤＮＡ副溝結合剤（エネジイン、レキシトロプシン、デュオカルマイシンとそれらの誘導体）、タキサン（パクリタキセルおよびドセタキセルとそれらの誘導体）、ポリケチド（ディスコデルモライドとその誘導体）、アントラキノン系（ミトキサントロンとその誘導体）、ベンゾジアゼピン（ピロロベンゾジアゼピン、インドリノベンゾジアゼピン、およびオキサゾリジノベンゾジアゼピンとそれらの誘導体）、ビンカアルカロイド（ビンクリスチン、ビンブラスチン、ビンデシン、およびビノレルビンとそれらの誘導体）、ドキソルビシン類（ドキソルビシン、モルホリノ－ドキソルビシン、およびシアノモルホリノ－ドキソルビシンとそれらの誘導体）、強心配糖体（ジギトキシンやその誘導体）、カレキアマイシン、エポチロン、クリプトフィシン、セマドチン、セマドチン、リゾキシン、ネトロプシン、コンブレタスタチン、エリュテロビン、エトポシド、Ｔ６７（チュラリク）、およびノコダゾール）、放射性同位体（例えば、32Ｐ、60Ｃ、90Ｙ、111Ｉｎ、131Ｉ、125Ｉ、153Ｓｍ、186Ｒｅ、188Ｒｅ、および212Ｂｉ）、および毒素（例えば、ジフテリアトキシンＡ、シュードモナスエンドトキシン、リシン、サポリン等）が挙げられ、本発明のＡＤＣにおける細胞傷害剤として用いることができる。細胞傷害剤はいずれも、がんの処置に用いられるものを用いることができる。 Therefore, according to the present invention, an antibody drug conjugate of an anti-IL-7R antibody or antigen-binding fragment thereof and a cytotoxic agent is provided. In the antibody drug conjugate of the present invention, an antibody that recognizes IL-7R expressed on the cell membrane can be used as the anti-IL-7R antibody. In one embodiment of the present invention, the antibody or antigen-binding fragment thereof and the cytotoxic agent in the antibody drug conjugate are linked via a linker. Examples of cytotoxic agents include chemotherapeutic agents (for example, anticancer agents such as commercially available anticancer agents such as auristatin (auristatin E, auristatin F phenylenediamine (AFP), monomethyl auristatin E, monomethyl auristatin). Statins F and their derivatives), maytansinoids DM1 and DM4 and their derivatives), camptothecin (SN-38, topotecan and exotecan and their derivatives), DNA minor groove binders (enzyin, lexitropsin, duocarmycin) And their derivatives), taxanes (paclitaxel and docetaxel and their derivatives), polyketides (discodermolide and their derivatives), anthraquinones (mitoxantrone and their derivatives), benzodiazepines (pyrrolobenzodiazepine, indolinobenzo) Azepine, and oxazolidinobenzodiazepines and their derivatives), vinca alkaloids (vincristine, vinblastine, vindesine, and vinorelbine and their derivatives), doxorubicins (doxorubicin, morpholino-doxorubicin, and cyanomorpholino-doxorubicin and their derivatives), Cardiac glycosides (digitoxin and its derivatives), calekiamycin, epothilone, cryptophycin, semadotin, semadotin, lysoxine, netropsin, combretastatin, eluterobin, etoposide, T67 (churaric), and nocodazole), radioisotopes (eg , 32 P, 60 C, 90 Y, 111 In, 131 I, 125 I, 153 Sm, 186 Re, 188 Re, and 212 Bi), and toxins (e.g., diphtheria butoxy A, Pseudomonas endotoxin, ricin, saporin and the like) can be mentioned, can be used as cytotoxic agents in the ADC of the present invention. As the cytotoxic agent, any of those used for the treatment of cancer can be used.

　本発明のある態様では、リンカーはＡＤＣの作製において当業者であれば適宜選択し、合成することができる。本発明のある態様では、リンカーは開裂性リンカーであり得る。例えば、開裂性リンカーとしては、バリン－シトルリン（Ｖａｌ－Ｃｉｔ）およびフェニルアラニン－リジン（Ｐｈｅ－ｌｙｓ）リンカーなどのペプチドリンカーや、ｐＨ依存的に開裂するヒドラゾンリンカーが挙げられる。開裂性リンカーとしてはまた、カルバメート結合またはエステル結合を含むリンカーが挙げられ、これらは酵素的に細胞内で分解されうる。これらのリンカーは組み合わせて用いてもよい。
　抗体とリンカーとの結合は、例えば抗体のスルフヒドリル基にマレイミド基を介して連結することができる。リンカーには必要に応じてポリエチレングリコールブロックが含まれていてもよい。 In one embodiment of the present invention, the linker can be appropriately selected and synthesized by those skilled in the art in the production of ADC. In certain embodiments of the invention, the linker can be a cleavable linker. For example, cleavable linkers include peptide linkers such as valine-citrulline (Val-Cit) and phenylalanine-lysine (Phe-lys) linkers, and hydrazone linkers that cleave in a pH-dependent manner. Cleaveable linkers also include linkers that contain carbamate or ester bonds, which can be enzymatically degraded intracellularly. These linkers may be used in combination.
The bond between the antibody and the linker can be linked to, for example, a sulfhydryl group of the antibody via a maleimide group. The linker may contain a polyethylene glycol block as necessary.

　また、本発明によれば、抗ＩＬ－７Ｒ抗体またはその抗原結合性断片と細胞傷害剤との抗体薬物コンジュゲートを含む、がんを処置することに用いるための医薬組成物が提供される。多くのがん細胞ではＩＬ－７Ｒが陽性である。従って、多くのがんが本発明の医薬の治療対象となり得る。また、有効性の高い対象集団を特定するために、本発明のある態様では、がんは、ＩＬ－７Ｒ陽性のがんまたはＩＬ－７Ｒ陽性であることが確認されたがんとすることができる。ＩＬ－７Ｒ陽性のがんとしては、白血病、リンパ腫、肺がん、膵臓がん、頭頸部がん、前立腺がん、膀胱がん、乳がん、食道がん、胃がん、大腸がん、子宮がん、卵巣がん、皮膚がん、甲状腺がん、胸腺がん、腎臓がん、精巣がん、陰茎がん、肝臓がん、胆道がん、脳腫瘍、骨軟部腫瘍、後腹膜腫瘍、血管・リンパ管肉腫、およびこれらの転移性のがん（例えば、転移性固形腫瘍）が挙げられる。多くのがん、例えば、白血病、リンパ腫、進行性のがん、および転移性のがんではＩＬ－７Ｒが陽性である。ＩＬ－７Ｒが陽性か陰性かは、免疫染色、抗ＩＬ－７Ｒ抗体を用いたＦＡＣＳなどの技術を用いて容易に決定することができる。 The present invention also provides a pharmaceutical composition for use in treating cancer, comprising an antibody drug conjugate of an anti-IL-7R antibody or antigen-binding fragment thereof and a cytotoxic agent. Many cancer cells are positive for IL-7R. Accordingly, many cancers can be treated by the medicament of the present invention. In order to identify a highly effective target population, in one embodiment of the present invention, the cancer may be an IL-7R-positive cancer or a cancer that has been confirmed to be IL-7R-positive. it can. IL-7R positive cancers include leukemia, lymphoma, lung cancer, pancreatic cancer, head and neck cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, stomach cancer, colon cancer, uterine cancer, ovary Cancer, skin cancer, thyroid cancer, thymic cancer, kidney cancer, testicular cancer, penile cancer, liver cancer, biliary tract cancer, brain tumor, bone soft tissue tumor, retroperitoneal tumor, angio-lymphatic sarcoma , And these metastatic cancers (eg, metastatic solid tumors). Many cancers, such as leukemia, lymphoma, advanced cancer, and metastatic cancer, are positive for IL-7R. Whether IL-7R is positive or negative can be easily determined using techniques such as immunostaining and FACS using an anti-IL-7R antibody.

　本発明の医薬組成物は、ＩＬ－７ＲとＩＬ－７との結合を遮断する治療に抵抗性を有するＩＬ－７Ｒ陽性のがんを処置することに用い得る。 The pharmaceutical composition of the present invention can be used for treating IL-7R-positive cancer having resistance to a therapy that blocks the binding between IL-7R and IL-7.

　さらに、本発明によれば、ＩＬ－７Ｒはステロイド抵抗性の付与と関与していることが明らかとなった。特に、ステロイド抵抗性細胞では、ＩＬ－７Ｒが陽性になる。従って、本発明によれば、ステロイド抵抗性の疾患または状態を処置することに用いるための医薬組成物であって、抗ＩＬ－７Ｒ抗体またはその抗原結合性断片と細胞傷害剤との抗体薬物コンジュゲートを含む、医薬組成物が提供される。ある態様では、疾患または状態のステロイド抵抗性を低減させることに用いるための医薬組成物であって、抗ＩＬ－７Ｒ抗体またはその抗原結合性断片と細胞傷害剤との抗体薬物コンジュゲートを含む、医薬組成物が提供される。ある態様では、ステロイド抵抗性の疾患または状態は、がんまたは炎症である。さらにまた、本発明によれば、ＩＬ－７Ｒはステロイド処置後、発現が向上することも明らかとなった。特にステロイド処置後は、ＩＬ－７Ｒ陽性細胞が生き残り易いため、本発明のコンジュゲートの有効性が高まる。仮にＩＬ－７Ｒ陰性細胞が生き残っていたとしても（例えば、ステロイド処置後の患者において）治療効果が期待できる。これは、ＰＤＣによるバイスタンダー効果によりＩＬ－７Ｒ陽性細胞近傍のＩＬ－７Ｒ陰性細胞を死滅させる効果が期待できるためである。従って、本発明によれば、ステロイド治療を受けた対象において、がんまたは炎症などの疾患または状態を処置することに用いるための医薬組成物であって、抗ＩＬ－７Ｒ抗体またはその抗原結合性断片と細胞傷害剤との抗体薬物コンジュゲートを含む、医薬組成物が提供される。 Furthermore, according to the present invention, it has been clarified that IL-7R is involved in imparting steroid resistance. In particular, IL-7R is positive in steroid resistant cells. Thus, according to the present invention, there is provided a pharmaceutical composition for use in treating a steroid resistant disease or condition comprising an antibody drug conjugate of an anti-IL-7R antibody or antigen-binding fragment thereof and a cytotoxic agent. A pharmaceutical composition comprising a gate is provided. In certain embodiments, a pharmaceutical composition for use in reducing steroid resistance in a disease or condition comprising an antibody drug conjugate of an anti-IL-7R antibody or antigen-binding fragment thereof and a cytotoxic agent. A pharmaceutical composition is provided. In certain embodiments, the steroid resistant disease or condition is cancer or inflammation. Furthermore, according to the present invention, it was also revealed that expression of IL-7R is improved after steroid treatment. Particularly after steroid treatment, IL-7R positive cells are more likely to survive, increasing the effectiveness of the conjugates of the invention. Even if IL-7R negative cells survive (for example, in patients after steroid treatment), a therapeutic effect can be expected. This is because the effect of killing IL-7R negative cells in the vicinity of IL-7R positive cells can be expected by the bystander effect of PDC. Therefore, according to the present invention, there is provided a pharmaceutical composition for use in treating a disease or condition such as cancer or inflammation in a subject who has received steroid therapy, wherein the anti-IL-7R antibody or antigen-binding property thereof is used. Pharmaceutical compositions comprising antibody drug conjugates of fragments and cytotoxic agents are provided.

　ある態様では、本発明の医薬組成物でステロイド抵抗性を低減しながら、ステロイドを投与することも可能である。従って、本発明のある態様では、ステロイド抵抗性の疾患または状態を処置することに用いるための医薬組成物であって、ステロイドと併用する、抗ＩＬ－７Ｒ抗体またはその抗原結合性断片と細胞傷害剤との抗体薬物コンジュゲートを含む医薬組成物が提供される。あるいは、本発明では、疾患または状態のステロイド抵抗性を低下させること（または疾患または状態のステロイド感受性を増大させること）に用いるための医薬組成物であって、抗ＩＬ－７Ｒ抗体またはその抗原結合性断片と細胞傷害剤との抗体薬物コンジュゲートを含む医薬組成物が提供される。これらの態様では、対象は、ステロイド抵抗性の疾患または状態を有する対象であり得る。 In one embodiment, it is possible to administer steroids while reducing steroid resistance with the pharmaceutical composition of the present invention. Accordingly, in one aspect of the invention, a pharmaceutical composition for use in treating a steroid resistant disease or condition, wherein the anti-IL-7R antibody or antigen-binding fragment thereof and cytotoxicity are used in combination with a steroid. Pharmaceutical compositions comprising an antibody drug conjugate with an agent are provided. Alternatively, in the present invention, a pharmaceutical composition for use in reducing steroid resistance of a disease or condition (or increasing steroid sensitivity of the disease or condition), comprising anti-IL-7R antibody or antigen binding thereof A pharmaceutical composition comprising an antibody drug conjugate of a sex fragment and a cytotoxic agent is provided. In these embodiments, the subject can be a subject with a steroid resistant disease or condition.

　さらにまた、本発明によれば、抗ＩＬ－７Ｒ抗体またはその抗原結合性断片と細胞傷害剤との抗体薬物コンジュゲートは、がんの対象においてリンパ節腫大を抑制することができる。従って、本発明によれば、がんの対象においてリンパ節腫大を抑制することに用いるための医薬組成物であって、抗ＩＬ－７Ｒ抗体またはその抗原結合性断片と細胞傷害剤との抗体薬物コンジュゲートを含む、医薬組成物が提供される。 Furthermore, according to the present invention, an antibody drug conjugate of an anti-IL-7R antibody or an antigen-binding fragment thereof and a cytotoxic agent can suppress lymphadenopathy in cancer subjects. Therefore, according to the present invention, a pharmaceutical composition for use in suppressing lymphadenopathy in a cancer subject, comprising an anti-IL-7R antibody or an antigen-binding fragment thereof and an antibody of a cytotoxic agent A pharmaceutical composition comprising a drug conjugate is provided.

