TW201834698A - 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

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

Anti-IL-7R antibody antibody drug conjugate and pharmaceutical composition containing anti-IL-7R antibody and cytotoxic agent antibody drug conjugate for treating cancer or inflammation

Pharmaceutical composition of the present invention with an anti-IL-7R antibody (anti-IL-7R receptor antibody) and an antibody-drug conjugate for treating cancer or inflammation containing an anti-IL-7R antibody and a cytotoxic agent Related.

The inhibitory effect of the topoisomerase 1 inhibitor SN-38 is expected to have an anticancer effect, but it is poorly soluble, and there is room for improvement from the viewpoint of blood mobility. Developed as a compound that improves blood mobility in SK-38, CPT-11. CPT-11 is a pre-SN-38 predrug. It has high conversion efficiency to active form in cancer tissues of solid tumors, but has low activity in blood and low activity in blood. Therefore, CPT-11 cannot be used in the treatment of leukemia.

IL-7 is known to be a cytokine necessary for the production of T cells and the maintenance of a constant state (Non-Patent Document 1). In addition, IL-7α slowly moves into the cell, and although it partially returns to the cell surface, it is partially decomposed (Non-Patent Document 1). The half-life of IL-7α is shortened from about 24 hours to 3 hours by IL-7 stimulation (Non-Patent Document 1). In addition, IL-7R was found to be the cause of steroid tolerance in cancer (Non-Patent Document 2). Non-patent document 2 suggests that suppressing the downstream signals of IL-7R is effective for treating steroid tolerance. [Prior Art Literature] [Non-Patent Literature]

[Non-Patent Document 1] Li et al., 2016, PLOS Medicine, 13 (12): e1002200 [Non-Patent Document 2] Henriques et al., 2010, Blood, 115 (16): 3269-3277

The present invention provides a pharmaceutical composition of an antibody-drug conjugate of an anti-IL-7R antibody and an antibody-drug conjugate containing an anti-IL-7R antibody and a cytotoxic agent for treating cancer or inflammation.

The inventors found that IL-7R is related to steroid tolerance and that the elimination of IL-7R inhibits the cell growth of cancer cell lines. The inventor group also found that the elimination of IL-7R makes the steroid sensitivity of cancer cells (especially lymphomas and leukemias) sharply improved, which can improve the cell killing effect of steroids and the IL-7R Performance will improve. The inventors group further found that in steroid tolerance, activation of STAT and BCL2 downstream signals of IL-7R was not found, and activation of NFκB occurred. The inventors have further discovered that the performance of IL-7R improves after chemotherapy treatment with steroid-based anti-inflammatory agents, and that IL-7R in cell lines treated with steroid-based anti-inflammatory drugs and obtained steroid resistance Improved performance. The inventors have further discovered that the anti-IL-7R antibody will accumulate in the lysate once it is taken into the cells, and will accumulate in the tumor tissue and lymph nodes after one week of administration. In addition, it was found that antibodies that block the binding of IL-7R and IL-7 have a weak effect on cancer cell survival function, but on the other hand, ADCs with anti-IL-7R antibodies showed significant effects and inhibited lymphadenopathy. It was found that the effect of ADC on tumors was similar in radioisotope-labeled anti-IL-7R antibodies. The inventors group has further discovered that the symptoms of inflamed patients (such as rheumatoid arthritis or ulcerative colitis) are reduced by reducing IL-7R positive cells of the immune system. The present invention is based on these discoverers.

That is, the following invention is provided by this invention. (1) A pharmaceutical composition, which is a pharmaceutical composition comprising an antibody-drug conjugate of an anti-IL-7R antibody or an antigen-binding fragment thereof and a cytotoxic agent, and is used for treating cancer or inflammation. (2) The pharmaceutical composition according to (1), which is used for treating inflammation. (3) The pharmaceutical composition according to (1) or (2), wherein the inflammation is inflammation after steroid treatment or steroid resistance. (4) The pharmaceutical composition according to (2) or (3), wherein the inflammation is ulcerative colitis or rheumatoid arthritis. (5) The pharmaceutical composition according to (1), which is used for treating cancer. (6) The pharmaceutical composition according to (1) or (5), wherein the cancer is leukemia, lymphoma, or metastatic cancer. (7) The pharmaceutical composition according to (1), (5) or (6), wherein the cancer is a steroid-treated cancer or a steroid-resistant cancer. (8) The pharmaceutical composition according to any one of (1) and (5) to (7), wherein the cancer is a metastatic cancer. (9) The pharmaceutical composition according to any one of (1) and (5) to (8), wherein the cancer is resistant to a treatment that neutralizes the combination of IL-7R and IL-7. (10) A pharmaceutical composition, which is a pharmaceutical composition comprising an anti-IL-7R antibody or an antigen-binding fragment thereof and an antibody-drug conjugate of a cytotoxic agent, and is a steroid-treated disease or a steroid-resistant disease or Status. (11) A pharmaceutical composition, which is a pharmaceutical composition comprising an anti-IL-7R antibody or an antigen-binding fragment thereof and an antibody-drug conjugate of a cytotoxic agent, and is used in combination with a steroid. (12) The pharmaceutical composition according to (11), which is used for treating cancer. (13) The pharmaceutical composition according to any one of (1) to (12), wherein the cytotoxic agent is a radioisotope, a chemotherapeutic agent, or a toxin. (14) An antibody drug conjugate, which is an antibody drug conjugate of an anti-IL-7R antibody or an antigen-binding fragment thereof and monomethyl orstatin E.

In the present invention, the "subject" means a mammal, and particularly a human.

In this specification, the use of the term "treatment" includes the meaning of treatment (therapeutic treatment) and prevention (prophylactic treatment). In the present specification, "treatment" means improvement in the treatment, cure, prevention, or alleviation of a disease or disorder, or a decrease in the rate of progression of a disease or disorder. In the present specification, "prevention" means reducing the possibility of the onset of a disease or a morbid state, or delaying the onset of a disease or a morbid state.

In this specification, "disease" means treating symptoms that are beneficial.

In the present specification, the "therapeutically effective amount" means a dose effective for the treatment (prevention or treatment) of a disease or condition. A therapeutically effective amount of a medicament can reduce the rate of deterioration of the symptoms of a disease or condition, prevent the deterioration of the aforementioned symptoms, improve the aforementioned symptoms, cure the aforementioned symptoms, or suppress the onset or progression of the aforementioned symptoms.

In the present specification, the term "resistance" means that it is not effective for treatment (for example, it does not exhibit significant effectiveness). In the present specification, "sensitivity" means the effectiveness of treatment performance. Decreasing resistance has the same meaning as increasing sensitivity, and increasing resistance has the same meaning as decreasing sensitivity. For example, the so-called steroid resistance in this specification can be defined as that in the case of renal disorders, there is no remission at all even if the adrenal corticosterone is administered daily for 4 weeks or more. In addition, other malignant diseases such as inflammation, autoimmune disease, and leukemia and malignant lymphoma in this manual can be regarded as various kinds even if administered to adrenocorticone, methyl adrenocorticone, dexamethasone, and betamethasone The symptoms of therapeutic steroids have not improved or worsened. Doctors can understand and grasp the resistance based on the knowledge in the field of steroid treatment, and make appropriate decisions on whether the treatment subject is resistant.

In the present specification, "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 phosphorylated receptors and undergoes dimerization via JAK phosphorylation. It is believed that gene transcription is activated via dimerized STAT. The IL-7R molecule itself has high internalization activity, which will be internalized on the membrane and be decomposed or recirculated on the membrane.

As used herein, "blocking" means neutralizing the binding of two proteins. Blocking includes partial dissociation in addition to complete dissociation. For example, reducing the binding of two proteins by more than 50% can also be referred to as blocking.

