Method for identifying targets responsive to the treatment of autoimmune diseases and composition for treating them.

Abstract

Kind Code: A1 Abstract: Methods are provided for identifying a subject having an autoimmune disease (eg, type 1 diabetes) as likely to respond to treatment with a tumor necrosis factor-α (TNF-α) receptor II activator. [Solution] The method includes measuring CD8 protein density on the surface of autoreactive CD8+ T cells and identifying the subject as likely to respond to the treatment if the CD8 protein density is reduced compared to reference CD8+ T cells. For type 1 diabetes, the method may include measuring C-peptide levels in an in vitro biological sample from the subject, identifying the subject as likely to respond to the treatment if the C-peptide level is detectable, and identifying the subject as unlikely to respond to the treatment if the C-peptide level is substantially undetectable. The invention also features pharmaceutical compositions of one or more TNFR2 activators for therapeutic use.

Description

[Technical Field]

[0001] In general, the present invention relates to a method for identifying subjects with autoimmune diseases that are likely to respond to treatment with TNF-α receptor II activator, and a method for treating subjects identified as likely to respond to such treatment. The present invention also provides a method for treating subjects determined to be less responsive to TNF-α receptor II activator therapy alone. [Background technology]

[0002] Autoimmune diseases are thought to involve an immune response against the body's own components that are not observed in a normal state, resulting in pathological conditions that cause various tissue damage and / or functional impairments. Autoimmune diseases are broadly classified into systemic autoimmune diseases and organ-specific autoimmune diseases according to their characteristics. Examples of autoimmune diseases include insulin-dependent diabetes mellitus (also known as type 1 diabetes), systemic lupus erythematosus, rheumatoid arthritis, Hashimoto's disease, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, Behçet's disease, bullous pemphigoid, cardiomyopathy, celiac plue dermatitis, chronic fatigue immunodeficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, scarring pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, ulcerative colitis, psoriatic arthritis, essential mixed cryoglobulinemia, fibromyalgia-fibromyitis, Graves' disease, Guillain-Barré syndrome, hypothyroidism, idiopathic pulmonary fibrosis, and idiopathic lacopene syndrome. These include purpura (ITP), IgA nephropathy, juvenile arthritis, lichen planus, lupus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjögren's syndrome, Stiffman syndrome, Devic's disease, Takayasu's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, Wegener's granulomatosis, and autoimmune neuropathy (e.g., autoimmune Alzheimer's disease, autoimmune Parkinson's disease, or amyotrophic lateral sclerosis).

[0003] In particular, type 1 diabetes is a severe childhood autoimmune disease characterized by insulin deficiency that disrupts the normal regulation of blood glucose levels. Insulin is a peptide hormone produced by beta cells (beta-islet cells) in the islets of Langerhans in the pancreas. Insulin promotes glucose utilization, protein synthesis, triglyceride formation and storage, and is the primary energy source for brain and muscle tissue. Type 1 diabetes is caused by an autoimmune reaction that leads to the complete destruction of pancreatic beta-islet cells, eliminating insulin production and ultimately resulting in hyperglycemia and ketoacidosis. While insulin injection therapy has been useful in preventing severe hyperglycemia and ketoacidosis, it cannot completely normalize blood glucose levels. Although insulin injection therapy is highly successful, it does not prevent early vascular deterioration, which is a major cause of modern diabetes. Diabetes-related vascular deterioration (including both microvascular deterioration and accelerated atherosclerosis) can ultimately lead to renal failure, retinal deterioration, angina pectoris, myocardial infarction, peripheral neuropathy, and atherosclerosis.

[0004] C-peptide (i.e., binding peptide) is a short 31-amino acid protein that binds the A and B chains of insulin within the proinsulin molecule. Pancreatic β-islet cells secrete preproinsulin, which contains the A chain, C-peptide, B chain, and signal sequence. The signal sequence is cleaved to produce proinsulin. Subsequently, the C-peptide is cleaved to produce mature insulin protein (containing disulfide-bonded A and B chains). Newly diagnosed diabetic patients are often classified into type 1 and type 2 diabetes based on their C-peptide levels. Measuring C-peptide levels in a subject constitutes a useful proxy test for insulin production in the subject's body, because variability in insulin levels within the subject's circulatory system due to hepatic metabolism makes insulin level measurement uncertain. On the other hand, C-peptide levels are less affected by hepatic metabolism. Therefore, peripheral C-peptide concentration reflects insulin secretion by β-islet cells more accurately than insulin concentration.

[0005] HbA1c (glycosylated hemoglobin) is a form of hemoglobin produced in vivo by the non-enzymatic glycation of hemoglobin in plasma. The ratio of HbA1c to non-glycated hemoglobin is directly correlated with plasma glucose concentration. Therefore, high levels of HbA1c observed over a long period in samples from diabetic patients may indicate serious symptoms, such as hyperglycemia (e.g., acute hyperglycemia).

[0006] Tumor necrosis factor-alpha (TNF-α) is a naturally occurring cytokine described in 1975 as a serum factor induced after Bacillus Calmette-Guerin (BCG) injection as a means of fighting tumors (Carswell et al., Proc. Natl. Acad. Sci. USA 72:3666-3670, 1975). Cloning of TNF-α and its two receptors revealed sequence homology to the genomes of microbial pathogens (e.g., Loetscher et al., Cell 62:351, 1990). This remarkable sequence duplication indicates a complex system of microbial responses that regulate host TNF-α secretion and the activity of its receptors (Rahman et al., PloS Pathogens 2:66, 2006).

[0007] TNF-α expression is induced by a variety of bacteria, parasites, and viruses as the first line of defense against host infection. Viruses (e.g., Epstein-Barr virus) encode receptors and proteins that even enhance TNF-α and TNF-α signaling (Liebowitz, New Engl. J. Med. 338:1461-1463, 1998; Guasparri et al., Blood 111:3813-3821, 2008; Wang et al., Cell 43:831-840, 1985). Alternatively, various viruses have been shown to express proteins that suppress TNF-α signaling activity and its function in the host (Rahman et al., PloS Pathogens 2:66, 2006). There is also evidence suggesting that viral infections (e.g., Epstein-Barr virus infection) can cause autoimmune diseases (Sairenji et al., Diabetologia 34:33-39, 1991). [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Carswell et al., Proc. Natl. Acad. Sci. USA 72:3666-3670, 1975. [Non-Patent Document 2] Loetscher et al., Cell 62:351, 1990. [Non-Patent Document 3] Rahman et al., PloS Pathogens 2:66, 2006. [Non-Patent Document 4] Liebowitz, New Engl. J. Med. 338:1461-1463, 1998 [Non-Patent Document 5] Guasparri et al., Blood 111:3813-3821, 2008. [Non-Patent Document 6] Wang et al., Cell 43:831-840, 1985. [Non-Patent Document 7] Rahman et al., PloS Pathogens 2:66, 2006. [Non-Patent Document 8] Sairenji et al., Diabetologia 34:33-39, 1991. [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] While many researched therapies for autoimmune diseases have demonstrated some pain-relieving or even therapeutic properties, these therapies cannot consistently produce beneficial outcomes for all subjects with autoimmune diseases (e.g., type 1 diabetes). Therefore, there remains a need for methods to accurately identify subjects likely to respond to a therapy or a series of therapies before treating them, as well as for therapies to treat those identified as likely to respond. [Means for solving the problem]

[0010] Summary of the Invention In a first embodiment, the present invention provides a method for determining the likelihood that a subject with an autoimmune disease will respond to treatment with tumor necrosis factor-α (TNF-α) receptor II (TNFR2) activator. This method involves the following steps: (i) CD8 derived from the above subject + The steps of contacting an in vitro biological sample containing a population of T cells with a composition containing a TNFR2 activator; and (ii) Autoreactive CD8 in the above group + A step of measuring the CD8 protein density on the surface of T cells; This includes, and here the standard CD8 + The above-mentioned autoreactive CD8 compared to T cells + A decrease in CD8 protein density on the surface of T cells indicates that the subject is more responsive to treatment. In some embodiments, reference CD8 +T cells are derived from a reference sample from a subject with an autoimmune disease who has not been treated or prior-treated with TNFR2 activator. In other embodiments, reference CD8 + T cells are derived from a reference sample from a healthy subject. In one embodiment, the measurement is performed using an anti-CD8 antibody which may be conjugated with a fluorescent dye. In certain embodiments, the biological sample is incubated with dasatinib before contact (e.g., the biological sample is incubated with dasatinib for at least 4 hours and / or the biological sample is incubated with dasatinib for up to 48 hours).

[0011] In some embodiments of the method of the present invention, autoimmune diseases include type 1 diabetes, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, Behçet's disease, bullous pemphigoid, cardiomyopathy, celiac plue dermatitis, chronic fatigue immunodeficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, scarring pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, systemic lupus erythematosus, ulcerative colitis, psoriatic arthritis, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hypothyroidism, and idiopathic disease. These include pulmonary fibrosis, idiopathic leptopenic purpura (ITP), IgA nephropathy, juvenile arthritis, lichen planus, lupus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, Stiffman syndrome, Devic's disease, Takayasu's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, Wegener's granulomatosis, or autoimmune neuropathy. In certain embodiments, autoimmune diseases include type 1 diabetes, celiac plue dermatitis, Crohn's disease, Graves' disease, hypothyroidism, lupus, multiple sclerosis, psoriasis, rheumatoid arthritis, sarcoidosis, Sjögren's syndrome, or ulcerative colitis. In certain embodiments, autoimmune neuropathy includes autoimmune-mediated injury to neurons, autoimmune Alzheimer's disease, autoimmune Parkinson's disease, or autoimmune-mediated amyotrophic lateral sclerosis.

[0012] In a second embodiment, the present invention provides a method for determining the likelihood of a subject with type 1 diabetes responding to treatment with tumor necrosis factor-α (TNF-α) receptor II (TNFR2) activator. The method comprises the step of contacting an in vitro biological sample derived from the subject with a device capable of detecting C peptide in the sample, where a detectable level of C peptide in the sample indicates that the subject is likely to respond to treatment, and a substantially undetectable level of C peptide indicates that the subject is unlikely to respond to treatment. In some embodiments, a substantially undetectable level of C peptide indicates that the subject should be excluded from treatment. In some embodiments, a substantially undetectable level is a C peptide level of less than about 1.5 pmol / L (e.g., less than about 1.0 pmol / L). In other embodiments, a detectable level is a C peptide level greater than about 1.5 pmol / L. In specific embodiments, a subject is identified as likely to respond to treatment if the C peptide concentration is in the range of about 1.5 pmol / L to about 4.0 pmol / L.

[0013] In one embodiment of this aspect of the present invention, the contact is made before treatment of the subject. The contact is made in the following steps: (i) A step of binding the C peptide in the sample to the immobilized capture agent by bringing the sample into contact with a device having an immobilized capture agent on its surface; (ii) The step of bringing the above surface into contact with the binding detection agent to bind the C peptide to the binding detection agent; and (iii) A step of measuring the concentration of C peptide in the sample using the above-mentioned binding detection agent. Includes.

[0014] In some embodiments, the binding detection agent comprises a peroxidase enzyme. In certain embodiments, the measurement comprises the step of contacting the surface with a solution containing hydrogen peroxide and a peroxidase substrate.

[0015] In some embodiments of this aspect of the present invention, the method of the present invention further includes the step of measuring a reference HbA1c level in a blood sample derived from the subject before initiating treatment. The method is as follows: (i) A step of measuring the HbA1c level in a blood sample taken from the subject after treatment, (ii) The step of comparing the above HbA1c level with a reference HbA1c level, (iii) If the above HbA1c level is equal to or greater than the reference HbA1c level, the step of identifying the subject as requiring repeated treatment with one or more TNFR2 activators. Includes.

[0016] In certain embodiments of the present invention, a blood sample is taken from the subject at least six months after treatment (for example, at least one year, at least two years, at least three years, or at least five years after treatment).

[0017] In certain embodiments of any aspect of the present invention, the sample comprises blood, blood components, or urine. In some embodiments, the blood component is serum or plasma. In other embodiments, the sample comprises urine.

[0018] In some embodiments of any aspect of the present invention, the TNFR2 activator is Bacillus calmette-Guélain (BCG), complete Freund's adjuvant, TNF-α, TNF-α receptor II agonist, TNF-α mutein, interleukin-1, interleukin-2, tissue plasminogen factor, lipopolysaccharide (LPS), lymphotoxin, or kaketin. In a particular embodiment, the TNFR2 activator is BCG. In a particular embodiment, the autoimmune disease is type 1 diabetes mellitus, and the above method is used in combination with one or more methods of a second aspect of the present invention.