　本発明によれば、潰瘍性大腸炎や関節リウマチなどの炎症性疾患において、抗ＩＬ－７Ｒ抗体と細胞傷害剤との抗体薬物コンジュゲートが症状の軽減効果を示した。これは、炎症疾患の発症または維持においてＩＬ－７Ｒ陽性細胞が強く関与していることを示唆するものであり、炎症状態でＩＬ－７Ｒ陽性細胞を殺傷することが炎症を緩和することに有用であることを意味するものである。従って、本明細書によれば、炎症性疾患を処置することに用いるための医薬組成物であって、抗ＩＬ－７Ｒ抗体またはその抗原結合性断片と細胞傷害剤との抗体薬物コンジュゲートを含む、医薬組成物が提供される。本明細書では、炎症性疾患には、膠原病が挙げられ、自己免疫疾患、Ｉ型糖尿病、多発性硬化症、全身性エリトマトーデス、アレルギー性疾患、臓器移植後の拒絶反応、および移植片対宿主病も含まれる。炎症性疾患にはさらにまた、クローン氏病などの炎症性腸疾患が含まれる。また、その他の自己免疫疾患および膠原病として、シェーグレン症候群、強皮症、皮膚筋炎、結節性多発動脈炎、混合性結合組織病、コーガン症候群、リウマチ性多発筋痛症、成人スティル病、巨細胞性動脈炎、抗リン脂質抗体症候群、ギラン・バレー症候群、重症筋無力症、自己免疫性肝炎、自己免疫性膵炎、原発性胆汁性胆管炎、高安動脈炎、 グッドパスチャー症候群、急速進行性糸球体腎炎、自己免疫性溶血性貧血、自己免疫性好中球減少症、特発性血小板減少性紫斑病、バセドウ病、橋本病、原発性甲状腺機能低下症、特発性アジソン病、 天疱瘡、膿疱性乾癬、尋常性乾癬、類天疱瘡、妊娠性疱疹、線状ＩｇＡ水疱性皮膚症、後天性表皮水疱症、円形脱毛症、尋常性白斑、原田病、自己免疫性視神経症、自己免疫性内耳障害、特発性無精子症、および習慣性流産から選択される自己免疫疾患または膠原病が挙げられる。リンパ球が関与する４型アレルギーとしては、臓器移植での拒絶反応、自然リンパ球が原因となる気管支喘息などが挙げられる。多くの炎症においてリンパ球がＩＬ－７Ｒ陽性である。具体的には、獲得免疫系リンパ球である、Ｔリンパ球およびＢリンパ球において陽性であると共に自然免疫系リンパ球においても陽性であることから、これらの細胞が原因となる多くの炎症性疾患が、本発明の医薬組成物の治療対象であり得る。 According to the present invention, in an inflammatory disease such as ulcerative colitis or rheumatoid arthritis, an antibody drug conjugate of an anti-IL-7R antibody and a cytotoxic agent showed a symptom reducing effect. This suggests that IL-7R positive cells are strongly involved in the onset or maintenance of inflammatory diseases, and killing IL-7R positive cells in an inflammatory state is useful for alleviating inflammation. It means that there is. Thus, according to the present specification, a pharmaceutical composition for use in treating an inflammatory disease, comprising an antibody drug conjugate of an anti-IL-7R antibody or antigen-binding fragment thereof and a cytotoxic agent A pharmaceutical composition is provided. As used herein, inflammatory diseases include collagen disease, autoimmune disease, type I diabetes, multiple sclerosis, systemic lupus erythematosus, allergic disease, rejection after organ transplantation, and graft versus host Diseases are also included. Inflammatory diseases also include inflammatory bowel diseases such as Crohn's disease. Other autoimmune diseases and collagen diseases include Sjogren's syndrome, scleroderma, dermatomyositis, polyarteritis nodosa, mixed connective tissue disease, Corgan syndrome, polymyalgia rheumatica, adult Still's disease, giant cells Arteritis, antiphospholipid antibody syndrome, Guillain-Barre syndrome, myasthenia gravis, autoimmune hepatitis, autoimmune pancreatitis, primary biliary cholangitis, Takayasu arteritis, Goodpasture syndrome, rapidly progressive glomeruli Nephritis, autoimmune hemolytic anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura, Graves' disease, Hashimoto's disease, primary hypothyroidism, idiopathic Addison's disease, pemphigus vulgaris, pustular psoriasis Psoriasis vulgaris, pemphigoid, gestational herpes zoster, linear IgA bullous dermatosis, acquired epidermolysis bullosa, alopecia areata, vulgaris vulgaris, Harada disease, autoimmune optic neuropathy, autoimmune inner ear disorder, Special occurrence Autoimmune diseases or collagen diseases selected from azoospermia and habitual abortion. Examples of type 4 allergy involving lymphocytes include rejection in organ transplantation and bronchial asthma caused by natural lymphocytes. Lymphocytes are IL-7R positive in many inflammations. Specifically, many inflammatory diseases caused by these cells because they are positive in acquired immune system lymphocytes, T lymphocytes and B lymphocytes, and also in innate immune system lymphocytes. Can be the subject of treatment with the pharmaceutical composition of the present invention.

　本発明では、炎症性疾患は、ステロイドに抵抗性である、またはＩＬ－７ＲとＩＬ－７との結合を遮断する治療に抵抗性である、疾患または状態であり得る。 In the present invention, the inflammatory disease can be a disease or condition that is resistant to steroids or resistant to treatment that blocks the binding of IL-7R to IL-7.

　本発明のある側面では、がんまたは炎症性疾患をその必要のある対象において処置する方法であって、該対象に抗ＩＬ－７Ｒ抗体またはその抗原結合性断片と細胞傷害剤との抗体薬物コンジュゲートを投与することを含む方法が提供される。コンジュゲートは、治療上有効量投与されうる。本発明のある態様では、前記対象は、ステロイド治療に耐性である対象であるか、またはステロイド治療を受けた対象であり得る。
　本発明のある態様では、がんまたは炎症性疾患をその必要のある対象において処置する方法であって、該対象に抗ＩＬ－７Ｒ抗体またはその抗原結合性断片と細胞傷害剤との抗体薬物コンジュゲートを投与すること、および該対象にステロイド系抗炎症薬を投与することを含む方法が提供される。本発明のある態様では、前記対象は、ステロイド治療に耐性である対象であるか、またはステロイド治療を受けた対象であり得る。 In one aspect of the invention, a method of treating cancer or an inflammatory disease in a subject in need thereof, comprising the step of administering to the subject an antibody drug conjugate of an anti-IL-7R antibody or antigen-binding fragment thereof and a cytotoxic agent. A method is provided that includes administering a gate. The conjugate can be administered in a therapeutically effective amount. In certain embodiments of the invention, the subject can be a subject that is resistant to steroid treatment or has received steroid treatment.
In one aspect of the invention, a method of treating cancer or an inflammatory disease in a subject in need thereof, comprising the step of administering an antibody drug conjugate of an anti-IL-7R antibody or antigen-binding fragment thereof to a cytotoxic agent. A method is provided comprising administering a gate and administering a steroidal anti-inflammatory drug to the subject. In certain embodiments of the invention, the subject can be a subject that is resistant to steroid treatment or has received steroid treatment.

　本発明のある態様では、ステロイド抵抗性の疾患または状態をその必要のある対象において処置する方法であって、該対象に抗ＩＬ－７Ｒ抗体またはその抗原結合性断片と細胞傷害剤との抗体薬物コンジュゲートを投与することを含む方法が提供される。 In one aspect of the invention, a method of treating a steroid-resistant disease or condition in a subject in need thereof, wherein the subject is an antibody drug comprising an anti-IL-7R antibody or antigen-binding fragment thereof and a cytotoxic agent. A method is provided that includes administering a conjugate.

　本発明のある態様では、疾患または状態のステロイド抵抗性をその必要のある対象において低下させる方法であって、該対象に抗ＩＬ－７Ｒ抗体またはその抗原結合性断片と細胞傷害剤との抗体薬物コンジュゲートを投与することを含む方法が提供される。 In one aspect of the present invention, a method of reducing steroid resistance of a disease or condition in a subject in need thereof, the subject comprising an antibody drug of an anti-IL-7R antibody or antigen-binding fragment thereof and a cytotoxic agent A method is provided that includes administering a conjugate.

　本発明のある側面では、抗ＩＬ－７Ｒ抗体またはその抗原結合性断片と細胞傷害剤との抗体薬物コンジュゲートを含む、がんまたは炎症性疾患を処置することに用いるための医薬の製造における、抗ＩＬ－７Ｒ抗体の使用が提供される。本発明のある側面では、抗ＩＬ－７Ｒ抗体またはその抗原結合性断片と細胞傷害剤との抗体薬物コンジュゲートを含む、がんまたは炎症性疾患を処置することに用いるための医薬の製造における、抗ＩＬ－７Ｒ抗体またはその抗原結合性断片と細胞傷害剤の使用が提供される。本発明のある側面では、がんまたは炎症性疾患を処置することに用いるための医薬の製造における、抗ＩＬ－７Ｒ抗体またはその抗原結合性断片と細胞傷害剤との抗体薬物コンジュゲートの使用が提供される。
　本発明のある側面では、抗ＩＬ－７Ｒ抗体またはその抗原結合性断片と細胞傷害剤との抗体薬物コンジュゲートの使用であって、がんまたは炎症性疾患を処置するための方法における使用が提供される。 In one aspect of the invention, in the manufacture of a medicament for use in treating cancer or an inflammatory disease comprising an antibody drug conjugate of an anti-IL-7R antibody or antigen-binding fragment thereof and a cytotoxic agent. The use of anti-IL-7R antibodies is provided. In one aspect of the invention, in the manufacture of a medicament for use in treating cancer or an inflammatory disease comprising an antibody drug conjugate of an anti-IL-7R antibody or antigen-binding fragment thereof and a cytotoxic agent. Use of an anti-IL-7R antibody or antigen-binding fragment thereof and a cytotoxic agent is provided. In one aspect of the invention, the use of an antibody drug conjugate of an anti-IL-7R antibody or antigen-binding fragment thereof and a cytotoxic agent in the manufacture of a medicament for use in treating cancer or an inflammatory disease. Provided.
One aspect of the invention provides the use of an antibody drug conjugate of an anti-IL-7R antibody or antigen-binding fragment thereof and a cytotoxic agent in a method for treating cancer or an inflammatory disease. Is done.

　本発明では、ＩＬ－７Ｒ発現細胞をＡＤＣにより殺傷することに代えて、ＩＬ－７Ｒ発現細胞においてＩＬ－７Ｒの発現を抑制してもよいであろう。従って、本発明のある側面では、ＩＬ－７Ｒの発現抑制剤を含む、がんまたは炎症性疾患を処置することに用いるための医薬組成物が提供される。本発明ではまた、ＩＬ－７Ｒの発現抑制剤を含む、ステロイド抵抗性の疾患または状態を処置することに用いるための医薬組成物が提供される。本発明ではさらに、ＩＬ－７Ｒの発現抑制剤を含む、疾患または状態のステロイド抵抗性を低下させることに用いるための医薬組成物が提供される。本発明ではさらにまた、ステロイド抵抗性の疾患または状態を処置することに用いるための医薬組成物であって、ステロイドと併用する、ＩＬ－７Ｒの発現抑制剤を含む、医薬組成物が提供される。本発明ではさらにまた、ＩＬ－７Ｒの発現抑制剤を含む、がんの対象においてリンパ節腫大を抑制することに用いるための医薬組成物が提供される。 In the present invention, instead of killing IL-7R-expressing cells by ADC, IL-7R expression may be suppressed in IL-7R-expressing cells. Accordingly, in one aspect of the present invention, there is provided a pharmaceutical composition for use in treating cancer or inflammatory disease, comprising an IL-7R expression inhibitor. The present invention also provides a pharmaceutical composition for use in treating a steroid resistant disease or condition, comprising an IL-7R expression inhibitor. The present invention further provides a pharmaceutical composition for use in reducing steroid resistance in a disease or condition, comprising an IL-7R expression inhibitor. The present invention further provides a pharmaceutical composition for use in treating a steroid resistant disease or condition, comprising an IL-7R expression inhibitor used in combination with a steroid. . The present invention further provides a pharmaceutical composition for use in suppressing lymphadenopathy in a cancer subject, comprising an IL-7R expression inhibitor.

　ＩＬ－７Ｒの発現抑制剤としては、ＩＬ－７Ｒに対するｓｉＲＮＡ、ｓｈＲＮＡ、アンチセンスオリゴが挙げられ、本発明で用いることができる。ＩＬ－７Ｒの発現抑制剤としては、ｓｉＲＮＡやｓｈＲＮＡをコードするＤＮＡを含む発現ベクターも挙げられる。 Examples of IL-7R expression inhibitors include siRNA, shRNA and antisense oligos against IL-7R, which can be used in the present invention. Examples of the IL-7R expression inhibitor include an expression vector containing a DNA encoding siRNA or shRNA.

　以下実施例では、断りの無い限り、以下記号の意味は、＊：Ｐ＜０．０５，＊＊：Ｐ＜０．０１，＊＊＊：Ｐ＜０．００１である。 In the following examples, unless otherwise specified, the meanings of the following symbols are **: P <0.05, **: P <0.01, ***: P <0.001.

実施例１：ＩＬ－７受容体と腫瘍細胞の生存活性
　本実施例では、各種腫瘍細胞におけるＩＬ－７受容体（ＩＬ－７Ｒ）の発現とＩＬ－７Ｒの生理機能について調べた。 Example 1: Survival activity of IL-7 receptor and tumor cells In this example, the expression of IL-7 receptor (IL-7R) and physiological functions of IL-7R in various tumor cells were examined.