As used herein, the term "steroids" means steroidal anti-inflammatory drugs (SAIDs). The active ingredients in steroids are mainly glucocorticoids or their derivatives. When steroids bind to the glucocorticoid receptor (GRa) in the cell, GRa dissociates from Hsp90, forms a dimer, moves within the nucleus, and regulates transcription. Examples of synthetic steroids include cortisone such as hydrocortisone and hydrocortisone succinate; corticosterone such as corticosterone, methyl adrenal cortexone, and methyl adrenal cortexone succinate; anti-inflammatory Pine and triamcinolone; dexamethasone and betamethasone. All of them are derivatives of glucocorticoids, which function in cells by the same mechanism as glucocorticoids.

In the present specification, the "antibody drug conjugate" (ADC) means a substance to which a single antibody or an antigen-binding fragment thereof (hereinafter sometimes referred to as "antibody", etc.) is linked to a drug. In ADCs, monoclonal antibodies and the like can be linked to drugs through appropriate linkers. ADC binds to membrane components on the cell membrane (such as transmembrane proteins such as receptors), is taken into cells via endocytosis and internalization, and is separated from antibodies and released into cells. By introducing a cleavage linker between the antibody and the drug into the cell in advance, the linker can be broken in the cell, for example, intracellularly, and the drug can be separated from the antibody and the like and released into the cytoplasm. The use of cytotoxic agents as drugs can cause the cells to which the drugs are delivered to die. As cytotoxic agents, chemotherapeutics, radioisotopes and toxins can be used.

With the present invention, it was found that the expression of IL-7R is improved in most tumor cells. By the present invention, tumor growth is significantly suppressed by reducing the performance of the enhanced IL-7R. IL-7R ADC shows strong killing ability to these cells. On the other hand, antibodies that block the binding of IL-7R to IL-7 have little effect on cell survival. Therefore, as long as the anti-IL-7R antibody used by the ADC of the present invention binds to the IL-7R on the cell membrane, the blocking ability of the IL-7R and IL-7 binding is not limited, and it can block IL. The antibody that binds -7R to IL-7 may also be an antibody that does not block the binding between IL-7R and IL-7.

Therefore, the present invention can provide an antibody-drug conjugate of an anti-IL-7R antibody or an antigen-binding fragment thereof and a cytotoxic agent. In the antibody-drug conjugate of the present invention, an antibody that recognizes IL-7R expressed on a cell membrane can be used as an anti-IL-7R antibody. Furthermore, in one aspect of the present invention, the antibody or the antigen-binding fragment thereof in the antibody-drug conjugate is linked to the cytotoxic agent 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), and monomethylol). Rostatin E, monomethyl orlistatin F and its derivatives), maytansinoids DM1 and DM4 and its derivatives), camptothecin (SN-38, topotecan and etheticon and its derivatives), DNA Small groove binders (enediyne, lexitoxin, diamycin and its derivatives), taxanes (paclitaxel and docetaxel and its derivatives), polyketene (round sponge sponge lactone and its derivatives Substances), anthraquinones (mitoxantrone and its derivatives), benzodiazepines (pyrrolobenzodiazepines, indoline benzodiazepines, and oxazoridinobenzodiazepines and their derivatives) Substances), vinca alkaloids (vincristine, vinblastine, vinblastine and vinorelbine and their derivatives), doxorubicin (adriamycin, morpholino-adriamycin and cyanomorpholino- Adriamycin and its derivatives), cardiac glycosides (digitoxin or its derivatives), calicheamicin, epothilone, candida, simadotin, simadotin, rhizomycin , Hammer streptozotocin, combretastatin, garnet Cello element Ai, etoposide, T67 (Tularik), and nocodazole), a radioisotope (e.g., ^{32 P, 60 C, 90 Y} , 111 In, 131 I, 125 I ¹⁵³ Sm, ¹⁸⁶ Re, ¹⁸⁸ Re, and ²¹² Bi) and toxins (such as diphtheria toxin A, Pseudomonas endotoxin, ricin, saponin, etc.) can be used as the ADC of the present invention Cell damage agent. Any cytotoxic agent can be used to treat cancer patients.

In a certain aspect of the present invention, as long as a person skilled in the art can appropriately select and synthesize a linker when making an ADC. In a certain aspect of the present invention, the linker may be a broken linker. Examples of cleavage linkers include dipeptide linkers such as valine-citrulline (Val-Cit) and phenylalanine-lysine (Phe-lys) linkers, or cleavage in a pH-dependent manner Zhilian linker. Examples of the cleavable linker include a linker containing a urethane chain or an ester chain, and these linkers can be broken down in a cell by an enzyme. These linkers can also be used in combination. The binding of the amidine antibody to a linker can be, for example, a thiol group of an antibody via a maleimidine imine group. The linker may optionally include polyethylene glycol blocks.

In addition, the present invention can provide a pharmaceutical composition for treating cancer including an antibody-drug conjugate of an anti-IL-7R antibody or an antigen-binding fragment thereof and a cytotoxic agent. IL-7R is positive in most cancer cells. Therefore, most cancers can be treated by the medicine of the present invention. In addition, in order to target a highly effective target group, in a certain aspect of the present invention, the cancer can be set as an IL-7R positive cancer or an IL-7R positive confirmed cancer. Examples of IL-7R positive cancers include leukemia, lymphoma, lung cancer, pancreatic cancer, head and neck cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, gastric cancer, colorectal cancer, uterine cancer, ovarian cancer, skin cancer, Thyroid cancer, thymic cancer, kidney cancer, testicular cancer, penile cancer, liver cancer, bile duct cancer, brain tumor, osteomalacia tumors, retroperitoneal tumors, vascular and lymphangitic sarcomas, and metastatic cancers such as metastatic solid tumors ). IL-7R is positive in most cancers, such as leukemia, lymphoma, progressive cancer, and metastatic cancer. Positive or negative IL-7R can be easily determined by techniques such as immunostaining, FACS using anti-IL-7R antibodies, and the like.

The pharmaceutical composition of the present invention can be used to treat an IL-7R-positive cancer that is resistant to treatment that blocks the combination of IL-7R and IL-7.

In addition, it was found by the present invention that IL-7R is related to the conferring of steroid resistance. In particular, IL-7R became positive in steroid-resistant cells. Therefore, the present invention can provide a pharmaceutical composition which is a pharmaceutical composition for treating steroid-resistant diseases or conditions, and which is an antibody-drug conjugate comprising an anti-IL-7R antibody or an antigen-binding fragment thereof and a cytotoxic agent. Pharmaceutical composition. In a certain aspect, a medicinal composition can be provided, which is a medicinal composition for reducing the steroid resistance of a disease or a condition, and is an antibody drug comprising an anti-IL-7R antibody or an antigen-binding fragment thereof and a cytotoxic agent Pharmaceutical composition of conjugate. In one aspect, the disease or state of steroid resistance is cancer or inflammation. In addition, it was discovered by the present invention that the performance of IL-7R was improved after steroid treatment. Especially after steroid treatment, the effectiveness of the conjugate of the present invention is increased because IL-7R-positive cells easily survive. Even if IL-7R-negative cells survive (for example, in patients after steroid treatment), therapeutic effects can still be expected. This is because the effect of dying IL-7R-negative cells in the vicinity of IL-7R-positive cells by the bystander effect of PDC is expected. Therefore, the present invention can provide a pharmaceutical composition which is a pharmaceutical composition for treating a disease or condition such as cancer or inflammation in a subject receiving steroid treatment, and which comprises an anti-IL-7R antibody or an antigen-binding fragment thereof and cells Pharmaceutical compositions of antibody-drug conjugates for nociceptors.