[0019] In a third embodiment, the present invention provides a pharmaceutical composition comprising one or more TNFR2 activators for use in the treatment of autoimmune diseases in subjects diagnosed as highly responsive to treatment. In certain embodiments, a sample derived from a subject contains a population of T cells having a reduced surface CD8 protein density compared to the surface CD8 protein density of reference T cells in a sample derived from a reference subject (e.g., a healthy subject, or a subject with an autoimmune disease and not treated or pre-treated with one or more TNFR2 activators) after exposure to a TNFR2 activator. In some embodiments, the subject is identified as highly responsive to treatment by a method according to a first embodiment of the present invention.

[0020] In some embodiments of this aspect of the present invention, autoimmune diseases include type 1 diabetes, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, Behçet's disease, bullous pemphigoid, cardiomyopathy, celiac plue dermatitis, chronic fatigue immunodeficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, scarring pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, systemic lupus erythematosus, ulcerative colitis, psoriatic arthritis, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hypothyroidism, and other specific conditions. These include pulmonary fibrosis, idiopathic leptopenic purpura (ITP), IgA nephropathy, juvenile arthritis, lichen planus, lupus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, Stiffman syndrome, Devic's disease, Takayasu's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, Wegener's granulomatosis, or autoimmune neuropathy. In some embodiments, autoimmune diseases are type 1 diabetes, celiac plue dermatitis, Crohn's disease, Graves' disease, hypothyroidism, lupus, multiple sclerosis, psoriasis, rheumatoid arthritis, sarcoidosis, Sjögren's syndrome, or ulcerative colitis. In certain embodiments, autoimmune neuropathy is autoimmune-mediated injury to neurons, autoimmune Alzheimer's disease, autoimmune Parkinson's disease, or autoimmune-mediated amyotrophic lateral sclerosis.

[0021] In a fourth embodiment, the present invention provides a pharmaceutical composition comprising one or more TNFR2 activators for use in the treatment of type 1 diabetes in subjects identified as likely to respond to treatment prior to treatment by determining the level of C peptide in an in vitro sample derived from the subject (where substantially undetectable C peptide levels indicate that the subject is unlikely to respond to treatment, and detectable C peptide levels indicate that the subject is likely to respond to treatment). In certain embodiments, substantially undetectable C peptide levels indicate that the subject should be excluded from treatment. In some embodiments, a substantially undetectable level is a C peptide level of less than 1.5 pmol / L. In other embodiments, a detectable level is a C peptide level not exceeding 1.5 pmol / L. In one embodiment, if a urine sample derived from the subject shows a C peptide-to-creatinine ratio of less than about 4.0 pmol / mmol, the subject is excluded from treatment. In yet another embodiment, if a urine sample derived from the subject shows a C peptide-to-creatinine ratio of about 4.0 pmol / mmol or more, the subject is identified as likely to respond to treatment. In certain embodiments, subjects are identified as being more likely to respond to treatment by the method of the second embodiment.

[0022] In some embodiments of this model, the pharmaceutical composition is characterized by a reduction of at least 0.1% in the subject's HbA1c level within approximately 4 years after administration of the composition. In other embodiments, the pharmaceutical composition is characterized by a reduction of at least 0.1% in the subject's HbA1c level within approximately 3 years after administration of the composition. In specific embodiments, the composition is intended for use in the second or subsequent treatment of type 1 diabetes in a subject, where the subject's HbA1c level is increased or remains unchanged compared to the subject's HbA1c level before the previous treatment. In certain embodiments, the subject is identified as requiring repeated treatment with one or more TNFR2 activators by the method of the second embodiment. In some embodiments, the subject is a human. In other embodiments, the subject is a long-term diabetic patient.

[0023] In some embodiments of some aspects of the present invention, one or more TNFR2 activators are selected from Bacillus Calmette-Guerin (BCG), complete Freund's adjuvant, TNF-α, TNF-α receptor II agonist, TNF-α mutant, interleukin-1, interleukin-2, tissue plasminogen factor, lipopolysaccharide (LPS), lymphotoxin and kakectin. In certain embodiments, the one or more TNFR2 activators is BCG. In certain embodiments, the composition is 2x10 6 CFU or more per BCG dose (e.g., 2.3x10 6 CFU per BCG dose). In specific embodiments, the composition is less than 4x10 6 CFU per BCG dose. In some embodiments, the composition comprises lyophilized BCG. In certain embodiments, the composition comprises an aqueous saline solution of BCG.

[0024] In certain embodiments of some aspects of the present invention, the composition is administered to a subject one or more times (e.g., two or more times, e.g., two times). In some embodiments, at least two administrations of the composition are spaced at least two weeks apart. In other embodiments, at least two administrations of the composition are spaced at least four weeks apart.

[0025] In certain embodiments of some aspects of the present invention, the composition is formulated for administration by a route selected from intradermal, intramuscular, parenteral, intravenous, intraarterial, intracranial, subcutaneous, intraorbital, intraventricular, intraspinal, intraperitoneal and intranasal. In specific embodiments, the composition is formulated for intradermal administration. In certain embodiments, the composition is formulated for administration as an aqueous saline solution. In some embodiments, the above solution has a volume of less than about 0.2 cc per dose (e.g., a volume of 0.1 cc per dose). In some embodiments, the pharmaceutical composition is formulated for separate administration of two or more TNFR2 activators. In other embodiments, the pharmaceutical composition is formulated for combined administration of two or more TNFR2 activators.

[0026] In some embodiments of the pharmaceutical compositions of the present invention, the pharmaceutical compositions are characterized by their ability to induce TNF-α expression in a target. In certain embodiments, the pharmaceutical compositions are characterized by their ability to induce TNF-α expression in the target's autoreactive immune cells (e.g., autoreactive CD8). + The present invention is characterized by its ability to induce activation of the NF-κB pathway in T cells. In a specific embodiment, the present invention is characterized by its ability to induce activation of the NF-κB pathway in autoreactive immune cells (e.g., autoreactive CD8 cells) in the target. + The present invention is characterized by causing the death of regulatory T cells (e.g., regulatory CD4 cells) in a target. In one embodiment, the present invention is characterized by the composition causing the death of regulatory T cells (e.g., regulatory CD4 cells) in a target. + It is characterized by causing the proliferation of T cells.

[0027] In one embodiment of any aspect of the present invention, the pharmaceutical composition is characterized in that the composition prevents complications arising from hyperglycemia in a subject. In some embodiments, complications arising from hyperglycemia are selected from renal impairment, neuropathy, cardiovascular injury, retinal injury, foot injury, leg injury, cardiac injury, and ketoacidosis. In specific embodiments, the composition comprises one or more pharmaceutically acceptable carriers or excipients.

[0028] definition As used herein, "approximately" means a value within ±10% of the given value.

[0029] As used herein, “antibody” means a whole antibody or immunoglobulin and any antigen-binding fragment or single chain thereof. As used herein, antibodies may be mammalian (e.g., human or mouse) antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, synthetically produced antibodies, or naturally isolated antibodies. In most mammals (including humans), a whole antibody has at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain has a heavy chain variable region (V in this specification). H It consists of a heavy chain constant region (abbreviated as C) and a heavy chain constant region. The heavy chain constant region consists of three domains CH 1. C H 2 and C H 3 and C H 1 and C H It consists of two hinge regions. Each light chain has a light chain variable region (V in this specification). L It consists of a domain C (abbreviated as C) and a light chain steady region. The light chain steady region is one domain C L It consists of V. H Region and V L The region can be further subdivided into highly variable regions called complementary determination regions (CDRs), which are divided by more conservative regions called framework regions (FRs). H and V L It consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors (including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system). The antibodies of the present invention encompass all known forms of antibodies and other protein scaffolds having antibody-like properties. For example, an antibody may be a monoclonal antibody, a polyclonal antibody, a human antibody, a humanized antibody, a bispecific antibody, a monovalent antibody, a chimeric antibody, or a protein scaffold having antibody-like properties (e.g., fibronectin or ankyrin repeat). Antibodies may exhibit one of the following isotypes: IgG (e.g., IgG1, IgG2, IgG3, and IgG4), IgM, IgA (e.g., IgA1, IgA2, and IgAsec), IgD, or IgE.

[0030] As used herein, “diabetic patient” means a person diagnosed with type 1 diabetes. In particular, a long-term diabetic patient is a person who has had type 1 diabetes for at least approximately 5 years (for example, at least approximately 6 years, at least approximately 7 years, at least approximately 8 years, at least approximately 9 years, at least 10 years, at least 11 years, at least approximately 12 years, at least approximately 13 years, or at least approximately 14 years) since the onset of type 1 diabetes.

[0031] "Hox11" as used herein + Splenocytes express the Hox11 gene and are pluripotent CD45 cells found in the spleen. - It means cell.

[0032] As used herein, “immune cells” means any cells involved in the generation, regulation, or effect of the acquired or innate immune system. Examples of immune cells include T cells (e.g., CD4). + Cells or CD8 + Examples include cells, B cells, natural killer (NK) cells, macrophages, monocytes and dendritic cells, and neutrophils.

[0033] As used herein, "mutein" refers to a polypeptide whose amino acid sequence differs by at least one amino acid. For example, a mutein may have an amino acid sequence that has more than 90% but less than 100% sequence identity with respect to the amino acid sequence of a reference polypeptide.

[0034] In this specification, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" as used interchangeably means any component other than the compounds described herein (e.g., a vehicle capable of suspending or dissolving the active compound) that has non-toxic and non-inflammatory properties in the patient. Examples of carriers and excipients include: anti-adhesion agents, antioxidants, binders, coating agents, compression aids, disintegrants, pigments (colorants), emollients, emulsifiers, fillers (diluents), film-forming agents or film coating agents, flavorings, fragrances, lubricants (flow enhancers), preservatives, printing inks, adsorbents, suspending agents or dispersants, sweeteners or hydration water. Examples of carriers and excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, dibasic calcium phosphate, calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycosides. Polyvinylpyrrolidone, povidone, pre-gelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, xylitol, water, phosphate-buffered saline (PBS), acetate-buffered saline (ABS), Ringer's solution, dextrose, glycerol, ethanol, etc., and combinations thereof.

[0035] As used herein, “sample” means any specimen taken from a subject (e.g., blood, blood components (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g., placenta or skin), pancreatic juice, chorionic villi samples, and cells). Preferably, the sample is blood, blood components (e.g., serum or plasma), or urine.

[0036] In this specification, the terms “subject” or “patient” as used interchangeably mean any animal, such as a mammal (e.g., human). Subjects treated with the pharmaceutical compositions described herein may be subjects diagnosed by a physician with such a condition (e.g., an autoimmune disease such as type 1 diabetes) or subjects at risk of developing such a condition. Diagnosis can be made by any technique or method known in the art. Those skilled in the art will understand that a subject could be diagnosed with an autoimmune disease (e.g., type 1 diabetes) using standard tests or examinations, or could be identified as a high-risk subject by the presence of one or more risk factors without performing any tests. For type 1 diabetes, such risk factors include, for example, autoreactive T cells (e.g., autoreactive CD8 + Examples include the presence of T cells, a fasting plasma glucose level of at least 6.1 mmol / L, a plasma glucose level of at least 11.1 mmol / L two hours after a 75 g oral glucose load, or a decrease in serum C-peptide levels. Prior to treatment with the pharmaceutical composition of the present invention, subjects with autoimmune diseases (e.g., type 1 diabetes) may be subjected to diagnostic tests described herein to determine whether the subject is likely to respond to or unlikely to respond to treatment with the pharmaceutical composition of the present invention.

[0037] As used herein, “substantially undetectable C-peptide levels” means that a sample derived from a subject may have a C-peptide level of less than approximately 1.5 pmol / L (e.g., less than approximately 1.0 pmol / L, or even less than approximately 0.5 pmol / L). The C-peptide level corresponding to a substantially undetectable C-peptide level does not depend on the feeding status of the subject at the time the sample was obtained. For example, the sample may be derived from a fasted subject or from a subject that has been stimulated, such as by a mixed food tolerance test or a glucagon test.

[0038] In this specification, the terms "tumor necrosis factor-α (TNF-α) inducer" or "TNF-α inducer" as used interchangeably mean a composition or molecule that induces the expression of tumor necrosis factor-α (TNF-α) in vivo or in vitro. The TNF-α inducer may be Bacillus calmette-Guélain (BCG), complete Freund's adjuvant, interleukin-1, interleukin-2, tissue plasminogen factor, lipopolysaccharide (LPS), lymphotoxin, or catechin. Preferably, the TNF-α inducer is BCG.