（１）ＩＬ－７Ｒの細胞表面発現
　ヒトＴ細胞悪性リンパ種細胞株ＳｕｐＴ１、肺がんリンパ節転移由来細胞株Ｈ２００９、肺がん肺胞転移由来細胞株Ｈ３５８、咽頭がんのがん性胸水由来細胞株Ｄｅｔｒｏｉｔｅ５６２、前立腺がん骨転移由来細胞株ＰＣ３、および骨転移を伴う膀胱がん細胞株ＴＣＣｓｕｐは、ＡＴＣＣ（米国細胞バンク）から入手した。ヒトＢ細胞リンパ性白血病細胞株ＮＡＬＭ６、肺がんのがん性胸水由来細胞株ＬＣ－２／ａｄ、膵臓がん肝転移由来細胞株ＰＫ－４５Ｈは、理研バイオリソースセンターから入手した。マウスリンパ性白血病細胞株ＣＹＧ８２は、下記参考文献１に記載の通りに樹立され、本実施例で使用した。
　抗ヒトＩＬ－７Ｒ抗体（eBioRDR5, eBioscience; R34-R34, TONB0）と抗マウスＩＬ－７Ｒ抗体（A7R34或いはSB/199 ,eBioscience）を用いてフローサイトメトリーでＩＬ－７Ｒの発現を測定した。ＩＬ－７Ｒ抗体（Ａ７Ｒ３４）は、下記参考文献２および３の通りに作製され、本実施例で使用した。結果は、図１Ａに示される通りであった。 (1) Cell surface expression of IL-7R Human T cell malignant lymphoma cell line SupT1, lung cancer lymph node metastasis derived cell line H2009, lung cancer alveolar metastasis derived cell line H358, cancerous pleural effusion cell line Detroite562 , Prostate cancer bone metastasis derived cell line PC3, and bladder cancer cell line TCCsup with bone metastasis were obtained from ATCC (US Cell Bank). Human B cell lymphocytic leukemia cell line NALM6, lung cancer cancer pleural effusion cell line LC-2 / ad, and pancreatic cancer liver metastasis cell line PK-45H were obtained from RIKEN BioResource Center. Mouse lymphocytic leukemia cell line CYG82 was established as described in Reference 1 below and used in this example.
IL-7R expression was measured by flow cytometry using an anti-human IL-7R antibody (eBioRDR5, eBioscience; R34-R34, TONB0) and an anti-mouse IL-7R antibody (A7R34 or SB / 199, eBioscience). IL-7R antibody (A7R34) was prepared as in References 2 and 3 below and used in this example. The result was as shown in FIG. 1A.

　図１Ａに示されるように、ヒトＴ細胞悪性リンパ腫細胞株ＳｕｐＴ１、ヒトＢ細胞リンパ性白血病細胞株ＮＡＬＭ６、およびマウスリンパ性白血病細胞株ＣＹＧ８２において、ＩＬ－７Ｒが陽性であった。また、肺がんリンパ節転移由来細胞株Ｈ２００９、肺がん肺胞転移由来細胞株Ｈ３５８、肺がんのがん性胸水由来細胞株ＬＣ－２／ａｄ、膵臓がん肝転移由来細胞株ＰＫ－４５Ｈ、咽頭がんのがん性胸水由来細胞株Ｄｅｔｒｏｉｔｅ５６２、前立腺がん骨転移由来細胞株ＰＣ３、および骨転移を伴う膀胱がん細胞株ＴＣＣｓｕｐにおいてＩＬ－７Ｒが陽性であった（各図の右側のヒストグラムがＩＬ－７Ｒ発現で、左側のヒストグラムは陰性コントロール）。このように、調べたリンパ性悪性疾患と転移性固形腫瘍のすべての細胞株でＩＬ－７Ｒが陽性であった。転移性の固形腫瘍において発現が確認され、固形腫瘍では、ＩＬ－７Ｒ発現と転移との関係が示唆された。 As shown in FIG. 1A, IL-7R was positive in human T cell malignant lymphoma cell line SupT1, human B cell lymphocytic leukemia cell line NALM6, and mouse lymphocytic leukemia cell line CYG82. Also, lung cancer lymph node metastasis derived cell line H2009, lung cancer alveolar metastasis derived cell line H358, lung cancer pleural effusion cell line LC-2 / ad, pancreatic cancer liver metastasis derived cell line PK-45H, pharyngeal cancer IL-7R was positive in the cancerous pleural effusion cell line Detroite 562, prostate cancer bone metastasis-derived cell line PC3, and bladder cancer cell line TCCsup with bone metastasis (the histogram on the right side of each figure shows IL- 7R expression, left histogram is negative control). Thus, IL-7R was positive in all cell lines of lymphoid malignancies and metastatic solid tumors examined. Expression was confirmed in metastatic solid tumors, suggesting a relationship between IL-7R expression and metastasis in solid tumors.

（２）ＩＬ－７Ｒノックダウン細胞株の作製
　ＳｕｐＴ１、ＮＡＬＭ６、ＣＹＧ８２、Ｈ２００９、ＰＫ－４５Ｈ細胞について、レンチウイルスベクターに組み込まれたショートヘアピンRNA(shRNA, Sigma)を発現させることで、ＩＬ－７Ｒ遺伝子ノックダウン（ＫＤ）細胞株（ＩＬ－７Ｒ－ＫＤ）を作製した。ＳｕｐＴ１、ＮＡＬＭ６、ＣＹＧ８２にはＧＦＰ遺伝子のｓｈＲＮＡ(Sigma)を用いて、Ｈ２００９、ＰＫ－４５Ｈには非特異的ｓｈＲＮＡ(Sigma)を導入して、比較用のコントロール細胞（ＣＴＲ）を作製した。抽出キット（QIAGEN）と合成キット(Thermo Fisher Scientific或いはTakara)を用いて細胞のｔｏｔａｌ　ＲＮＡからｃＤＮＡを合成した。定量性ＲＴ－ＰＣＲを行った。すなわち、９μｌのｃＤＮＡ希釈液、1 μl TaqMan primers/probe mixture (Thermo Fisher Scientific)と10 μl TaqMan Fast Universal PCR Master Mix (Thermo Fisher Scientific)の合計20 μlの反応液に対して、ABI7500Fast（Thermo Fisher Scientific）を用いて測定した。各遺伝子の発現は比較ＣＴ法を用いて、ヒト遺伝子の場合はＧＡＰＤＨを、マウス遺伝子の場合はＡＣＴｂの発現で標準化した。 (2) Preparation of IL-7R knockdown cell line For SupT1, NALM6, CYG82, H2009, PK-45H cells, IL-7R is expressed by expressing short hairpin RNA (shRNA, Sigma) incorporated into a lentiviral vector. A gene knockdown (KD) cell line (IL-7R-KD) was generated. A control cell (CTR) for comparison was prepared by introducing shRNA (Sigma) of the GFP gene into SupT1, NALM6, and CYG82, and non-specific shRNA (Sigma) into H2009 and PK-45H. CDNA was synthesized from the total RNA of the cells using an extraction kit (QIAGEN) and a synthesis kit (Thermo Fisher Scientific or Takara). Quantitative RT-PCR was performed. That is, ABI7500Fast (Thermo Fisher Scientific) was used in a total of 20 μl of the reaction mixture of 9 μl of cDNA dilution, 1 μl TaqMan primers / probe mixture (Thermo Fisher Scientific) and 10 μl TaqMan Fast Universal PCR Master Mix (Thermo Fisher Scientific). ). The expression of each gene was standardized by the comparative CT method using GAPDH for human genes and ACTb for mouse genes.

　図１Ｂに示されるように、作製されたＳｕｐＴ１、ＮＡＬＭ６、ＣＹＧ８２、Ｈ２００９、ＰＫ－４５ＨのＩＬ－７Ｒ－ＫＤ細胞株でＩＬ－７Ｒ遺伝子の発現量低下を確認した。 As shown in FIG. 1B, a decrease in the expression level of the IL-7R gene was confirmed in the prepared SupT1, NALM6, CYG82, H2009, and PK-45H IL-7R-KD cell lines.

（３）細胞増殖におけるＩＬ－７Ｒノックダウンの影響
　ＩＬ－７Ｒ－ＫＤ細胞とＣＴＲ細胞の試験管内での培養液中での増殖活性を調べた。細胞１０００もしくは３０００個を９６穴プレートにまき、連日細胞数をカウントした。結果は、図１Ｃに示される通りであった。
　図１Ｃに示されるように、ＳｕｐＴ１、ＮＡＬＭ６、ＣＹＧ８２、Ｈ２００９、ＰＫ－４５Ｈ　のＩＬ－７Ｒ－ＫＤ細胞株は、いずれもＣＴＲ細胞株よりも、試験管内の細胞増殖能が低下していた。 (3) Effect of IL-7R knockdown on cell proliferation The proliferation activity of IL-7R-KD cells and CTR cells in culture in vitro was examined. 1000 or 3000 cells were seeded in a 96-well plate, and the number of cells was counted every day. The result was as shown in FIG. 1C.
As shown in FIG. 1C, the IL-7R-KD cell lines of SupT1, NALM6, CYG82, H2009, and PK-45H all had a lower cell proliferation ability in vitro than the CTR cell line.

（４）腫瘍増殖活性とＩＬ－７Ｒノックダウンとの関係
　ＳｕｐＴ１、ＮＡＬＭ６、ＣＹＧ８２、Ｈ２００９細胞を雌のヌードマウス（１群５匹）の皮下に移植して、生着後に腫瘍体積を計測することで、腫瘍増殖活性を測定した。結果は図１Ｄに示される通りであった。
　図１Ｄに示されるように、ＳｕｐＴ１、ＮＡＬＭ６、ＣＹＧ８２、Ｈ２００９のＩＬ－７Ｒ－ＫＤ細胞株は、ＣＴＲ細胞に比較してヌードマウスへの移植実験で腫瘍体積の増大率が低下していた。 (4) Relationship between tumor growth activity and IL-7R knockdown SupT1, NALM6, CYG82, H2009 cells are implanted subcutaneously in female nude mice (5 per group), and tumor volume is measured after engraftment The tumor growth activity was measured. The result was as shown in FIG. 1D.
As shown in FIG. 1D, SupT1, NALM6, CYG82, and H2009 IL-7R-KD cell lines showed a decrease in tumor volume increase rate in nude mouse transplantation experiments compared to CTR cells.

（５）細胞の状態の観察
　ＰＫ－４５ＨのＩＬ－７Ｒ－ＫＤ細胞株の試験管内での細胞生存状態を培養倒立顕微鏡ELIPSE TS100(ニコン)で観察した。結果は、図１Ｅに示される通りであった。
　図１Ｅに示されるように、ＰＫ－４５ＨのＩＬ－７Ｒ－ＫＤ細胞では、ＣＴＲ細胞と比較して細胞生存能が著明に低下していた。 (5) Observation of cell state The cell viability of the PK-45H IL-7R-KD cell line in vitro was observed with an inverted culture microscope ELIPSE TS100 (Nikon). The result was as shown in FIG. 1E.
As shown in FIG. 1E, cell viability was markedly reduced in PK-45H IL-7R-KD cells compared to CTR cells.

実施例２：ＩＬ－７Ｒの生理機能
　本実施例では、ＩＬ－７Ｒの更なる生理機能を調べた。 Example 2: Physiological function of IL-7R In this example, further physiological function of IL-7R was examined.

　リンパ系悪性腫瘍ではステロイドの抗がん作用を利用した治療が行われるが、ステロイドに体制の腫瘍細胞が出現することが知られている。ステロイド耐性の獲得とＩＬ－７Ｒとに何らかの生理学的関連があるかについてまず調べた。 In lymphoid malignant tumors, treatment using the anticancer effect of steroids is performed, but it is known that systemic tumor cells appear in steroids. We first examined whether there was any physiological link between acquiring steroid resistance and IL-7R.

　ＳｕｐＴ１、ＮＡＬＭ６、ＣＹＧ８２、Ｈ２００９、およびＰＫ４５ＨのそれぞれにおけるＩＬ－７Ｒシグナルとステロイド耐性との関係を調べるために、試験管内での各細胞におけるデキサメタゾンの殺細胞効果（ＩＣ５０；５０％細胞障害濃度）を確認した。細胞１０００～３０００個を９６穴プレートに１００μＬ／ウエルでまき、デキサメタゾンを０．１μＭから１ｍＭの濃度で各ウエルに添加した。７２時間培養後、ＷＳＴ－８（同仁化学、Cell Counting Kit-8）を１０μＬ／ウエルで添加し、３時間培養後にマイクロプレートリーダー（Molecular Devices）で４５０ｎｍの吸光度を測定した。各濃度における細胞生存率を計算して、ＩＣ５０を算出した。結果は、図２Ａに示される通りであった。 To examine the relationship between IL-7R signal and steroid resistance in each of SupT1, NALM6, CYG82, H2009, and PK45H, the cytotoxic effect (IC50; 50% cytotoxic concentration) of dexamethasone in each cell in vitro was examined. confirmed. 1000 to 3000 cells were seeded at 100 μL / well in a 96-well plate, and dexamethasone was added to each well at a concentration of 0.1 μM to 1 mM. After culturing for 72 hours, WST-8 (Dojindo, Cell-Counting-Kit-8) was added at 10 μL / well, and after culturing for 3 hours, absorbance at 450 nm was measured with a microplate reader (Molecular Devices). IC50 was calculated by calculating the cell viability at each concentration. The result was as shown in FIG. 2A.

　図２Ａに示されるように、ＳｕｐＴ１、ＮＡＬＭ６、およびＣＹＧ８２のいずれにおいても、ＩＬ－７Ｒ－ＫＤ細胞で、ステロイド（デキサメタゾン）のＩＣ５０が低下しており、ステロイド感受性が増加していることが判明した。Ｈ２００９、およびＰＫ４５Ｈでは、ＩＬ－７Ｒ－ＫＤ細胞とＣＴＲ細胞に差を認めなかった。
　このように、リンパ系悪性腫瘍においては、ステロイド感受性に対してＩＬ－７Ｒが大きく関与していることが明らかであるが、転移性固形腫瘍であるＨ２００９、およびＰＫ４５Ｈにおいては、ステロイド感受性に対してＩＬ－７Ｒの関与がほとんどないことが明らかとなった。 As shown in FIG. 2A, in SupT1, NALM6, and CYG82, the IC50 of steroid (dexamethasone) was decreased and the steroid sensitivity was increased in IL-7R-KD cells. . In H2009 and PK45H, there was no difference between IL-7R-KD cells and CTR cells.
Thus, it is clear that IL-7R is greatly involved in steroid sensitivity in lymphoid malignant tumors, whereas in metastatic solid tumors H2009 and PK45H, steroid sensitivity is It became clear that IL-7R was hardly involved.

