BRPI0720928A2 - A method for treating a human being, a disease or condition, and using an anti-IL-1-BETA antibody or a fragrance thereof. - Google Patents

Description

I “METHOD TO TREAT ON A HUMAN, A DISEASE OR CONDITION, AND, USE OF ANTI-IL-I-BETA ANTIBODY OR FRAGMENT OF THE SAME”

This claim claims benefit under 35 USC § 119 of the 5th Provisional Application No. 60 / 871,046, filed December 20, 2006, 60 / 908,389 filed March 27, 2007 and 60 / 911,033 filed April 10, 2007 , the disclosures of which are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION The invention relates to methods for treating and / or

prevention of Type 2 diabetes, obesity, hyperglycemia, hyperinsulinemia, Type 2 diabetes, insulin resistance and disease states and conditions characterized by insulin resistance. Such methods may be used to treat a mammalian patient suffering from Type 2 diabetes, obesity, hyperglycemia, hyperinsulinemia, Type 2 diabetes, insulin resistance and disease states and conditions characterized by insulin resistance or to prevent its occurrence in a patient at risk. BACKGROUND OF THE INVENTION

The present disclosure is directed to methods for the treatment and / or prevention in mammals of Type 2 diabetes, obesity, hyperglycemia, hyperinsulinemia, Type 1 diabetes, insulin resistance, and disease states and conditions characterized by insulin resistance. be used to treat a mammalian patient (e.g., a human being) suffering from Type 2 diabetes, obesity, hyperglycemia, hyperinsulinemia, Type 1 diabetes, insulin resistance and disease states and conditions characterized by insulin resistance or to prevent its occurrence in a patient at risk.

Diabetes mellitus is a metabolic disorder in humans with a prevalence of approximately one percent in the general population (Foster, DW, Harrison's Principles of International Medicine, Chap. 114, pp. 661 - 678, 10th Ed., McGraw-Hill, New York). The disease manifests itself as a series of hormone-induced metabolic abnormalities that eventually leads to serious, long-lasting and debilitating complications involving various organ systems including the eyes, kidneys, nerves, and blood vessels. Pathologically, the disease is characterized by lesions of the base membranes, demonstrable under electron microscopy. Diabetes mellitus can be divided into two clinical syndromes, Type 1 and Type 2 diabetes mellitus. Type 1 or insulin-dependent diabetes mellitus (IDDM), also referred to as the juvenile onset, is a chronic autoimmune disease characterized by extensive loss. of beta cells in the pancreatic islets of Langerhans, which produce insulin. Since these cells are progressively destroyed, the amount of insulin secreted decreases, eventually leading to hyperglycemia (abnormally high blood glucose level) when the amount of insulin secreted falls below the normally required blood glucose levels. Although the exact trigger for this immune response is not known, IDDM patients have high levels of antibodies to proteins expressed in pancreatic beta cells. However, not all patients with high levels of these antibodies develop IDDM.

Characteristically Type 1 diabetics have very low or immeasurable high glucagon plasma insulin. Regardless of what the exact etiology is, most Type 1 patients have circulating antibodies directed against their own pancreatic cells including antibodies to insulin, the Langerhans islet cell cytoplasm, and the glutamic acid decarboxylase enzyme. A specifically targeted immune response against beta cells (insulin producing cells) leads to Type 2 diabetes. Current treatment for Type 1 diabetic patients is insulin injection and may also include dietary modifications to minimize the hyperglycaemia that results from Lack of natural insulin, which in turn is the result of damaged beta cells. The diet is also modified with respect to insulin administration to counteract the hypoglycemic effects of the hormone.

Type 2 diabetes (also referred to as non-insulin-dependent diabetes mellitus (NIDDM), onset of maturity, onset of adulthood) develops when muscle, fat and liver cells fail to respond normally to insulin. This failure to respond (called insulin resistance) may be due to reduced numbers of insulin receptors on these cells or a dysfunction of signaling pathways within cells or both. Beta cells initially compensate for this insulin resistance by increasing insulin production. Over time, these cells become unable to produce enough insulin to maintain normal glucose levels, indicating progression to Type 2 diabetes. Type 2 diabetes is caused by a combination of genetic and acquired risk factors, including a diet. high fat, lack of exercise and aging. More than 90% of the diabetic population suffers from Type 2 diabetes and the incidence continues to rise, leading to a leading cause of mortality, morbidity and health care spending worldwide (Amos et al., Diabetic Med. 14: 51 -85, 1997).

Type 2 diabetes is a complex disease characterized by defects in glucose and lipid metabolism. Disturbances typically occur in many metabolic parameters including increases in fasting plasma glucose levels, free fatty acid levels, and triglyceride levels, as well as a decrease in HDL / LDL ratio. As discussed above, one of the major underlying causes of diabetes is considered to be an increase in insulin resistance in peripheral tissues, particularly muscle and fat. The causes of Type 2 diabetes are not well understood. Both tissue resistance to insulin action and decreased insulin secretion ("β cell insufficiency") are considered to occur. The main insulin tissues responsible for glucose homeostasis are the liver, where insulin stimulates glycogen synthesis and inhibits gluconeogenesis; muscle, where insulin stimulates glucose uptake and glycogen stimulates glucose uptake and inhibits lipolysis. Thus, as a consequence of the diabetic condition, there are elevated blood glucose levels which can lead to glucose-mediated cell toxicity and subsequent morbidity (nephropathy, neuropathy, retinopathy, etc.). Insulin resistance is strongly correlated with the development of Type 2 diabetes.

There are currently several pharmacological methods for the treatment of Type 2 diabetes (Scheen et al., Diabetes Care, 22 (9): 1568-1577, 1999). They act through different modes of action: 1) 15 sulfonylureas (eg, glimepiride, glisentide, sulfonylurea, AY31637) essentially stimulate insulin secretion; 2) biguanides (eg, metformin) act by promoting glucose utilization, reducing hepatic glucose production and decreased intestinal glucose production; 3) alpha-glucosidase inhibitors (e.g. acarbose, miglitol) decrease carbohydrate digestion and consequently intestinal absorption and reduce postprandial hyperglycemia; 4) thiazolidinediones (eg troglitazone, pioglitazone, rosiglitazone, glipizide, balaglitazone, rivoglitazone, netoglitazone, troglitazone, englitazone, AD 5075, T 174, YM 268, R 102380, NC 2100, NIP 223, NIP761, NIP 221 , 25 adaglitazone, CLX 0921, darglitazone, CP 92768, BM 152054) enhance the action of insulin, thereby promoting the utilization of glucose in peripheral tissues; 5) glucagon equivalent peptides including DPP4 inhibitors (e.g. sitagliptin); and 6) insulin stimulates tissue glucose utilization and inhibits hepatic glucose production. The pharmacological methods mentioned above may be used individually or in combination therapy. However, each method has its limitations and adverse effects. Over time, a large percentage of Type 2 diabetic patients lose their response to these agents.

Insulin treatment is typically instituted after

diet, exercise, and oral medications have failed to properly control blood glucose. The disadvantages of insulin treatment are the need for drug injection, the potential for hypoglycemia and weight gain.

IL-1β is a proinflammatory cytokine secreted by several

different cell types including monocytes and macrophages. When released as part of an inflammatory reaction, IL-1β produces a range of biological effects, mainly mediated through the induction of other inflammatory mediators such as corticotropin, platelet factor 4, prostaglandin E2 15 (PGE2), IL-6 and IL-8. AIL-1β induces both local and systemic inflammatory effects through activation of the IL-1 receptor found in almost all cell types. The interleukin-1 (IL-I) family of cytokines has been implicated in various disease states. Members of the IL-1 family include IL-1α, IL-1β and IL-I Ra. Although related by their ability to bind to 20 IL-I receptors (IL-IR1 and IL-1R2), each of these cytokines is different, being expressed by a different gene and having a different primary amino acid sequence. In addition, the physiological activities of these cytokines can be distinguished from each other. Experiments indicating the evident involvement of IL-1 β in diabetes have been published.

Maedler et al, J Clin Invest (2002) 110: 851-860 suggested

whereas in Type 2 diabetes chronic hyperglycemia can be harmful to pancreatic β cells, causing deteriorated insulin secretion and mentioned that IL-1β is a proinflammatory cytokine that acts during the autoimmune process of Type 1 diabetes and inhibits β cell function. . In particular, they tested the hypothesis that IL-β may mediate the deleterious effects of high glucose levels. Treatment of diabetic animals with normalized plasma glucose with florizine and prevented IL-1β β cell expression. This was said to imply an inflammatory process in the pathogenesis of glycotoxicity in type 2 diabetes and they identified the pathway of IL-I β / NF-KB as a target to preserve β cell mass and function in this condition.

Donath et al, J Mol med (2003) 81: 455-470 mentioned the evident significance of IL-I β on the pathway to apoptosis of pancreatic islet β cell death, leading to insulin deficiency and diabetes, and proposed therapeutic methods. anti-inflammatory drugs designed to block β-cell apoptosis in Type 1 and 2 diabetes.

WO 2004/002512 is directed to the use of an IL-I receptor antagonist (IL-IRa) and / or pyrrolidine dithiocarbamate (PDTC) for the treatment or prophylaxis of type 2 diabetes. However, the frequent dosage suggested for Therapeutic use of IL-Ra polypeptide in the treatment of Type 2 diabetes (injection every 24 hours) may result in problems with patient compliance, thereby diminishing the effectiveness of this treatment modality and / or limiting its desirability. Thus, there remains a need for effective means for treating Type 2 diabetes, particularly those that do not require daily injections.

Larsen et al, New England Journal of Medicine (2007) 356: 1517-1526 describes the use of a recombinant IL-I receptor antagonist (IL-1 Ra, anakinra) for the treatment of type 2 diabetes mellitus. Dosage of 100 mg anakinra once daily for 13 weeks may result in problems with patient compliance, thereby diminishing the effectiveness of this treatment modality or limiting its desirability. Thus, there remains a need for effective means for treating Type 2 diabetes, particularly treatment means that do not require frequent injections (e.g., daily). US 2005/0256197 and US 2005/0152850 are directed to a method for facilitating metabolic control (eg, glucose) in a patient (eg, a patient with diabetes) comprising lowering the level of IL-1 β in fluid. gingival crevicular disease such that the circulating TNF level is decreased, particularly by the use of an anti-inflammatory agent, such as an anti-inflammatory ketorolac mouthwash.