[0039] In this specification, the terms "TNF-α receptor II (TNFR2) agonist" or "TNFR2 agonist," as used interchangeably, mean a composition or molecule that activates TNFR2 upon binding in vivo or in vitro. Examples of TNFR2 agonists include TNFR2 agonist antibodies and TNF mutains that specifically bind to and activate TNFR2.

[0040] In this specification, the terms "TNF-α receptor II (TNFR2) activator" or "TNFR2 activator" are interchangeable and refer to compositions that directly or indirectly activate TNFR2 in vivo or in vitro. TNFR2 activators can directly activate TNFR2, for example, by binding to TNFR2, or indirectly by inducing TNF-α expression, for example. TNFR2 activators encompass TNF-α, TNF-α inducers, and TNFR2 agonists. [Brief explanation of the drawing]

[0041] [Figure 1A] Figure 1A is a set of three graphs presenting time-series data showing the appearance of EBV-specific T cells in subjects 1, 2, and 3. In each graph, time point 0 represents the estimated occurrence of EBV infection, based on the timing of the appearance of antibodies against EBV. [Figure 1B] Figure 1B is a set of three dot plots showing the results of flow cytometry analysis of a sample containing EBV-specific autoreactive CD8+ T cells and a negative control, stained with tetramers. The levels of nonspecific tetramers bound by CD8+ T cells were determined using tetramers loaded with unrelated peptide sequences (negative control, left panel). The center and right panels show examples of positive staining using EBV-specific tetramers. [Figure 2] Figure 2 is a set of graphs showing the time course of C-peptide levels in three EBV-infected subjects compared to a reference population of subjects with type 1 diabetes without active EBV infection. Subjects 1 and 2 showed a statistically significant transient increase in C-peptide compared to the reference population (p<0.04 and p=0.00134, respectively). Subject 3 did not show an increase in C-peptide levels. [Figure 3A]Figure 3A is a set of graphs showing the increased presence of circulating insulin B autoreactive T cells in type 1 diabetic subjects after EBV infection, quantifying the presence of insulin B autoreactive T cells and comparing them to the concentration of autoreactive T cells in uninfected type 1 diabetic subjects. The background fluorescence of peripheral T cells from the same subjects, stained with unrelated peptides including a class I reagent, was 0.2%. [Figure 3B] Figure 3B shows a set of flow cytometry histograms from samples derived from subjects with or without EBV infection and type 1 diabetes, quantifying CD8 protein density on the surface of T cells. The decrease in CD8 protein density on the surface of T cells was specific to newly emerging insulin B autoreactive T cells in EBV-infected subjects. Long-term type 1 diabetes patients without EBV infection had fewer peripheral insulin B autoreactive T cells, but their CD8 protein density on the surface of T cells was within the normal range. Similarly, EBV-specific T cells from EBV-infected subjects also had normal density of CD8 protein. [Modes for carrying out the invention]

[0042] Detailed explanation The inventors have found a method for determining whether a subject with an autoimmune disease is likely to respond to or unlikely to respond to treatment with TNF-α receptor II (TNFR2) activators. According to the method of the present invention, autoreactive CD8 proteins in a sample from a subject with an autoimmune disease that show a decrease in surface CD8 protein density are those exposed in vivo or in vitro to one or more TNF-α receptor II (TNFR2) activators. + The presence of a T cell population may indicate that the subject is more likely to respond to treatment with one or more TNFR2 activators. Autoreactive CD8 exposed to one or more TNFR2 activators. + A decrease in CD8 protein density on the surface of T cells is a reference CD8 + This may be due to a decrease in the CD8 protein density on the surface of T cells. Reference CD8+ T cells may be derived from samples of subjects with autoimmune diseases who have not been treated or pre-treated in vivo or in vitro with TNFR2 activators. Alternatively, reference CD8 + T cells may be derived from samples from healthy subjects. According to the method of the present invention, CD8 + The CD8 protein density on the surface of T cells can be determined using an anti-CD8 antibody.

[0043] The inventors have also found a method for distinguishing between subjects with type 1 diabetes who are responsive to treatment with TNF-α receptor II (TNFR2) activators and subjects with type 1 diabetes who are not responsive to treatment with TNFR2 activators. The inventors have found that the C-peptide level in a subject can be used to assess the likelihood that the subject will respond to treatment, particularly treatment with a TNFR2 activator. Subjects with type 1 diabetes who have detectable levels of C-peptide (e.g., levels of about 1.5 pmol / L or higher) are responsive to treatment with a pharmaceutical composition containing a TNFR2 activator. In particular, the response to treatment with a TNFR2 activator can be assessed by detecting an increase in C-peptide levels in the subject after treatment with a TNFR2 activator. On the other hand, subjects with type 1 diabetes who have substantially undetectable levels of C-peptide (e.g., levels of less than about 1.5 pmol / L) are not responsive to treatment with a pharmaceutical composition containing a TNFR2 activator (for example, the subject may be excluded from such treatment, or TNFR2 therapy may be replaced with cell therapy (e.g., pluripotent cells (e.g., Hox11)). + (May be administered in combination with cell-based therapies.)

[0044] C peptide levels can be assayed using samples from the target organism (e.g., blood, blood components (serum or plasma), or urine). C peptide levels can be measured using any method known in the art, for example, an enzyme-linked immunosorbent assay (ELISA) as described in Thermo Scientific Pierce Assay Development Technical Handbook, 2nd edition, 2011 (disclosures of this document are incorporated herein by reference).

[0045] For example, the use of TNFR2 activators in the treatment of type 1 diabetic patients with detectable C-peptide levels is predicted based on the finding that if damage to β-islet cells in the pancreas can be reduced (e.g., by reducing the number of autoreactive T cells that target β-islet cells), these cells can be repaired or regenerated over time. Subjects with type 1 diabetes with substantially undetectable C-peptide levels, unlike subjects with detectable C-peptide levels, are unable to repair or regenerate β-islet cells in the pancreas. This is likely due to the loss of many or substantially all β-islet cells as a result of the disease (e.g., subjects with substantially undetectable C-peptide levels are likely to have an insufficient number of β-islet cells remaining in their bodies, and therefore may be unable to establish normal blood glucose even in the absence of cytotoxicity by autoreactive immune cells). Furthermore, the repair or regeneration of β-islet cells depends on the presence of pluripotent cells (e.g., Hox11) in the subject that can provide a cell source capable of giving rise to various cell types (including β-islet cells). + This may be related to the differentiation potential of splenic cells. Substantially undetectable C-peptide levels in the subject indicate that the subject has a sufficient number of these pluripotent cells (e.g., Hox11) to repair or regenerate β-islet cells and establish euglycemia. + This could also indicate a deficiency of splenic cells.

[0046] The beneficial activity of TNFR2 activator in subjects with type 1 diabetes who have detectable C peptide levels is linked to their in vivo autoreactive CD8+ Related to its ability to kill T cells (see, e.g., Ban et al., Proc. Nat. Acad. Sci, USA, 105:13644-13649, 2008 (this document is incorporated herein by reference)), this ability reduces or minimizes tissue damage caused by these cells (e.g., loss of β-islet cells). TNFR2 activators also have beneficial regulatory properties that modulate the inflammatory components of disease in vivo. + It also promotes T cell proliferation. While not adhering to any particular theory, TNFR2 agonism is thought to activate intracellular NF-κB signaling, which induces apoptosis in autoreactive T cells, thereby treating type 1 diabetes in subjects (e.g., humans) treated with TNFR2 activators.

[0047] The present invention's diagnostic method CD8 surface protein assay The present invention is characterized by a method for identifying subjects (e.g., humans) with autoimmune diseases who are likely to respond to treatment with one or more TNFR2 activators. In some embodiments, the method involves the following steps: (i) one or more CD8s derived from the subject. + (ii) a step of contacting an in vitro biological sample containing T cells (e.g., blood) with a composition containing TNFR2 activator, and (ii) an anti-CD8 antibody to autoreactive CD8 + Includes a step of detecting T cells. Reference CD8 + One or more autoreactive CD8 cells compared to T cells + A decrease in CD8 protein density on the surface of T cells indicates that the subject is more responsive to treatment. Reference CD8 + T cells (e.g. autoreactive CD8 + The T cells may be derived from samples of subjects with autoimmune diseases who have not been treated or pre-treated in vivo or in vitro with TNFR2 activators. Alternatively, reference CD8 + T cells (e.g., non-autoreactive CD8 +The T cells may be derived from samples from healthy subjects.

[0048] Autoreactive CD8 + For the analysis of CD8 protein density on the surface of T cells, one or more self-reactive CD8 + Samples containing T cells (e.g., blood) can be obtained from subjects with autoimmune diseases. The samples can be stored with a tyrosine kinase inhibitor that metabolically "freezes" the cells in the sample, such as dasatinib (Axon Medchem BV, Groningen, Netherlands) (see Lissina et al., J. Immunol. Methods, 340:11-24, 2009 (this document is incorporated herein by reference)). The final concentration of dasatinib in the sample may be at least about 10 nM (e.g., at least 50 nM, at least about 100 nM, at least about 200 nM, at least about 500 nM, or at least about 1 μM). CD8 + To preserve T cells, a sample containing these cells and a tyrosine kinase inhibitor can be incubated for at least approximately 4 hours (e.g., at least approximately 6 hours, at least approximately 8 hours, at least approximately 10 hours, at least approximately 12 hours, or at least approximately 14 hours) and up to approximately 48 hours (e.g., up to approximately 40 hours, up to approximately 36 hours, up to approximately 32 hours, up to approximately 28 hours, up to approximately 24 hours, or up to approximately 20 hours) before determining the cell surface CD8 protein density using the cells in the sample. Dasatinib is a metabolic inhibitor that does not alter the cell surface structure. Therefore, cells preserved with dasatinib can be accurately stained even after being stored for 4 to 48 hours.

[0049] Samples (whether fresh or stored with a tyrosine kinase inhibitor (e.g., dasatinib)) can be analyzed, for example, by contacting the sample with an anti-CD8 antibody conjugated with any fluorescent dye known in the art. Non-limiting examples of fluorescent dyes include FITC, RD1, allophycocyanin (APC), CF® dye (Biotium, Hayward, California), BODIPY (Invitrogen®, Life Technologies, Carlsbad, California), Alexa Fluor® (Invitrogen®, Life Technologies, Carlsbad, California), DyLight fluorescent labeling (Protein Biology Products, Thermo Scientific Pierce, Rockford, Illinois), ATTO (ATTO-TEC GmbH, Siegen, Germany), Fluo probe (Interchim SA, Montorcon, France), and Abberier probe (Abberior GmbH, Göttingen, Germany). Using methods such as flow cytometry, fluorescence derived from a fluorescent dye conjugated to an anti-CD8 antibody bound to the CD8 protein on the surface of autoreactive T cells can be detected. The intensity of the fluorescence provides a quantitative measurement of the CD8 protein density on the cell surface.

[0050] Autoreactive CD8 after exposure to TNFR2 activator + Detection of decreased cell surface CD8 protein density in T cell populations indicates that the subjects are more likely to respond to treatment of autoimmune diseases with TNFR2 activators. Reference CD8 + Autoreactive CD8 cells compared to T cells + A decrease in the cell surface CD8 protein density on T cells indicates that self-reactive CD8 + This shows that T cells undergo apoptosis as a result of treatment with TNFR2 activator. Reference CD8 + T cells are non-self-reactive CD8 derived from healthy subjects. + It can be a T cell. Alternatively, a reference CD8 +T cells are autoreactive CD8 cells that have not been exposed to TNFR2 activator in vivo or in vitro. + It may be T cells. Thus, the decrease in CD8 cell surface density detected using the above assay indicates that the target is TNFR2 activator-mediated in vivo therapy (autoreactive CD8 during treatment). + This indicates that the cells are more responsive to (which is expected to induce T cell death) and that viable autoreactive CD8 cells are readily available. + The absence of T cells may reduce the cellular damage caused by these cells and potentially promote the regeneration of damaged tissue, which could improve the subject's health.