　さらに、ステロイド耐性株を得て、これに対してＩＬ－７Ｒのノックダウン株と、比特異的ｓｈＲＮＡによる対照株（ＣＴＲ）とを作製し、デキサメタゾン存在下におけるＩＬ－７Ｒノックダウンの影響を確認した。ＣＹＧ８２親株からＣＹＧ８２ステロイド耐性株が容易に得られることから、高濃度のステロイドを添加した状態での細胞の増殖性を観察した。６穴プレートに細胞１０6個を３ｍｌ／ウエルにまき、デキサメタゾンを１０ｎＭまたは１００ｎＭを添加して、１週間後と２週間後の細胞数をカウントした。結果は、図２Ｂに示される通りであった。 Furthermore, a steroid resistant strain was obtained, and an IL-7R knockdown strain and a control strain (CTR) with a specific shRNA were prepared to confirm the effect of IL-7R knockdown in the presence of dexamethasone. did. Since a CYG82 steroid resistant strain can be easily obtained from the CYG82 parental strain, the growth of cells in a state where a high concentration of steroid was added was observed. 10 6 cells were seeded at 3 ml / well in a 6- well plate, 10 nM or 100 nM dexamethasone was added, and the number of cells after 1 week and 2 weeks was counted. The result was as shown in FIG. 2B.

　図２Ｂに示されるように、ステロイド耐性株に非特異的ｓｈＲＮＡを導入したＣＴＲ細胞は、デキサメタゾン存在下で最初の１週間は増殖が抑制されたが、その後、細胞が増殖を示した。このことは、これに対して、ステロイド耐性株においてＩＬ－７Ｒをノックダウンした株では、細胞数が顕著に減少した。このことは、ＩＬ－７Ｒによりステロイド耐性が解除されること、および、ＩＬ－７Ｒノックダウン株では、耐性株の出現が抑えられることを示す。 As shown in FIG. 2B, the growth of CTR cells into which a non-specific shRNA had been introduced into a steroid resistant strain was suppressed for the first week in the presence of dexamethasone, but thereafter the cells showed growth. In contrast, in the steroid resistant strain, the number of cells was significantly decreased in the strain in which IL-7R was knocked down. This indicates that steroid resistance is released by IL-7R, and that the emergence of resistant strains is suppressed in IL-7R knockdown strains.

　まず、デキサメタゾンのＩＣ５０の違いを比較した。さらに、ステロイド耐性の獲得とＩＬ－７Ｒとの関係を調べた。具体的には、抽出キット（QIAGEN）と合成キット(Thermo Fisher Scientific或いはTakara)を用いて細胞のｔｏｔａｌ　ＲＮＡからｃＤＮＡを合成して、定量性ＰＣＲを行い、ＩＬ－７Ｒ遺伝子とＣＤ１９遺伝子の発現を調べた。結果は、図２Ｃに示される通りであった。 First, the difference in IC50 of dexamethasone was compared. Furthermore, the relationship between acquisition of steroid resistance and IL-7R was examined. Specifically, cDNA is synthesized from total RNA of cells using an extraction kit (QIAGEN) and a synthesis kit (Thermo Fisher Fisher or Takara), quantitative PCR is performed, and the expression of IL-7R gene and CD19 gene is performed. Examined. The result was as shown in FIG. 2C.

　図２Ｃに示されるように、上記で得られたＣＹＧ８２細胞のステロイド耐性株は、デキサメタゾンに対して高いＩＣ５０を示す。そして、非耐性の親株と耐性株とでＩＬ－７Ｒの発現量を比較すると、耐性株においてＩＬ－７Ｒの発現が有意に向上していることが明らかとなった。一方で、ＣＤ１９の発現量については、親株と耐性株との間で有意な差は認められなかった。 As shown in FIG. 2C, the steroid resistant strain of CYG82 cells obtained above shows a high IC50 against dexamethasone. Then, when the expression level of IL-7R was compared between the non-resistant parent strain and the resistant strain, it was revealed that the expression of IL-7R was significantly improved in the resistant strain. On the other hand, regarding the expression level of CD19, no significant difference was observed between the parent strain and the resistant strain.

実施例３：ＩＬ－７Ｒシグナルの活性化とステロイド耐性との関係
　本実施例では、ステロイド耐性におけるＩＬ－７Ｒシグナルの活性化との関係を調べた。 Example 3 Relationship between IL-7R Signal Activation and Steroid Resistance In this example, the relationship between IL-7R signal activation and steroid resistance was examined.

　ＳｕｐＴ１、ＮＡＬＭ６、ＣＹＧ８２　、ＲＡＧ２－／－細胞について、ＩＬ－７Ｒ下流のシグナル分子であるＪＡＫ／ＳＴＡＴ、ＢＣＬ２、およびＮＦκβの活性化に対する、ＩＬ－７Ｒ－ＫＤの影響を調べた。ＪＡＫ／ＳＴＡＴに関しては、細胞を４％パラホルムアルデヒドで固定してメタノール処理を行い、抗phospho-STAT5抗体（eBioscience）を用いてリン酸化ＳＴＡＴ５を検出することで測定した。フローサイトメトリー検出装置であるGuava easyCyte 10HT (Merck Millipore) 或いは Aria flow cytometer (BD Biosciences)を用いて測定を行った。死細胞はPropidium iodide (Thermo Fisher Scientific)で染色して測定時に除外した。 データ解析にはFlowJo program (Tree Star)を用いた。
　ＢＣＬ２のシグナルは、各細胞からｔｏｔａｌ　ＲＮＡ抽出・ｃＤＮＡ合成を経て、定量性ＰＣＲで、ＢＣＬ２の発現量を調べた。
　ＮＦκβについては、細胞を４％パラホルムアルデヒドで固定して、抗phospho-p65/NF-kβ 抗体 (Abcam) を反応させた。2次抗体はAlexa-488/555/647-標識した抗ウサギＩｇＧ抗体(Thermo Fisher Scientific)を用いた。核はＤＡＰＩ (Thermo Fisher Scientific)で染色した。画像は共焦点レーザー顕微鏡LSM-710 (Carl Zeiss)もしくはＨＳオールインワン蛍光顕微鏡BZ-9000 (Keyence Co.)で撮影した。ＲＡＧ２－／－細胞は参考文献３の通りに樹立・使用した。 SupT1, NALM6, CYG82, RAG2 − / − cells were examined for the effect of IL-7R-KD on the activation of IL-7R downstream signaling molecules JAK / STAT, BCL2, and NFκβ. JAK / STAT was measured by fixing cells with 4% paraformaldehyde, treating with methanol, and detecting phosphorylated STAT5 using an anti-phospho-STAT5 antibody (eBioscience). Measurement was performed using a flow cytometry detection device, Guava easyCyte 10HT (Merck Millipore) or Aria flow cytometer (BD Biosciences). Dead cells were stained with Propidium iodide (Thermo Fisher Scientific) and excluded during measurement. FlowJo program (Tree Star) was used for data analysis.
The BCL2 signal was subjected to total RNA extraction and cDNA synthesis from each cell, and the expression level of BCL2 was examined by quantitative PCR.
For NFκβ, cells were fixed with 4% paraformaldehyde and reacted with anti-phospho-p65 / NF-kβ antibody (Abcam). As the secondary antibody, Alexa-488 / 555 / 647-labeled anti-rabbit IgG antibody (Thermo Fisher Scientific) was used. Nuclei were stained with DAPI (Thermo Fisher Scientific). Images were taken with a confocal laser microscope LSM-710 (Carl Zeiss) or an HS all-in-one fluorescence microscope BZ-9000 (Keyence Co.). RAG2 − / − cells were established and used as described in Reference 3.

　結果、ＪＡＬ／ＳＴＡＴとＢＣＬ２シグナルにはＩＬ－７Ｒのノックダウンの影響は無かったことから、ステロイド耐性とＩＬ－７Ｒシグナルの活性化とは関連性がほとんど無いであろうことが示唆された。
　また、図３に示されるように、ＮＦｋＢシグナルがＣＴＲ細胞では核に認められたのに対して、ＩＬ－７Ｒ－ＫＤ細胞ではシグナルがほとんど認められなかった。このことから、ステロイド耐性株でＮＦκＢの活性化が生じていたのに対して、ＩＬ－７ＲのノックダウンによりＮＦκＢの活性化が抑制されたことが分かった。
　さらに、増殖因子としてＩＬ－７が必要なＲＡＧ２－／－細胞では、ＩＬ－７の非存在下では、ＪＡＫ／ＳＴＡＴ、ＢＣＬ２、ＮＦｋＢのいずれのシグナルも抑制されていた。これらのことから、ステロイド耐性にはＪＡＫ／ＳＴＡＴおよびＢＣＬ２シグナルとの関係は認められないことが分かった。
　さらにまた、ＮＦｋＢは、いずれの細胞でもＩＬ－７Ｒのノックダウンにより低下したがＨ２００９、ＰＫ－４５Ｈ細胞のＩＬ－７Ｒ－ＫＤでも低下した。これらの細胞では図２Ａに示されるようにステロイド耐性との関係が無かったことから、ＮＦｋＢはステロイド耐性よりも細胞増殖・生存に強く関与している可能性が高いと思われた。 As a result, JAL / STAT and BCL2 signals were not affected by IL-7R knockdown, suggesting that steroid resistance and IL-7R signal activation would have little relevance.
Further, as shown in FIG. 3, NFkB signal was observed in the nucleus in CTR cells, whereas almost no signal was observed in IL-7R-KD cells. From this, it was found that activation of NFκB occurred in the steroid resistant strain, whereas activation of NFκB was suppressed by knockdown of IL-7R.
Furthermore, in RAG2 − / − cells that require IL-7 as a growth factor, all signals of JAK / STAT, BCL2, and NFkB were suppressed in the absence of IL-7. From these results, it was found that steroid resistance was not associated with JAK / STAT and BCL2 signals.
Furthermore, NFkB was decreased by IL-7R knockdown in all cells, but also decreased in IL-7R-KD of H2009 and PK-45H cells. Since these cells had no relationship with steroid resistance as shown in FIG. 2A, NFkB seemed to be more likely to be more strongly involved in cell proliferation and survival than steroid resistance.

実施例４：ステロイド耐性の獲得と遺伝子発現の変化
　本実施例では、臨床サンプルにおけるIL-7R遺伝子発現とステロイド耐性との関係を調べた。 Example 4: Acquisition of steroid resistance and change in gene expression In this example, the relationship between IL-7R gene expression and steroid resistance in clinical samples was examined.

　まず、Gene Expression Omnibus (GEO)から、GSE39339とGSE32962の臨床データを入手し、また、Array ExpressからE-MEXP-3916の臨床データを入手した。データは解析ソフトＲ（オープンソースフリーウエア）を用いて、ＲＭＡ処理で標準化した後に統計解析を行った。統計解析にはSPSS Statistics バージョン20 (IBM社)を用いて、P<0.05以下を判定の基準とした。結果は、図４に示される通りであった。図４Ａでは、GSE39339の解析結果が示され、図４Ｂでは、GSE32962の解析結果が示され、図４Ｃでは、E-MEXP-3916の解析結果が示されている。 First, clinical data of GSE39339 and GSE32962 were obtained from Gene® Expression® Omnibus® (GEO), and clinical data of E-MEXP-3916 was also acquired from Array® Express. The data was subjected to statistical analysis after being standardized by RMA processing using analysis software R (open source freeware). For statistical analysis, SPSS Statistics Version 20 (IBM) was used, and P <0.05 or less was used as a criterion. The result was as shown in FIG. 4A shows the analysis result of GSE39339, FIG. 4B shows the analysis result of GSE32962, and FIG. 4C shows the analysis result of E-MEXP-3916.

　図４Ａに示されるように、ヒト急性リンパ性白血病のステロイド（デキサメタゾン）を含む化学療法後でリンパ球マーカーのＴｄＴ、ＣＤ１９、ＣＤ２２は有意に低下した。一方で、ＩＬ－７Ｒの発現は、治療後に上昇する傾向が見られた。化学療法は、デキサメタゾン以外には、アントラサイクリン、ビンクリスチン、およびＬ－アスパラキナーゼが含まれていた。 As shown in FIG. 4A, lymphocyte markers TdT, CD19, and CD22 significantly decreased after chemotherapy including steroid (dexamethasone) for human acute lymphoblastic leukemia. On the other hand, the expression of IL-7R tended to increase after treatment. Chemotherapy included anthracycline, vincristine, and L-asparakinase in addition to dexamethasone.

　図４Ｂに示されるように、ヒト急性Ｂリンパ性白血病の患者由来細胞からステロイド（プレドニゾロン）感受性と耐性株を樹立して、ＤＮＡマイクロアレイ解析を行ったものである。両群間で、ＴｄＴ、ＣＤ１９、ＣＤ２２に変化はないが、ステロイド（プレドニゾロン）耐性株でＩＬ－７Ｒが有意に高値を示した。 As shown in FIG. 4B, a steroid (prednisolone) sensitive and resistant strain was established from cells derived from a patient with human acute B lymphoblastic leukemia and subjected to DNA microarray analysis. There was no change in TdT, CD19, and CD22 between the two groups, but IL-7R was significantly higher in the steroid (prednisolone) resistant strain.