Obesity is a chronic disease that is highly prevalent and is associated not only with social stigma, but also with shortened life span and numerous medical problems including adverse psychological development, dermatological disorders such as infections, varicose veins, exercise intolerance, diabetes mellitus, insulin resistance, hypertension, hypercholesterolemia, and coronary heart disease (Rissanen et al., British Medical Journal, 301: 835-837, 1990). Obesity is highly correlated with insulin resistance and diabetes in experimental animals and humans. In fact, obesity and insulin resistance, the latter of which is usually accompanied by hyperinsulinemia or hyperglycemia or both, are hallmarks of Type 2 diabetes. In addition, Type 2 diabetes is associated with a two to fourfold risk. of coronary artery disease. Despite decades of research into these serious health problems, the etiology of obesity and insulin resistance is unknown.

Insulin resistance is associated with various disease states and conditions and is present in approximately 30 to 40% of non-diabetic individuals. These disease states and conditions include, but are not limited to, pre-diabetes and metabolic syndrome (also referred to as insulin resistance syndrome). Pre-diabetes is a state of abnormal glucose tolerance characterized by impaired glucose tolerance (IGT) or impaired fasting glucose (IFG). Patients with pre-diabetes are insulin resistant and are at high risk for future progression to observable Type 2 diabetes. Metabolic syndrome is an associated group of traits that include, but are not limited to, hyperinsulinemia, abnormal glucose tolerance, obesity, fat redistribution to the abdominal or upper body compartment, hypertension, dysfibrinolysis, and a dyslipidemia characterized by high triglycerides, cholesterol. Low HDL and small dense LDL particles. Insulin resistance was linked to each μπι of the traits, suggesting that metabolic syndrome and insulin resistance are closely related. The diagnosis of metabolic syndrome is a powerful risk factor for the future development of Type 2 diabetes, as well as accelerated atherosclerosis that results in heart attacks, strokes, and peripheral vascular disease. Inflammatory cytokines, including IL-1, have been shown to mediate inflammation within adipose tissue that appears to be involved in adipocyte insulin resistance (Trayhum et al., Br. J. Nutt '. 92: 347-355, 2004; Wisse, J. Am. Soc. Nephrol 15: 2792-2800, 2004; Fantuzzi, J. Allergy Clin. Immunol 115: 911-919, 2005; Matsuzawa, FEBS Lett. 580: 2917-2921, 2006; Greenberg et al. Eur J. Clin Invest 32 Suppl 3: 24-34, 2002; Jager et al., Endocrinology 148: 241-251, 2007). Adipocytes are cells that store fat and secrete adipokines (ie a subset of cytokines) and are a major component of adipose tissue. Macrophages, which are inflammatory cells and major producers of inflammatory cytokines, IL-1, TNF-α and IL-6, also exist within adipose tissue, especially inflamed adipose associated with obesity (Kem et al., Diabetes 52: 1779 - 1785, 2003). TNF-α and IL-6 have previously been known to desensitize adipocytes for insulin stimulation (ie, insulin resistance).

Because of problems with current treatments, new therapies to treat Type 2 diabetes and other indications of disease such as those disclosed herein are required to replace or complement the available pharmaceutical methods. The present invention provides a method for treating Type 2 diabetes. In addition, the present invention also provides a method for treating obesity, hyperglycemia, hyperinsulinemia, Type 2 diabetes, insulin resistance and disease states and conditions characterized by resistance to insulin. The methods disclosed herein comprise, for example, administering an anti-IL-ββ antibody or fragment thereof. Methods that directly target IL-1β ligand with an antibody, particularly antibodies that exhibit high affinity, provide advantages over other potential treatment methods, such as IL-1β receptor antagonists (eg, IL-I Ra, Anakinra, Kineret). ®). The challenge for IL-I receptor antagonist-based therapeutic products is the need for such therapeutic products to occupy large number of receptors, which is a formidable task as these receptors are widely expressed in all cells except cells. red blood cells (Dinarello, Curr. Opin. Pharmacol. 4: 378-385, 2004). In most immune-mediated diseases, such as the diseases disclosed herein, the amount of cytokine IL-1 β that is measurable in body fluids or associated with activated cells is relatively low. Thus, a method of treatment and / or prevention that directly targets IL-10 ligand is a superior strategy, particularly when administering a high affinity IL-Iβ antibody.

SUMMARY OF THE INVENTION

The present disclosure is directed to methods for the treatment and / or prevention in mammals of Type 2 diabetes, obesity, hyperglycemia, hyperinsulinemia, decreased insulin production, Type 25 1 diabetes, insulin resistance and / or disease states and conditions characterized by Insulin resistance, such methods may be used to treat a mammalian (e.g., human) patient suffering from or at risk for diabetes, obesity, hyperglycemia, hyperinsulinemia, impaired insulin production, insulin resistance and / or conditions and conditions. of disease characterized by insulin resistance. The methods may also be used to prevent the occurrence of Type 2 diabetes, Type 1 diabetes, obesity, hyperglycemia, hyperinsulinemia, decreased insulin production, insulin resistance, and disease states and conditions characterized by insulin resistance in an at-risk patient. .

In one aspect, the invention is a method of treating in a human being, a disease or condition selected from the group consisting of Type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin production, Type 1 diabetes, and insulin resistance. method 10 comprising administering an anti-IL-ββ antibody or fragment thereof to the human. In a preferred embodiment, the disease or condition is selected from the group consisting of Type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin production, and insulin resistance. Preferably, the disease or condition is Type 2 diabetes, obesity, decreased insulin production or insulin resistance. More preferably the disease or condition is Type 2 diabetes, obesity or insulin resistance. More preferably the disease or condition is Type 2 diabetes. In one embodiment, the method does not increase a cardiovascular disease or condition. In certain embodiments, the antibody or fragment is used to treat two or more of the aforementioned diseases or conditions in the same patient (e.g., human patient).

In another aspect, the invention provides a method for treating or preventing a disease or condition by administering an anti-IL-ββ antibody or fragment, wherein the disease or condition is pre-diabetes, dyslipidemia, hyperlipidemia, hypertension, metabolic syndrome or unhealthy behavior. In another aspect, the method reduces or prevents in a human being a complication or condition associated with Type 2 diabetes selected from the group consisting of retinopathy, renal failure, cardiovascular disease, and wound healing, the method comprising administering an anti-human antibody. -IL-Ιβ or fragment thereof to human. In certain embodiments, the antibody or fragment is used to treat two or more of the aforementioned diseases or conditions in the same patient (e.g., human patient). In another embodiment, the antibody or fragment is used to treat renal failure (e.g., kidney disease) that may result from a condition other than Type 2 diabetes. In another embodiment, the antibody or fragment are used to lower the level of C-reactive protein (CRP) in 10 a patient who exhibits elevated CRP levels.

In another aspect, the invention provides IL-1β antibodies or antibody fragments thereof for use in treating or preventing a disease or condition as disclosed herein. In another aspect, the invention provides IL-1β antibodies or antibody fragments thereof for use in the treatment or prevention of a disease or conditions selected from the group consisting of Type 2 diabetes, obesity, decreased insulin production, and resistance to insulin. In another aspect, the invention provides IL-1β antibodies or antibody fragments thereof for use in the treatment or prevention of Type 2 diabetes.

In another aspect, the invention provides compositions

Pharmaceutical compositions comprising IL-1β antibodies or antibody fragments thereof and optionally at least one pharmaceutically acceptable excipient for use in the treatment or prevention of a disease or condition as disclosed herein. In another aspect, the invention provides pharmaceutical compositions comprising IL-1β antibodies or antibody fragments thereof and optionally at least one pharmaceutically acceptable excipient for use in the treatment or prevention of a disease or conditions selected from the group consisting of diabetes mellitus. 2, obesity, decreased insulin production and insulin resistance. In another aspect, the invention provides pharmaceutical compositions comprising IL-1β antibodies or antibody fragments thereof and optionally at least one pharmaceutically acceptable excipient for use in the treatment or prevention of Type 2 diabetes.

5 Used anti-IL-Ιβ antibodies or antibody fragments

in the methods of the invention generally bind to high affinity IL-1β. In preferred embodiments, the antibody or antibody fragment binds IL-1β with a dissociation constant of about 10 nM or less, about 5 nM or less, about 1 nM or less, about 500 pM or less. at least about 250 pM or less, about 100 pM or less, about 50 pM or less or about 25 pM or less. In particularly preferred embodiments, the antibody or antibody fragment binds human IL-1β with a dissociation constant of about 100 pM or less, about 50 pM or less, about 10 pM or less, about 5 pM. about 3 pM or less, about 1 pM or less, about 0.75 pM or less, about 0.5 pM or less, about 0.3 pM or less, about 0.2 pM or less or about 0.1 pM or less. In particularly preferred embodiments, the antibody or antibody fragment binds to human IL-1β with a dissociation constant of about 10 pM or less.

In another aspect of the invention, the anti-IL-ββ antibody or antibody fragment is a neutralizing antibody. In another aspect, the anti-IL-ββ antibody or antibody fragment binds an IL-1β epitope such that the bound antibody or fragment substantially permits the binding of 25 IL-Iβ to the IL-I I receptor (IL-1β). 1RI). In another aspect, the anti-IL-β antibody or antibody fragment binds to IL-1β, but does not substantially prevent IL-1β binding from IL-I I receptor (IL-1 RI) binding. In another aspect, the antibody or antibody fragment does not detectably bind IL-Iα, IL-IR or IL-I Ra. In yet another aspect of the invention, the antibody or antibody fragment binds to an epitope contained in the sequence ESVDPKNYPKKKMEKRFVFNKIE (SEQ ID NO: 1). In yet another aspect of the invention, the antibody or antibody fragment binds to an epitope incorporating IL-1β Glu64. In yet another aspect of the invention, the antibody or antibody fragment binds to amino acids 1 through 34 of the IL-1β N-terminus. Preferably, the antibody or antibody fragment is human, humanized or human engineered.

In another aspect, the invention provides a method of treating a human being who exhibits symptoms of or at risk for developing any of the aforementioned diseases or conditions (e.g., Type 1 diabetes, Type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin production, insulin resistance), the method comprising administering an anti-IL-β antibody or fragment thereof to the human in one or more doses.

In another aspect of the invention, a method is provided.

to treat in a human being a disease or condition selected from the group consisting of Type 1 diabetes, Type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin production, and insulin resistance, the method comprising administering an anti-IL-1 antibody. Ιβ or fragment thereof to human, wherein administration of an initial dose of the IL-1β antibody or antibody fragment is followed by administration of one or more subsequent doses. In one embodiment, administration of an initial dose of antibody or antibody fragment is followed by administration of two or more subsequent doses. In another embodiment, administration of an initial dose of antibody or antibody fragment is followed by administration of one or more subsequent doses and wherein said one or more subsequent doses are in an amount that is approximately the same or more. less than the initial dose. In another embodiment, administration of an initial dose of antibody or antibody fragment is followed by administration of one or more subsequent doses and wherein at least one of the subsequent doses is in an amount that is greater than the initial dose. .