[0051] C-peptide assay The present invention also features a method for identifying subjects (e.g., humans) with type 1 diabetes who are likely to respond to treatment with one or more TNFR2 activators. This method includes the step of contacting an in vitro biological sample (e.g., blood, blood components (e.g., serum or plasma), or urine) derived from a subject (e.g., human) with a device capable of detecting C peptides in the sample. Detection of C peptide levels in a sample greater than about 1.5 pmol / L (e.g., greater than about 2.5 pmol / L) indicates that the subject (e.g., human) is likely to respond to treatment. Detection of C peptide levels in a sample less than about 1.5 pmol / L indicates that the subject is less likely to respond to treatment (e.g., the subject may be excluded from such treatment, or TNFR2 therapy may be replaced with cell therapy (e.g., pluripotent cells (e.g., Hox11)). +It may be administered in combination with splenocytes. Detection of C peptide levels in a sample in the range of approximately 1.5 pmol / L to approximately 900 pmol / L (e.g., approximately 500 pmol / L, approximately 200 pmol / L, approximately 100 pmol / L, approximately 50 pmol / L, or approximately 4.0 pmol / L) indicates that the subject is likely to respond to treatment. According to the method of the present invention, the subject may be a long-term diabetic patient. C peptide levels greater than 1.5 pmol / L indicate that the subject is likely to respond to treatment with TNFR2 activator. C peptide levels can be measured in a sample derived from a fasted subject (fasting C peptide level) or in a sample derived from a subject stimulated by a mixed food tolerance test or a glucagon test. Treatment is likely to be autoreactive CD8 + If it induces T cell death and / or proliferation or regeneration of endogenous β-islet cells, thereby resulting in increased insulin levels and decreased mean plasma glucose levels compared to pre-treatment levels (e.g., establishing euglycemia), the subject will respond to treatment with TNFR2 activators.

[0052] According to the method of the present invention, the contact step can be performed by: (i) conjugating the C peptide in the sample to the immobilized capture agent (e.g., an antibody that binds to C peptide) by contacting the sample with a device having an immobilized capture agent on its surface; (ii) conjugating the C peptide to the conjugation detection agent (e.g., an antibody that specifically binds to C peptide) by contacting the surface with the conjugation detection agent; and (iii) detecting the level of C peptide in the sample using the conjugation detection agent. The conjugation detection agent may be specific to C peptide. The conjugation detection agent can be conjugated to a peroxidase enzyme, which can be detected by contacting the surface of the device with a hydrogen peroxide solution. The hydrogen peroxide solution may further contain a peroxidase substrate. According to the method of the present invention, any peroxidase substrate known in the art can be used. Some common peroxidase substrates include 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 3,3',5,5'-tetramethylbenzidine (TMB), o-phenylenediamine dihydrochloride (OPD), 10-acetyl-3,7-dihydroxyphenoxazine (ADHP), luminol, p-nitrophenyl disodium phosphate (PNPP), o-nitrophenyl-β-galactopyranosidase (ONPG), QuantaBlu fluorescence-emitting peroxidase substrate (Thermo Scientific Pierce, Protein Biology Products, Rockford, Illinois), QuantaRed enhanced chemifluorescence peroxidase substrate (Thermo Scientific Pierce, Protein Biology Products, Rockford, Illinois), SuperSignal ELISA femto highest sensitivity substrate (Thermo Scientific Pierce, Protein Biology Products, Rockford, Illinois), and SuperSignal ELISA pico chemiluminescent substrate (Thermo Scientific This is a protein biology product manufactured by Pierce, Inc. (Rockford, Illinois). The reaction between the peroxidase substrate and peroxidase can be detected by spectrophotometrics.

[0053] Detection of C peptide in a sample derived from the target can be performed using any assay known in the art. For example, the ultra-sensitive C peptide enzyme-conjugated immunosorbent assay (ELISA) (e.g., ELISA manufactured by Mercodia AB (Uppsala, Sweden)) provides a solid-phase two-site enzyme immunoassay for the quantitative determination of C peptide in a sample. The immunoassay is based on a direct sandwich technique in which two monoclonal antibodies are directed to separate antigenic determinants on the C peptide molecule. During incubation, the C peptide in the sample reacts with an anti-C peptide antibody conjugated in a microtitration well. After washing, a peroxidase (e.g., horseradish peroxidase) conjugated to the anti-C peptide antibody is added. After a second incubation and a single washing step, the conjugated conjugate can be detected by the reaction of hydrogen peroxide with 3,3',5,5'-tetramethylbenzidine (TMB). The reaction is stopped by the addition of acid, giving an endpoint read by spectrophotometric analysis. Alternatively, after the second incubation, the bound conjugate can be detected by any other peroxidase substrate known in the art (e.g., the substrates listed above).

[0054] According to the method of the present invention, substantially undetectable levels of C peptide in a sample derived from a subject with type 1 diabetes may indicate that the subject has lost the ability to regenerate β-islet cells, particularly when the C peptide level is lower than about 1.5 pmol / L. Substantially undetectable C peptide levels indicate that the subject has lost the ability to regenerate β-islet cells. + This could also indicate a deficiency of splenic cells.

[0055] The long-term efficacy of TNFR2 activator-mediated treatment of type 1 diabetes can be determined by the subject's ability to regenerate β-islet cells and / or reduce the incidence of hyperglycemia and hypoglycemia compared to the pre-treatment incidence of hyperglycemia and hypoglycemia in the subject (for example, efficacy can be determined by an increase in the period of time the subject is in a normal blood glucose state). The treatment involves providing the subject with the pharmaceutical composition of the present invention (e.g., a composition comprising one or more TNFR2 activators), one or more pluripotent cells (e.g., Hox11). +The procedure includes the step of administering a composition containing splenocytes, or a combination thereof. C peptide levels can be used as a proxy to evaluate the effectiveness of treatment with the pharmaceutical composition of the present invention in a treated subject. A change (e.g., an increase) in C peptide levels in a subject treated with the pharmaceutical composition of the present invention indicates that the subject is responding to the treatment. An increase of about 1% (e.g., about 2%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 100%, about 200%, about 50%, about 100%, about 200%, about 500%, about 100%, about 200%, about 200%, about 500%, about 1000%, about 2000%, about 500%, about 500%, about 500%, about 200%, about 200%, about 500%, about 500%, about 2 A decrease in or lack of change in C-peptide levels in a subject at least one month after treatment of the subject (e.g., at least three months, at least six months, at least one year, at least two years, at least three years, at least four years, at least five years, or at least six years) may indicate that the subject requires repeated administration of the pharmaceutical composition of the present invention. C-peptide levels can be detected at least one month after treatment of the subject (e.g., at least three months, at least six months, at least one year, at least two years, at least three years, at least four years, at least five years, or at least six years). Changes in C-peptide levels can be evaluated in comparison to a reference C-peptide level. The reference C-peptide level may be the C-peptide level detected in a sample taken from the subject before the first (or subsequent) treatment with the pharmaceutical composition of the present invention.

[0056] HbA1c assay The present invention also measures the HbA1c level in a blood sample derived from the subject before, during, or after treatment of the subject, thereby determining whether the subject is treated with TNFR2 activators (alone or with one or more pluripotent cells (e.g., Hox11)). +The method also features a method for determining whether a subject is in a state of receptiveness to or is likely to be receptive in the future to a composition containing splenocytes. The HbA1c level measured before treatment of the subject can be used as a reference HbA1c level. Further measurement of HbA1c levels in blood samples taken from the subject can be performed at least one month (e.g., at least three months, at least six months, at least one year, at least two years, at least three years, at least four years, at least five years, or at least six years) after treatment of the subject. The method of the present invention may further include the steps of comparing the HbA1c level measured after treatment to a reference HbA1c level, and identifying the subject as requiring repeated treatment with the pharmaceutical composition of the present invention if the HbA1c level is equal to or greater than the reference HbA1c level. After administration of one or more TNFR2 activators, HbA1c levels can be measured in samples derived from the subject to determine an endpoint and evaluate the long-term success of the treatment. A decrease of at least approximately 0.1% (e.g., 0.15%, 0.2%, 0.25%, 0.3%, 0.4%, or 0.5%) in HbA1c levels compared to a baseline HbA1c level indicates a high probability of long-term success of treatment. An increase of at least approximately 0.1% (e.g., 0.2%, 0.3%, 0.4%, or 0.5%) in HbA1c levels exceeding the baseline HbA1c level may indicate that one or more repeated treatments with the pharmaceutical composition of the present invention should be administered.

[0057] HbA1c levels in blood samples can be measured by methods known in the art, such as high-performance liquid chromatography, immunoassays, enzyme assays (direct enzyme HbA1c assay, Diazyme Laboratories, Poway, California), capillary electrophoresis (Sebia, Norcross, Georgia), or boronic acid affinity chromatography (Trinity Biotech Plc, Bray, Ireland). Methods for measuring HbA1 in samples derived from subjects are described, for example, in Little et al., Clin. Chem. 54:1277-1282, 2008 (this document is incorporated herein by reference). Standard criteria used to describe HbA1c levels are described, for example, in Goodall, I., Clin. Biochem. Rev. 26:5-20, 2005 (this document is incorporated herein by reference).

[0058] Pharmaceutical composition of the present invention The present invention also relates to the treatment of subjects (e.g., humans) with autoimmune diseases (e.g., type 1 diabetes), and in particular to subjects (e.g., humans) who are more responsive to treatment by one or more diagnostic methods of the present invention (e.g., CD8 + The invention also features pharmaceutical compositions containing one or more TNFR2 activators for use in the treatment of subjects identified as susceptible to T cell death and / or tissue damage due to autoimmune diseases (e.g., β-islet cells in subjects with type 1 diabetes) and / or regeneration.

[0059] A pharmaceutical composition containing one or more TNFR2 activators can be used in the treatment of autoimmune diseases (e.g., type 1 diabetes) in subjects that are likely to respond to treatment. Autoreactive CD8 in samples from subjects with autoimmune diseases compared to CD8 protein density on the surface of reference T cells. +A decrease in CD8 protein density on the surface of T cells indicates that the subject is more responsive to treatment with TNFR2 activators. Reference T cells can be obtained from the subject being treated or from a different subject. Reference T cells may be derived from a healthy subject or from a subject with an autoimmune disease that has not been treated with one or more TNFR2 activators.

[0060] Examples of autoimmune diseases that can be treated with the pharmaceutical compositions of the present invention in subjects identified as likely to respond to treatment by one or more diagnostic methods of the present invention include type 1 diabetes, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, Behçet's disease, bullous pemphigoid, cardiomyopathy, celiac plue dermatitis, chronic fatigue immunodeficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, scarring pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, systemic lupus erythematosus, ulcerative colitis, psoriatic arthritis, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, and Guillain-Barré syndrome. These include Hashimoto's thyroiditis, hypothyroidism, idiopathic pulmonary fibrosis, idiopathic leptopenic purpura (ITP), IgA nephropathy, juvenile arthritis, lichen planus, lupus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, Stiffman syndrome, Devic's disease, Takayasu's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, Wegener's granulomatosis, and autoimmune neuropathy. In particular, the diagnostic method of the present invention can treat subjects with type 1 diabetes, celiac plue dermatitis, Crohn's disease, Graves' disease, hypothyroidism, lupus, multiple sclerosis, psoriasis, rheumatoid arthritis, sarcoidosis, Sjögren's syndrome, or ulcerative colitis, which are identified as being more responsive to the treatment. Autoimmune neuropathy can be an autoimmune-mediated disorder that causes damage to neurons (e.g., motor neurons). Specific autoimmune neuropathy include autoimmune Alzheimer's disease, autoimmune Parkinson's disease, and autoimmune-mediated amyotrophic lateral sclerosis (ALS).

[0061] Substantially undetectable C-peptide levels in samples from subjects with type 1 diabetes (e.g., blood, blood components (e.g., serum or plasma), or urine) indicate that the subject is less responsive to treatment with a pharmaceutical composition containing one or more TNFR2 activators (e.g., the subject may be excluded from such treatment, or TNFR2 therapy may be replaced with cell therapy (e.g., pluripotent cells (e.g., Hox11)). + (May be administered in combination with cells). A detectable C-peptide level (e.g., above about 1.5 pmol / L) in a sample derived from a subject with type 1 diabetes indicates that the subject is likely to respond to treatment with a pharmaceutical composition containing one or more TNFR2 activators. The subject may be a long-term diabetic patient. The compositions of the present invention can be used to treat subjects who have type 1 diabetes and are identified as likely to respond to treatment with TNFR2 activators by any one or more of the methods of the present invention.

[0062] Furthermore, a C-peptide to creatinine ratio of less than approximately 4 pmol / mmol in urine samples from subjects with type 1 diabetes may also indicate that the subject is less responsive to treatment with a pharmaceutical composition containing one or more TNFR2 activators (for example, the subject may be excluded from such treatment, or TNFR2 therapy may be replaced with cell therapy (e.g., pluripotent cells (e.g., Hox11)). +It may be administered in combination with cells. A C-peptide-to-creatinine ratio greater than approximately 4 pmol / mmol in urine samples from subjects with type 1 diabetes may indicate that the subject is likely to respond to treatment with one or more TNFR2 activators. A C-peptide-to-creatinine ratio of approximately 4 pmol / mmol to approximately 11 pmol / mmol in urine samples from subjects with type 1 diabetes may indicate that the subject is likely to respond to treatment with one or more TNFR2 activators. The C-peptide-to-creatinine ratio in urine samples can be determined as described by Besser et al. (Diabetes Care 34:607-609, 2011) (this document is incorporated herein by reference). These subjects identified as likely to respond to treatment with a TNFR2 activator (e.g., BCG) can then be treated with a TNFR2 activator (e.g., BCG) one or more times.