In a certain aspect, the pharmacological composition of the present invention can also be administered while reducing steroid resistance. Therefore, in a certain aspect of the present invention, a pharmaceutical composition can be provided, which is a pharmaceutical composition for treating a steroid-resistant disease or condition, and is used in combination with a steroid and contains an anti-IL-7R antibody or an antigen binding agent thereof. A pharmaceutical composition of a fragment and an antibody drug conjugate of a cytotoxic agent. Alternatively, the present invention can provide a pharmaceutical composition which is a pharmaceutical composition for reducing the steroid resistance (or increasing the steroid sensitivity of a disease or condition) of a disease or condition, and comprising an anti-IL-7R antibody or A pharmaceutical composition comprising an antigen-binding fragment thereof and an antibody drug conjugate of a cytotoxic agent. In these aspects, the subject may be a subject having a steroid-resistant disease or condition.

In addition, according to the present invention, an anti-IL-7R antibody or an antigen-binding fragment thereof and an antibody drug conjugate of a cytotoxic agent can suppress lymphadenopathy in a subject with cancer. Therefore, the present invention can provide a pharmaceutical composition which is a pharmaceutical composition for suppressing lymphadenopathy in a subject of cancer, and is an antibody drug comprising an anti-IL-7R antibody or an antigen-binding fragment thereof and a cell damaging agent. Pharmaceutical composition of yoke.

According to the present invention, an antibody-drug conjugate of an anti-IL-7R antibody and a cytotoxic agent exhibits a symptom-reducing effect in inflammatory diseases such as ulcerative colitis and rheumatoid arthritis. This suggests that IL-7R-positive cells are strongly related to the onset or maintenance of inflammatory diseases, and represents that killing IL-7R-positive cells in an inflammatory state is useful for alleviating inflammation. Therefore, the present specification can provide a pharmaceutical composition which is a pharmaceutical composition for treating inflammatory diseases, and which is a pharmaceutical composition comprising an anti-IL-7R antibody or an antigen-binding fragment thereof and an antibody-drug conjugate of a cell damaging agent. Thing. In this specification, inflammatory diseases include collagen, and also include autoimmune diseases, type 1 diabetes, multiple sclerosis, systemic lupus erythematosus, allergic diseases, rejection after organ transplantation, and graft resistance. Host disease. The inflammatory disease further includes an inflammatory bowel disease such as Crohn's disease. In addition, as other autoimmune diseases and collagen diseases, they can be selected from the group consisting of Schlegel's syndrome, scleroderma, dermatomyositis, nodular multiple arteritis, mixed connective tissue disease, Kougan's syndrome, and rheumatic multiple muscles. Pain, Adult Still's Disease, Giant Cell Arteritis, Antiphospholipid Syndrome, Grubb's Syndrome, Myasthenia Gravis, Autoimmune Hepatitis, Autoimmune Pancreatitis, Primary Biliary cholangitis, Gao'an's arteritis, Good Pasteur syndrome, rapid glomerulonephritis, autoimmune hemolytic anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura , Brazil's disease, Hashimoto's disease, primary hypothyroidism, idiopathic Edison's disease, pemphigus, psoriasis psoriasis, psoriasis vulgaris, pemphigoid, pregnancy herpes, linear IgA vesicular dermatosis , Acquired epidermal vesicular disease, alopecia areata, leukoplakia vulgaris, Harada disease, autoimmune optic neurosis, autoimmune inner ear abnormality, idiopathic azoospermia, and autoimmune disease or habitual abortion Disease. Examples of type 4 allergies involving lymphocytes include rejection of organ transplantation and bronchial asthma caused by congenital lymphocytes. Lymphocytes are positive for IL-7R in most inflammations. Specifically, if it is positive in the acquired immune system lymphocytes T lymphocytes and B lymphocytes, it will also be positive in the innate immune system lymphocytes, so most of the inflammatory diseases caused by these cells can be based on Subject of the invention's medicinal composition.

In the present invention, an inflammatory disease may be a disease or condition that is resistant to steroids, or resistant to treatment that blocks the combination of IL-7R and IL-7.

In a certain aspect of the present invention, there is provided a method for treating cancer or an inflammatory disease in a subject in need thereof, and the method comprising administering to the subject an anti-IL-7R antibody or an antigen-binding fragment thereof and a cytotoxic agent Methods for antibody drug conjugates. The conjugate can be administered in a therapeutically effective amount. In a certain aspect of the present invention, the aforementioned subject may be a subject that is tolerant to steroid therapy, or a subject that has received steroid therapy. In a certain aspect of the present invention, there is provided a method for treating cancer or an inflammatory disease in a subject in need thereof, and comprising administering an anti-IL-7R antibody or an antigen-binding fragment thereof and a cytotoxic agent to the subject. Antibody drug conjugates, and methods of administering a steroid-based anti-inflammatory drug to the subject. In one aspect of the present invention, the aforementioned subject may be a subject that is tolerant to steroid therapy, or a subject that has received steroid therapy.

In a certain aspect of the present invention, there is provided a method for treating a steroid-resistant disease or condition in a subject in need thereof, and comprising administering an anti-IL-7R antibody or an antigen-binding fragment thereof to the subject and cells. Methods for antibody-drug conjugates of noxious agents.

In a certain aspect of the present invention, there is provided a method for reducing steroid resistance to a disease or condition in a subject in need thereof, and comprising administering an anti-IL-7R antibody or an antigen-binding fragment thereof to the subject. Method for conjugate of antibody drug with cytotoxic agent.

In one aspect of the present invention, there is provided a use in the manufacture of an antibody-drug conjugate comprising an anti-IL-7R antibody or an antigen-binding fragment thereof and a cytotoxic agent, and a medicine for treating cancer or an inflammatory disease. Use of anti-IL-7R antibodies. In one aspect of the present invention, there is provided a use in the manufacture of an antibody-drug conjugate comprising an anti-IL-7R antibody or an antigen-binding fragment thereof and a cytotoxic agent, and a medicine for treating cancer or an inflammatory disease. Use of anti-IL-7R antibodies or antigen-binding fragments thereof and cytotoxic agents. In one aspect of the present invention, there is provided a use of an anti-IL-7R antibody or an antigen-binding fragment thereof and an antibody drug conjugate of a cytotoxic agent in the manufacture of a medicine for treating cancer or inflammatory diseases. In a certain aspect of the present invention, there is provided a use which is the use of an antibody-drug conjugate of an anti-IL-7R antibody or an antigen-binding fragment thereof and a cytotoxic agent, and is a method for treating cancer or an inflammatory disease. use.

In the present invention, it should be possible to suppress the expression of IL-7R in IL-7R-expressing cells instead of killing IL-7R-expressing cells with ADC. Therefore, in a certain aspect of the present invention, there is provided a pharmaceutical composition comprising an expression inhibitor of IL-7R and a pharmaceutical composition for treating cancer or inflammatory diseases. In addition, the present invention provides a pharmaceutical composition comprising a IL-7R expression inhibitor and a pharmaceutical composition for treating a steroid-resistant disease or condition. Furthermore, the present invention provides a pharmaceutical composition comprising a IL-7R expression inhibitor and a pharmaceutical composition for reducing the steroid resistance of a disease or a state. Furthermore, in the present invention, there is provided a pharmaceutical composition which is a pharmaceutical composition for treating a steroid-resistant disease or condition, and which is a pharmaceutical composition which is used in combination with a steroid and contains an anti-IL-7R performance inhibitor. Furthermore, in the present invention, there is provided a pharmaceutical composition comprising a IL-7R expression inhibitor and a pharmaceutical composition for suppressing lymphadenopathy in a subject of cancer.