5 In one embodiment, two or more, three or more,

four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more or eleven or more subsequent doses of the antibody are administered. In another embodiment the administration of the starting dose and each or more subsequent doses are separated from each other 10 by an interval of at least about two weeks, at least about three weeks, at least about one month, by at least one month. at least about two months, at least about three months, at least about four months, at least about five months, at least about six months, at least about seven months, at least about eight months, at least about 15 nine months, at least about ten months, at least about eleven months or at least about twelve months.

In another embodiment, the antibody or fragment is administered at one or more doses of 5 mg / kg or less antibody or fragment, 3 mg / kg or less antibody or fragment, 2 mg / kg or less antibody. or fragment, 1 mg / kg or less of antibody or fragment, 0.75 mg / kg or less of antibody or fragment, 0.5 mg / kg or less of antibody or fragment, 0.3 mg / kg or less of antibody or fragment, 0.1 mg / kg or less of antibody or fragment or 0.03 mg / kg or less of antibody or fragment. Preferably, in each of the aforementioned embodiments, the antibody or fragment is administered in one or more doses of at least 0.01 mg / kg antibody or at least 0.03 mg / kg antibody or fragment. fragment, at least 0.05 mg / kg antibody or fragment or at least 0.09 mg / kg antibody or fragment. The above dosage amounts refer to mg (antibody or fragment) / kg (weight of subject to be treated).

In another embodiment, the starting dose and one or more subsequent doses of antibody or fragment are each from about 0.01 mg / kg to about 10 mg / kg of antibody, from about 0.05 to about 55 mg / kg antibody, from about 0.05 mg / kg to about 3 mg / kg antibody, from about 0.1 mg / kg to about 3 mg / kg antibody, from about 0.1 mg / kg to about 1 mg / kg antibody, from about 0.1 mg / kg to about 0.5 mg / kg antibody, from about 0.3 mg / kg to about 5 mg / kg antibody, from about 0.3 mg / kg to about 3 mg / kg antibody, from about 0.3 mg / kg to about 10 from 1 mg / kg antibody, from about 0, 5 mg / kg to about 5 mg / kg antibody, from about 0.5 mg / kg to about 3 mg / kg antibody, from about 0.5 mg / kg to about 1 mg / kg antibody, from about 1 mg / kg to about 5 mg / kg antibody or from about 1 mg / kg to about 3 mg / kg antibody. In certain embodiments, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or eleven subsequent doses of the antibody are administered. The above dosage amounts refer to mg (antibody or fragment) / kg (weight of subject to be treated). The same applies next if a dosage amount is mentioned.

In another aspect, the invention provides a method of treating

in a human being, a disease or condition selected from the group consisting of Type 1 diabetes, Type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin production, and insulin resistance, the method comprising administering a therapeutically effective amount of an antibody. anti-IL-β or fragment thereof as a starting dose of about 5 mg / kg or less of antibody or fragment and a plurality of subsequent doses of antibody or fragment in an amount about the same or less than starting dose, wherein subsequent doses are separated by a time interval of at least 2 weeks.

In another aspect, the invention provides a method of treating in a human being a disease or condition selected from the group consisting of Type 1 diabetes, Type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin production, and insulin resistance. , the method comprising administering a therapeutically effective amount of an anti-IL-ββ antibody or fragment thereof to human as an initial dose of about 3 mg / kg or less of antibody or fragment and a plurality of subsequent doses of antibody or fragment. in an amount about the same or less than the starting dose, wherein subsequent doses are separated by a time interval of at least 2 weeks.

In another aspect, the invention provides a method of treating in a human being, a disease or condition selected from the group consisting of Type 1 diabetes, Type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin production, and insulin resistance. , the method comprising administering a therapeutically effective amount of an anti-IL-ββ antibody or fragment thereof to human as an initial dose of about 1 mg / kg or less of antibody or fragment and a plurality of subsequent doses of antibody or fragment in an amount around the same or less than the initial dose, wherein subsequent doses are separated by a time interval of at least 2 weeks.

In another aspect, the invention provides a method of treating in a human being, a disease or condition selected from the group consisting of Type 1 diabetes, Type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin production, and insulin resistance. , the method comprising administering a therapeutically effective amount of an anti-IL-β antibody or fragment thereof to human as an initial dose of about 0.5 mg / kg or less of antibody or fragment and a plurality of subsequent doses of antibody. or fragment in an amount about the same or less than the starting dose, wherein subsequent doses are separated by a time interval of at least 25 weeks.

In another aspect, the invention provides a method of treating in a human being a disease or condition selected from the group consisting of Type 1 diabetes, Type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin production, and insulin resistance. Method 10 comprising administering a therapeutically effective amount of an anti-IL-β antibody or fragment thereof to human as an initial dose of about 0.3 mg / kg or less of antibody or fragment and a plurality of subsequent doses of antibody. or fragment in an amount about the same or less than the initial dose, wherein the subsequent 15 doses are separated by a time interval of at least 2 weeks.

In another aspect, the invention provides a method of treating in a human being a disease or condition selected from the group consisting of Type 1 diabetes, Type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin production, and insulin resistance. , the method comprising administering a therapeutically effective amount of an anti-IL-β antibody or fragment thereof to human as an initial dose of about 0.1 mg / kg or less of antibody or fragment and a plurality of subsequent doses of antibody. or fragment in an amount about the same or less than the initial dose, wherein subsequent doses are separated by a time interval of at least 2 weeks.

Preferably, in the aforementioned embodiments wherein the antibody or fragment is administered as a starting dose and a plurality of subsequent doses, the antibody or fragment dose is at least 0.01 mg / kg of antibody or fragment at least 0.03 mg / kg antibody or fragment, at least 0.05 mg / kg antibody or fragment or at least 0.09 mg / kg antibody or fragment.

In another aspect of the invention, the antibody or fragment

are given as a fixed dose, regardless of a dose by weight ratio of the patient. In one embodiment, the antibody or fragment is administered in one or more fixed doses of 1000 mg or less antibody or fragment, 750 mg or less antibody or fragment, 500 mg 10 or less antibody or fragment, 250 mg or less. less than antibody or fragment, 100 mg or less antibody or fragment or about 25 mg or less antibody or fragment. In another embodiment, the antibody or fragment is administered in one or more fixed doses of at least about 1 mg of antibody or fragment, at least about 515 mg of antibody or fragment or at least about 10 mg of antibody or fragment.

In certain embodiments, the fixed dose is from about 1 mg to about 10 mg, about 1 mg to about 25 mg, about 10 mg to about 25 mg, about 10 mg to about 50 mg. , about 10 mg to about 100 mg, about 25 mg to about 50 mg, about 25 mg to about 100 mg, about 50 mg to about 100 mg, about 50 mg to about 150 mg, about 100 mg to about 150 mg, about 100 mg to about 200 mg, about 150 mg to about 200 mg, about 150 mg to about 250 mg, about 200 mg to about 250 mg, about 200 mg to about 300 mg, about 250 mg to about 25 300 mg, about 250 mg to about 500 mg, about 300 mg to about 400 mg, about 400 mg to about 500 mg, about 400 mg to about 600 mg, about 500 mg to about 750 mg, about 600 mg to about 750 mg, about 700 mg to about 800 mg, about 750 mg to about 1000 mg. In a preferred embodiment, the fixed dose is selected from the group consisting of about 1 mg to about 10 mg, about 1 mg to about 25 mg, about 10 mg to about 25 mg, about 10 mg. mg to about 100 mg, about 25 mg to about 50 mg, about 50 mg to about 100 mg, about 100 mg to about 150 mg, about 150 mg to about 200 mg, about 200 mg to about 5 250 mg.

In another aspect, the invention provides a method of treating in a human being a disease or condition selected from the group consisting of Type 1 diabetes, Type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin production, and insulin resistance. method 10 comprising administering a therapeutically effective amount of an anti-IL-ββ antibody or fragment thereof to the human, wherein administration of an initial dose of the antibody or antibody fragment is followed by administration of one or more subsequent doses and in whereas the plasma concentration of said antibody or antibody fragment in humans is allowed to decrease below a level of about 0.1 μg / ml for a period of time greater than about 1 week and less than about 6 months between administrations during a course of treatment with said initial dose and one or more doses subsequent. In one embodiment, the plasma concentration of said antibody or antibody fragment is allowed to decrease below a level of about 0.07 μg / ml, about 0.05 μg / ml, about 0.03 μg / ml. ml or about 0.01 μg / ml for a period of time greater than about 1 week and less than about 5 months, about 4 months, about 3 months, about 2 months, about 1 month about 3 weeks or about 2 weeks between administrations. In one embodiment, these plasma values refer to the values obtained for an individual who is treated with the fragment antibody according to the invention. In one embodiment, such an individual may be a patient suffering from one of the following diseases such as Type 2 diabetes. The invention considers that an anti-IL-ββ antibody or fragment used according to the methods herein may be administered at any of the aforementioned dose amounts, subsequent administration numbers and dosing intervals between administrations and that any of the disclosed dose amounts, subsequent administration numbers and dosing intervals between administrations may be combined with each other in alternative regimens. to modulate the therapeutic benefit. In certain embodiments, one or more subsequent doses are in an amount that is approximately the same or less than the first dose administered. In another embodiment, one or more subsequent doses are in an amount that is approximately greater than the first dose administered. Preferably the anti-IL-ββ antibody or fragment is administered by subcutaneous, intramuscular or intravenous injection. The invention considers that each antibody dose or fragment may be administered at one or more sites.

In another aspect, the invention provides a method of treating or preventing a disease or condition in a human using an anti-IL-ββ antibody or antibody fragment, wherein the disease or condition is Type 2 diabetes and wherein a dose of antibody or fragment is sufficient to obtain at least one 0.5, at least one 1.0, at least one 1.5, at least one 2.0, at least one 2.5 or at least one. .0 percentage points improvement in hemoglobin Alc. In one embodiment, these parameter values refer to values obtained for an individual who is treated with the antibody or fragment according to the invention. In one embodiment, such an individual may be a patient suffering from one of the following diseases such as Type 2 diabetes.

In a preferred embodiment, the improvement in hemoglobin Alc is sufficient to achieve regulatory guidelines for the approval of therapeutic agents in the treatment of Type 2 diabetes. Assay methods for the determination of hemoglobin Alc are well known in the art. The invention considers that the dose of antibody or fragment sufficient to achieve improvement in hemoglobin Alc may comprise any of the aforementioned dose amounts, subsequent administration numbers and dosing intervals between administrations, as well as any combination of dose amount numbers of subsequent administrations and dosage ranges between the antibody or fragment administrations described herein. In addition, the improvement in hemoglobin Alc may be at a time point of at least about 1 month, about 2 months, about 3 months, about 4 months or about 5 months and preferably about 6 months or so. more, about 7 months or more, about 8 months or more, about 9 months 15 or more, about 10 months or more, about 11 months or more, or about 12 months or so after an initial administration of one or more doses of antibody or fragment.