[0063] The pharmaceutical compositions of the present invention may include one or more TNFR2 activators, such as Bacillus calmette-Guélain (BCG), complete Freund's adjuvant, TNF-α, TNF-α receptor II agonists (non-limiting examples of TNFR2 agonists (e.g., antibodies) are described in U.S. Patent Nos. 7,582,313, 8,017,392, and 8,173,129 (these documents are incorporated herein by reference)), TNF-α mutein (non-limiting examples of TNF-α mutein are described in U.S. Patent No. 5,597,899 (this document is incorporated herein by reference)), interleukin-1, interleukin-2, tissue plasminogen factor, lipopolysaccharide (LPS), lymphotoxin, and kekectin. Some of the pharmaceutical compositions of the present invention can induce TNF-α expression in a subject when administered. Other compositions of the present invention can induce autoreactive immune cells (e.g., autoreactive CD8) in a subject when administered. +The composition can specifically activate TNF-α receptor II (as an agonist) in T cells and / or induce activation of the NF-κB pathway. Preferably, administration of the composition of the present invention also induces the death of one or more autoreactive immune cells in the subject. The composition of the present invention also induces the death of regulatory T cells (e.g., regulatory CD4) in the subject. + It may also induce proliferation of T cells. The compositions of the present invention may also reduce or treat hyperglycemia-related complications (e.g., renal impairment, neuropathy, cardiovascular injury, retinal injury, foot injury, leg injury, cardiac injury, or ketoacidosis) in subjects with type 1 diabetes.

[0064] The TNFR2 activator-containing pharmaceutical composition of the present invention can be used to treat type 1 diabetes in a subject (e.g., a human) for a second or subsequent dose if, after the first dose, the HbA1c level in a sample derived from the subject (e.g., blood, blood component (e.g., serum or plasma), or urine) increases or does not change compared to the baseline HbA1c level derived from the subject before the previous treatment. A decrease in the HbA1c level in the treated subject may indicate that the subject is responding to TNFR2 activator therapy (e.g., the subject may not require a second or subsequent dose). A decrease of at least approximately 0.1% (e.g., at least approximately 0.15%, at least approximately 0.2%, at least approximately 0.25%, at least approximately 0.3%, at least approximately 0.4%, at least approximately 0.5%, at least approximately 0.6%, at least approximately 0.7%, at least approximately 0.8%, at least approximately 0.9%, at least approximately 1.0%) in HbA1c levels in a subject within approximately 4 years (e.g., within approximately 3 years) following administration of TNFR2 activator to the subject indicates that the subject is responding to treatment.

[0065] cell therapy The identification of subjects having type 1 diabetes and substantially undetectable C-peptide levels (e.g., C-peptide levels less than about 1.5 pmol / L) by the method of the present invention is made possible by identifying subjects that are β-islet cells or cells capable of regenerating β-islet cells (e.g., pluripotent cells (e.g., Hox11)).+ It may indicate that treatment with spleen cells may be beneficial or necessary. Hox11 + Splenocytes are an endogenous source of cells that can regenerate β-islet cells in vivo in subjects, as described in International Publication No. WO 2005 / 042727 (this document is incorporated herein by reference). Therefore, subjects with type 1 diabetes can be treated with one or more TNFR2 activators to kill autoreactive T cells and further regenerate endogenous β-islet cells (e.g., through proliferation of endogenous β-islet cells) or pluripotent cells (e.g., Hox11). + If regeneration (by differentiation of splenic cells) into β-islet cells is possible, long-term benefits from cell therapy (e.g., within 4 years (e.g., within 3 years)) may be obtained. Other types of pluripotent cells capable of differentiating into β-islet cells in subjects (e.g., pluripotent cells described in U.S. Patent No. 7,432,104 and No. 8,008,075 (these documents are incorporated herein by reference)) may be administered to subjects. Substantially undetectable C-peptide levels (e.g., less than about 1.5 pmol / L) in samples derived from subjects with type 1 diabetes may indicate that the subject has, for example, Hox11 + The splenic cell compartment may have lost its ability to regenerate β-islet cells. In this way, the subject may benefit from the administration of an exogenous source of β-islet cells.

[0066] Non-limiting examples of pluripotent cells that can be administered to subjects requiring pluripotent cells according to the present invention include, for example, the pluripotent cells described in WO 2002 / 059278, WO 2003 / 026584, WO 2005 / 042727, WO 2006 / 074308, WO 2012 / 152717, US 7,432,104, and US 8,008,075 (these documents are incorporated herein by reference). Compositions containing one or more pluripotent cells (e.g., Hox11) + Splenocytes may be administered before, after, or simultaneously with the administration of the TNFR2 activator-containing composition, or the two compositions may be combined for administration in a single dosage form.

[0067] Formulation of the pharmaceutical composition of the present invention The pharmaceutical composition of the present invention, comprising one or more TNFR2 activators, can be formulated for administration via any route, such as intradermal, intramuscular, parenteral, intravenous, intra-arterial, intracranial, subcutaneous, intraorbital, intraventricular, intraspinal, intraperitoneal, or intranasal administration. The composition of the present invention can be formulated for intradermal administration.

[0068] The pharmaceutical compositions of the present invention may contain pharmaceutically acceptable carriers or excipients. Such carriers or excipients can be selected from, for example, water, phosphate-buffered saline (PBS), acetate-buffered saline (ABS), Ringer's solution, dextrose, glycerol, ethanol, and combinations thereof. Furthermore, if desired, the composition for administration to a subject may contain small amounts of auxiliary substances (e.g., wetting agents or emulsifiers) or pH buffers to enhance the efficacy of the composition.

[0069] Similarly, TNFR2 activators can be administered in multiple, varying numbers and / or frequencies. For example, one or more doses of TNFR2 activators (e.g., one, two, three, four, or five or more doses) can be administered daily, weekly, monthly, or annually (e.g., twice daily, once every two weeks, four times a year, twice a year, or three times a year).

[0070] The TNFR2 activator (e.g., BCG)-containing composition of the present invention can be administered once or more (e.g., twice or more (e.g., twice)) to subjects who have type 1 diabetes and are identified as likely to respond to TNFR2 activator therapy by the diagnostic methods described herein. The TNFR2 activator-containing composition can be administered twice or more with intervals of, for example, about one week (e.g., about two weeks, about three weeks, about four weeks, about five weeks, about six weeks, about seven weeks or about eight weeks or longer) between administrations. In some embodiments, administrations of the TNFR2 activator composition can be spaced at least two weeks apart (e.g., four weeks apart).

[0071] The TNFR2 activator can be a TNF-α inducer (e.g., BCG). This pharmaceutical composition can contain more than 2x10 6 CFU per BCG dose (e.g., more than 2.3x10 6 CFU per BCG dose). This composition can contain less than 4x10 6 CFU per BCG dose. This composition can contain approximately 2x10 6 to approximately 2.5x10 6 CFU / BCG dose, approximately 2.5x10 6 to approximately 3x10 6 CFU / BCG dose, approximately 3x10 6 to approximately 3.5x10 6 CFU / BCG dose, or approximately 3.5x10 6 to approximately 4x10 6 CFU / BCG dose. This composition can contain approximately 2x10 6 to approximately 4x10 6 CFU / BCG dose. This composition can contain approximately 2.3x10 6 to approximately 4x10 6 CFU / BCG dose. This composition can contain approximately 2.5x10 6 to approximately 4x10 6 CFU / BCG dose. In one embodiment, the BCG composition can be lyophilized. Alternatively, this composition can contain an aqueous physiological saline solution of BCG. The aqueous physiological saline solution of BCG can be prepared by reconstituting lyophilized BCG in an aqueous physiological saline solution. The solution can have a volume of less than approximately 0.2 cc per dose (e.g., approximately 0.1 cc per dose).

[0072] The compositions of the present invention, comprising one or more pluripotent cells, can be administered after a subject has been identified as unresponsive to treatment with TNFR2 activators. Compositions comprising pluripotent cells, administered to subjects requiring pluripotent cells, can be formulated for administration via any route, e.g., intradermal, intramuscular, parenteral, intravenous, intra-arterial, intracranial, subcutaneous, intraorbital, intraventricular, intraspinal, intraperitoneal, or intranasal administration. Preferably, compositions comprising one or more pluripotent cells are administered parenterally, for example, by injection (e.g., intravenous or intramuscular injection) or surgical transplantation. Compositions comprising pluripotent cells can be administered to subjects requiring them one or more times (e.g., one, two, three, four, or five or more times). These one or more doses can be spaced apart by one week, two weeks, three weeks, four weeks, five weeks, six weeks, two months, six months, one year, 1.5 years, two years, or three years or longer. TNFR2 activator may be administered one week, two weeks, three weeks, four weeks, five weeks, six weeks, two months, six months, one year, one.5 years, two years, or three years or more after administration of the composition containing pluripotent cells. Alternatively, TNFR2 activator may be administered simultaneously with or after administration of the composition containing pluripotent cells via any of the above-mentioned routes (e.g., the routes may be the same or different), or the compositions may be combined for administration in a single dosage form.

[0073] The present invention kit CD8 surface protein assay kit The present invention provides a kit comprising a container for a sample derived from a subject, a preservative (for example, a container containing a final solution of dasatinib (for example, at least about 10 nM (e.g., at least about 50 nM, at least about 100 nM, at least about 200 nM, at least about 500 nM, or at least about 1 μM) of dasatinib in the sample; for example, an amount of dasatinib sufficient to produce a 1 mL solution having at least about 10 mM of dasatinib), or a container containing at least about 1.0 μg to about 25 mg of dasatinib in solid form), an instrument for collecting and / or placing the sample in the container, and instructions for use for collecting and storing the sample. This kit may further include a CD8 antibody conjugated to a fluorescent dye and / or a TNFR2 activator (e.g., Bacillus calmette-Guélain (BCG), complete Freund's adjuvant, TNF-α, TNF-α receptor II agonist, TNF-α mutein, interleukin-1, interleukin-2, tissue plasminogen factor, lipopolysaccharide (LPS), lymphotoxin, or caquetin). The instructions for use in the above kit may also describe how a practitioner (e.g., a physician, nurse, or laboratory assistant) may use a sample derived from the subject to determine whether the subject is likely to respond to treatment with a TNFR2 activator. This kit may further include equipment for collecting samples and transferring them to the above container (e.g., a needle or catheter), and components for maintaining cells at a specified temperature (e.g., approximately 4°C to approximately 27°C), such as a coolant (e.g., an ice pack, dry ice, cooling pouch, or cooling plate). This kit may also include a cooler box or insulating carrier for transporting cell samples, if necessary.

[0074] C-peptide detection kit The present invention also provides a kit comprising a device on which a capture agent (e.g., an antibody that binds to C peptide) is immobilized on its surface, a binding detection agent (e.g., an antibody that selectively binds to C peptide) conjugated to a peroxidase enzyme, a peroxidase substrate, and instructions for use describing how a practitioner (e.g., a physician, nurse, or laboratory assistant) may use the contents of the kit for the analysis of a sample derived from a subject to determine whether the subject is likely to respond to treatment with a TNFR2 activator.

[0075] HbA1c assay kit The present invention also provides a container for storing a sample derived from a subject, and a kit including instructions for use to determine the HbA1c level in the sample and to use this HbA1c level to determine whether the subject requires administration of a TNFR2 activator-containing composition.

[0076] The following examples are intended to illustrate the present invention. These examples are not intended to limit the present invention in any way. [Examples]

[0077] Example 1. Identification of the likelihood of responding to TNFR2 activator therapy in subjects with type 1 diabetes. Prior to treatment with the pharmaceutical composition of the present invention (e.g., TNFR2 activator), a subject (e.g., a human) may be tested to determine whether the subject is likely to respond to the treatment. The test may be performed using a sample taken from the subject (e.g., blood, blood components, or urine). For example, to prepare a serum sample, blood may be collected by venous puncture and allowed to coagulate, and the serum may be separated by centrifugation. To prepare a plasma sample, blood may be collected by venous puncture into a tube containing heparin or EDTA as an anticoagulant; the plasma fraction may then be separated from the sample. To prepare a urine sample, a 24-hour urine sample (without preservatives) may be collected. Cellular debris may be removed from the sample by filtration or centrifugation before testing.