Examples of IL-7R expression inhibitors include siRNA, shRNA, and antisense oligonucleotides directed against IL-7R, and can be used in the present invention. As the expression inhibitor of IL-7R, expression vectors containing DNA encoding siRNA or shRNA can also be mentioned. [Example]

In the following examples, unless otherwise stated, the meaning of the following symbols is *: P <0.05, **: P <0.01, ***: P <0.001.

Example 1: Survival activity of IL-7 receptor and tumor cells In this example, the expression of IL-7 receptor (IL-7R) and the physiological function of IL-7R in various tumor cells were investigated.

(1) The cell surface of IL-7R was obtained from ATCC (American Cell Bank), a 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, and cancerous pleural effusion of pharynx Derived cell line Detroite562, prostate cancer bone metastasis-derived cell line PC3, and bladder cancer cell line TCCsup with bone metastasis. Self-Research Institute of Biological Resources Research Center obtained human B-cell lymphocytic leukemia cell line NALM6, lung cancer-derived pleural fluid-derived cell line LC-2 / ad, and pancreatic cancer liver metastasis-derived cell line PK-45H. A mouse lymphocytic leukemia cell line CYG82 was established according to the following reference 1 and used in this example. The anti-human IL-7R antibody (eBioRDR5, eBioscience; R34-R34, TONBO) and anti-mouse IL-7R antibody (A7R34 or SB / 199, eBioscience) were used and the performance of IL-7R was measured by flow cytometry. An IL-7R antibody (A7R34) was prepared according to the following references 2 and 3, and used in this example. The results are shown in Figure 1A.

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. In addition, IL-7R was found in lung cancer lymph node metastasis-derived cell line H2009, lung cancer alveolar metastasis-derived cell line H358, lung cancer-derived pleural effusion cell line LC-2 / ad, pancreatic cancer liver metastasis-derived cell line PK-45H, and pharynx The cancerous pleural fluid-derived cell line Detroite562, the prostate cancer bone metastasis-derived cell line PC3, and the bladder cancer cell line TCCsup with bone metastasis were positive (the histogram on the right side of each figure shows the IL-7R performance, and the histogram on the left side is the negative control group). Therefore, IL-7R is positive in all lymphatic malignancies and metastatic solid tumor cell lines investigated. Confirmation of performance in metastatic solid tumors suggests that IL-7R expression is associated with metastasis in solid tumors.

(2) Production of IL-7R knockout cell line For Supt1, NALM6, CYG82, H2009, PK-45H cells, the expression of short hairpin RNA (shRNA, Sigma) incorporated into the lentiviral vector was performed to make IL-7R gene knockout. (KD) cell line (IL-7R-KD). The GFP gene shRNA (Sigma) was used for Supt1, NALM6, and CYG82, and non-specific shRNA (Sigma) was introduced into H2009 and PK-45H to prepare control cells (CTR) for comparison. CDNA was synthesized from the total RNA of the cells using an extraction kit (QIAGEN) and a synthetic kit (Thermo Fisher Scientific or Takara). Quantitative RT-PCR was performed. That is, ABI7500 Fast (Thermo Fisher Scientific) was used to measure a total of 20 ml of the reaction solution of 9 ml of cDNA dilution, 1 ml of TaqMan primers / probe mixture (Thermo Fisher Scientific), and 10 ml of TaqMan Fast Universal PCR Master Mix (Thermo Fisher Scientific). The comparative CT method was used to normalize the expression of each gene, in the case of human genes, the expression of GAPDH was used to normalize, and in the case of mouse genes, the expression of ACTb was used to normalize.

As shown in FIG. 1B, it was confirmed that the expression level of the IL-7R gene was decreased in the prepared IL-7R-KD cell lines of SupT1, NALM6, CYG82, H2009, and PK-45H.

(3) Elimination of the effects of IL-7R on cell growth Investigate the growth activity of IL-7R-KD cells and CTR cells in the culture medium in the test tube. 1000 or 3000 cells were seeded into 96-well plates and the number of cells was counted daily. The results are shown in Figure 1C. As shown in FIG. 1C, compared with the CTR cell line, the cell growth energy in the test tube of any of the IL-7R-KD cell lines of SupT1, NALM6, CYG82, H2009, and PK-45H decreased.

(4) Relationship between tumor growth activity and IL-7R exclusion SupT1, NALM6, CYG82, H2009 cells were transplanted into the skin of female nude mice (a group of five), and tumor volume was measured after implantation to determine tumor growth activity. The results are shown in Figure 1D. As shown in Figure 1D, compared with CTR cells, the tumor volume increase rate of the IL-7R-KD cell lines of SupT1, NALM6, CYG82, and H2009 decreased in nude mice transplantation experiments.

(5) Observation of cell state The cell survival state of the IL-7R-KD cell line of PK-45H in a test tube was observed with a culture inverted microscope ELIPSE TS100 (Nikon). The results are shown in Figure 1E. As shown in Figure 1E, compared with CTR cells, the cell viability of IL-7R-KD cells of PK-45H was significantly reduced.

Example 2: Physiological functions of IL-7R In this example, further physiological functions of IL-7R were investigated.

Lymphoid malignancies are treated with anti-cancer effects of steroids, but tumor cells that are resistant to steroids are known to appear. First, we investigated how the acquisition of steroid tolerance is related to the physiology of IL-7R.

In order to investigate the relationship between the IL-7R signal of each of SupT1, NALM6, CYG82, H2009, and PK45H and steroid tolerance, confirm the cytotoxic effect of dexamethasone in each cell in the test tube (IC50; 50% cytotoxic concentration ). 1000 to 3000 cells were seeded into a 96-well plate at 100 mL / well, and dexamethasone was added to each well at a concentration of 0.1 mM to 1 mM. After 72 hours of incubation, WST-8 (Dojindo Laboratories, Cell Counting Kit-8) was added at 10 mL / well, and after 3 hours of incubation, the absorbance at 450 nm was measured with a microplate analyzer (Molecular Devices). The cell survival rate at each concentration was calculated to calculate the IC50. The results are shown in Figure 2A.

As shown in FIG. 2A, in any of SupT1, NALM6, and CYG82, the IC50 of steroids (dexamethasone) decreased in IL-7R-KD cells, indicating an increase in steroid sensitivity. There were no differences between IL-7R-KD cells and CTR cells in H2009 and PK45H. Therefore, it has been shown that in lymphoid malignancies, IL-7R is closely related to steroid susceptibility, but in metastatic solid tumors H2009 and PK45H, IL-7R is hardly related to steroid susceptibility.

Furthermore, a steroid-tolerant strain was obtained, and then an IL-7R knockout strain and a non-specific shRNA control strain (CTR) were produced to confirm the effect of IL-7R knockout in the presence of dexamethasone. Since CYG82 steroid-resistant strains are easily obtained from the parent strain of CYG82, the growth of cells was observed in the state of high concentration of steroids. 10 ⁶ cells were seeded into a 6-well plate at 3 ml / well, and dexamethasone 10 nM or 100 nM was added, and the number of cells after 1 week and 2 weeks was counted. The results are shown in Figure 2B.

As shown in FIG. 2B, when non-specific shRNA was introduced into CTR cells of a steroid-resistant strain, growth was inhibited in the first week in the presence of dexamethasone, but cell growth was thereafter exhibited. In contrast, among the steroid-resistant strains, the number of cells in which the IL-7R-excluded cell line was significantly reduced. This represents that steroid tolerance is released by IL-7R and that the emergence of resistant strains is suppressed in the IL-7R knockout strain.