In another aspect of the aforementioned methods for treating or preventing Type 2 diabetes, the method is sufficient to achieve at least one of the following modifications: reduced fasting blood sugar, decreased insulin resistance, reduced insulin hyperinsulinemia, improved glucose tolerance, reduced C-reactive peptide (CRP), increased insulin production and decreased hyperglycemia, reduced need for diabetes medication, reduced BMT, 25 change in glucose / insulin C-peptide AUC , decreased urine glucose level, decreased acute phase reagents, decreased serum lipids with improved lipid profile with respect to cardiovascular risk. Test methods for determining any of the above modifications are well known in the art. Further, the invention considers that obtaining one of the aforementioned modifications may be at a time point of at least about 1 month, about 2 months, about 3 months, about 4 months or about 5 months and preferably at least about 6 months or more, about 7 months or more, about 8 months or 5 more, about 9 months or more, about 10 months or more, about 11 months or more or about 12 months or more following an initial administration of one or more doses of antibody or fragment.

In another aspect of the invention, the method provided herein reduces or prevents a complication or condition associated with Type 10 diabetes selected from the group consisting of retinopathy, renal failure, cardiovascular disease and wound healing, the method comprising administering an anti-antibody. -IL-Ιβ or fragment thereof to human. In one embodiment, the complication or condition is cardiovascular disease and wherein said cardiovascular disease is atherosclerosis or peripheral vascular disease. In another embodiment, the complication or condition is wound healing and wherein said wound healing condition is diabetic ulcer. In another aspect, the method prevents or delays diabetic stage kidney disease or diabetic neuropathy. In one embodiment, the anti-IL-ββ antibody or fragment is administered in combination with at least one other medically accepted treatment for the disease, condition or complication. In another embodiment, the at least one other accepted drug treatment for the disease, condition or complication is reduced or discontinued, while treatment with the anti-IL-ββ antibody or fragment is maintained at a constant dosage regimen. . In another embodiment, the at least one other medically accepted treatment for the disease, condition or complication is reduced or discontinued and treatment with the anti-IL-ββ antibody or fragment is reduced. In another embodiment, the at least one other medically accepted treatment for the disease, condition or complication is reduced or discontinued and treatment with the anti-IL-ββ antibody or fragment is increased. In another embodiment, at least one other medically accepted treatment for the disease, condition or complication is maintained and treatment with the anti-IL-β antibody or fragment is reduced or discontinued. In another embodiment, at least one other medically accepted treatment for the disease, condition or complication and treatment with the anti-IL-β antibody or fragment is reduced or discontinued.

In another aspect of the invention, a method of reducing the amount of C-reactive protein in a patient is provided, the method comprising administering an anti-IL-ββ antibody or fragment thereof to the patient. In one embodiment, the antibody or antibody fragment is administered at one or more doses of 1 mg / kg or less of antibody or fragment, 0.75 mg / kg or less of antibody or fragment, 0.5 mg / kg. or less of antibody or fragment, 0.3 mg / kg or less of antibody or fragment, 0.1 mg / kg or less of antibody or fragment or 0.03 mg / kg or less of antibody or fragment. Preferably, the antibody or fragment is administered in one or more doses of at least 0.01 mg / kg antibody or fragment, at least 0.03 mg / kg antibody or fragment, at least 0.05 mg / kg. of antibody or fragment or at least 0.09 mg / kg antibody or fragment. In another embodiment, the antibody or fragment is administered as one or more fixed doses, independent of a patient weight ratio dose of 500 mg or less antibody or fragment, 250 mg or less antibody or fragment, 100 mg or less of antibody or fragment or about 25 mg or less of antibody or fragment. Preferably, the antibody or fragment is administered in one or more fixed doses of at least about 1 mg antibody or fragment, at least about 5 mg antibody or fragment or at least about 10 mg antibody or fragment. In another embodiment, the antibody or antibody fragment binds IL-1β with a dissociation constant of about 500 pM or less, about 250 pM or less, about 100 pM or less, about 50 pM or less. or about 25 pM or less, about 10 pM or less, about 5 pM or less, about 3 pM or less, about 1 pM or less, about 0.75 pM or less, about 0, 5 pM or less, about 0.3 pM or less, about 0.2 pM or less or about 0.1 pM or less. In another embodiment, administration of an initial dose is followed by administration of one or more subsequent doses, separated from each other by at least about 10 weeks, at least about 3 weeks, at least about 3 weeks. one month, at least about two months, at least about three months, at least about four months, at least about five months, at least about six months, at least about seven months, at least about eight months, at least about nine months, at least about ten months, at least about eleven months or at least about twelve months. In another embodiment, said method of reducing the amount of C-reactive protein in a patient is provided, wherein the patient is suffering from kidney disease (eg, chronic kidney disease, kidney failure). In another embodiment, you are suffering from Type 2 diabetes, Type 1 diabetes, obesity, hyperglycemia, hyperinsulinemia, decreased insulin production, insulin resistance, and / or disease states and conditions characterized by insulin resistance. In another embodiment, you are suffering from a disease or condition of pre-diabetes, dyslipidemia, hyperlipidemia, hypertension, metabolic syndrome or unhealthy behavior. The above dosage amounts refer to mg (antibody or fragment) / kg (weight of subject to be treated). Administration of the antibodies or fragments with the aforementioned dissociation constants may be carried out according to any of the aforementioned dose amounts and dosage ranges (when administering two or more doses).

In another aspect, the methods provided herein are in conjunction with at least one additional treatment method, said further treatment method comprising administering at least one pharmaceutical composition comprising an active agent other than an IL-1β antibody or fragment. . In another aspect, the methods prevent or delay the need for at least one additional treatment method, said additional treatment method comprising administering at least one pharmaceutical composition comprising an active agent.

other than an IL-1 β antibody or fragment. In yet another aspect, the methods reduce the amount, frequency or duration of at least one additional treatment method, said further treatment method comprising administering at least one pharmaceutical composition comprising an active agent other than an IL-1β antibody. or fragment. In one embodiment, the at least one pharmaceutical composition comprising an active agent other than an IL-1β antibody or fragment is selected from the group consisting of a sulfonylurea, a meglitinide, a biguanide, an alpha inhibitor. glycosidase, a thiazolidinedione, a glucagon and insulin equivalent peptide. In another embodiment, the active agent is a sulfonylurea. In another embodiment, the active agent is a meglitinide. In another embodiment, the active agent is a biguanide. In another embodiment, the active agent is an alpha glycosidase inhibitor. In another embodiment, the active agent is a thiazolidinedione. In another embodiment, the active agent is a glucagon equivalent peptide. In another embodiment, the active agent is insulin. In another embodiment, the at least one pharmaceutical composition comprising an active agent comprises two active agents. In one embodiment, the two active agents are a sulfonylurea and a biguanide. In another embodiment, the two active agents are a thiazolidinedione and a biguanide. In another embodiment, treatment with at least one active agent is maintained. In another embodiment, treatment with at least one active agent is reduced or discontinued, while treatment with the anti-IL-1β antibody or fragment is maintained at a constant dosage regimen. In another embodiment, treatment with at least one active agent is reduced or discontinued and treatment with anti-IL-ββ antibody or fragment is reduced. In another embodiment, treatment with IO at least one active agent is reduced or discontinued and treatment with

the anti-IL-β antibody or fragment is increased. In another embodiment, treatment with at least one active agent is maintained and treatment with the anti-IL-β antibody or fragment is reduced or discontinued. In another embodiment, treatment with at least one active agent and treatment with the anti-IL-β antibody or fragment is reduced or discontinued.

In another aspect, the invention provides a method of treating in a human being a disease or condition selected from the group consisting of Type 1 diabetes, Type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin production, and insulin resistance. , the method comprising administering a therapeutically effective amount of an anti-IL-ββ antibody or fragment thereof to the human, wherein administration of an initial dose of the antibody or antibody fragment is followed by administration of one or more subsequent doses and in that the plasma concentration of said antibody or antibody fragment in humans is maintained at a level of at least about 0.03 μg / ml, at least about 0.05 μg / ml, at least about 0.08 μg / ml, at least about 0.1 μg / ml, at least about 0.15 μg / ml, at least about 0.2 μg / ml, at least about 0.25 μg / ml, at least about 0.3 μg / ml, at least about 0.4 μ§ / ηι1, at least about 0.5 μg / ml, at least about 0.6 μg / ml, at least about 0 , 8 μg / ml, at least about 1 μg / ml, at least about 1.5 μg / ml, at least about 2 μg / ml, at least about 3 μg / ml, at least about 4 μg / ml or at least about 5 μg / ml during a course of treatment with said initial dose and one or more subsequent doses. In one embodiment, these plasma values refer to values obtained for an individual who is treated with the antibody or fragment according to the invention. In one embodiment, such an individual may be a patient suffering from one of the following diseases such as Type 2 diabetes.

In another aspect, the invention provides a method of treating in a human being a disease or condition selected from the group consisting of Type 1 diabetes, Type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin production, and insulin resistance. method 15 comprising administering a therapeutically effective amount of an anti-IL-ββ antibody or fragment thereof to human, wherein administration of the anti-IL-ββ antibody or fragment thereof to human results in a decrease in the production of one or more more gene products selected from the group consisting of leptin, resistin, visfatin, RANTES, 20 IL-6, MCP-I, PAI-1, acylation stimulating protein, SAA3, Pentraxin-3, macrophage migration inhibiting factor, IL-1RA, IL-12, IL-8 and TNF-a. In one embodiment, administration of the anti-IL-ββ antibody or fragment thereof to humans results in a decrease in the production of one or more gene products selected from the group consisting of leptin, resistin and visfatin. In another embodiment, administration of the anti-IL-ββ antibody or fragment thereof to human results in a decrease in the production of one or more gene products selected from the group consisting of MCP-1, RANTES, IL-6. , TNF-α and Pentraxin-3. In another embodiment, said decrease in the production of one or more gene products is from adipose tissue. In another embodiment, said decrease is detected in human blood.

In another aspect, the invention provides a method of treating in a human being a disease or condition selected from the group that

5 consists of Type 1 diabetes, Type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin production, and insulin resistance, the method comprising administering a therapeutically effective amount of an anti-IL-ββ antibody or fragment thereof to humans. administration of the anti-IL-ββ antibody or fragment thereof to human 10 results in an increase in adiponectin production. In one embodiment, said increase in adiponectin production is from adipose tissue. In another embodiment, said increase is detected in the human's blood.