[0078] Next, the prepared sample derived from the subject may be assayed using, for example, the Mercodia Ultra-Sensitive C-Peptide ELISA Kit (Mercodia AB, Uppsala, Sweden) to determine the C-peptide level in the sample derived from the subject. Subjects with measured C-peptide levels lower than approximately 1.5 pmol / L are identified as less responsive to TNFR2 activator therapy. Subjects identified as having substantially undetectable C-peptide levels can be excluded from treatment with TNFR2 activator-containing compositions. In particular, such subjects may have an insufficient number of remaining endogenous β-islet cells or an insufficient amount of pluripotent cells (e.g., Hox11) that have not yet differentiated into β-islet cells. + The subject has splenic cells, and therefore it can be determined that it is unlikely to show recovery or improvement of normal blood glucose after treatment with TNFR2 activator. The determination that the subject has substantially undetectable C peptide levels also indicates that the subject has β-islet cells or pluripotent cells that can differentiate into β-islet cells (e.g., Hox11). + The potential benefits of transplantation of splenocytes may also be demonstrated. Subjects may be judged to be more responsive to treatment with TNFR2 activators if their measured C-peptide levels are greater than approximately 1.5 pmol / L; these subjects may be treated with the TNFR2 activator composition of the present invention, with the expectation that such treatment will result in the regeneration of β-islet cells and / or the restoration or improvement of normal blood glucose levels.

[0079] The test may be repeated one or more times to correct for possible single-measurement variability in the data.

[0080] Example 2. Treatment of type 1 diabetes in humans with the pharmaceutical composition of the present invention. Prior to treatment with a TNFR2 activator (e.g., BCG), a sample (e.g., blood) can be collected from subjects identified in Example 1 as likely to respond to treatment. The sample can be analyzed for HbA1c levels to determine a baseline HbA1c level for the study.

[0081] A pharmaceutical composition containing BCG reconstituted in physiological saline can be administered to a subject at a dose of 1.0 - 2.3x10 6 CFU / dose (volume = 0.1 cc / dose) as two intradermal injections given four weeks apart. After treatment, samples obtained from the subject can be tested using the functional assays (autoreactive CD8 + T cell assay and regulatory CD4 + T cell assay) further described below to monitor the subject over time.

[0082] After the above treatment, one year (e.g., two years, three years, four years, five years or six years) after administration of a TNFR2 activator (e.g., BCG), samples from the subject can be evaluated for HbA1c levels in order to determine an endpoint and confirm long - term success of the treatment. A decrease in HbA1c level of at least about 0.1% (e.g., 0.15%, 0.2%, 0.25%, 0.3%, 0.4% or 0.5%) compared to the baseline HbA1c level indicates long - term success of the treatment.

[0083] The subject can be continuously monitored for changes in HbA1c level. An increase in HbA1c level of at least about 0.1% (e.g., 0.2%, 0.3%, 0.4% or 0.5%) above the baseline HbA1c level may indicate that the pharmaceutical composition of the present invention must be readministered to the subject.

[0084] Immunotherapy 1. T cell assay CD4 + and CD8 + T cells can be isolated from fresh human blood within 1.5 hours of venipuncture using Dynal CD4 positive isolation kit and Dynal CD8 positive isolation kit (Invitrogen, Carlsbad, California). This method is unique in that cells are obtained without using magnetic particles or positive selection by antibodies.

[0085] 2. Autoreactive CD8 in type 1 diabetes+ Detection of T cells As previously described (Verginis et al., Proc. Natl. Acad. Sci. USA 105:3479-3484, 2008), highly purified, high-yield viable CD8 + T cells can be used for tetramer staining. A tetramer is a diagnostic reagent composed of a binding region of a specific HLA class 1 protein having a peptide loaded in an external binding groove. The tetramer is then made fluorescent and functions as a diagnostic reagent that can bind to T cells that are specifically reactive to the presented peptide fragment. For the detection of insulin-reactive T cells, HLA class 1 proteins having the HLVEALYLV fragment can be used. * The tetramer for insulin β10-18 (Beckman Coulter, #T02001) can be used. For the negative control tetramer, the following tetramer reagent can be used: HLA with a sequence for KIFGSLAFL. * 0201 Her-2 / neu (Beckman Coulter, #T02001), breast cancer peptide, HLA without nonspecific peptide fragments * CMV virus HLA-A having the sequence 0201 null (Beckman Coulter, #T01010) and / or NLVPMVATV * Tetramer for 0201 CMGPP65 (Beckman Coulter, #T01009).

[0086] Tetramer reagent staining may be performed after incubation at 26°C for 12 hours, followed by 6 hours at 37°C, and / or after standing at 26°C for 1 hour, followed by 12 hours at 37°C. The cells can then be stained with Sytox-Green (MBL International Co., Woburn, Massachusetts) and / or CD8 antibody (BD Biosciences, San Jose, California). All cells can be stained in the dark at 4°C for 30 minutes, and then washed twice in Hanks buffer containing 2% heat-inactivated bovine serum. An average of 100,000 high-purity CD8 antibodies are obtained. + Analyzing T cells can secure clear data points and enable the detection of rare autoreactive T cells. All cells may be fresh to prevent fixation artifacts and allow for the quantification of dead cells relative to live cells. Cell viability can be quantified using one of two fluorescent stains that label dead cells: Sytox (MBL International Co., Woburn, Massachusetts) or propidium iodide (PI).

[0087] 3. T in type 1 diabetes reg CD4 + Cell detection T REG Two different methods can be used to detect cells. REG Cells have CD4, CD25 bright , and using Foxp3 staining, or CD4, CD25 bright , and CD127 lowIt can be detected by antibody staining. In short, isolated CD4-positive cells can be incubated with CD4-PE-Cy5 (clone RPA-T4) antibody and CD25-PE (clone BC96) antibody at room temperature for 20 minutes. After washing, the cells can be fixed with Foxp3 Fix / Perm solution (Biolegend) at room temperature for 20 minutes. The cells can be washed and permeabilized with Biolegend's Foxp3 Perm buffer at room temperature for 15 minutes. Then, the cells can be stained with Foxp3 Alexa Fluor477 antibody (clone 259D) for 30 minutes. Isotype controls can be performed for each sample before flow cytometry analysis. Alternatively, staining with CD4 antibody (clone RPA-T4, BD Biosciences, San Jose, California) and anti-human CD127 antibody (clone hIL-7R-M21, BD Biosciences) can also be performed to detect T regulatory cells. REG Other methods for detecting cells are described in U.S. Patent Application No. 61 / 763,217 (this document is incorporated herein by reference in its entirety).

[0088] Example 3. CD8 in a sample from a subject with EBV infection + Detection of T cells Materials and methods subject Patients with type 1 diabetes were recruited at Massachusetts General Hospital. Diabetic patients were routinely screened to characterize the disease course and exclude those with potentially interfering medical conditions. During screening, three patients were identified as having long-term type 1 diabetes and having recently developed EBV. In this study, diabetic patients without EBV infection (N=66) were used as the reference population. Blood from all patients and controls was collected in BD Vacutainer® tubes containing EDTA (BD Corporation, Franklin Lake, New Jersey).

[0089] ethics statement This study was approved by the Institutional Review Board of Massachusetts General Hospital (IRB Protocol No. 2001P-001379). Written informed consent was obtained from all blood donors.

[0090] Epitope-specific CD8 + Detection of T cells To detect specific subpopulations of T cells with restricted antigen specificity, commercially available HLA class I reagents loaded with small peptide fragments were used. These commercially available reagents are commonly called tetramers (MBL International, Des Plains, Illinois; formerly Beckman Coulter, Fullerton, California) or dextramers (Immudex, Fairfax, Virginia). The two T cell detection methods differ in the skeletal structure of the detection reagents, but their binding specificity to autoreactive T cells is the same. The tetramer or dextramer reagents are purchased and fluorescently labeled for detection of the reagent bound to antigen-specific T cells by flow cytometry.

[0091] In the studies included in this book, two types of antigen-specific T cells were detected: EBV-specific T cells (HLA class I loaded with the peptide GLCTLVAML) or insulin B autoreactive T cells (HLA class I loaded with insulin B chain HLVEALYLV). To reduce background fluorescence of the T cells, unrelated peptides were loaded into matched HLA class I structures (Beckman Coulter, Immudex).

[0092] Isolated CD8 + T cell method CD8 from fresh blood +To directly isolate T cells, a "Detach-a-bead" CD8-positive isolation kit based on paramagnetic beads coated with anti-CD8 antibody was used (Life Technologies, Carlsbad, California) (see Burger et al., PLoS One, 6:e22430, 2011 (this document is incorporated herein by reference)). The beads were attached to a tumbler under continuous agitation at room temperature for 1 hour. The bead / cell complex was then immobilized using a magnet, and any unbound (non-CD8) cells were removed by repeated washing with HBSS (Hank's Balanced Salt Solution, Invitrogen, Grand Island, New York) containing 2% FBS (fetal bovine serum). Subsequently, the beads were removed using the Detach-A-Bead reagent supplied in the isolation kit to remove any remaining CD8 cells. + The reagent was isolated from the cells. This reagent is a polyclonal antibody against the antigen recognition site of a CD8 antibody coated on a bead. This reagent isolates the antibody / bead complex from the cell by competition for the CD8 antibody binding site, leaving the cells essentially virgin.

[0093] Next, isolated CD8 + T cells were labeled with PE (phycoerythrin)-labeled tetramer or dextramer (in the dark, at room temperature for 20 minutes), then labeled with APC-anti-CD8 antibody (in the dark, at room temperature for 10 minutes; clone SK1, BD Biosciences, San Jose, California) to determine the purity of the isolated cell prep. Next, for flow cytometry, the samples were fixed with HBSS 0.1% formaldehyde buffer, washed with HBSS, and resuspended in HBSS / 0.05% formaldehyde.

[0094] whole blood method Blood samples were first washed with 50x volume HBSS containing 2% FBS. These were then labeled with tetramers or dextramers (at room temperature (RT) in the dark for 20 minutes), followed by labeling with APC-anti-CD8 antibody (at room temperature (RT) in the dark for 10 minutes), and then CD8 + This enabled gating on T cells. Subsequently, for flow cytometry, the samples were simultaneously lysed, fixed with NH4Cl / formaldehyde buffer, washed with HBSS, and resuspended in HBSS / formaldehyde.

[0095] Flow cytometry Cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, California), and data were collected in list mode. Data analysis was performed using Cell Quest software (BD Biosciences). The flow gate was set to "open" to capture all cells. The open gate captured cells of all sizes but excluded cell debris, erythrocytes, fragmented cells, and apoptotic bodies. PE fluorescence and APC fluorescence were detected in FL2 and FL7, respectively. Percent CD8 + T cells were defined as the ratio of the number of CD8-positive events to the total number of events in the lymphocyte gate.

[0096] ELISA C-peptide levels were determined using Elisa in the blood serum of EBV-infected patients. The ultra-sensitive C-peptide Elisa was purchased from Mercodia (Uppsala, Sweden). The kit was used according to the manufacturer's instructions. Serum levels of VCA IgM, early antigen D, and EBNA were determined by Massachusetts General Hospital Clinical Laboratory Services.

[0097] Statistical values Statistical significance was determined using an independent one-sided Student t-test with a confidence level of 0.05 (Figures 3A and 2B) or a one-sided Kolmogorov-Smirnov test (Figure 2).

[0098] result Clinical Profile In the course of routine medical visits to patients with type 1 diabetes, we identified three patients who had recently developed EBV infection, clinically known as mononucleosis or "mono" (see Table 1). These patients were carefully followed for at least 15 weeks after the presentation of infection. Recent onset of EBV infection provides an opportunity to study the morbidity and characteristics of autoimmune T cells and EBV-specific T cells both during and after the clinical manifestation of EBV infection. Recent onset of EBV infection also provides an opportunity to demonstrate the reproducibility of past observations in clinical trials of BCG therapy, in which EBV infection caused a transient increase in pancreatic insulin production (measured as co-secreted C-peptide) (see Faustman et al., PLoS One, 7:e41756, 2012 (this document is incorporated herein by reference)).

[0099] [Table 1]

[0100] EBV infection was initially diagnosed clinically by standard serological methods and symptoms. The clinical characteristics of these subjects are summarized in Table 1. All subjects had established type 1 diabetes and elevated HbA1c at periods of 6, 19, and 30 years. Two of the subjects (subjects 1 and 2) were positive for glutamate decarboxylase GAD65 autoantibody, a pancreatic islet-specific marker for type 1 diabetes.