First compare the IC50 differences of dexamethasone. The relationship between steroid tolerance and IL-7R was investigated. Specifically, cDNA was synthesized from total RNA of cells using an extraction kit (QIAGEN) and a synthetic kit (Thermo Fisher Scientific or Takara), and quantitative RT-PCR was performed to investigate the expression of the IL-7R gene and the CD19 gene. The results are shown in Figure 2C.

As shown in FIG. 2C, the steroid-resistant strain of CYG82 cells obtained as described above showed a high IC50 against dexamethasone. In addition, comparing the expression levels of IL-7R in the parent and non-tolerant strains showed that the expression of IL-7R in the tolerant strains was significantly improved. On the other hand, regarding the amount of CD19 found, no significant difference was found between the parent strain and the tolerant strain.

Example 3: Relationship between activation of IL-7R signal and steroid tolerance In this example, the relationship between steroid tolerance and activation of IL-7R signal was investigated.

The effects of IL-7R-KD on the activation of JAK / STAT, BCL2, and NFκB, the downstream signaling molecules of IL-7R, were investigated in SupT1, NALM6, CYG82, and RAG2-/-cells. For the determination of JAK / STAT, cells were fixed with 4% paraformaldehyde and treated with methanol, and then phosphorylated STAT5 was detected using an anti-phospho-STAT5 antibody (eBioscience). The measurement was performed using a flow cytometry detection device Guava easyCyte 10HT (Merck Millipore) or Aria flow cytometer (BD Bioscience). Dead cells were stained with Propidium iodide (Thermo Fisher Scientific) and excluded from the assay. Data analysis was performed using the FlowJo program (Tree Star). The signal of BCL2 is to extract total RNA and synthesize cDNA from each cell, and then investigate the expression level of BCL2 by quantitative PCR. About NFkb, cells were fixed with 4% paraformaldehyde, and then phospho-65 / NF-kb antibody (Abcam) was reacted. The secondary antibody was an Alexa-488 / 555/647 labeled anti-rabbit IgG antibody (Thermo Fisher Scientific). Nuclei were stained with DAPI (Thermo Fisher Scientific). Images were taken with a conjugate cross-laser microscope LSM-710 (Carl Zeiss) or an HS multifunctional fluorescence microscope BZ-9000 (Keyence Co.). RAG2-/-cells were established and used according to reference 3.

As a result, because the elimination of IL-7R had no effect on JAL / STAT and BCL2 signaling, it was suggested that steroid tolerance and activation of IL-7R signaling should have little correlation. Furthermore, as shown in Fig. 3, NFkB signal was observed in the nucleus of CTR cells, but almost no signal was observed in IL-7R-KD cells. From this, it can be seen that NFκB activation occurs in steroid-resistant strains, but NFκB activation is inhibited by deletion of IL-7R. In addition, in RAG2-/-cells that require IL-7 as a growth factor, no signal in JAK / STAT, BCL2, or NFkB is suppressed in the absence of IL-7. This shows that there is no relationship between JAK / STAT and BCL2 signals and steroid tolerance. In addition, NFkB decreased in any cell line due to the elimination of IL-7R, but IL-7R-KD in H2009 and PK-45H cells also decreased. As shown in Figure 2, these cells are not related to steroid tolerance. Therefore, it is believed that the relationship between NFkB and cell growth and survival may be stronger than the relationship with steroid tolerance.

Example 4: Obtaining steroid tolerance and changes in gene expression In this example, the relationship between IL-7R gene expression and steroid tolerance in clinical samples was investigated.

First, clinical data of GSE39339 and GSE32962 were obtained from Gene Expression Omnibus (GEO), and clinical data of E-MEXP-3916 were obtained from ArraYExpress. The data was analyzed using analysis software R (open source free software), and then statistically analyzed after being standardized by RMA processing. For statistical analysis, SPSS Statistics version 20 (IBM Corporation) was used, and P <0.05 or less was used as a criterion for determination. The results are shown in Figure 4. FIG. 4A illustrates the analysis result of GSE39339, FIG. 4B illustrates the analysis result of GSE32962, and FIG. 4C illustrates the analysis result of E-MEXP-3916.

As shown in FIG. 4A, after chemotherapeutics including steroids (dexamethasone) in human acute lymphoblastic leukemia, lymphoid markers TdT, CD19, and CD22 decreased significantly. On the other hand, it was observed that the expression of IL-7R tended to increase after treatment. In addition to dexamethasone, chemotherapy also includes anthracycline, vincristine, and L-asparaginase.

As shown in FIG. 4B, the results of establishing a steroid (adrenocortical hormone) susceptible and tolerant strain from cells from human patients with acute B lymphocytic leukemia and analyzing the results by DNA microarray. Although there were no changes in TdT, CD19, and CD22 between the two groups, IL-7R showed significant high values in steroid (adrenocorticone) tolerant strains.

As shown in FIG. 4C, the results of DNA microarray analysis of the non-treatment group and the steroid (dexamethasone) -containing treatment group in patients with human acute T-lymphocytic leukemia are shown. The performance of IL-7R showed a significant high value in the treatment population. In addition to dexamethasone, chemotherapy also includes daunorubicin, vincristine, and L-asparaginase.

As shown in Figs. 4A to 4C, it can also be found from clinical data that the steroid tolerance obtained by steroid treatment is related to the performance of IL-7R.

Example 5: Characteristics of anti-IL-7R antibody Based on this example, the dynamics when the anti-IL-7R antibody was brought into contact with the cells were investigated.

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 was observed after 10 minutes and 30 minutes. The solution was visualized with Lyso Tracker (Thermo Fisher Scientific). The results are shown in Figure 5A.

As shown in FIG. 5A, it was found that the anti-IL-7R antibody was taken into cells within 30 minutes after administration, and was localized in the lysosomal body.

Next, the behavior of the anti-IL-7R antibody in vivo was confirmed. Specifically, Alexa647-labeled anti-IL-7R antibody (A7R34) or a control antibody was administered from the tail vein of the mouse after the parental CYG82 strain was transplanted subcutaneously in a female nude mouse to allow tumor formation. One week after the administration, tumors, lymph nodes, spleen, bone, and liver were removed to confirm the accumulation of anti-IL-7R antibody (A7R34) in each tissue. The results are shown in Figure 5B.

As shown in FIG. 5B, compared with the accumulation of anti-IL-7R antibody (A7R34) in tumors and diseased lymph nodes, the control antibody did not accumulate in these tissues.

CYG82 cells and IL-7-dependent RAG2-/-cells were each added to a 96-well plate culture in the form of 2000 or 4000 cells / 100 mL / well. Anti-IL-7R antibody A7R34 was added in a serial dilution of 10 ^-4 to 10 ² mg / ml. After 72 hours, WST-8 (Dojindo Laboratories, Cell Counting Kit-8) was added at 10 mL / well, and cultured for 3 hours. The absorbance at 450 nm was then measured with a microdisk analyzer (Molecular Devices). Cell survival was calculated to determine the cell killing effect due to the neutralizing activity of the antibody's IL-7 signal. The results are shown in Figure 5C.

As shown in FIG. 5C, the anti-IL-7R antibody (A7R34) had a cell-killing effect on IL-7-dependent RAG2-/-cells, but had almost no effect on CYG82 cells. It can be seen that CYG82 cells do not require IL-7. It is speculated that CYG82 cells maintain cell growth and survival through self-activation of IL-7R in order to avoid the influence of the neutralizing activity of IL-7R antibody (A7R34).

Therefore, the cell-killing effect of the antibody-drug conjugate (ADC, using SN-38 as a drug) of the IL-7R antibody (A7R34) was compared with the SN-38 precursors CPT-11 and free SN-38. The cell killing effect in the test tube was confirmed in the same manner as in FIG. 5C. The ADC is prepared according to the following references 6-9.