In another aspect, the invention provides a method of treating in a human being, a disease or condition selected from the group consisting of Type 1 diabetes, Type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin production, and insulin resistance. , the method comprising administering a therapeutically effective amount of an anti-IL-1β antibody or fragment thereof to the human, wherein the antibody or fragment thereof has a lower IC50 than an IL-1β receptor antagonist in a human whole blood IL-1β inhibition assay that measures IL-1β induced production of IL-8. In one embodiment, the antibody or fragment has an IC50 that is less than about 90%, 80%, 70%, 60%, 50% of the IC50 of an IL-25 1β receptor antagonist in one assay. of IL-Iβ inhibition in human whole blood measuring IL-1β-induced production of IL-8. In another embodiment, the antibody or fragment has an IC50 that is less than about 40%, 30%, 20%, 10% of the IC50 of an IL-1β Receptor Antagonist in an IL inhibition assay. - 1β in human whole blood measuring IL-Iβ induced production of IL-8. In a preferred embodiment, the antibody or fragment has an IC50 that is less than about 8%, 5%, 4%, 3%, 2%, 1% of the IC50 of an IL-1β receptor antagonist. in a human whole blood IL1 β inhibition assay that measures IL-1 β 5 induced production of IL-8. In one embodiment, the IL-1β receptor antagonist is anaquin (i.e. Kineret®).

In another aspect, the invention provides a method of treating in a human being a disease or condition selected from the group consisting of Type 1 diabetes, Type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin production and insulin resistance. , the method comprising administering a therapeutically effective amount of an anti-IL-ββ antibody or fragment thereof to the human, wherein the antibody or fragment thereof provides in vivo inhibition of IL-β-stimulated release.

1-β IL-6 in mice compared to a control antibody using an assay that is described by Economides et al., Nature Med., 9: 47-52 (2003) which is incorporated by reference. In one embodiment the antibody or fragment provides in vivo inhibition of IL-1β-stimulated release of IL-1β.

6 in mice of at least about 10%, 20%, 30%, 40%, 50% compared with the control antibody. In another embodiment, the antibody or fragment provides in vivo inhibition of IL-18 stimulated release of IL-6 in mice of at least about 60%, 70%, 80%, 90%, 95% compared. with the control antibody. In one embodiment, the control antibody is an isotype control antibody.

In another aspect, the invention provides a method of treating in a human being, a disease or condition selected from the group consisting of Type 1 diabetes, Type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin production, and insulin resistance. A method comprising administering a therapeutically effective amount of an anti-IL-1β antibody or fragment thereof to a human, wherein the antibody or fragment thereof inhibits Staphilococcus epidermidis-induced cytokine production in human whole blood compared to a control in which No antibodies are used. In one embodiment the antibody or fragment provides a higher level of inhibition of Staphilococcus epidermidis-induced cytokine production in human whole blood by at least about 10%, 20%, 30%, 40%, 50% compared to control. In another embodiment, the antibody or fragment provides a higher level of inhibition of Staphilococcus epidermidis-induced cytokine production in human whole blood by at least about 60%, 70%, 10 80%, 90%, 95% compared with the control. In one embodiment, the inhibited cytokines are IL-Iβ, IL-βα, M-6, IL-8, IL-1Ra, TNFα or IFNγ.

In another aspect, the invention discloses the use of an anti-IL-ββ antibody or fragment thereof which as a lower IC50 than an IL-1β receptor antagonist in a blood IL-1β inhibition assay. human body that measures IL-1β-induced production of IL-8 in the manufacture of a composition for use in the treatment of Type 1 diabetes, Type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin production, and insulin resistance. In one embodiment, the IL-1β receptor antagonist is anaquin (i.e. Kineret®)

In another aspect of the invention, the use of IL-1β antibodies

or binding fragments is contemplated in the manufacture of a medicament for treating or preventing a disease or condition as disclosed herein. In either use, the drug may be coordinated with treatment using a second active agent. In another embodiment of the invention, the use of a synergistic combination of an antibody of the invention for the preparation of a medicament for treating a patient exhibiting symptoms at risk for developing a disease or condition as disclosed herein, wherein the medicament It is coordinated with treatment using a second active agent is considered. In yet another related embodiment, the composition is provided wherein the second active agent is another antibody, growth factor, cytokine or insulin. Embodiments of any of the aforementioned uses are considered wherein the amount of the IL-1β binding antibody or fragment in the medicament is in an effective dose to reduce the dosage of the second active agent required to achieve a therapeutic effect. .

In yet another aspect of the invention, an article of manufacture is provided, comprising a container, a composition within the container comprising an anti-IL-ββ antibody or fragment thereof and a packaging insert containing instructions for administering the antibody or fragment to a human in need of treatment according to the aforementioned methods of the invention. In one embodiment, the container further comprises a pharmaceutically suitable carrier, excipient or diluent. In a related embodiment, the composition within the container further comprises a second active agent. In yet another related embodiment, the composition is provided wherein the second active agent is another antibody, growth factor, cytokine or insulin.

Kits are also considered by the present invention. In one embodiment, a kit comprises a therapeutically or prophylactically effective amount of an anti-IL-ββ antibody or fragment, packaged in a container, such as a vial or bottle, and further comprising a label attached to or packaged with the container. , the label describing the contents of the container and providing indications and / or instructions with respect to the use of the contents of the container for treating or preventing a disease or condition according to the aforementioned methods of the invention. In one embodiment, the container further comprises a pharmaceutically suitable carrier, excipient or diluent. In a related embodiment, the container further contains a second active agent. In yet another related embodiment, the second active agent comprises another antibody, growth factor, cytokine or insulin.

In one embodiment, the article of manufacture, kit or medicament is for treating or preventing a disease or condition in a human being, said disease or condition selected from the group consisting of Type 1 diabetes, Type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin production and insulin resistance. In a preferred embodiment, the disease or condition is selected from the group consisting of Type 2 diabetes, obesity and insulin resistance. In another embodiment, the instructions of a packaging insert for an article of manufacture or label of a kit comprise instructions for administration of the antibody or fragment according to any of the dose amounts, subsequent administration numbers and dosing intervals. Dosage levels between administrations mentioned above, as well as any combination of amounts of dose numbers of administrations and subsequent dosing intervals between administrations described herein. In another embodiment, the kit container or article of manufacture is a pre-filled syringe.

It should be understood that where this descriptive report

mentions methods of treatment by making use of antibodies or fragments thereof with certain properties (such as Kd values or IC50 values), this also means incorporating the use of such antibodies or fragments thereof in the manufacture of a medicament for use in these methods. Furthermore, the invention also encompasses antibodies or fragments thereof having these properties as well as pharmaceutical compositions comprising these antibodies or fragments thereof for use in the treatment methods discussed below. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a graph showing the results of an in vitro IL-Iβ inhibition experiment for the antibody designated AB7 and for Kineret® involving IL-I induced production of IL-8.

Fig. 2 is a graph showing the results of an in vivo IL-1β inhibition experiment for antibodies designated AB5 and AB7 involving IL-I stimulated release of IL-6.

Fig. 2B is a graph showing the results of an in vivo IL-1β inhibition experiment for antibodies designated AB7 involving IL-I stimulated release of IL-6 and comparing inhibition of human IL-1β ( panel A) versus mouse (panel B).

Fig. 3 is a graph showing serum concentrations following administration of 0.1, 1 or 10 mg / kg of an anti-IL-1β antibody to rats.

Fig. 4 is a graph showing serum concentrations following administration of 0.3 or 3 mg / kg of an anti-IL-ββ antibody to Cinomolgus monkeys.

Fig. 5 is a graph modeling the plasma concentration profiles of an anti-IL-ββ antibody in Cinomolgus monkeys following five monthly doses of 0.1, 0.3, 1 or 3 mg / kg.

Fig. 6 is a table showing the reduction of Staphiloccus epidermidis-induced cytokine production in human whole blood by treatment with an anti-IL-ββ antibody.

Fig. 7 is a graph showing the pharmacokinetics of AB7 in humans following administration of a 0.01 mg / kg dose of antibody.

Fig. 8 is a graph showing the effect on reducing CRP levels in humans following administration of a 0.01 mg / kg dose of AB7. Fig. 9 is a graph showing the effect on reducing CRP levels in humans following administration of a 0.03 mg / kg dose of AB7.

Fig. 10 is a graph showing CRP levels in placebo controls of the 0.01 and 0.03 mg / kg dose groups.

Fig. 11 is a graph modeling the effect on CRP levels in humans following administration of various doses of an antibody with similar properties to AB7 at 28 day intervals.

Fig. 12 is a graph showing the effect of AB7 on a mouse model of Type 2 diabetes diet-induced obesity. DETAILED DESCRIPTION

IL-1β is a proinflammatory cytokine secreted by several different cell types including monocytes and macrophages. When released as part of an inflammatory reaction, IL-1β produces a range of biological effects, mainly mediated through the induction of other inflammatory mediators such as corticotropin, platelet factor 4, prostaglandin E2 (PGE2), IL-6 and IL-8. IL-1 β induces both local and systemic inflammatory effects through activation of the IL-I receptor found in almost all cell types.

Interleukin-1 (IL-I) family of cytokines has been implicated

in various disease states such as rheumatoid arthritis (RA), osteoarthritis, Crohn's disease, ulcerative colitis (UC), septic shock, chronic obstructive pulmonary disease (COPD), asthma, graft versus host disease, atherosclerosis, T-cell leukemia adult disease, multiple myeloma, multiple sclerosis, stroke and Alzheimer's disease. IL-I family members include IL-Ia, IL-1 β, and IL-I Ra. Although related to their ability to bind to IL-I receptors (IL-IR1, IL-1R2), each of these cytokines is expressed by a different gene and has a different primary amino acid sequence. In addition, the physiological activities of these cytokines can be distinguished from each other.

Compounds that disrupt IL-I receptor signaling have been investigated as therapeutic agents for treating IL-1 mediated diseases, such as for example some of the aforementioned diseases. These compounds include recombinant IL-IRa (Amgen Inc., Thousand Oaks, CA), IL-I receptor trap peptide (Regeneron Inc., Tarrytown, NY) as well as animal-derived IL-1β antibodies and IL antibodies. -1 β recombinants and fragments thereof.