　図４Ｃに示されるように、ヒト急性Ｔリンパ性白血病の患者で、無治療群とステロイド（デキサメタゾン）を含む治療群でＤＮＡマイクロアレイ解析を行ったものである。治療群でＩＬ－７Ｒの発現が有意に高値を示した。化学療法は、デキサメタゾン以外には、ダウノルビシン、ビンクリスチン、Ｌ－アスパラキナーゼが含まれていた。 As shown in FIG. 4C, DNA microarray analysis was performed in a non-treated group and a treated group containing steroid (dexamethasone) in a patient with human acute T lymphocytic leukemia. IL-7R expression was significantly higher in the treatment group. Chemotherapy included daunorubicin, vincristine, and L-asparakinase in addition to dexamethasone.

　図４Ａ～４Ｃにおいて示されたように、臨床データからも、ステロイド治療によるステロイド耐性の獲得とＩＬ－７Ｒの発現とが関連することが分かる。 As shown in FIGS. 4A to 4C, it can be seen from the clinical data that the acquisition of steroid resistance by the steroid treatment is related to the expression of IL-7R.

実施例５：抗ＩＬ－７Ｒ抗体の特性
　本実施例では、抗ＩＬ－７Ｒ抗体を細胞と接触させたときの動態について調べた。 Example 5: Characteristics of anti-IL-7R antibody In this example, the kinetics when an anti-IL-7R antibody was brought into contact with cells were examined.

　Ａｌｅｘａ６４７(Thermo Fisher Scientific)を抗ＩＬ－７Ｒ抗体（Ａ７Ｒ３４）に標識した。試験管内の培養条件下でＣＹＧ６２細胞にＡｌｅｘａ６４７標識した抗ＩＬ－７Ｒ抗体（Ａ７Ｒ３４）を投与して、１０分後と３０分後の細胞内取り込みを観察した。ライソゾームはライソトラッカー(Thermo Fisher Scientific)で可視化した。結果は、図５Ａに示される通りであった。 Alexa647 (Thermo Fisher Fisher Scientific) was labeled with an anti-IL-7R antibody (A7R34). Alexa647-labeled anti-IL-7R antibody (A7R34) was administered to CYG62 cells under in vitro culture conditions, and intracellular uptake after 10 and 30 minutes was observed. Lysosomes were visualized with a lyso tracker (Thermo Fisher Fisher Scientific). The result was as shown in FIG. 5A.

　図５Ａに示されるように、抗ＩＬ－７Ｒ抗体は、投与後３０分以内に細胞内に取り込まれ、ライソゾーム内に局在することが分かった。 As shown in FIG. 5A, it was found that the anti-IL-7R antibody was taken up into cells within 30 minutes after administration and localized in lysosomes.

　次に、インビボでの抗ＩＬ－７Ｒ抗体の動態を確認した。具体的には、ＣＹＧ８２親株を雌のヌードマウスの皮下移植後に腫瘍を形成させた後に、Ａｌｅｘａ６４７標識抗ＩＬ－７Ｒ抗体（Ａ７Ｒ３４）或いはコントロール抗体をマウス尾静脈から投与した。投与１週間後に腫瘍、リンパ節、脾臓、骨、および肝臓を摘出して、各組織における抗ＩＬ－７Ｒ抗体（Ａ７Ｒ３４）の集積を確認した。結果は、図５Ｂに示される通りであった。 Next, the kinetics of anti-IL-7R antibody in vivo was confirmed. Specifically, after the CYG82 parent strain was formed into a tumor after subcutaneous implantation of female nude mice, Alexa647-labeled anti-IL-7R antibody (A7R34) or control antibody was administered from the tail vein of the mouse. One week after administration, the tumor, lymph node, spleen, bone, and liver were removed, and accumulation of anti-IL-7R antibody (A7R34) in each tissue was confirmed. The result was as shown in FIG. 5B.

　図５Ｂに示されるように、抗ＩＬ－７Ｒ抗体（Ａ７Ｒ３４）は腫瘍と病変リンパ節に集積したのに対して、コントロール抗体はこれらの組織への集積は認めなかった。 As shown in FIG. 5B, the anti-IL-7R antibody (A7R34) accumulated in the tumor and the diseased lymph node, whereas the control antibody did not accumulate in these tissues.

　ＣＹＧ８２細胞とＩＬ－７依存性のＲＡＧ２－／－細胞とをそれぞれ２０００或いは４０００個／１００μＬ／ｗｅｌｌで各々９６ウェルプレートに添加し培養した。ＩＬ－７Ｒ抗体Ａ７Ｒ３４を１０-4～１０2μｇ／ｍｌの希釈系列で添加して、７２時間後にＷＳＴ－８（同仁化学、Ｃｅｌｌ　Ｃｏｕｎｔｉｎｇ　Ｋｉｔ－８）を１０μＬ／ｗｅｌｌで添加し、３時間培養後にマイクロプレートリーダー（Ｍｏｌｅｃｕｌａｒ　Ｄｅｖｉｃｅｓ）で４５０ｎｍの吸光度を測定した。細胞生存率を算出して、抗体のＩＬ－７シグナルの中和活性による殺細胞効果を測定した。結果は、図５Ｃに示される通りであった。 CYG82 cells and IL-7-dependent RAG2 − / − cells were added to each 96-well plate at 2000 or 4000 cells / 100 μL / well and cultured. IL-7R antibody A7R34 was added in a dilution series of 10 −4 to 10 2 μg / ml, and 72 hours later, WST-8 (Dojindo, Cell Counting Kit-8) was added at 10 μL / well and cultured for 3 hours. Later, the absorbance at 450 nm was measured with a microplate reader (Molecular Devices). The cell viability was calculated and the cell killing effect due to the neutralizing activity of IL-7 signal of the antibody was measured. The result was as shown in FIG. 5C.

　図５Ｃに示されるように、抗ＩＬ－７Ｒ抗体（Ａ７Ｒ３４）はＩＬ－７依存性のＲＡＧ２－／－細胞には中和活性による殺細胞効果を認めたが、ＣＹＧ８２細胞には効果がほとんど認められなかった。このことから、ＣＹＧ８２細胞はＩＬ－７を必要としていなことが明らかとなった。ＣＹＧ８２細胞中では、ＩＬ－７Ｒの自己活性化により細胞増殖・生存が保たれており、ＩＬ－７Ｒ抗体（Ａ７Ｒ３４）の中和活性の影響を回避していると考えられる。 As shown in FIG. 5C, the anti-IL-7R antibody (A7R34) showed a cell-killing effect due to neutralizing activity in IL-7-dependent RAG2 − / − cells, but almost no effect in CYG82 cells. I couldn't. This revealed that CYG82 cells do not require IL-7. In CYG82 cells, cell proliferation and survival are maintained by autoactivation of IL-7R, and it is considered that the influence of the neutralizing activity of IL-7R antibody (A7R34) is avoided.

　そこで、ＩＬ－７Ｒ抗体（Ａ７Ｒ３４）の抗体－薬物コンジュゲート（ＡＤＣ，ＳＮ－３８を薬剤として使用）の殺細胞効果をＳＮ－３８のプロドラッグであるＣＰＴ－１１とフリーのＳＮ－３８を対照にして、図５Ｃと同様の方法で試験管内での殺細胞効果を確認した。ＡＤＣは、下記参考文献６～９に従い作製した。 Therefore, the cell-killing effect of IL-7R antibody (A7R34) antibody-drug conjugate (ADC, using SN-38 as a drug) was compared with CPT-11, a prodrug of SN-38, and free SN-38. Thus, the cell killing effect in the test tube was confirmed by the same method as in FIG. 5C. The ADC was made according to the following references 6-9.

Figure JPOXMLDOC01-appb-C000001
Figure JPOXMLDOC01-appb-C000001

　すなわち、リンカーと薬剤は抗体への結合用にマレイミドを使用している。Mal‐PEG12‐OSuはQuanta Biodesign社もしくは、IRIS Biotech GMBH社から購入して極性を高めるために使用した。細胞内で薬剤をリリースさせるための切断用部位としてリンカー部位にカルバメート結合を導入した。ＳＮ－３８はジメチルスルホキシドに１０ｍＭの濃度で溶解して、マイナス８０℃で保存した。ＡＤＣは抗体内のＳＳ結合を還元して、リンカーのマレイミド基と薬剤を反応させることで作製した。抗体１個あたり４～６個の薬剤が結合しているＡＤＣを得ることができた。結果は、図５Ｄに示される通りであった。 That is, the linker and drug use maleimide for binding to the antibody. Mal-PEG 12 -OSu was purchased from Quanta Biodesign or IRIS Biotech GMBH and used to increase polarity. A carbamate bond was introduced into the linker site as a cleavage site for releasing the drug inside the cell. SN-38 was dissolved in dimethyl sulfoxide at a concentration of 10 mM and stored at −80 ° C. The ADC was prepared by reducing the SS bond in the antibody and reacting the maleimide group of the linker with the drug. It was possible to obtain an ADC in which 4 to 6 drugs were bound per antibody. The result was as shown in FIG. 5D.

　図５Ｄに示されるように、Ａ７Ｒ３４－ＡＤＣ（ＳＮ－３８）はＣＰＴ－１１の１００倍効果が強く、フリーのＳＮ－３８と同様の殺細胞効果を示した。このことから、ＩＬ－７に依存せずに生存するＣＹＧ８２に対してはＡ７Ｒ３４－ＡＤＣが効果的であることが示された。
　このことは、ＩＬ－７Ｒは、ＩＬ－７シグナル非存在下であっても恒常的にインターナライズすることを示す。
　また、抗ＩＬ－７Ｒ抗体（Ａ７Ｒ３４）は、ＩＬ－７とＩＬ－７Ｒとの中和活性は細胞生存率や腫瘍体積の抑制効果に大きな影響を与えなかった。抗ＩＬ－７Ｒ抗体と細胞傷害剤とのＡＤＣにより顕著な抗腫瘍効果が奏された。 As shown in FIG. 5D, A7R34-ADC (SN-38) was 100 times more potent than CPT-11 and showed a cell killing effect similar to free SN-38. This indicates that A7R34-ADC is effective against CYG82 that survives independent of IL-7.
This indicates that IL-7R constitutively internalizes even in the absence of IL-7 signal.
In addition, with the anti-IL-7R antibody (A7R34), the neutralizing activity between IL-7 and IL-7R did not significantly affect the cell viability and tumor volume suppression effects. A remarkable antitumor effect was exhibited by the ADC of the anti-IL-7R antibody and the cytotoxic agent.

実施例６：白血病モデルにおける抗腫瘍効果
　本実施例では、抗ＩＬ－７Ｒ抗体と薬剤とのＡＤＣによる抗腫瘍効果を白血病モデルで確認した。 Example 6: Anti-tumor effect in leukemia model In this example, the anti-tumor effect by ADC of anti-IL-7R antibody and drug was confirmed in leukemia model.

　ＣＹＧ８２親株を雌のヌードマウス（１群５匹）の皮下移植後に腫瘍体積が約５０ｍｍ3となったマウスを白血病モデルマウスとした。ＡＤＣは、実施例５で作製したＡ７Ｒ３４－ＡＤＣ（ＳＮ－３８）を用いた。Ａ７Ｒ３４－ＡＤＣ（ＳＮ－３８）をマウス尾静脈より０，４，８日目にＡＤＣ量として５０ｍｇ／ｋｇ、ＳＮ－３８換算量では０．６ｍｇ／ｋｇを投与した。対照として生理食塩水、５０ｍｇ／ｋｇのＡ７Ｒ３４抗体、５０ｍｇ／ｋｇのアイソタイプ抗体ＡＤＣ（＝コントロール抗体のＡＤＣ）［ＳＮ－３８換算量では０．６ｍｇ／ｋｇ］、１０ｍｇ／ｋｇのデキサメタゾン、または２０ｍｇ／ｋｇのＣＰＴ－１１［ＳＮ－３８換算量では１１．６ｍｇ／ｋｇ］を投与した。２日間隔で腫瘍体積を計測して投与後２６日目まで計測した。同じくＣＹＧ８２のステロイド耐性株を移植後に、腫瘍体積が約５０ｍｍ3となったところで、５０ｍｇ／ｋｇのＡ７Ｒ３４－ＡＤＣ［ＳＮ－３８換算量では０．６ｍｇ／ｋｇ］をマウス尾静脈より０，４，８日目に投与した。対照として生理食塩水、１０ｍｇ／ｋｇのデキサメタゾン、２０ｍｇ／ｋｇのＮＦｋβ阻害剤カフェイン酸フェネチルエステル（ＣＰＡＥ）を投与した。２日間隔で腫瘍体積を計測して投与後１８日目まで計測した。結果は、図６Ａおよび６Ｂに示される通りであった。 A mouse having a tumor volume of about 50 mm 3 after subcutaneous implantation of female nude mice (5 mice per group) with the CYG82 parent strain was used as a leukemia model mouse. As the ADC, A7R34-ADC (SN-38) prepared in Example 5 was used. A7R34-ADC (SN-38) was administered as an ADC amount of 50 mg / kg on days 0, 4 and 8 from the tail vein of the mouse, and 0.6 mg / kg as an SN-38 equivalent amount. As control, physiological saline, 50 mg / kg A7R34 antibody, 50 mg / kg isotype antibody ADC (= ADC of control antibody) [0.6 mg / kg in SN-38 equivalent], 10 mg / kg dexamethasone, or 20 mg / kg kg CPT-11 [SN-38 equivalent 11.6 mg / kg] was administered. Tumor volume was measured at 2-day intervals and measured until day 26 after administration. Similarly, after transplantation of a steroid resistant strain of CYG82, when the tumor volume reached about 50 mm 3 , 50 mg / kg of A7R34-ADC [0.6 mg / kg in terms of SN-38] was transferred to the mouse via the tail vein. Administered on day 8. As a control, physiological saline, 10 mg / kg dexamethasone, and 20 mg / kg NFkβ inhibitor caffeic acid phenethyl ester (CPAE) were administered. Tumor volume was measured at 2-day intervals and measured until day 18 after administration. The results were as shown in FIGS. 6A and 6B.