That is, the linker and the drug are bound to the antibody using maleimine. Mal-PEG ₁₂ -Osu was purchased from Quanta Biodesign or IRIS Biotech GMBH and was used to increase polarity. A urethane bond is introduced into the linker site as a cutting site for releasing a drug in the cell. SN-38 was dissolved in dimethyl sulfene at a concentration of 10 mM and stored at minus 80 ° C. ADC is produced by reducing the SS bond in the antibody and reacting the maleimide group of the linker with a drug. ADCs are obtained in which each antibody binds to 4 to 6 agents. The results are shown in Figure 5D.

As shown in Figure 5D, the effect of A7R34-ADC (SN-38) is 100 times stronger than that of CPT-11, and it shows the same cell killing effect as dissociated from SN-38. This shows that A7R34-ADC is effective for CYG82 that does not depend on IL-7 survival. This shows that even in the absence of the IL-7 signal, IL-7R is constantly internalized. In addition, anti-IL-7R antibody (A7R34) has no significant effect on the neutralizing activity of IL-7 and IL-7R, the cell survival rate, and the inhibitory effect of tumor volume. Anti-IL-7R antibody and cytotoxic ADC have a more significant anti-tumor effect.

Example 6: Antitumor effect in leukemia model In this example, the antitumor effect of ADC with anti-IL-7R antibody and drug was confirmed in a leukemia model.

Female nude mice (a group of five) were subcutaneously transplanted with a parental strain of CYG82 to obtain a tumor volume of about 50 mm ³ as a leukemia model mouse. The ADC uses the A7R34-ADC (SN-38) manufactured in Example 5. On days 0, 4, and 8, the amount of ADC administered from the tail vein of mice was calculated as 50 mg / kg, and the amount of A7R34-ADC (SN-38) was calculated as 0.6 mg / kg in terms of SN-38 conversion. As a control, physiological saline, A7R34 antibody at 50 mg / kg, ADC of 50 mg / kg isotype antibody (= ADC of control antibody) [0.6 mg / kg in terms of SN-38], 10 mg / kg Dexamethasone or CPT-11 at 20mg / kg [calculated at SN-38 as 11.6mg / kg]. Tumor volume was measured every two days until the 26th day after administration. Similarly, after transplanting a steroid-resistant strain such as CYG82, when the tumor volume reached approximately 50 mm ³ , 50 mg / kg of A7R34-ADC was administered from the tail vein of mice on days 0, ⁴ , and 8 [based on the amount of SN-38 Calculated as 0.6 mg / kg]. As controls, physiological saline, dexamethasone at 10 mg / kg, and phenethyl caffeate caffeic acid (CPAE) were administered at 20 mg / kg. Tumor volume was measured every two days and until the 18th day after administration. The results are shown in Figures 6A and 6B.

As shown in FIG. 6A, the ADC of the anti-IL-7R antibody and the drug showed the most excellent effect of suppressing tumor volume increase. On the other hand, anti-IL-7R antibody alone has little effect. In addition, as shown in FIG. 6B, ADCs with anti-IL-7R antibodies and agents also showed a strong antitumor effect on steroid-resistant lymphocytic leukemia models.

Example 7: Inhibition of lymph node enlargement in a leukemia model CY CYG82 cells were transplanted to the soles of the front feet of female nude mice (a group of five), and the average value of lymph node volume was measured on the 16th day after transplantation. The results are shown in Figure 7A. As shown in Figure 7A, the swelling of axillary lymph nodes in IL-7R knockout mice was significantly suppressed.

Next, after female nude mice (a group of three) were subcutaneously transplanted with a parental CYG82 strain and tumors formed, lymph nodes in the abdominal cavity were enlarged and enlarged by regional lymph nodes. It was evaluated whether the ADC of the anti-IL-7R antibody (A7R34) had the effect of suppressing lymphadenopathy. As in FIG. 5, A7R34-ADC (SN-38) was administered from the tail vein of mice on days 0, 4, and 8. As a control, any one of physiological saline, A7R34 antibody, isotype ADC (SN-38), dexamethasone, and CPT-11 was administered. Lymph node volume was measured on the 16th day after administration. The results are shown in Figure 7B. As shown in Figure 7B, A7R34-ADC (SN-38) has a strong inhibitory effect on lymphadenopathy. A7R34 alone has almost no therapeutic effect.

Example 8: Radioisotope-labeled anti-IL-7R antibody In this example, a radioisotope-labeled anti-IL-7R antibody was used in place of ADC administration, and the effect of the antibody was confirmed.

A ⁹⁰ Y-labeled antibody was prepared according to the following reference 10. 1M acetate buffer (pH 6.0) and ⁹⁰ YCl ₃ were mixed in a 1: 1 manner, and allowed to stand at room temperature for 5 minutes. Then, a commercially available anti-IL-7R antibody (40131 strain, R & D company) was added and left to stand at room temperature for 30 minutes. After completion of the reaction, purification was performed using a Sephadex G50 (manufactured by GE Medical Co., Ltd.) column (swelled with 0.1 M acetate buffer (pH 6.0)). Thereafter, the dose was adjusted so that the amount was converted to 20 mg of unlabeled anti-human IL-7R antibody. ⁹⁰ Y-labeled anti-IL-7R antibody was administered to the tail vein of nude mice that had been subcutaneously transplanted with either of SupT1 and H2009, and the efficacy was confirmed. 20 mg of ⁹⁰ Y-labeled-anti-IL-7R antibody was administered at a time. The results are shown in Figures 8A and 8B.

As shown in FIGS. 8A and 8B, it was found that ⁹⁰ Y-labeled anti-IL-7R antibody exhibited an antitumor effect at 100 mCi. It is known that anti-IL-7R antibodies can be used in radioimmunotherapy.

Example 9: Effect of ADC on immune response Based on the above examples, the effect of ADC on tumors was confirmed. In this embodiment, it is investigated whether ADC can be used for the treatment of abnormal immune response.

Healthy normal female BALB / c mice were administered physiological saline, A7R34-ADC (SN-38) at 50 mg / kg [0.6 mg / kg in terms of SN-38], and 10 mg from the tail vein of the mice. dexamethasone per kg or dexamethasone at 40 mg / kg.

In A7R34-ADC, IL-7R positive T cells and B cells were reduced to less than 1/10 in thymus, bone marrow and spleen. In the case of dexamethasone administration, the total number of T cells containing IL-7R-positive T cells in the thymus was reduced, while the number of IL-7R-positive B cells in the bone marrow increased more than twice. In addition, although the number of whole cells in the spleen decreased, the proportion of IL-7R-positive T cells increased, suggesting that physiologically IL-7R is also related to steroid tolerance of T cells and B cells. In addition, the difference in pharmacodynamics between A7R34-ADC and steroids (dexamethasone) indicates that the two can control immune cells through different mechanisms. In particular, it is speculated that the control of A7R34-ADC on steroid-resistant IL-7R-positive cells is effective.

Example 10: Increase of IL-7R-positive cells in immune diseases In this example, the gene expression of IL-7R in clinical samples was investigated in the same manner as in Example 4.

The raw data of GSE55235 and GSE14580 were obtained from Gene Expression Omnibus (GEO). Data analysis was performed using the analysis software Qlucore Omics Explorer (Filgen), and P &lt; 0.05 or less was used as a criterion for determination. The results are shown in Figure 10.

As shown in FIG. 10, in patients with chronic rheumatoid arthritis (RA), it was confirmed that the gene expression of IL-7R tended to be higher than that of healthy normal people. In addition, in patients with ulcerative colitis (UA), it has been confirmed that the expression of IL-7R gene is higher than that of healthy normal people. In particular, the expression of IL-7R gene is enhanced in patients with unsuccessful anti-TNF therapy. .