As mentioned above, IL-I receptor antagonist polypeptide (IL-IRa) has been suggested for use in the treatment of Type 2 diabetes (WO 2004/002512), but there remains a need for effective means to treat Type 1 diabetes. 2, particularly those that do not require daily, repeated injections. An additional challenge for IL-I receptor antagonist-based therapeutic products is the need for such therapeutic products to occupy a large number of receptors, which is a formidable task as these receptors are widely expressed in all cells except red blood cells (Dinarello, Curr. Opin. Pharmacol. 4: 378-385, 2004). In most immune-mediated diseases, such as the diseases disclosed herein, the amount of cytokine IL-1 β that is measurable in body fluids or associated with activated cells is relatively low. Thus, a method of treatment and / or prevention that directly targets IL-1β ligand is a superior strategy, particularly when administering a high affinity IL-1β antibody.

The present invention provides methods and related articles of

manufacture for the treatment and / or prevention in mammals of Type 1 diabetes, Type 2 diabetes, obesity, hyperglycemia, hyperinsulinemia, decreased insulin production, insulin resistance, and / or disease states and conditions characterized by insulin resistance, using an antibody. or fragment of this specific for IL-1β.

As shown in Example 1 below, we have surprisingly found that such an antibody (e.g., with high affinity) may be a much more potent inhibitor of the IL-I pathway than is IL-Ra (e.g., Kinere®) and as shown in Example 9 below provides an opportunity to achieve a therapeutic effect at a lower dose and / or less frequent administration than is required for other drugs, such as recombinant IL-IRa.

Such methods as described herein with an IL-1β antibody or fragment may include treating a patient suffering from Type 1 diabetes, Type 2 diabetes, obesity, hyperglycemia, hyperinsulinemia, decreased insulin production, hypoinsulinemia, insulin resistance and / or disease states and conditions characterized by insulin resistance. Methods may also include preventing the occurrence of Type 1 diabetes, Type 15 2 diabetes, obesity, hyperglycemia, hyperinsulinemia, decreased insulin production, insulin resistance, and disease conditions and conditions characterized by insulin resistance or to prevent its occurrence in a patient at risk. As used herein, hyperinsulinemia refers to relative hyperinsulinemia due to insulin resistance.

Antibodies, Humanized Antibodies and Human Engineered Antibodies

IL-I binding antibodies (e.g., IL-Iβ) of the present invention may be provided as polyclonal antibodies, monoclonal antibodies (mAbs), recombinant antibodies, chimeric antibodies, CDR-grafted antibodies, fully human antibodies, single chain, and / or bispecific antibodies, as well as fragments, including variants and derivatives thereof, provided by known techniques, including, but not limited to enzymatic cleavage, peptide synthesis or recombinant techniques. Antibodies generally comprise two heavy chain polypeptides and two light chain polypeptides, however single domain antibodies having one heavy and one light chain and heavy chain antibodies devoid of light chains are also considered, there are five five types of heavy chains. , called alpha, delta, epsilon, gamma, and mu, based on the amino acid sequence of the heavy chain constant domain. These different heavy chain types give rise to five antibody classes, IgA (including IgA1 and IgA2), IgD, IgE, IgG and IgM, respectively, including four IgG subclasses, namely IgGi, IgG2, IgG310 and IgG4. There are also two types of light chains called cape (k) or lambda (λ) based on the amino acid sequence of the constant domains. A life-size antibody includes a constant domain and a variable domain. The constant region need not be present in an antigen binding fragment of an antibody. Antigen binding fragments of an antibody disclosed herein may include Fab, Fab ', F (ab') 2 and F (v) antibody fragments. As discussed in more detail below, IL-1β binding fragments encompass antibody fragments and antigen binding polypeptides that will bind IL-1β.

Each of the heavy chain and light chain sequences of an antibody or antigen binding fragment thereof includes a variable region with three complementarity determining regions (CDRs) as well as non-CDR matrix regions (FRs). The terms "heavy chain" and "light chain," as used herein, mean the heavy chain variable region and the light chain variable region, respectively, unless otherwise noted. Heavy chain CDRs are alluded to herein as CDR-H1, CDR-H2 and CDR-H3. Light chain CDRs are alluded to herein as CDR-L1, CDR-L2 and CDR-L3. Variable regions and CDRs in an antibody sequence can be identified (i) according to the general rules that have been developed in the art or (ii) by aligning the sequences against a known variable region database. Methods for identifying these regions are described in Kontermann and Dubel, eds., Antibody Engineering, Springer, New York, NY, 2001 and Dinarello et al., Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken, NJ, 2000. Antibody sequence databases are described and can be accessed through the “The Kabatman” database at www.bioinf.org.uklabs (maintained by AC Martin at the Department of Biochemistry & Molecular Biology University College London, London, England) and VBASE2 at www.vbase2.org as described in Retter et al., Nucl. Acids Res., 33 (Database Issue): D671-D674 (2005). The Kabatman database website also includes general handling rules for identifying CDRs. The term "CDR," as used herein, is as defined in Kabat et al., Sequences of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, 1991, unless otherwise indicated.

Polyclonal antibodies are preferably elicited in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. An improved antibody response can be obtained by conjugating the relevant antigen to a protein that is immunogenic in the species to be immunized, for example, keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin or soybean trypsin inhibitor. bifunctional agent or derivative, for example maleimidobenzoyl sulfosuccinimide ester (conjugation via cysteine residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic aldehyde or other agents known in the art.

Animals are immunized against the antigen, immunogenic conjugates or derivatives by combining, for example, 100 μg or 5 μg protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally into multiple sites. One month later, the animals are boosted with 1/5 (fraction (1/10)) of the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. the animals are bled and the serum is assayed for antibody titer .The animals are boosted to the titer plateau. Preferably the animal is boosted with the conjugate of the same antigen but conjugated to a different protein and / or through conjugates may also be made in recombinant cell culture as protein fusions Also, aggregating agents such as alum are suitably used to enhance the immune response.

Monoclonal antibody refers to an antibody obtained from a substantially homogeneous antibody population. Monoclonal antibodies are generally highly specific and can be directed against a single antigenic site, as opposed to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes). In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by homogeneous culture, uncontaminated by other immunoglobulins with different specificities and characteristics.

Monoclonal antibodies to be used according to the present invention may be made by the hybridoma method first described by Kohler et al. (Nature, 256: 495-7, 1975) or may be made by recombinant DNA methods (see, for example, US Patent No. 25 4,816,567). Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described, for example, in Clackson et al. (Nature 352: 624-628, 1991) and Marks et al. (J. Mol. Biol 222: 581-597, 1991).

In the hybridoma method, a mouse or other appropriate host animal, such as a hamster or monkey, is immunized as described herein to evoke lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro.

Lymphocytes are then fused to myeloma cells using a suitable fusion agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). .

Hybridoma cells thus prepared are seeded and cultured in a suitable culture medium which preferably contains one or more substances that inhibit the growth or survival of the precursor unfused myeloma cells. For example, if precursor myeloma cells lack the hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT) enzyme, the culture medium for hybridomas will typically include hypoxanthine, aminopterin and thymidine (HAT medium), which inhibit cell growth. HGPRT disabled.

Preferred myeloma cells are those that fuse efficiently, sustain stable high-level antibody production by selected antibody-producing cells, and are sensitive to a medium. The 20 human myeloma cell lines and mouse heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal. Antibody Production Techniques and Applications, pp. 51-63 (Mareei Dekker, Inc., New York, 1987)). Exemplary murine myeloma strains include those derived from MOP-21 and MG-11 mouse tumors available from Salk Institute Cell Distribution Center, San Diego, CalifUSA and available SP-2 or X63-Ag8- 653 cells from American Type Culture Collection, Rockville, Md. USA.

The culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or an in vitro binding assay such as radioimmunoassay (RIA) or enzyme linked immunosorbent assay (ELISA). Binding affinity of monoclonal antibody, for example, can be determined by Scatchard analysis (Munson et al., Anal. Biochem., 107: 220 (1980)).

After hybridoma cells are identified that produce antibodies of the desired specificity, affinity and / or activity, clones can be subcloned by limiting dilution procedures and cultured by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59- 103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, DMEM or RPM-1640 medium. In addition, hybridoma cells can be cultured in vivo as ascites tumors in an animal. Monoclonal antibodies secreted by subclones are suitably separated from the culture medium, ascites fluid or serum by standard immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or chromatography. affinity.

It is further contemplated that the antibodies of the invention may be used as minor antibody antigen binding fragments well known in the art and described herein.

The present invention encompasses IL-Iβ binding antibodies (for example, IL-1β) which include two life size heavy chains and two life size light chains. Alternatively, IL-1β binding antibodies may be constructs such as single chain antibodies or "mini" antibodies that retain IL-Iβ binding activity. Such constructs may be prepared by methods known in the art such as, for example, PCR-mediated cloning and assembly of single chain antibodies for expression in E. coli (as described in Antibody Engineering, The Practical Approach Series, J. McCafferty , HR Hoogenboom and DJ Chiswell, publishers, Oxford University Press, 1996).

In this type of construct, the heavy and light chain variable portions of an antibody molecule are amplified by PCR from cDNA. The resulting amplicons are then assembled, for example, in a second PCR step, via a linker DNA encoding a flexible protein linker composed of the amino acids Gly and Ser. This linker allows the variable heavy and light chain portions to fold in such a way that the antigen binding pouch is regenerated and the antigen is bound with affinities often comparable with the precursor life-size dimeric immunoglobulin molecule.

IL-I (e.g., IL-1β binding antibodies and fragments of the present invention encompass exemplary antibody variants, fragments and sequences disclosed herein. Variants include peptides and polypeptides comprising one or more substitutions, deletions and / or amino acid sequence additions that have the same or substantially the same affinity and epitope binding specificity as one or more of the exemplary antibodies, fragments, and sequences disclosed herein Thus, variants include peptides and polypeptides comprising one or more substitutions, amino acid sequence deletions and / or additions to the exemplary antibodies, fragments and sequences disclosed herein where such substitutions, deletions and / or additions do not cause substantial changes in epitope binding affinity and specificity, for example, a variant of an antibody or fra Such a sequence may result from one or more changes to an antibody or fragment, where the changed antibody or fragment has the same or substantially the same affinity and epitope binding specificity as the starting sequence. Variants may be naturally occurring, such as allelic or junction variants, or may be artificially constructed. Variants may be prepared from corresponding nucleic acid molecules encoding said variants. Variants of the present antibodies and IL-Iβ binding fragments may have changes in the naturally occurring or light chain and / or heavy chain amino acid sequences or are introduced by in vitro engineering of native sequences using recombinant DNA techniques. Naturally occurring variants include "somatic" variants that are generated in vivo in the corresponding germline nucleotide sequences during the generation of an antibody response to a foreign antigen.