[0101] Time course of EBV infection Following the presentation of EBV infection symptoms and clinical diagnosis, more detailed EBV antibody serology was performed, compared with the serial blood studies conducted here, to determine the details of the infection's time course and estimate the onset of infection (Table 2).

[0102] [Table 2]

[0103] Subclasses of EBV antibodies against different parts of the EBV virus particle peak at different time points after infection is detected. Combinations of VCA IgM, early antigen D, and EBNA were used. These markers peak at 0–6 weeks, 4–8 weeks, and 6–8 weeks after EBV infection, respectively (Table 2). All subsequent data reported in this study, particularly the time series of immunological events, were plotted and extrapolated to infection day "0". For Subject 1, it was estimated that the subject was 2 weeks post-infection at the time of presentation to this study. For Subject 2, it was estimated that the subject was 4 weeks post-infection at the time of presentation. For Subject 3, it was estimated that the subject was 2 weeks post-infection at the time of presentation.

[0104] EBV-specific CD8 + Detection of T cells While typical clinical methods for diagnosing EBV infection involve antibody testing, in this study setting, infection can be confirmed by direct monitoring of newly created EBV-specific T cells. EBV-specific tetramers (i.e., HLA class I proteins loaded with the synthetic peptide sequence GLCTLVAML) were used. In this manner, EBV-specific CD8 cells were identified using flow cytometry. + T cells were detected (Figures 1A and 1B). In Subject 1, a slightly different method was used for detecting these EBV-specific T cells compared to Subjects 2 and 3 (CD8 in lysed blood). + For direct observation of T cells, isolated CD8 + In all subjects, EBV-specific T cells showed an early increase in fluorescence beyond the background level (Figure 1B).

[0105] Temporal course of C-peptide in EBV-infected subjects Studies over the past 15 years have shown that systemic increases in TNF or induction of TNF by either BCG or EBV lead to pancreatic regeneration, along with C-peptide restoration (see Kodama et al., Science, 302:1223-1227, 2003; Faustman et al., PLoS One, 7:e41756, 2012; and Faustman, J. Clin. Immunol. 13:1-7, 1993 (incorporated herein by reference)). C-peptide is a protein co-secreted with insulin and is a highly sensitive method for measuring insulin secretion from the pancreas in the presence of extracorporeally administered insulin.

[0106] To determine the impact of EBV infection on insulin secretion, serial serum C-peptide levels were monitored for at least 15 weeks after subjects with EBV visited the research hospital. A control group of long-term diabetic patients without EBV infection was also monitored for 15 weeks. Monitoring of fasting morning C-peptide levels in the control group demonstrated variability between subjects and the assay.

[0107] In subjects 1 and 2, who were infected with EBV, C-peptide levels showed statistically significant increases (p=0.04 and p=0.0013, respectively) above those in the control population. Subject 3 did not show a significant increase in pancreatic C-peptide (Figure 2).

[0108] The effects of EBV on insulin B autoreactive T cells in patients with type 1 diabetes. Previous data show that treatment of NOD mice or diabetic humans with BCG (or non-cGMP-compatible full Freund's adjuvant (CFA)) results in a transient increase in dead autoreactive T cells. In NOD mice, autoreactive T cells present in the pancreas are observed to undergo direct apoptosis by TNF, BCG, or CFA in the upper part of the insulin-secreting islets in the pancreas (Kuhtreiber et al., J. Mol. Endocrinol. 31:373-399, 2003; this document is incorporated herein by reference). In humans, the effect of TNF on autoreactive T cells can be monitored by rapidly releasing dead autoreactive T cells into circulation (see Faustman et al., PLoS One, 7:e41756, 2012 (this document is incorporated herein by reference)). Increased TNF or TNFR2 agonist antibodies are known to induce apoptosis in mouse and human diabetic autoreactive T cells in culture medium (see Ban et al., Proc. Nat. Acad. Sci. USA, 105:13644-13649, 2008 (this document is incorporated herein by reference)).

[0109] To determine whether EBV infection similarly kills or damages autoreactive T cells, insulin B autoreactive CD8 cells were examined in the peripheral blood of three recently EBV-infected subjects. + T cells were detected and compared to those in uninfected long-term diabetic patients. Tracking insulin B autoreactive T cells in long-term diabetic patients is feasible. According to a highly sensitive monitoring method, approximately 41% (21 out of 51) of randomly selected long-term type 1 diabetic patients had detectable insulin B autoreactive T cells (0.28%–0.65%, Figure 3A). The same type 1 diabetic CD8 cells were stained with negative dextramer reagent. + T cells showed a background signal range of 0.19% to 0.27% (Figure 3A).

[0110] As previously shown in long-term diabetic patients exposed to TNF-induced infection, EBV-infected subjects exhibited an excess of insulin B autoreactive T cells in the peripheral blood post-infection. The mean percentage of insulin B autoreactive T cells in EBV-infected subjects was higher than that in negative background staining (p=0.002) and in the corresponding group of non-infected standard diabetic patients (p=0.02). This finding supports the conclusion that TNF-boosting infection leads to the release of these autoreactive T cells into circulation.

[0111] Newly released insulin B autoreactive T cells have abnormally low CD8 counts. + Having density Further analysis of insulin B autoreactive T cells in long-term diabetic patients infected with EBV virus is needed to identify antigen-specific CD8 + Several further notable characteristics of T cells were revealed (Figure 3B). More insulin B autoreactive CD8 cells were found in circulation after EBV infection. + Not only were T cells present, but the density of CD8 markers on the cells was also dramatically lower. As shown in the three characteristic histograms in Figure 3B, the mean log fluorescence densities of CD8 for insulin B autoreactive T cells in EBV-infected subjects were 1408, 1157, and 1516. In contrast, the mean log antigen densities of CD8 markers for insulin B autoreactive T cells from uninfected long-term diabetic subjects were 2976, 4003, and 4948. The lower density of CD8 protein in EBV-infected subjects was specific only to autoreactive T cells and not a generalized trend across all antigen-specific T cells. For these same infected subjects, the log CD8 of EBV-specific T cells... + T cell density was normal. Cell surface CD8 protein densities were 4308, 3530, and 4416 (similar to the CD8 protein densities on insulin B autoreactive T cells in non-infected diabetic patients) (Figure 3B). +Loss of the marker indicates T cell damage or apoptosis (see Diaz et al., J. Leukoc. Biol., 76:609-615, 2004 (this document is incorporated herein by reference)). Thus, loss of CD8 protein on the surface of autoreactive T cells in response to TNFR2 activators (e.g., TNF-α or BCG) can be used as a proxy to diagnose the possibility of treating subjects with autoimmune diseases using TNFR2 activators.

[0112] Other Embodiments All publications, patents, and patent applications referenced in the above specification are incorporated herein by reference. Various modifications and variations of the apparatus and methods of use described in the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention is described in relation to specific embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. In fact, various modifications of the described style for carrying out the invention, which will be apparent to those skilled in the art, are intended to be within the scope of the invention.

[0113] Other embodiments are within the scope of the following claims. In other words, the present invention includes the following embodiments. [1] A method for determining the likelihood of a subject with an autoimmune disease responding to treatment with tumor necrosis factor-α (TNF-α) receptor II (TNFR2) activator, comprising the following steps: (i) CD8 derived from the subject + The steps of contacting an in vitro biological sample containing a population of T cells with a composition containing a TNFR2 activator; and (ii) Autoreactive CD8 in the population + A step of measuring the CD8 protein density on the surface of T cells; This includes, and here the standard CD8 + The self-reactive CD8 compared to T cells +The method wherein a decrease in the CD8 protein density on the surface of T cells indicates that the subject is more likely to respond to the treatment. [2] The aforementioned standard CD8 + The method according to [1] above, wherein the T cells are derived from a reference sample from a subject having an autoimmune disease and who has not been treated or prior-treated with TNFR2 activator. [3] The aforementioned standard CD8 + The method described in [1] above, wherein the T cells are derived from a reference sample derived from a healthy subject. [4] The method according to any one of [1] to [3] above, wherein the measurement is performed using an anti-CD8 antibody. [5] The method according to [4] above, wherein the antibody is conjugated with a fluorescent dye. [6] The method according to any one of [1] to [5] above, wherein the biological sample is incubated with dasatinib prior to contact. [7] The method according to [6] above, wherein the biological sample is incubated with dasatinib for at least 4 hours prior to the contact. [8] The method according to [7] above, wherein the biological sample is incubated with dasatinib for up to 48 hours prior to the contact. [9] The autoimmune diseases mentioned above include type 1 diabetes, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, Behçet's disease, bullous pemphigoid, cardiomyopathy, celiac plue dermatitis, chronic fatigue immunodeficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, scarring pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, systemic lupus erythematosus, ulcerative colitis, psoriatic arthritis, essential mixed cryoglobulinemia, fibromyalgia-fibromyitis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hypothyroidism, idiopathic pulmonary fibrosis, idiopathic leptopenic purpura (ITP), Ig The method according to any one of the above [1] to [8], selected from the group consisting of A nephropathy, juvenile arthritis, lichen planus, lupus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, Stiffman syndrome, Devic's disease, Takayasu's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, Wegener's granulomatosis, and autoimmune neuropathy.

[10] The method according to [9] above, wherein the autoimmune disease is selected from the group consisting of type 1 diabetes, celiac plue dermatitis, Crohn's disease, Graves' disease, hypothyroidism, lupus, multiple sclerosis, psoriasis, rheumatoid arthritis, sarcoidosis, Sjögren's syndrome, and ulcerative colitis.

[11] The method according to [9] above, wherein the autoimmune neurological disorder is autoimmune-mediated injury to neurons, autoimmune Alzheimer's disease, autoimmune Parkinson's disease, or autoimmune-mediated amyotrophic lateral sclerosis.

[12] A method for determining the likelihood of a subject having type 1 diabetes responding to treatment with tumor necrosis factor-α (TNF-α) receptor II (TNFR2) activator, the method comprising the step of contacting an in vitro biological sample derived from the subject with a device capable of detecting a C peptide in the sample, wherein a detectable level of C peptide in the sample indicates that the subject is likely to respond to the treatment, and a substantially undetectable level of C peptide indicates that the subject is unlikely to respond to the treatment.

[13] The method according to

[12] above, wherein a substantially undetectable level of the C peptide indicates that the subject should be excluded from the treatment.

[14] The method according to

[12] or

[13] above, wherein the substantially undetectable level is a C-peptide level of less than about 1.5 pmol / L.

[15] The method according to any one of the above

[12] to

[14] , wherein the substantially undetectable level is a C peptide level of less than about 1.0 pmol / L.

[16] The method according to

[12] above, wherein the detectable level is a C peptide level greater than approximately 1.5 pmol / L.

[17] The method according to any one of

[12] to

[16] above, wherein the concentration of the C peptide is in the range of about 1.5 pmol / L to about 4.0 pmol / L, and the subject is identified as being likely to respond to the treatment.

[18] The method according to any one of

[12] to

[17] above, wherein the contact is performed before the treatment of the subject.

[19] The method according to any one of the above

[12] to

[18] , further comprising the step of measuring a reference HbA1c level in a blood sample derived from the subject before initiating the treatment.

[20] The following steps: (i) A step of measuring the HbA1c level in a blood sample taken from the subject after the treatment, (ii) the step of comparing the HbA1c level with the reference HbA1c level, (iii) If the HbA1c level is equal to or greater than the reference HbA1c level, the step of identifying the subject as requiring repeated treatment with one or more TNFR2 activators. The method described in

[18] above, including the method described above.

[21] The method according to

[20] , wherein the blood sample is taken from the subject at least six months after the treatment.

[22] The method according to

[21] , wherein the blood sample is taken from the subject at least one year after the treatment.

[23] The method according to

[22] , wherein the blood sample is taken from the subject at least two years after the treatment.

[24] The method according to

[23] , wherein the blood sample is taken from the subject at least three years after the treatment.

[25] The method according to

[24] , wherein the blood sample is taken from the subject at least five years after the treatment.

[26] The contact is as follows: (i) The step of bringing the sample into contact with the device having an immobilized scavenging agent on its surface, thereby binding the C peptide in the sample to the immobilized scavenging agent; (ii) The step of bringing the surface into contact with the binding detection agent to bind the C peptide to the binding detection agent; and (iii) The method according to any one of the above

[12] to

[25] , comprising the step of measuring the level of the C peptide in the sample using the binding detection agent.

[27] The method according to

[26] , wherein the binding detection agent comprises a peroxidase enzyme.

[28] The method according to

[26] , wherein the measurement comprises the step of bringing the surface into contact with a solution comprising hydrogen peroxide and a peroxidase substrate.

[29] The method according to any one of [1] to

[28] above, wherein the sample comprises blood, blood components, or a urine sample.