Example 11: Effect of using anti-IL-7R antibody to treat rheumatoid arthritis model A conjugate of an anti-IL-7R antibody and a drug was administered to a rheumatoid arthritis model and its effect was investigated.

A7R34-ADC (MMAE) linked with A7R34 monoclonal antibody and monomethyl auristatin E (MMAE) with Val-Cit linkage (valine-citrulline dipeptide) as the anti-IL-7R antibody and Conjugate of Elixir. Val-Cit linkage and MMAE are linked by p-aminobenzylcarbamate (PABC). MMAE is known to be more cytotoxic than SN-38. A7R34-ADC (MMAE) was produced in the same manner as in Example 5 as described in References 6 to 9 below. The chemical structural formula of A7R34-ADC (MMAE) is as follows.

A rheumatoid arthritis model is produced according to the method described in Reference 7 below. That is, 2 mg of anti-collagen 4 antibody (Chondrex) was administered to female DBA / 1J mice into the abdominal cavity (day 0). On the third day, 50 mg of LPS (Chondrex) was administered into the abdominal cavity. On the 5th day, the obtained mice were administered A7R34-ADC (MMAE) at 50 mg / kg [1 mg / kg in terms of MMAE conversion] or A7R34-ADC (SN-38) at 50 mg / kg [as SN-38 The conversion amount is calculated as 0.6 mg / kg]. As controls, use normal saline, 1 mg / kg MMAE, 50 mg / kg isotype control antibody ADC (MMAE) [1 mg / kg in MMAE conversion], 10 mg / kg dexamethasone, or 50 mg / kg An anti-TNF formulation, etanercept, was administered in place of ADC. In the case of 50 mg / kg of A7R34-ADC (SN-38) [0.6 mg / kg calculated based on the amount of SN-38], use physiological saline, 50 mg / kg of anti-IL-7R antibody (A7R34), 50 mg / kg of anti-CD19 antibody ADC (SN-38) [0.6 mg / kg calculated based on the amount of SN-38 conversion] and 10 mg / kg of dexamethasone were used as controls. The arthritis score of each individual was measured in accordance with the instructions (a full score of 16 and the higher the score, the more severe the symptoms) to evaluate the severity of arthritis symptoms. The results are shown in Figures 11A ~ C. In this example, the arthritis score is obtained by observing the three joints of the fingers, claws and wrists of each limb. Specifically, the arthritis score of each limb was calculated based on the following criteria. Criteria for the evaluation of arthritis scores of each limb 0: In the case of no inflammation in any joint, score 1: In the case of inflammation in any joint, Score 2: In the case of inflammation in any two joints, Score 3: In the case where all joints are inflamed, score 4: In the case where all joints are inflamed and the entire limb is red and swollen.测定 Based on the above-mentioned scoring points, the arthritic scores are measured for each limb (4 points out of each limb), and the limbs are totaled (16 points out of total) to obtain the individual arthritis score.

As shown in FIG. 11A, A7R34-ADC (MMAE) is the same as dexamethasone and significantly inhibits the deterioration of the arthritis coefficient in a rheumatoid arthritis model. After more detailed confirmation, it was found that individuals with more prominent forelimb inflammation than hindlimbs were present in the dexamethasone administration group (examples of poorly controlled inflammation). In the A7R34-ADC (MMAE) administration group, stable and strong anti-inflammatory effects were observed in both forelimbs and hind limbs. This anti-inflammatory effect is superior to the anti-TNF therapy of etanercept. In addition, when investigating the joint tissue of a case of dexamethasone with poor inflammation control, a strong inflammation condition was observed, and many IL-7R-positive cells were observed. In addition, as shown in Figure 11B, the A7R34-ADC (SN-38) has the same result. Moreover, although the effect of suppressing the deterioration of the arthritis coefficient in the rheumatoid arthritis model was also observed using the anti-IL-7R antibody (A7R34 antibody) alone, the effect was limited (see FIG. 11B). (C) In FIG. 11C, a tissue section of the lesion area of the forefoot of the mouse on the 9th day after the administration of the drug was prepared, and observed by hematoxylin-eosin staining (HE staining). As shown in FIG. 11C, it can be seen that the inflammation of the ADC administration group is greatly improved.

Therefore, it was confirmed that the effect of neutralizing the binding of IL-7R and IL-7 was weak, and that the effect of ADC with an anti-IL-7R antibody and a cell damaging agent was strong.

Example 12: Effect of using anti-IL-7R antibody to treat ulcerative colitis In this example, the effect of anti-IL-7R antibody on ulcerative colitis was investigated.

A model of ulcerative colitis was made in accordance with reference material 11 below. That is, female BALB / c mice were given 5% dextran sulfate (DSS; MP Biomedicals). From the 2nd day after DSS drinking, the A7R34-ADC (MMAE) or A7R34-ADC (SN-38) is administered into the abdominal cavity in the same way as the rheumatoid arthritis. As controls, saline, MMAE, isotype control antibody ADC (MMAE), dexamethasone, or anti-TNF preparation etanercept was used instead of ADC. In the case of A7R34-ADC (SN-38), physiological saline, anti-IL-7R antibody ADC (A7R34), anti-CD19 antibody ADC (SN-38), and dexamethasone were used as controls in the same manner. Rheumatoid arthritis model. The large intestine on the 8th day after drinking was taken out and the full length was measured. In addition, a tissue specimen was prepared and stained with HE to confirm a pathological image. The severity of ulcerative colitis is observed by weight loss (the more severe the weight loss, the more severe the symptoms), the full length of the large intestine (the shorter the full length of the large intestine, the more severe the symptoms), and histological changes. The results of weight change are shown in Figures 12A and B. The results of histological changes in the full length of the large intestine are shown in FIG. 13.

As shown in FIG. 12A, A7R34-ADC (MMAE) significantly inhibited weight loss after the 5th day, showing little therapeutic efficacy relative to dexamethasone. This suppressive effect was significantly higher than the weight loss suppressive effect of the etanercept-administered group. MMAE and the control antibody ADC showed almost no inhibitory effect on weight loss. In addition, as shown in Figure 12B, A7R34-ADC (SN-38) also significantly inhibited the weight loss of model mice. In addition, as shown in FIG. 13, the total length of the negative control group of ulcerative colitis was significantly shortened compared with the DSS untreated group, but the full length of the large intestine of the A7R34-ADC (MMAE) administration group recovered to the same level as the DSS untreated group. In addition, the A7R34-ADC (SN-38) administration group also observed a tendency to increase the total length.

Therefore, it was confirmed that the effect of neutralizing the binding of IL-7R and IL-7 was weak, and that the effect of ADC with an anti-IL-7R antibody and a cell damaging agent was strong.

The pathological tissue images of the large intestine removed specimens 6 days after the DSS administration group were further observed. As a result, as shown in Fig. 14, the colonic ulcer formation and mucosal destruction of the raw food group were obvious. In addition, the effects of dexamethasone and A7R34 alone were weak. In addition, the anti-TNF preparation etanercept did not sufficiently inhibit inflammation. In contrast, however, A7R34-ADC (MMAE) completely inhibits inflammation. Although A & R34-ADC (SN-38) also suppressed inflammation, some ulcerative lesions were observed in the center. Although anti-CD19 antibody-ADC (SN-38) inhibited inflammation, the entire mucosa changed into granulomatous. It can be seen that MMAE, which is highly cytotoxic, is more effective than SN-38 in the colorectal cancer model.