IL-I binding antibody variants (e.g. IL-1β) and binding fragments can also be prepared by mutagenesis techniques. For example, amino acid changes may be randomly introduced throughout an antibody coding region and the resulting variants may be screened for binding affinity for TL-β or for another property. Alternatively, amino acid changes can be introduced into selected regions of an IL-1 β antibody, such as light and / or heavy chain CDRs and / or matrix regions, and the resulting antibodies can be screened for MAP binding. or some other activity. Amino acid changes encompass one or more amino acid substitutions in a CDR, ranging from a single amino acid difference to the introduction of multiple amino acid exchanges within a given CDR, such as CDR3. In another method, the contribution of each residue within a CDR to IL-1β binding can be assessed by replacing at least one residue within the CDR with alanine. Lewis et al. (1995), Mol. Immunol. 32: 1065-72. Residues that are not ideal for IL-1β binding can then be changed to determine a more optimal sequence. Also encompassed are variants generated by amino acid insertion to increase the size of a CDR, such as CDR3. For example, most light chain CDR3 sequences are nine amino acids in length. Light chain sequences in an antibody that are shorter than nine residues can be optimized for binding to IL-1 β by inserting appropriate amino acids to increase CDR length.

Variants can also be prepared by "chain shuffling" of light or heavy chains. Marks et al. (1992), Biotechnology 10: 779-83. A single (or heavy) light chain may be combined with a library having a repertoire of heavy (or light) chains and the resulting population is screened for a desired activity, such as binding to IL-1β. This allows screening of a larger sample of different heavy (or light) chains in combination with a single light (or heavy) chain than is possible with libraries comprising both heavy and light chain repertoires.

IL-I-binding antibodies (e.g., IL-Iβ) and fragments of the present invention encompass derivatives of the exemplary antibodies, fragments and sequences disclosed herein. Derivatives include polypeptides or peptides or variants, fragments or derivatives thereof which have been chemically modified. Examples include covalent bonding of one or more polymers, such as water-soluble polymers, N-linked or O-linked carbohydrates, sugars, phosphates and / or other such molecules. Derivatives are modified in a manner that is different from naturally occurring or starting peptide or polypeptides in the type or location of the bound molecules. Derivatives further include deletion of one or more chemical groups that are naturally present in the peptide or polypeptide.

IL-1β binding antibodies and fragments of the present invention may be bispecific. Bispecific antibodies or fragments may be of various configurations. For example, bispecific antibodies may resemble single antibodies (or antibody fragments) but have two different antigen binding sites (variable regions). Bispecific antibodies can be produced by chemical techniques (Kranz et al. (1981), Proc. Natl. Acad. Sci. USA, 78: 5807), by "polidoma" techniques (US Pat. No. 4,474,893) or by recombinant DNA techniques. Bispecific antibodies of the present invention may have binding specificities for at least two different epitopes, at least one of which is an IL-1β epitope. IL-10β binding antibodies may also be heteroantibodies. Heteroantibodies are two or more antibodies or binding antibody (Fab) fragments linked together, each antibody or fragment having a different specificity.

Techniques for creating recombinant DNA versions of the antigen binding regions of antibody molecules that divert the generation of monoclonal antibodies are considered for the present IL-I binding antibodies (e.g., IL-1β) and fragments. DNA is cloned into a bacterial expression system. An example of such a suitable technique for practicing this invention uses a lambda bacteriophage vector system having a leader sequence that causes the expressed Fab protein to migrate to the periplasmic space (between the bacterial cell membrane and the cell wall) or be secreted. Larger numbers of functional Fab fragments can be generated and screened in place of those that bind IL-Iβ. Such IL-1β binding agents (Fab fragments with specificity for an IL-Iβ polypeptide) are specifically encompassed within the IL-Iβ binding antibodies and fragments of the present invention.

The present IL-I-binding antibodies (e.g., IL-1β) and fragments may be humanized or engineered human antibodies. As used herein, a humanized antibody or antigen binding fragment thereof is a recombinant polypeptide comprising a portion of an antigen binding site of a non-human antibody and a portion of the matrix and / or constant regions of a human antibody. A human engineered antibody or antibody fragment is a non-human (e.g., mouse) antibody that has been engineered by modifying (e.g., deleting, inserting or substituting) amino acids at specific positions to reduce or eliminate any detectable immunogenicity. of the modified antibody in a human.

Humanized antibodies include chimeric antibodies and CDR-grafted antibodies. Chimeric antibodies are antibodies that include a non-human antibody variable region bound to a human constant region. Thus, in chimeric antibodies, the variable region is most often non-human and the constant region is human. Chimeric antibodies and methods for making them are described in Morrison, et al., Proc. Natl. Acad. Know. USA, 81: 6841-6855 (1984), Boulianne, et al., Nature, 312: 643-646 (1984) and PCT Application Publication WO 86/01533. Although they may be less immunogenic than a mouse monoclonal antibody, chimeric antibody administrations have been associated with human anti-mouse antibody (HAMA) responses to the non-human portion of the antibodies. Chimeric antibodies can also be produced by joining genes from a mouse antibody molecule of appropriate antigen binding specificity together with genes from a human antibody molecule of appropriate biological activity, such as the ability to activate human complement and complement. mediate ADCC. Morrison et al. (1984), Proc. Natl. Acad. Sci. 81: 6851; Neuberger et al. (1984), Nature, 312: 604. An example is the replacement of an Fc region with that of a different isotype.

CDR-grafted antibodies are antibodies that include the CDRs of a non-human "donor" antibody bound to the matrix region of a human "receptor" antibody. In general, CDR-grafted antibodies include more human antibody sequences than chimeric antibodies because they include both the constant region sequences and the variable region (matrix) sequences of human antibodies. Thus, for example, a CDR-grafted humanized antibody of the invention may comprise a heavy chain comprising a contiguous amino acid sequence (e.g., about 5 or more, 10 or more or even 15 or more contiguous amino acid residues) of the invention. matrix region of a human antibody (e.g. FR-1, FR-2 or FR-3 of a human antibody) or optionally most or all of the entire matrix region 10 of a human antibody. CDR-grafted antibodies and methods for making them are described in, Jones et al., Nature, 321: 522-525 (1986), Riechmann et al., Nature, 332: 323-327 (1988) and Verhoeyen et al. ., Science, 239: 1534-1536 (1988)). Methods that can be used to produce humanized antibodies are also described in U.S. Patent Nos. 4,816,567, 5,721,367, 5,837,243 and 6,180,377. CDR-grafted antibodies are considered less likely than chimeric antibodies to induce an immune reaction against non-human antibody moieties. However, it has been reported that donor antibody matrix sequences are required for donor antibody affinity and / or binding specificity, 20 presumably because these matrix sequences affect the fold of the donor antibody antigen binding portion. Therefore, when non-human donor CDR sequences are grafted onto unaltered human matrix sequences, the resulting CDR-grafted antibodies may exhibit, in some cases, loss of binding avidity relative to the original non-human donor antibody. See, for example, Riechmann et al., Nature, 332: 323-327 (1988) and Verhoeyen et al., Science, 239: 1534-1536 (1988).

Human engineered antibodies include, for example, "embedded" antibodies and antibodies prepared using HUMAN ENGINEERING technology (U.S. Patent 5,869,619). HUMAN ENGINEERING® technology is commercially available and involves altering a non-human antibody or antibody fragment, such as a mouse or chimeric antibody or antibody fragment, making specific changes to the amino acid sequence of the antibody to produce 5 a modified antibody with reduced immunogenicity in a human that nonetheless retains the desirable binding properties of the original non-human antibodies. In general, the technique involves classifying amino acid residues of a non-human (eg mouse) antibody as "low risk", "moderate risk" or "high risk" residues. Scoring is performed using an overall risk / reward calculation that assesses the predicted benefits of doing particular substitution (eg for immunogenicity in humans) against the risk that substitution will affect antigen folding and / or binding properties. of the resulting antibody. Thus, a low risk position is one for which a substitution is predicted to be beneficial because it is predicted to reduce immunogenicity without significantly affecting antigen binding properties. A moderate risk position is one for which a substitution is predicted to reduce immunogenicity, but is most likely to affect protein folding and / or antigen binding. The 20 high-risk positions contain residues that are likely to be involved in proper antigen folding or ligation. In general, low-risk positions in a non-human antibody are replaced with human residues, high-risk positions are rarely replaced, and humanization substitutions at moderate risk positions are sometimes made, although not indiscriminately. Proline positions in the non-human antibody variable region sequence are usually classified as at least moderate risk positions.

The particular human amino acid residue to be substituted at a given low or moderate risk position of a non-human (e.g., mouse) antibody sequence may be selected by aligning an amino acid sequence of the non-human antibody variable regions with the corresponding region of a specific or consensus human antibody sequence. Amino acid residues at low or moderate risk in the non-human sequence may be substituted in place of the corresponding residues in the human antibody sequence according to alignment. Techniques for making human engineered proteins are described in more detail in Studnicka et al., Protein Engineering, 7: 805-814 (1994), US Patents 5,766,886, 5,770,196, 5,821,1123 and 5,869,619 and Publication. PCT Application WO 93/11794.

"Embedded" antibodies are non-human or humanized antibodies (e.g., chimeric or CDR-grafted antibodies) that have been engineered to replace certain solvent-exposed amino acid residues to further reduce their immunogenicity or enhance their function. Since surface residues of a chimeric antibody are supposed to be less likely to affect the appropriate antibody fold and more likely to evoke an immune reaction, embedding of a chimeric antibody may include, for example, identifying solvent exposed residues in the region. non-human matrix of a chimeric antibody and substituting at least one of them with the corresponding surface residues of a human matrix region. Embedding may be performed by any suitable engineering techniques, including the use of the HUMAN ENGINEERING® technology described above.

In a different method, a recovery of binding activity can be achieved by "dehumanizing" a CDR-grafted antibody. Dehumanization may include restoring residues of the donor antibody matrix regions to the CDR-grafted antibody, thereby restoring the appropriate fold. Similar to "dehumanization" can be obtained by (i) including portions of the "donor" matrix region in the "receptor" antibody or (ii) grafting portions of the "donor" antibody matrix region into the receptor antibody (together with the CDRs grafted donors).

For further discussion of antibodies, humanized antibodies, human engineers and methods for their preparation, see Kontermann and Dube], eds., Antibody Engineering, Springer, New York, NY, 2001.

Exemplary humanized or engineered human antibodies include IgG, IgM, IgE, IgA and IgD antibodies. The present antibodies may be of any class (IgG, IgA, IgM, IgE, IgD, etc.) or isotype and may comprise a kappa or lambda light chain. For example, a human antibody may comprise an IgG heavy chain or defined fragment, such as at least one of the isotypes, IgG1, IgG2, IgG3 or IgG4. As another example, the present antibodies or fragments may comprise an IgG1 heavy chain and an IgG1 light chain.