　図６Ａに示されるように抗ＩＬ－７Ｒ抗体と薬剤とのＡＤＣが最も優れた腫瘍体積増大の抑制効果を示した。一方で、抗ＩＬ－７Ｒ抗体単独では大きな効果は認められなかった。また、図６Ｂに示されるように抗ＩＬ－７Ｒ抗体と薬剤とのＡＤＣは、ステロイド耐性のリンパ性白血病モデルに対しても強い抗腫瘍効果を示した。 As shown in FIG. 6A, ADC of anti-IL-7R antibody and drug showed the most excellent inhibitory effect on tumor volume increase. On the other hand, no significant effect was observed with the anti-IL-7R antibody alone. Further, as shown in FIG. 6B, the ADC of the anti-IL-7R antibody and the drug showed a strong antitumor effect even against a steroid-resistant lymphocytic leukemia model.

実施例７：白血病モデルにおけるリンパ節腫大抑制
　ＣＹＧ８２細胞を雌のヌードマウス（１群５匹）の前足底に移植して、移植後１６日目にリンパ節の体積の平均を計測した。結果は、図７Ａに示される通りであった。図７Ａに示されるように、ＩＬ－７Ｒノックダウンマウスでは、腋窩リンパ節の腫大が著明に抑制された。 Example 7: Lymph node enlargement suppression CYG82 cells in a leukemia model were transplanted into the front sole of female nude mice (5 mice per group), and the average volume of lymph nodes was measured on the 16th day after transplantation. The result was as shown in FIG. 7A. As shown in FIG. 7A, the enlargement of axillary lymph nodes was markedly suppressed in IL-7R knockdown mice.

　次に、ＣＹＧ８２親株を雌のヌードマウス（１群３匹）の皮下移植後に腫瘍が形成されるが、所属リンパ節の腫大を経て腹腔内のリンパ節が腫大する。抗ＩＬ－７Ｒ抗体（Ａ７Ｒ３４）のＡＤＣに、リンパ節腫大抑制効果があるかどうかを評価した。図５同様に、Ａ７Ｒ３４－ＡＤＣ（ＳＮ－３８）をマウス尾静脈より０，４，８日目に投与した。対照として生理食塩水、Ａ７Ｒ３４抗体、アイソタイプ抗体ＡＤＣ（ＳＮ－３８）、デキサメタゾン、およびＣＰＴ－１１のいずれかを投与した。投与後１６日目にリンパ節の体積を測定した。結果は図７Ｂに示される通りであった。図７Ｂに示されるように、Ａ７Ｒ３４－ＡＤＣ（ＳＮ－３８）が、強いリンパ節腫大の抑制効果を認めた。Ａ７Ｒ３４単独療法で治療効果をほとんど認めなかった。 Next, a tumor is formed after subcutaneous implantation of female nude mice (3 mice per group) with the CYG82 parent strain, but the lymph nodes in the abdominal cavity swell after the enlargement of the regional lymph nodes. It was evaluated whether the ADC of the anti-IL-7R antibody (A7R34) has a lymph node enlargement suppressing effect. Similarly to FIG. 5, A7R34-ADC (SN-38) was administered on days 0, 4 and 8 from the mouse tail vein. As a control, physiological saline, A7R34 antibody, isotype antibody ADC (SN-38), dexamethasone, or CPT-11 was administered. On the 16th day after administration, the volume of the lymph nodes was measured. The result was as shown in FIG. 7B. As shown in FIG. 7B, A7R34-ADC (SN-38) showed a strong lymph node enlargement suppressing effect. Almost no therapeutic effect was observed with A7R34 monotherapy.

実施例８：放射線同位体ラベルされた抗ＩＬ－７Ｒ抗体
　本実施例では、ＡＤＣの代わりに、放射線同位体ラベルされた抗ＩＬ－７Ｒ抗体を投与して抗体の効果を確認した。 Example 8: Radioisotope-labeled anti-IL-7R antibody In this example, instead of ADC, a radioisotope-labeled anti-IL-7R antibody was administered to confirm the effect of the antibody.

　90Ｙ標識抗体の作製は下記参考文献１０に従い行った。１Ｍ酢酸緩衝液（ｐＨ６．０）と90ＹＣｌ3を１：１で混合し、室温で５分静置した。その後、市販の抗ＩＬ－７Ｒ抗体（クローン４０１３１，　Ｒ＆Ｄ社）を加えて室温で３０分静置した。反応終了後、Ｓｅｐｈａｄｅｘ　Ｇ５０（ＧＥヘルスケア社製）カラム（０．１Ｍ酢酸緩衝液（ｐＨ６．０）で膨潤）を用いて精製した。さらにその後、未標識の抗ヒトＩＬ－７Ｒ抗体換算で投与量を２０μｇになるように調整した。90Ｙ標識抗ＩＬ－７Ｒ抗体を、ＳｕｐＴ１およびＨ２００９のいずれかを皮下移植したヌードマウス尾静脈に投与し薬効を確認した。90Ｙ標識－抗ＩＬ－７Ｒ抗体２０μｇを単回投与した。結果は、図８ＡおよびＢに示される通りであった。 The preparation of 90 Y-labeled antibody was performed according to Reference Document 10 below. 1M acetate buffer (pH 6.0) and 90 YCl 3 were mixed at 1: 1 and allowed to stand at room temperature for 5 minutes. Thereafter, a commercially available anti-IL-7R antibody (clone 40131, R & D) was added and allowed to stand at room temperature for 30 minutes. After completion of the reaction, purification was performed using a Sephadex G50 (GE Healthcare) column (swelled with 0.1 M acetate buffer (pH 6.0)). Thereafter, the dose was adjusted to 20 μg in terms of unlabeled anti-human IL-7R antibody. 90 Y-labeled anti-IL-7R antibody was administered to the tail vein of nude mice transplanted subcutaneously with either SupT1 or H2009 to confirm the drug efficacy. A single dose of 90 μg of 90 Y-labeled anti-IL-7R antibody was administered. The results were as shown in FIGS. 8A and B.

　図８Ａおよび８Ｂに示され得ように、１００μＣｉで90Ｙ標識抗ＩＬ－７Ｒ抗体が抗腫瘍効果を示すことが明らかとなった。このことから、抗ＩＬ－７Ｒ抗体はラジオイムノセラピーに用い得ることが明らかとなった。 As can be seen in FIGS. 8A and 8B, it was revealed that at 90 μCi, 90 Y-labeled anti-IL-7R antibody exhibits an anti-tumor effect. This revealed that the anti-IL-7R antibody can be used for radioimmunotherapy.

実施例９：免疫応答に対するＡＤＣの効果
　上記実施例では、腫瘍に対するＡＤＣの効果を確認した。本実施例では、ＡＤＣを免疫反応の異常の治療に用い得るかを検討した。 Example 9: Effect of ADC on immune response In the above example, the effect of ADC on tumor was confirmed. In this example, it was examined whether ADC could be used for treatment of abnormal immune response.

　健常の雌ＢＡＬＢ／ｃマウスに、生理食塩水、５０ｍｇ／ｋｇのＡ７Ｒ３４－ＡＤＣ（ＳＮ－３８）［ＳＮ－３８換算量では０．６ｍｇ／ｋｇ］、１０ｍｇ／ｋｇデキサメタゾン、または４０ｍｇ／ｋｇデキサメタゾンをそれぞれマウスの尾静脈から投与した。 Healthy female BALB / c mice receive saline, 50 mg / kg A7R34-ADC (SN-38) [0.6 mg / kg in SN-38 equivalent], 10 mg / kg dexamethasone, or 40 mg / kg dexamethasone. Each was administered from the tail vein of mice.

　Ａ７Ｒ３４－ＡＤＣでは、ＩＬ－７Ｒ陽性のＴ細胞およびＢ細胞は、胸腺、骨髄、脾臓のいずれにおいても１／１０以下に減少していた。一方デキサメタゾン投与では、胸腺のＩＬ－７Ｒ陽性Ｔ細胞を含むＴ細胞が全般的に減少していたが、逆に骨髄のＩＬ－７Ｒ陽性Ｂ細胞は２倍以上に増加していた。さらに、脾臓では全細胞数の減少にもかかわらず、ＩＬ－７Ｒ陽性のＴ細胞の比率は増加しており、生理的にもＩＬ－７ＲはＴ細胞およびＢ細胞のステロイド耐性に関与していることが示された。また、このＡ７Ｒ３４－ＡＤＣとステロイド（デキサメタゾン）との薬効性の違いは、両者が異なる機序で免疫細胞の制御ができることを示している。特に、ステロイド耐性のＩＬ－７Ｒ陽性細胞の制御にＡ７Ｒ３４－ＡＤＣが有効と考えられる。 In A7R34-ADC, IL-7R positive T cells and B cells were reduced to 1/10 or less in any of the thymus, bone marrow, and spleen. On the other hand, with dexamethasone administration, T cells including IL-7R positive T cells in the thymus were generally decreased, but conversely, IL-7R positive B cells in the bone marrow increased more than twice. Furthermore, in the spleen, despite the decrease in the total number of cells, the ratio of IL-7R positive T cells is increased, and physiologically IL-7R is involved in steroid resistance of T cells and B cells. It was shown that. The difference in drug efficacy between A7R34-ADC and steroid (dexamethasone) indicates that both can control immune cells by different mechanisms. In particular, A7R34-ADC is considered effective in controlling steroid-resistant IL-7R positive cells.

実施例１０：免疫疾患におけるＩＬ－７Ｒ陽性細胞の増加
　本実施例では、実施例４と同様にして臨床サンプルにおけるＩＬ－７Ｒ遺伝子発現を調べた。 Example 10: Increase in IL-7R positive cells in immune diseases In this example, IL-7R gene expression in clinical samples was examined in the same manner as in Example 4.

　Gene Expression Omnibus (GEO)からGSE55235とGSE14580の生データを入手した。データは解析ソフトQlucore Omics Explorer（Filgen）を用いて解析を行い、P<0.05以下を判定の基準とした。結果は図１０に示される通りであった。 Raw data of GSE55235 and GSE14580 were obtained from Gene Expression Omnibus (GEO). Data was analyzed using analysis software Qlucore Omics Explorer (Filgen), and P <0.05 or less was used as a criterion for judgment. The result was as shown in FIG.

　図１０に示されるように、慢性関節リウマチ（ＲＡ）患者において、ＩＬ－７Ｒの遺伝子発現が健常者よりも高い傾向が確認された。また、潰瘍性大腸炎（ＵＡ）患者においても、、ＩＬ－７Ｒの遺伝子発現が健常者よりも高い傾向が確認され、特に抗ＴＮＦ両方が奏功しなかった患者においてＩＬ－７Ｒ遺伝子の発現は亢進していた。 As shown in FIG. 10, it was confirmed that IL-7R gene expression tended to be higher in normal rheumatoid arthritis (RA) patients than in healthy subjects. In addition, in patients with ulcerative colitis (UA), IL-7R gene expression tended to be higher than that in healthy subjects, and in particular, IL-7R gene expression was enhanced in patients who did not succeed in both anti-TNF. Was.

実施例１１：抗ＩＬ－７Ｒ抗体を用いた関節リウマチモデルの治療効果
　関節リウマチ炎モデルに対して、抗ＩＬ－７Ｒ抗体と薬剤とのコンジュゲートを投与してその効果を調べた。 Example 11: Therapeutic effect of rheumatoid arthritis model using anti-IL-7R antibody The anti-IL-7R antibody-drug conjugate was administered to a rheumatoid arthritis model, and the effect was examined.

　抗ＩＬ－７Ｒ抗体と薬剤とのコンジュゲートとしては、Ａ７Ｒ３４モノクローナル抗体とモノメチルオーリスタチンＥ（ＭＭＡＥ）とをＶａｌ－Ｃｉｔ結合（バリン－シトルリンジペプチド）によって連結したＡ７Ｒ３４－ＡＤＣ（ＭＭＡＥ）を用いた。Ｖａｌ－Ｃｉｔ結合とＭＭＡＥとは、ｐ－アミノベンジルカルバメート（ＰＡＢＣ）により連結した。ＭＭＡＥはＳＮ－３８より細胞障害活性が強いことが知られている。
　Ａ７Ｒ３４－ＡＤＣ（ＭＭＡＥ）は、実施例５と同じように以下参考文献６～９の記載の通り作製した。Ａ７Ｒ３４－ＡＤＣ（ＭＭＡＥ）の化学構造式は以下の通りであった。 As a conjugate of the anti-IL-7R antibody and the drug, A7R34-ADC (MMAE) in which A7R34 monoclonal antibody and monomethyl auristatin E (MMAE) are linked by Val-Cit bond (valine-citrulline dipeptide) was used. The Val-Cit bond and MMAE were linked by p-aminobenzylcarbamate (PABC). MMAE is known to have stronger cytotoxic activity than SN-38.
A7R34-ADC (MMAE) was prepared as described in References 6 to 9 as in Example 5. The chemical structural formula of A7R34-ADC (MMAE) was as follows.