[30] The method according to

[29] above, wherein the blood component is serum or plasma.

[31] The method according to

[29] above, wherein the sample includes urine.

[32] The method according to any one of the above [1] to

[31] , wherein the TNFR2 activator is selected from the group consisting of Bacillus calmette-Guélain (BCG), complete Freund's adjuvant, TNF-α, TNF-α receptor II agonist, TNF-α mutein, interleukin-1, interleukin-2, tissue plasminogen factor, lipopolysaccharide (LPS), lymphotoxin, and caquetin.

[33] The method according to

[32] above, wherein the TNFR2 activator is BCG.

[34] The method according to

[10] above, wherein the autoimmune disease is type 1 diabetes mellitus, and the method is used in combination with one or more of the methods described in any of

[12] to

[33] above.

[35] A pharmaceutical composition comprising one or more TNFR2 activators for use in the treatment of an autoimmune disease in a subject diagnosed as being responsive to the aforementioned treatment.

[36] The pharmaceutical composition according to

[35] , wherein the sample derived from the subject includes a population of T cells that show a decrease in surface CD8 protein density after exposure to a TNFR2 activator, compared to the surface CD8 protein density of reference T cells in a sample derived from a reference subject.

[37] The pharmaceutical composition according to

[36] above, wherein the reference subject is a healthy subject.

[38] The pharmaceutical composition according to

[36] above, wherein the subject has an autoimmune disease and has not been treated or prior treated with one or more TNFR2 activators.

[39] The autoimmune diseases mentioned above include type 1 diabetes, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, Behçet's disease, bullous pemphigoid, cardiomyopathy, celiac plue dermatitis, chronic fatigue immunodeficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, scarring pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, systemic lupus erythematosus, ulcerative colitis, psoriatic arthritis, essential mixed cryoglobulinemia, fibromyalgia-fibromyitis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hypothyroidism, idiopathic pulmonary fibrosis, idiopathic leptopenic purpura (ITP), and IgA A pharmaceutical composition according to any one of the above

[35] to

[38] , selected from the group consisting of nephropathy, juvenile arthritis, lichen planus, lupus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, Stiffman syndrome, Devic's disease, Takayasu's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, Wegener's granulomatosis, and autoimmune neuropathy.

[40] The pharmaceutical composition according to

[39] above, wherein the autoimmune disease is selected from the group consisting of type 1 diabetes, celiac plue dermatitis, Crohn's disease, Graves' disease, hypothyroidism, lupus, multiple sclerosis, psoriasis, rheumatoid arthritis, sarcoidosis, Sjögren's syndrome, and ulcerative colitis.

[41] The pharmaceutical composition according to

[39] , wherein the autoimmune neurological disorder is autoimmune-mediated injury to neurons, autoimmune Alzheimer's disease, autoimmune Parkinson's disease, or autoimmune-mediated amyotrophic lateral sclerosis.

[42] A pharmaceutical composition according to any one of

[35] to

[41] , wherein the subject is identified as being likely to respond to the treatment by any one of the methods described in [1] to

[11] above.

[43] A pharmaceutical composition comprising one or more TNFR2 activators for use in the treatment of type 1 diabetes in a subject identified as being responsive to the treatment by determining the level of C peptide in an in vitro sample derived from the subject prior to the treatment, wherein a substantially undetectable C peptide level indicates that the subject is not responsive to the treatment, and a detectable C peptide level indicates that the subject is responsive to the treatment.

[44] The pharmaceutical composition according to

[43] , wherein the substantially undetectable C-peptide level indicates that the subject should be excluded from the treatment.

[45] The pharmaceutical composition according to

[43] or

[44] above, wherein the substantially undetectable level is a C-peptide level of less than 1.5 pmol / L.

[46] The pharmaceutical composition according to

[43] or

[44] above, wherein the detectable level of the C peptide does not exceed 1.5 pmol / L.

[47] The pharmaceutical composition according to any one of

[43] to

[46] above, wherein if a urine sample derived from the subject shows a C-peptide to creatinine ratio of less than approximately 4.0 pmol / mmol, the subject is excluded from the treatment.

[48] ​​A pharmaceutical composition according to any one of

[43] to

[46] above, wherein a urine sample derived from the subject shows a C-peptide to creatinine ratio of approximately 4.0 pmol / mmol or higher, and the subject is identified as being likely to respond to the treatment.

[49] A pharmaceutical composition according to any one of

[43] to

[46] , wherein the subject is identified as being likely to respond to the treatment by any one of the methods described in

[11] to

[33] above.

[50] The pharmaceutical composition according to any one of

[43] to

[46] above, characterized in that the HbA1c level of the subject decreases by at least 0.1% within about 4 years after administration of the composition.

[51] The pharmaceutical composition according to

[50] , characterized in that the HbA1c level of the subject decreases by at least 0.1% within approximately 3 years after administration of the composition.

[52] The pharmaceutical composition according to any one of

[43] to

[49] above, which is for use in a second or subsequent treatment of type 1 diabetes in the subject, wherein the HbA1c level in the subject is increased or remains unchanged compared to the HbA1c level in the subject prior to the previous treatment.

[53] The pharmaceutical composition according to

[52] , wherein the subject is identified as requiring repeated treatment with one or more TNFR2 activators by any of the methods described in

[20] to

[25] above.

[54] The pharmaceutical composition according to any of

[35] to

[53] above, wherein the subject is a human.

[55] The pharmaceutical composition according to any one of

[35] to

[54] above, wherein the subject is a long-term diabetic patient.

[56] The pharmaceutical composition according to any one of the above

[35] to

[55] , wherein the one or more TNFR2 activators are selected from the group consisting of Bacillus calmette-Guélain (BCG), complete Freund's adjuvant, TNF-α, TNF-α receptor II agonist, TNF-α mutein, interleukin-1, interleukin-2, tissue plasminogen factor, lipopolysaccharide (LPS), lymphotoxin, and caquetin.

[57] The pharmaceutical composition according to

[56] , wherein one or more TNFR2 activators are BCG.

[58] The composition is 2 x 10 6 The pharmaceutical composition described above

[57] , comprising a dose exceeding CFU / BCG.

[59] The composition is 2.3 x 10 6 The pharmaceutical composition according to

[57] or

[58] above, comprising a dose exceeding CFU / BCG.

[60] The composition is 4x10 6 A pharmaceutical composition according to any of the above

[57] to

[59] , comprising less than a CFU / BCG dose.

[61] The pharmaceutical composition according to any one of

[57] to

[60] above, wherein the composition comprises freeze-dried BCG.

[62] The pharmaceutical composition according to any one of

[57] to

[61] above, wherein the composition comprises a physiological saline solution of BCG.

[63] The pharmaceutical composition according to any one of

[56] to

[62] above, wherein the composition is administered to the subject once or more times.

[64] The pharmaceutical composition according to

[63] , wherein the composition is administered to the subject two or more times.

[65] The pharmaceutical composition according to

[63] or

[64] above, wherein the composition is administered twice to the subject.

[66] The pharmaceutical composition according to

[64] or

[65] , wherein at least two doses of the composition are administered at intervals of at least two weeks.

[67] The pharmaceutical composition according to any one of

[64] to

[66] , wherein at least two doses of the composition are administered at an interval of at least four weeks.

[68] The pharmaceutical composition according to any one of

[35] to

[67] above, wherein the composition is formulated for administration by a route selected from the group consisting of intradermal, intramuscular, parenteral, intravenous, intraarterial, intracranial, subcutaneous, intraorbital, intraventricular, intraspinal, intraperitoneal and intranasal.

[69] The pharmaceutical composition according to

[68] above, wherein the composition is formulated for intradermal administration.

[70] The pharmaceutical composition according to any one of

[35] to

[69] above, wherein the composition is formulated for administration as a physiological saline solution.

[71] The pharmaceutical composition according to

[70] , wherein the solution has a volume of less than about 0.2 cc per dose.

[72] The pharmaceutical composition according to

[71] , wherein the solution has a volume of 0.1 cc per dose.

[73] A pharmaceutical composition according to any one of

[35] to

[72] above, formulated for the individual administration of two or more TNFR2 activators.

[74] A pharmaceutical composition according to any one of

[35] to

[73] above, formulated for the combined administration of two or more TNFR2 activators.

[75] The pharmaceutical composition according to any one of

[35] to

[74] above, characterized in that the composition can induce the expression of TNF-α in the subject.

[76] The pharmaceutical composition according to any one of

[35] to

[75] , characterized in that the composition can induce activation of the NF-κB pathway in the target autoreactive immune cells.

[77] The autoreactive immune cells are autoreactive CD8 + The pharmaceutical composition described above

[76] , wherein T cells.

[78] The pharmaceutical composition according to any one of

[35] to

[75] above, characterized in that the composition causes the death of autoreactive immune cells in the subject.

[79] The pharmaceutical composition according to any one of

[35] to

[78] above, characterized in that the composition induces proliferation of regulatory T cells in the subject.

[80] The regulatory T cells may contain regulatory CD4 + The pharmaceutical composition described above

[79] , wherein T cells.

[81] The pharmaceutical composition according to any one of

[35] to

[80] above, characterized in that the composition prevents complications arising from hyperglycemia in the subject.

[82] The pharmaceutical composition according to

[81] , wherein the complications arising from the hyperglycemia are selected from the group consisting of renal impairment, neuropathy, cardiovascular injury, retinal injury, foot injury, leg injury, heart injury, and ketoacidosis.

[83] The pharmaceutical composition according to any one of

[35] to

[82] above, wherein the composition comprises one or more pharmaceutically acceptable carriers or excipients.

Claims

1. A pharmaceutical composition comprising Bacillus calmette-Guélain (BCG) for use in a method of treating type 1 diabetes in a subject having a blood C-peptide level of 1.5 pmol / L to 4.0 pmol / L, The method described above is (i) The glycated hemoglobin A1c (HbA1c) level in a blood sample derived from the subject is measured at least 3 years after the administration of the first BCG treatment to the subject. (ii) After detecting that the HbA1c level is equal to or higher than the reference HbA1c level, administer a second treatment with BCG to the subject. The pharmaceutical composition comprising the above.

2. The pharmaceutical composition according to claim 1, wherein the blood sample is taken from the subject at least three years after the first treatment of BCG.

3. The pharmaceutical composition according to claim 2, wherein the blood sample is taken from the subject at least five years after the first treatment of BCG.

4. The pharmaceutical composition according to claim 1, wherein the subject exhibits an increase in insulin levels and a decrease in mean plasma glucose levels after administration of the first treatment of BCG to the subject.

5. The pharmaceutical composition according to claim 1, wherein, prior to the one or more repeated treatments of BCG, a urine sample derived from the subject is identified to have a C-peptide-to-creatinine ratio of 4.0 pmol / mmol or more.

6. The pharmaceutical composition according to claim 1, wherein the subject is a human.

7. The pharmaceutical composition according to claim 6, wherein the subject is a long-term diabetic patient.

8. The above composition is 2 × 10 6 The pharmaceutical composition according to claim 1, comprising a CFU / BCG dose exceeding [a certain value].

9. The above composition is 2.3 × 10 6 The pharmaceutical composition according to claim 8, comprising a CFU / BCG dose exceeding [a certain value].

10. The composition is 4 × 10 6 The pharmaceutical composition according to claim 1, comprising a CFU / BCG dose of less than 1.

11. The pharmaceutical composition according to claim 1, wherein the composition comprises freeze-dried BCG.

12. The pharmaceutical composition according to claim 1, wherein the composition comprises a physiological saline solution of BCG.

13. The pharmaceutical composition according to claim 1, wherein the composition is formulated for one or more doses to the subject.

14. The pharmaceutical composition according to claim 13, wherein the composition is formulated for two or more administrations to the subject.

15. The pharmaceutical composition according to claim 14, wherein the composition is formulated for two administrations to the subject.

16. The pharmaceutical composition according to claim 1, wherein the composition is formulated for administration by a route selected from the group consisting of intradermal, intramuscular, parenteral, intravenous, intraarterial, intracranial, subcutaneous, intraorbital, intraventricular, intraspinal, intraperitoneal, and intranasal.

17. The pharmaceutical composition according to claim 16, wherein the composition is formulated for intradermal administration.

18. The pharmaceutical composition according to claim 1, wherein the composition is formulated for administration as a physiological saline solution.

19. The pharmaceutical composition according to claim 18, wherein the solution has a volume of less than 0.2 cc per dose.

20. The pharmaceutical composition according to claim 19, wherein the solution has a volume of 0.1 cc per dose.

21. The pharmaceutical composition according to claim 1, wherein the composition comprises one or more pharmaceutically acceptable carriers or excipients.

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