IL-7R is expressed in acquired immune system lymphocyte T cells and B cells, and in particular innate immune system lymphocytes. Since the inflammation can be sedated by ADC against IL-7R, it is suggested that this ADC is effective for sedation of innate immune system lymphocyte T cells and B cells, and in particular, innate immune system lymphocyte-mediated inflammation. of. Therefore, the present invention shows that the antibody-drug conjugates of anti-IL-7R antibodies and cytotoxic agents are effective against diseases such as autoimmune diseases and graft resistance caused by various inflammations and innate immune hypertrophies mediated by innate immune system lymphocytes. Host disease, etc.) and other diseases or conditions mentioned above are effective.

[Fig. 1A] Fig. 1A is a result of investigating the expression level of IL-7R in various cancer cell lines by flow cytometry. [FIG. 1B] FIG. 1B is a graph showing that IL-7R expression levels of various cancer cells actually decreased due to the IL-7R knockout test. [Fig. 1C] Fig. 1C is a graph showing the effect on the growth of a cancer cell line in which IL-7R is eliminated. [Fig. 1D] Fig. 1D is a graph showing the effect on growth of a cancer cell line in which a subcutaneously transplanted IL-7R is removed in a nude mouse subcutaneous transplantation model. [Fig. 1E] Fig. 1E is a microscope photograph showing the viability of a cancer cell line in which IL-7R has been eliminated. [Fig. 2A] Fig. 2A is a graph showing that IC50 of dexamethasone (a steroidal anti-inflammatory drug) of various cancer cell lines decreases due to the elimination of IL-7R. [Fig. 2B] Fig. 2B is a graph showing the effect of IL-7R deletion on dexamethasone tolerance in vitro after obtaining a dexamethasone-resistant strain. [Fig. 2C] Fig. 2C is a graph showing an improvement in the expression of IL-7R in a dexamethasone-resistant strain. FIG. 3 is a graph showing the results of staining various cancer cell lines and their IL-7R knockout lines with anti-phosphorylated p65 / NFκB antibodies. [Fig. 4A] Fig. 4A is a graph showing changes in gene expression of B-cell acute leukemia before and after chemotherapy with steroids. [Fig. 4B] Fig. 4B is a graph showing changes in gene expression of B-cell acute leukemia before and after steroid resistance is obtained with steroid treatment. 4C is a graph showing changes in gene expression of T-cell acute leukemia before and after chemotherapy with steroids. [Fig. 5A] Fig. 5A shows the results of contacting an Alexa647-labeled anti-IL-7R antibody with a cancer cell line in vitro and confirming the local presence with the lysate. [Fig. 5B] Fig. 5B shows the results of administering a fluorescently labeled anti-IL-7R antibody to a tumor subcutaneous transplantation model, and investigating the accumulation of anti-IL-7R antibodies in various tissues one week later. [FIG. 5C] FIG. 5C illustrates that an anti-IL-7R antibody having the ability to neutralize the binding of IL-7R and IL-7 exerts an effect on RAG2-/-cells that survive in an IL-7 dependent manner, but in another On the other hand, it has almost no effect on the cancer cell line CYG82. [Fig. 5D] Fig. 5D shows that the antibody drug conjugate (ADC) of an anti-IL-7R antibody and a cytotoxic agent exerts a killing effect on a cancer cell line CYG82. 6A is a graph showing the effect of an antibody-drug conjugate of an anti-IL-7R antibody and a cytotoxic agent on a tumor volume increase in a subcutaneous transplantation model of a cancer cell line. [Fig. 6B] Fig. 6B is a graph showing an effect of an increase in tumor volume in a subcutaneous transplantation model of an anti-IL-7R antibody and a cytotoxic agent antibody drug conjugate in an existing steroid-resistant cell line. Fig. 7A is a graph showing the effect of the removal of IL-7R on lymph node enlargement in a subcutaneous transplantation model of a cancer cell line CYG82. 7B is a graph showing the effect of ADCs against IL-7R and SN-38 on intra-abdominal lymphadenopathy caused by regional lymphadenopathy in a subcutaneous transplantation model of a cancer cell line CYG82. 8A is a graph showing the effect of a radioisotope-labeled ( ⁹⁰ Y-labeled) anti-IL-7R antibody on a T-cell lymphoma cell line Sup-T1. 8B is a graph showing the effect of a radioisotope-labeled ( ⁹⁰ Y-labeled) anti-IL-7R antibody on a lung cancer cell line H2009. [Fig. 9] Fig. 9 is a graph showing the effect of ADC against IL-7R on the immune system. [Fig. 10] Fig. 10 shows an increase in the expression of IL-7R in chronic rheumatoid arthritis and ulcerative colitis. [Fig. 11A] Fig. 11A illustrates the effect of ADCs of anti-IL-7R antibody and monomethyl orstatin E (MMAE) on a rheumatoid arthritis model. [Fig. 11B] Fig. 11B illustrates the effect of anti-IL-7R antibody and ADC of SN-38 on rheumatoid arthritis model. [FIG. 11C] FIG. 11C illustrates the results of making a tissue section of the forefoot lesion of a rheumatoid arthritis model mouse and performing hematoxylin-eosin staining. [Fig. 12A] Fig. 12A is a graph showing the effect of ADC of anti-IL-7R antibody and MMAE on ulcerative colitis model. 12B is a graph showing the effect of anti-IL-7R antibody and ADC of SN-38 on a model of ulcerative colitis. [Fig. 13] Fig. 13 is a graph showing the improvement of colonic tissue type in an ulcerative colitis model with an anti-IL-7R antibody and MMAE ADC or an anti-IL-7R antibody and SN-38 ADC. [Fig. 14] Fig. 14 is a graph showing the effect of anti-IL-7R antibody and ADC of MMAE or ADC of anti-IL-7R antibody and SN-38 in pathological tissue images of a large intestine specimen extracted from an ulcerative colitis model. .

Claims (14)

A pharmaceutical composition is a pharmaceutical composition comprising an anti-IL-7R antibody or an antigen-binding fragment thereof and an antibody drug conjugate of a cytotoxic agent, and is used for treating cancer or inflammation. For example, the pharmaceutical composition in the scope of patent application No. 1 is used for treating inflammation. For example, the application of the pharmaceutical composition of the first or second patent scope, wherein the inflammation is steroid-resistant inflammation. For example, the pharmaceutical composition of the second or third item of the patent application scope, wherein the inflammation is ulcerative colitis or rheumatoid arthritis. For example, the pharmaceutical composition in the scope of patent application No. 1 is used for treating cancer. For example, the pharmaceutical composition of the first or fifth scope of the patent application, wherein the cancer is leukemia, lymphoma or metastatic cancer. For example, the application of the pharmaceutical composition in the first, the fifth or the sixth of the patent scope, wherein the cancer is a steroid-resistant cancer. For example, the application of the pharmaceutical composition of any one of items 1 and 5 to 7 in which the cancer is metastatic cancer. For example, the application of the pharmaceutical composition of any one of items 1 and 5 to 8 in which the cancer is resistant to the treatment that neutralizes the combination of IL-7R and IL-7. A pharmaceutical composition is a pharmaceutical composition comprising an anti-IL-7R antibody or an antigen-binding fragment thereof and an antibody drug conjugate of a cytotoxic agent, and is used for treating a steroid-resistant disease or condition. A pharmaceutical composition is a pharmaceutical composition comprising an anti-IL-7R antibody or an antigen-binding fragment thereof and an antibody-drug conjugate of a cytotoxic agent, and is used in combination with a steroid. For example, the pharmaceutical composition under the scope of patent application No. 11 is used for treating cancer. For example, 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. An antibody-drug conjugate is an antibody-drug conjugate of an anti-IL-7R antibody or an antigen-binding fragment thereof and monomethyl auristatin E.