Figure JPOXMLDOC01-appb-C000002
Figure JPOXMLDOC01-appb-C000002

　関節リウマチ炎モデルは、下記参考文献７に記載の方法により作製した。すなわち、メスのＤＢＡ／１Ｊマウスに２ｍｇの抗コラーゲン４抗体（Chondrex）を腹腔内に投与した（０日目）。３日目に５０μｇのＬＰＳ（Chondrex）を腹腔内に投与した。
　得られたマウスに対して、５日目に、５０ｍｇ／ｋｇのＡ７Ｒ３４－ＡＤＣ（ＭＭＡＥ）［ＭＭＡＥ換算量では１ｍｇ／ｋｇ］または５０ｍｇ／ｋｇのＡ７Ｒ３４－ＡＤＣ（ＳＮ－３８）［ＳＮ－３８換算量では０．６ｍｇ／ｋｇ］を投与した。対照としては、生理食塩水、１ｍｇ／ｋｇＭＭＡＥ、５０ｍｇ／ｋｇのアイソタイプコントロール抗体ＡＤＣ（ＭＭＡＥ）［ＭＭＡＥ換算量では１ｍｇ／ｋｇ］、１０ｍｇ／ｋｇのデキサメタゾン、または５０ｍｇ／ｋｇの抗ＴＮＦ製剤エタネルセプトをＡＤＣの代わりに投与した。５０ｍｇ／ｋｇのＡ７Ｒ３４－ＡＤＣ（ＳＮ－３８）［ＳＮ－３８換算量では０．６ｍｇ／ｋｇ］の場合には、対照に生理食塩水、５０ｍｇ／ｋｇの抗ＩＬ－７Ｒ抗体（Ａ７Ｒ３４）、５０ｍｇ／ｋｇ抗ＣＤ１９抗体ＡＤＣ（ＳＮ－３８）［ＳＮ－３８換算量では０．６ｍｇ／ｋｇ］、１０ｍｇ／ｋｇデキサメタゾンを用いた。指示書に従い各個体の関節炎スコア（１６点満点で高いほど重症）を測定することで、関節炎の重症度を評価した。結果は、図１１Ａ～Ｃに示される通りであった。
　本実施例では、関節炎スコアは、各肢の指、甲、および手首の３つの関節を観察することにより求めた。具体的には、各肢の関節炎スコアは以下の基準に基づいて求めた。
各肢の関節炎スコアの評価基準
　スコア０：いずれの関節にも炎症が認められない場合、
　スコア１：いずれかの関節の１つで炎症が認められた場合、
　スコア２：いずれかの関節の２つで炎症が認められた場合、
　スコア３：全ての関節で炎症が認められた場合、
　スコア４：すべての関節で炎症が認められ、肢全体が赤く腫れた場合。
　上記スコアリングに基づいて各肢について関節炎スコアを測定（各肢４点満点）して、四肢について合計（１６点満点）して、個体の関節炎スコアを求めた。 A rheumatoid arthritis model was prepared by the method described in Reference 7 below. That is, 2 mg of anti-collagen 4 antibody (Chondrex) was intraperitoneally administered to female DBA / 1J mice (day 0). On day 3, 50 μg of LPS (Chondrex) was administered intraperitoneally.
On the 5th day, 50 mg / kg of A7R34-ADC (MMAE) [MMAE equivalent 1 mg / kg] or 50 mg / kg of A7R34-ADC (SN-38) [SN-38 equivalent] The dose was 0.6 mg / kg]. As controls, physiological saline, 1 mg / kg MMAE, 50 mg / kg isotype control antibody ADC (MMAE) [1 mg / kg in MMAE equivalent amount], 10 mg / kg dexamethasone, or 50 mg / kg anti-TNF preparation etanercept Was administered instead. In the case of 50 mg / kg A7R34-ADC (SN-38) [0.6 mg / kg in terms of SN-38], physiological saline as a control, 50 mg / kg anti-IL-7R antibody (A7R34), 50 mg / Kg anti-CD19 antibody ADC (SN-38) [0.6 mg / kg in SN-38 equivalent amount], 10 mg / kg dexamethasone was used. The severity of arthritis was evaluated by measuring the arthritis score of each individual according to the instructions (the higher the score is, the more severe it is). The results were as shown in FIGS. 11A-C.
In this example, the arthritis score was determined by observing three joints: fingers, back and wrists of each limb. Specifically, the arthritis score of each limb was determined based on the following criteria.
Evaluation criteria score of arthritis score of each limb 0: When there is no inflammation in any joint,
Score 1: If inflammation is observed in one of the joints,
Score 2: If inflammation is observed in any two of the joints,
Score 3: If inflammation is observed in all joints,
Score 4: All joints are inflamed and the entire limb is swollen red.
Based on the above scoring, the arthritis score was measured for each limb (4 points maximum for each limb), and the limbs were summed (16 points maximum) to determine the arthritis score of the individual.

　図１１Ａに示されるように、Ａ７Ｒ３４－ＡＤＣ（ＭＭＡＥ）は、デキサメタゾン同様に関節リウマチ炎モデルにおける関節炎係数の増悪を顕著に抑制した。より詳細に確認したところ、デキサメタゾン投与群では、後肢と比較して前肢の炎症が著明な個体（炎症のコントロール不良例）が存在した。一方、Ａ７Ｒ３４－ＡＤＣ（ＭＭＡＥ）投与群では、前肢および後肢共に安定して強い抗炎症作用を認めた。この抗炎症作用は、エタネルセプトによる抗ＴＮＦ療法よりも優れていた。また、デキサメタゾンによる炎症のコントロール不良例の関節組織を調べたところ、強い炎症所見が認められ、また、ＩＬ－７Ｒ陽性細胞が多く観察された。
　また、図１１Ｂに示されるように、Ａ７Ｒ３４－ＡＤＣ（ＳＮ－３８）でも、同様の結果であった。また、抗ＩＬ－７Ｒ抗体（Ａ７Ｒ３４抗体）単独では、関節リウマチ炎モデルにおける関節炎係数の増悪の抑制効果が観察されたが、その効果は限定的であった（図１１Ｂ参照）。
　図１１Ｃでは、薬剤投与後９日目のマウス前足病変部の組織切片を作製し、ヘマトキシリン・エオシン染色（ＨＥ染色）して観察した。図１１Ｃに示される通り、ＡＤＣ投与群では、炎症が大幅に改善していることが分かる。 As shown in FIG. 11A, A7R34-ADC (MMAE) remarkably suppressed the exacerbation of the arthritic coefficient in the rheumatoid arthritis model, like dexamethasone. When confirmed in more detail, in the dexamethasone-administered group, there was an individual (an example of poor control of inflammation) in which inflammation in the forelimbs was marked compared to the hindlimbs. On the other hand, in the A7R34-ADC (MMAE) administration group, both the forelimbs and hindlimbs showed stable and strong anti-inflammatory effects. This anti-inflammatory effect was superior to anti-TNF therapy with etanercept. In addition, when examining the joint tissues of patients with poor control of inflammation due to dexamethasone, strong inflammatory findings were observed, and many IL-7R positive cells were observed.
In addition, as shown in FIG. 11B, similar results were obtained with A7R34-ADC (SN-38). In addition, anti-IL-7R antibody (A7R34 antibody) alone was observed to suppress the deterioration of arthritis coefficient in a rheumatoid arthritis model, but the effect was limited (see FIG. 11B).
In FIG. 11C, a tissue section of a mouse forefoot lesion on day 9 after drug administration was prepared and observed with hematoxylin and eosin staining (HE staining). As shown in FIG. 11C, it can be seen that inflammation was significantly improved in the ADC administration group.

　このことから、ＩＬ－７ＲとＩＬ－７との結合を中和することでは効果が弱く、抗ＩＬ－７Ｒ抗体と細胞傷害剤とのＡＤＣにおいて強い効果が確認された。 Therefore, neutralizing the binding between IL-7R and IL-7 was weak in effect, and a strong effect was confirmed in ADC of anti-IL-7R antibody and cytotoxic agent.

実施例１２：抗ＩＬ－７Ｒ抗体を用いた潰瘍性大腸炎の治療効果
　本実施例では、潰瘍性大腸炎に対する抗ＩＬ－７Ｒ抗体の効果を調べた。 Example 12: Treatment effect of ulcerative colitis using anti-IL-7R antibody In this example, the effect of anti-IL-7R antibody on ulcerative colitis was examined.

　潰瘍性大腸炎モデルは、下記参考資料１１の通りに作製した。すなわち、メスのＢＡＬＢ／ｃマウスに、５％デキストラン硫酸塩（ＤＳＳ；MP Biomedicals）を飲水させた。ＤＳＳ飲水後２日目から、関節リウマチ炎モデル同様の投与方法・投与量でＡ７Ｒ３４－ＡＤＣ（ＭＭＡＥ）またはＡ７Ｒ３４－ＡＤＣ（ＳＮ－３８）を腹腔内に投与した。対照としては、ＡＤＣの代わりに生理食塩水、ＭＭＡＥ、アイソタイプのコントロール抗体ＡＤＣ（ＭＭＡＥ）、デキサメタゾン、または抗ＴＮＦ製剤エタネルセプトを投与した。Ａ７Ｒ３４－ＡＤＣ（ＳＮ－３８）の場合には、対照に生理食塩水、抗ＩＬ－７Ｒ抗体（Ａ７Ｒ３４）、抗ＣＤ１９抗体ＡＤＣ（ＳＮ－３８）、デキサメタゾンを関節リウマチ炎モデル同様に用いた。飲水後８日目の大腸を摘出して全長を測定した。さらに、組織標本を作製してＨＥ染色で病理像を確認した。潰瘍性大腸炎の重症度は体重減少（体重低下が大きいほど重症）、大腸全長（大腸の全長が短くなっているほど重症）と組織学的変化で観察した。体重変化の結果は、図１２ＡおよびＢに示される通りであった。また、大腸全長の組織学的変化の結果は、図１３に示される通りであった。 The ulcerative colitis model was prepared as shown in Reference Material 11 below. That is, female BALB / c mice were allowed to drink 5% dextran sulfate (DSS; MP Biomedicals). From the second day after drinking DSS, A7R34-ADC (MMAE) or A7R34-ADC (SN-38) was intraperitoneally administered in the same manner and dose as in the rheumatoid arthritis model. As a control, physiological saline, MMAE, isotype control antibody ADC (MMAE), dexamethasone, or anti-TNF preparation etanercept was administered instead of ADC. In the case of A7R34-ADC (SN-38), physiological saline, anti-IL-7R antibody (A7R34), anti-CD19 antibody ADC (SN-38), and dexamethasone were used as controls as in the rheumatoid arthritis model. The large intestine on the 8th day after drinking was removed and the total length was measured. Furthermore, a tissue specimen was prepared and a pathological image was confirmed by HE staining. The severity of ulcerative colitis was observed in terms of weight loss (the greater the weight loss, the greater the severity), the entire length of the large intestine (the more severe the length of the large intestine), and histological changes. The results of changes in body weight were as shown in FIGS. 12A and B. Moreover, the result of the histological change of the entire length of the large intestine was as shown in FIG.

　図１２Ａに示されるように、デキサメタゾンはほとんど治療有効性を示さなかったのに対して、Ａ７Ｒ３４－ＡＤＣ（ＭＭＡＥ）は、５日目以降は体重低下が顕著に抑制された。この抑制効果は、エタネルセプト投与群における体重低下の抑制よりも顕著に高かった。ＭＭＡＥやコントロール抗体ＡＤＣでは体重低下の抑制効果はほとんど示されなかった。
　また、図１２Ｂに示されるように、Ａ７Ｒ３４－ＡＤＣ（ＳＮ－３８）も、モデルマウスにおける体重低下が顕著に抑制された。
　さらに図１３に示されるように、潰瘍性大腸炎のネガティブコントロールでは、ＤＳＳ未処置群と比較して全長が大幅に縮小していたが、Ａ７Ｒ３４－ＡＤＣ（ＭＭＡＥ）投与群では、大腸全長がＤＳＳ未処置群と同等レベルに開腹していた。また、Ａ７Ｒ３４－ＡＤＣ（ＳＮ－３８）投与群でも、全長は長くなる傾向が見られた。 As shown in FIG. 12A, dexamethasone showed little therapeutic efficacy, whereas A7R34-ADC (MMAE) significantly suppressed weight loss after the fifth day. This inhibitory effect was significantly higher than the suppression of weight loss in the etanercept administration group. MMAE and control antibody ADC showed almost no inhibitory effect on weight loss.
In addition, as shown in FIG. 12B, A7R34-ADC (SN-38) also remarkably suppressed weight loss in model mice.
Further, as shown in FIG. 13, in the negative control for ulcerative colitis, the total length was significantly reduced compared to the DSS-untreated group, but in the A7R34-ADC (MMAE) administration group, the total length of the large intestine was DSS. The laparotomy was at the same level as the untreated group. In the A7R34-ADC (SN-38) administration group, the total length tended to be long.

　このことから、ＩＬ－７ＲとＩＬ－７との結合を中和することでは効果が弱く、抗ＩＬ－７Ｒ抗体と細胞傷害剤とのＡＤＣにおいて強い効果が確認された。 Therefore, neutralizing the binding between IL-7R and IL-7 was weak in effect, and a strong effect was confirmed in ADC of anti-IL-7R antibody and cytotoxic agent.

　さらに、上記においてＤＳＳ投与６日後の大腸摘出標本の病理組織像を観察した。その結果、図１４に示されるように、生食群では大腸の潰瘍形成と粘膜破壊が著しかった。また、デキサメタゾンやＡ７Ｒ３４単独療法の効果も弱かった。さらに、抗ＴＮＦ製剤エタネルセプトも炎症を抑えているが十分ではなかった。しかし、これらとは対照的に、Ａ７Ｒ３４－ＡＤＣ（ＭＭＡＥ）は、完全に炎症を抑えている。Ａ７Ｒ３４－ＡＤＣ（ＳＮ－３８）も炎症を抑えているが中心に一部潰瘍性病変を認める。抗ＣＤ１９抗体－ＡＤＣ（ＳＮ－３８）は炎症を抑えるどころか、粘膜全体が肉芽腫様に変化していた。関節炎モデル同様に、大腸炎モデルでもＳＮ－３８より細胞障害の強いＭＭＡＥの方が有効であることが判明した。 Furthermore, the histopathological image of the colectomy specimen 6 days after DSS administration was observed in the above. As a result, as shown in FIG. 14, colon ulceration and mucosal destruction were remarkable in the saline group. The effects of dexamethasone and A7R34 monotherapy were also weak. Furthermore, the anti-TNF preparation etanercept also suppressed inflammation but was not sufficient. However, in contrast, A7R34-ADC (MMAE) completely suppresses inflammation. A7R34-ADC (SN-38) also suppresses inflammation, but some ulcerative lesions are observed in the center. The anti-CD19 antibody-ADC (SN-38) did not suppress inflammation, but the entire mucosa changed to granulomas. Similar to the arthritis model, it was found that MMAE, which has stronger cell damage than SN-38, is more effective in the colitis model.

　ＩＬ－７Ｒは、獲得免疫系リンパ球であるＴ細胞およびＢ細胞、および、特に自然免疫系リンパ球に発現する。ＩＬ－７Ｒに対するＡＤＣにより炎症を鎮静化できたことから、当該ＡＤＣは、獲得免疫系リンパ球であるＴ細胞およびＢ細胞、および、特に自然免疫系リンパ球を介した炎症の鎮静化に対して有効であることが示唆された。従って、本発明によれば、自然免疫系リンパ球を介したあらゆる炎症、獲得免疫系の亢進による疾患（例えば、自己免疫疾患や移植片対宿主病等）およびその他上記の疾患または状態に対して、抗ＩＬ－７Ｒ抗体と細胞傷害剤との抗体薬物コンジュゲートが有効であることが示された。 IL-7R is expressed on acquired immune system lymphocytes, T cells and B cells, and in particular on innate immune system lymphocytes. Since the inflammation could be sedated by the ADC against IL-7R, the ADC is effective against the sedation of inflammation via the acquired immune system lymphocytes, T cells and B cells, and in particular the innate immune system lymphocytes. It was suggested to be effective. Therefore, according to the present invention, against all inflammation via innate immune system lymphocytes, diseases caused by enhancement of acquired immune system (for example, autoimmune diseases, graft-versus-host diseases, etc.) and other diseases or conditions described above It has been shown that antibody drug conjugates of anti-IL-7R antibodies and cytotoxic agents are effective.

文献リスト
参考文献１
Ogawa, M., K. Ikuta, Y. Katsura, and S. Nishikawa. 1989. Stepwise progression of
B cell malignancy occurred in a bone marrow stromal cell-dependent pre-B cell clone. Leukemia. 3:282-288.
参考文献２
Sudo, T., S. Nishikawa, N. Ohno, N. Akiyama, M. Tamakoshi, H. Yoshida, and S. Nishikawa. 1993. Expression and function of the interleukin 7 receptor in murine lymphocytes. Proceedings of the National Academy of Sciences of the United States
of America. 90:9125-9129.
参考文献３
Yasunaga, M., F. Wang, T. Kunisada, S. Nishikawa, and S. Nishikawa. 1995. Cell cycle control of c-kit+IL-7R+ B precursor cells by two distinct signals derived from IL-7 receptor and c-kit in a fully defined medium. The Journal of experimental medicine. 182:315-323.
参考文献４
Yasunaga, M., S. Tada, S. Torikai-Nishikawa, Y. Nakano, M. Okada, L.M. Jakt, S. Nishikawa, T. Chiba, T. Era, and S. Nishikawa. 2005. Induction and monitoring of
definitive and visceral endoderm differentiation of mouse ES cells. Nature biotechnology. 23:1542-1550.
参考文献５
Yasunaga, M., and Y. Matsumura. 2014. Role of SLC6A6 in promoting the survival and multidrug resistance of colorectal cancer. Scientific reports. 4:4852.
参考文献６
Yasunaga, M., S. Manabe, D. Tarin, and Y. Matsumura. 2011. Cancer-stroma targeting therapy by cytotoxic immunoconjugate bound to the collagen 4 network in the tumor tissue. Bioconjugate chemistry. 22:1776-1783.
参考文献７
Yasunaga, M., S. Manabe, D. Tarin, and Y. Matsumura. 2013. Tailored immunoconjugate therapy depending on a quantity of tumor stroma. Cancer science. 104:231-237.
参考文献８
Koga, Y., S. Manabe, Y. Aihara, R. Sato, R. Tsumura, H. Iwafuji, F. Furuya, H. Fuchigami, Y. Fujiwara, Y. Hisada, Y. Yamamoto, M. Yasunaga, and Y. Matsumura. 2015. Antitumor effect of antitissue factor antibody-MMAE conjugate in human pancreatic tumor xenografts. International journal of cancer. Journal international du cancer. 137:1457-1466.
参考文献９
Fujiwara, Y., M. Furuta, S. Manabe, Y. Koga, M. Yasunaga, and Y. Matsumura. 2016. Imaging mass spectrometry for the precise design of antibody-drug conjugates. Scientific reports. 6:24954.
参考文献10
Yoshida C, Tsuji AB, Sudo H, Sugyo A, Kikuchi T, Koizumi M, Arano Y, Saga T. Therapeutic efficacy of c-kit-targeted radioimmunotherapy using 90Y-labeled anti-c-kit antibodies in a mouse model of small cell lung cancer. PLoS One. 2013;8(3):e59248.
参考文献11
Chassaing, B., J.D. Aitken, M. Malleshappa, and M. Vijay-Kumar. 2014. Dextran sulfate sodium (DSS)-induced colitis in mice. Current protocols in immunology. 104:Unit 15 25.
参考文献12
van Meeteren, M.E., M.A. Meijssen, and F.J. Zijlstra. 2000. The effect of dexamethasone treatment on murine colitis. Scandinavian journal of gastroenterology. 35:517-521. Reference list Reference 1
Ogawa, M., K. Ikuta, Y. Katsura, and S. Nishikawa. 1989. Stepwise progression of
B cell malignancy occurred in a bone marrow stromal cell-dependent pre-B cell clone. Leukemia. 3: 282-288.
Reference 2
Sudo, T., S. Nishikawa, N. Ohno, N. Akiyama, M. Tamakoshi, H. Yoshida, and S. Nishikawa. 1993. Expression and function of the interleukin 7 receptor in murine lymphocytes. Proceedings of the National Academy of Sciences of the United States
of America. 90: 9125-9129.
Reference 3
Yasunaga, M., F. Wang, T. Kunisada, S. Nishikawa, and S. Nishikawa. 1995. Cell cycle control of c-kit + IL-7R + B precursor cells by two distinct signals derived from IL-7 receptor and c -kit in a fully defined medium.The Journal of experimental medicine.182: 315-323.
Reference 4
Yasunaga, M., S. Tada, S. Torikai-Nishikawa, Y. Nakano, M. Okada, LM Jakt, S. Nishikawa, T. Chiba, T. Era, and S. Nishikawa. 2005. Induction and monitoring of
definitive and visceral endoderm differentiation of mouse ES cells.Nature biotechnology.23: 1542-1550.
Reference 5
Yasunaga, M., and Y. Matsumura. 2014. Role of SLC6A6 in promoting the survival and multidrug resistance of colorectal cancer. Scientific reports. 4: 4852.
Reference 6
Yasunaga, M., S. Manabe, D. Tarin, and Y. Matsumura. 2011. Cancer-stroma targeting therapy by cytotoxic immunoconjugate bound to the collagen 4 network in the tumor tissue. Bioconjugate chemistry. 22: 1776-1783.
Reference 7
Yasunaga, M., S. Manabe, D. Tarin, and Y. Matsumura. 2013. Tailored immunoconjugate therapy depending on a quantity of tumor stroma.Cancer science. 104: 231-237.
Reference 8
Koga, Y., S. Manabe, Y. Aihara, R. Sato, R. Tsumura, H. Iwafuji, F. Furuya, H. Fuchigami, Y. Fujiwara, Y. Hisada, Y. Yamamoto, M. Yasunaga, and Y. Matsumura. 2015. Antitumor effect of antitissue factor antibody-MMAE conjugate in human pancreatic tumor xenografts. International journal of cancer. Journal international du cancer. 137: 1457-1466.
Reference 9
Fujiwara, Y., M. Furuta, S. Manabe, Y. Koga, M. Yasunaga, and Y. Matsumura. 2016. Imaging mass spectrometry for the precise design of antibody-drug conjugates. Scientific reports. 6: 24954.
Reference 10
Yoshida C, Tsuji AB, Sudo H, Sugyo A, Kikuchi T, Koizumi M, Arano Y, Saga T. Therapeutic efficacy of c-kit-targeted radioimmunotherapy using 90Y-labeled anti-c-kit antibodies in a mouse model of small cell lung cancer.PLoS One. 2013; 8 (3): e59248.
Reference 11
Chassaing, B., JD Aitken, M. Malleshappa, and M. Vijay-Kumar. 2014. Dextran sulfate sodium (DSS) -induced colitis in mice.Current protocols in immunology. 104: Unit 15 25.
Reference 12
van Meeteren, ME, MA Meijssen, and FJ Zijlstra. 2000.The effect of dexamethasone treatment on murine colitis.Scandinavian journal of gastroenterology. 35: 517-521.

Claims (14)

　抗ＩＬ－７Ｒ抗体またはその抗原結合性断片と細胞傷害剤との抗体薬物コンジュゲートを含む医薬組成物であって、がんまたは炎症を処置することに用いるための医薬組成物。 A pharmaceutical composition comprising an antibody drug conjugate of an anti-IL-7R antibody or an antigen-binding fragment thereof and a cytotoxic agent, for use in treating cancer or inflammation. 　炎症を処置することに用いるための、請求項１に記載の医薬組成物。 The pharmaceutical composition according to claim 1, for use in treating inflammation. 　炎症が、ステロイド抵抗性の炎症である、請求項１または２に記載の医薬組成物。 The pharmaceutical composition according to claim 1 or 2, wherein the inflammation is steroid resistant inflammation. 　炎症が、潰瘍性大腸炎または関節リウマチである、請求項２または３に記載の医薬組成物。 The pharmaceutical composition according to claim 2 or 3, wherein the inflammation is ulcerative colitis or rheumatoid arthritis. 　がんを処置することに用いるための請求項１に記載の医薬組成物。 The pharmaceutical composition according to claim 1 for use in treating cancer. 　がんが、白血病、リンパ腫、または転移性のがんである、請求項１または５に記載の医薬組成物。 The pharmaceutical composition according to claim 1 or 5, wherein the cancer is leukemia, lymphoma, or metastatic cancer. 　がんが、ステロイド耐性のがんである、請求項１、５または６に記載の医薬組成物。 The pharmaceutical composition according to claim 1, 5 or 6, wherein the cancer is a steroid-resistant cancer. 　がんが、転移性のがんである、請求項１および５～７のいずれか一項に記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 and 5 to 7, wherein the cancer is metastatic cancer. 　がんが、ＩＬ－７ＲとＩＬ－７との結合を中和する治療に対して抵抗性である、請求項１および５～８のいずれか一項に記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 and 5 to 8, wherein the cancer is resistant to a treatment that neutralizes the binding between IL-7R and IL-7. 　抗ＩＬ－７Ｒ抗体またはその抗原結合性断片と細胞傷害剤との抗体薬物コンジュゲートを含む医薬組成物であって、ステロイド耐性の疾患または状態を処置することに用いるための医薬組成物。 A pharmaceutical composition comprising an antibody drug conjugate of an anti-IL-7R antibody or antigen-binding fragment thereof and a cytotoxic agent, for use in treating a steroid-resistant disease or condition. 　抗ＩＬ－７Ｒ抗体またはその抗原結合性断片と細胞傷害剤との抗体薬物コンジュゲートを含む医薬組成物であって、ステロイドと併用される、医薬組成物。 A pharmaceutical composition comprising an antibody drug conjugate of an anti-IL-7R antibody or an antigen-binding fragment thereof and a cytotoxic agent, which is used in combination with a steroid. 　がんを処置することに用いるための、請求項１１に記載の医薬組成物。 The pharmaceutical composition according to claim 11 for use in treating cancer. 　細胞傷害剤が、放射性同位体、化学療法剤、または毒素である、請求項１～１２のいずれか一項に記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 to 12, wherein the cytotoxic agent is a radioisotope, a chemotherapeutic agent, or a toxin. 　抗ＩＬ－７Ｒ抗体またはその抗原結合性断片とモノメチルオーリスタチンＥとの抗体薬物コンジュゲート。 Antibody drug conjugate of anti-IL-7R antibody or antigen-binding fragment thereof and monomethyl auristatin E
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025089399A1 (en) * 2023-10-26 2025-05-01 国立研究開発法人国立がん研究センター Antibody against interleukin-7 receptor (il-7r), and antibody-drug conjugate (adc) including said antibody

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009102367A (en) * 2001-04-06 2009-05-14 Univ Of Bristol Use of CD25 binding molecules in steroid resistant patients
JP2012024095A (en) * 2004-08-10 2012-02-09 Cardiff Biologicals Ltd Method and kit for prognosis of breast cancer
WO2012057315A1 (en) * 2010-10-29 2012-05-03 株式会社ペルセウスプロテオミクス High-affinity anti-cdh3 antibody
JP2014012657A (en) * 2007-03-20 2014-01-23 Clavis Pharma Asa Fatty acid esters of glucocorticoids as anti-inflammatory and anti-cancer agents

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009102367A (en) * 2001-04-06 2009-05-14 Univ Of Bristol Use of CD25 binding molecules in steroid resistant patients
JP2012024095A (en) * 2004-08-10 2012-02-09 Cardiff Biologicals Ltd Method and kit for prognosis of breast cancer
JP2014012657A (en) * 2007-03-20 2014-01-23 Clavis Pharma Asa Fatty acid esters of glucocorticoids as anti-inflammatory and anti-cancer agents
WO2012057315A1 (en) * 2010-10-29 2012-05-03 株式会社ペルセウスプロテオミクス High-affinity anti-cdh3 antibody

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
COSENZA, L. ET AL.: "Inter leukin-7 receptor expression and activation in nonhaematopoietic neoplastic cell lines", CELLULAR SIGNALLING, vol. 14, 2002, pages 317 - 325, XP055538436, ISSN: 0898-6568 *
YAMAZAKI, M. ET AL.: "Mucosal T Cells Expressing High Level of IL -7 Receptor Are Potential Targets for Treatment Chronic Coli tis", THE JOURNAL OF IMMUNOLOGY, vol. 171, 2013, pages 1556 - 1563, XP002560032, ISSN: 0022-1767 *
YASUNAGA, MASAHIRO: "Development on anti- IL -7 receptor antibody, SN-38 complex for targeting acute lymphocytic leukemia", COMPREHENSIVE RESEARCH REPORT OF CANCER RESEARCH AND DEVELOPMENT COST, 2014, pages 1 - 5 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025089399A1 (en) * 2023-10-26 2025-05-01 国立研究開発法人国立がん研究センター Antibody against interleukin-7 receptor (il-7r), and antibody-drug conjugate (adc) including said antibody

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