The present antibodies and fragments may be human antibodies, such as antibodies that bind IL-1β polypeptides and are encoded by nucleic acid sequences that are naturally occurring somatic variants of the human germline immunoglobulin nucleic acid sequence and fragments, synthetic variants. , derivatives and fusions thereof. Such antibodies may be produced by any method known in the art, such as through the use of transgenic mammals (such as transgenic mice) wherein the native immunoglobulin repertoire has been replaced with human V genes on the mammalian chromosome. Such mammals appear to carry VDJ recombination and somatic hypermutation of human germline antibody genes in a normal manner, thus producing high affinity antibodies with fully human sequences.

Human antibodies to target protein can also be produced using transgenic animals that have no endogenous immunoglobulin production and are engineered to contain human immunoglobulin sites. For example, WO 98/24893 discloses transgenic animals having a human Ig site in which animals do not produce functional endogenous immunoglobulins due to inactivation of endogenous heavy and light chain sites. WO 91/00906 also discloses transgenic non-primate mammalian hosts capable of mounting an immune response to an immunogen, wherein the antibodies have primate constant and / or variable regions and wherein the endogenous immunoglobulin coding sites are substituted or inactivated. WO 96/30498 and US Patent No. 6,091,001 disclose the use of the Cre / Lox System to modify the immunoglobulin site in a mammal, such as to replace all or a portion of the constant or variable region to form a molecule. modified antibody. WO 94/02602 discloses non-human mammalian hosts having inactivated endogenous Ig sites and functional human Ig sites. U.S. Patent No. 5,939,598 discloses methods of manufacturing transgenic mice wherein the mice lack endogenous heavy chains and express an exogenous immunoglobulin site comprising one or more xenogenic constant regions. See also U.S. Patent Nos. 6,114,598, 6,657,103 and 6,833,268.

Using a transgenic animal described above, an immune response can be produced for a selected antigenic molecule and antibody-producing cells can be removed from the animal and used to produce hybridomas that secrete human monoclonal antibodies. Immunization, adjuvant and other protocols are known in the art and are used in immunization, for example, of a transgenic mouse as described in WO 96/33735. This publication discloses monoclonal antibodies against a variety of antigenic molecules including IL-6, IL-8, TNFα, human CD4, L selectin, gp39 and tetanus toxin. Monoclonal antibodies may be tested for their ability to inhibit or neutralize the biological activity or physiological effect of the corresponding protein. WO 96/33735 discloses that IL-8 monoclonal antibodies derived from immune cells from IL-8 immunized transgenic mice blocked IL-8 induced functions of 5 neutrophils. Human monoclonal antibodies with antigen specificity used to immunize transgenic animals are also disclosed in WO 96/34096 and U.S. Patent Application No. 20030194404; and U.S. Patent Application No. 20030031667.

Additional transgenic animals useful for making monoclonal antibodies include Medarex's HuMAb-MOUSE®, described in U.S. Pat. No. 5,770,429 and Fishwild, et al. (Nat. Biotechnol. 14: 845-851, 1996), which contains non-rearranged human antibody gene gene sequences that encode the human antibody heavy and light chains. Immunization of a HuMAb-MOUSE® enables the production of fully human monoclonal antibodies to the target protein.

Also, Ishida et al. (Cloning Stem Cells. 4: 91-102, 2002) describes the TransChromo Mouse (TCMOUSE®) which comprises megabase-sized segments of human DNA and incorporates whole human immunoglobulin (hlg) sites. TCMOUSE® has a totally diverse repertoire of hlgs, including all IgG subclasses (IgGl-G4). Immunization of TCMOUSE® with various human antigens produces antibody responses that comprise human antibodies.

See also Jakobovits et al., Proc. Natl. Acad. Know. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255-258 (1993); Bruggermann et 25 al., Year in Immunol., 7: 33 (1993); and Pat. No. 5,591,669, U.S. Patent No. 5,589,369, U.S. Patent No. 5,545,807; and U.S. Patent Publication No. 20020199213. U.S. Patent Publication No. 20030092125 describes methods for polarizing an animal's immune response to the desired epitope. Human antibodies can also be generated by in vitro activated B cells (see U.S. Patent Nos. 5,567,610 and 5,229,275).

Human antibodies can also be generated by screening for in vitro antibody demonstration libraries. See Hoogenboom et al. (1991), J. Mol. Biol. 227: 381; and Marks et al. (1991), J.

5 Mol. Biol. 222: 581. Several antibody-containing phage demonstration libraries have been described and can be readily prepared. Libraries may contain a variety of human antibody sequences, such as human Fab, Fv and scFv fragments, which may be screened against an appropriate target. Phage demonstration libraries may comprise peptides or proteins other than antibodies that may be screened to identify IL-1β selective binding agents.

The development of technologies for making recombinant human antibody gene repertoires and the demonstration of antibody fragments encoded on the filamentous bacteriophage surface has provided a means for making human antibodies directly. Antibodies produced by phage technology are produced as antigen binding fragments - usually Fv or Fab fragments - in bacteria and thus lack effector functions. Effector functions can be introduced by one of two strategies: Fragments can be engineered into complete antibodies for expression in mammalian cells or bispecific antibody fragments with a second binding site capable of triggering an effector function.

The invention provides a method for producing target specific antibody or antigen binding portion thereof comprising the steps of synthesizing a human phage antibody library, sorting the library with the target protein or a portion thereof, isolating the phage that bind to the target and obtain the phage antibody. By way of example, a method for preparing the antibody library for use in phage demonstration techniques comprises the steps of immunizing a non-human animal comprising human immunoglobulin sites with target antigen or an antigenic portion thereof to create an immune response. extracting antibody producing cells from the immunized animal; isolate RNA from extracted cells, reverse transcribe RNA to produce cDNA, amplify cDNA using an primer, and insert cDNA into a phage display vector such that antibodies are expressed in the phage. The recombinant target specific antibodies of the invention may be obtained in this manner.

Phage demonstration processes mimic immune selection by demonstrating antibody repertoires on the surface of filamentous bacteriophages and subsequent selection of phage by their binding to an antigen of choice. One such technique is described in WO 99/10494, which describes the isolation of high affinity and functional agonistic antibodies to MPL and msk receptors using such a method. Antibodies of the invention may be isolated by screening for a recombinant combinatorial antibody library, preferably a scFv phage demonstration library prepared using human V1 and Vh cDNAs prepared from human lymphocyte derived mRNA. Methodologies for preparing and sorting such libraries are known in the art. See for example, U.S. Patent No. 5,969,108. There are commercially available kits for generating phage demonstration libraries (for example, the Pharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01; and Stratagene SurfZAP® phage demonstration kit, catalog no. 240612). There are also other methods and reagents that may be used in the generation and screening of antibody demonstration libraries (see, for example, Ladner et al. US Pat. No. 5,223,409; Kang et al. PCT Publication No. WO 92/18619; Dower et al. PCT Publication No. WO 91/17271; Winter et al. PCT Publication No. WO 92/20791; Markland et al. PCT Publication No. WO 92/15679; Breitling et al. PCT Publication No. WO 93/01288; McCafferty et PCT Publication No. WO 92/01047 Garrard et al PCT Publication No. WO 92/09690 Fuchs et al (1991) Bio / Technology 9: 1370-1372 Hay et al (1992) Hum Antibod. Hybridomas 3: 81-85; Huse et al. (1989) Science 246: 1275-1281; McCafferty et al., Nature (1990) 348: 552-554; Griffiths et al. (1993) EILIBO 5 J 12: 725- 734; Hawkins et al. (1992) J. Mol. Biol 226: 889-896; Clackson et al. (1991) Nature 352: 624-628; Gram et al. (1992) Proc. Natl. Acad. Sci. USA 89: 3576-3580; Garrad et al. (1991) Bio / Technology 9: 1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19: 4133-4137; and Barbas et al. (1991) Proc. Natl. Acad. Know. USA 88: 7978-7982.

In one embodiment, to isolate human antibodies

Specific to the target antigen with the desired characteristics, a human Vr and Vl library is screened to select for antibody fragments having the desired specificity. Antibody libraries used in this method are preferably prepared and screened scFv libraries as described herein and in the art (McCafferty et al., PCT Publication No. WO 92/01047, McCafferty et al., (Nature 348: 552-554, Griffiths et al., (EMBO J 12: 725-734, 1993) The scFv antibody libraries are preferably screened using target protein as the antigen.

Alternatively, the Fd fragment (Vh-ChI) and antibody light chain 20 (Vl-Cl) are separately cloned by PCR and randomly recombined into combinatorial phage display libraries, which can then be selected for binding to a particular antigen. . Fab fragments are expressed on the phage surface, that is, physically linked to the genes encoding them. Thus, selection of Fab by antigen binding co-selects for Fab-encoding sequences, which can be subsequently amplified. Through several rounds of antigen binding and re-amplification, a procedure called panning, antigen-specific Fab, is enriched and finally isolated.

In 1994, a method for antibody humanization, called "targeted selection", was described. Targeted selection utilizes the energy of phage demonstration techniques for humanization of mouse monoclonal antibody (See, Jespers, L. S., et al., Bio / Technology 12, 899-903 (1994)). For this, the Fd fragment of mouse monoclonal antibody 5 can be demonstrated in combination with a human light chain library and the resulting hybrid Fab library can then be selected with antigen. The mouse Fd fragment thus provides a pattern for guiding selection. Subsequently, the selected human light chains are combined with a library of 10 human Fd fragment. Selection of the resulting library produces fully human Fab.

A variety of procedures have been described for deriving human antibodies from phage demonstration libraries (See, for example, Hoogenboom et al., J. Mol. Biol., 227: 381 (1991); Marks et al., J. 15 Mol. Biol, 222: 581-597 (1991); US Pat. Nos. 5,565,332 and 5,573,905; Clackson, T. and Wells, JA, TIBTECH 12, 173-184 (1994)). In particular, in vitro selection and evolution of antibodies derived from phage demonstration libraries has become a powerful tool (See Burton, DR and Barbas III, CF, Adv. Immunol. 57, 191-280 (1994); Winter, G., et al., Annu, Rev. Immunol., 12, 433-455 (1994); US Patent Publication No. 20020004215 and WO 92101047; US Patent Publication No. 20030190317; and US Patent No. 6,054,287 and 5,877,293.

Watkins, “Screening of Phage-Expressed Antibody Libraries by Capture Lift,” Methods in Molecular Biology, Antibody Phage Display: 25 Methods and Protocols 178: 187-193 (2002) and US Patent Publication No. 20030044772, published March 6 2003, describes methods for screening antibody libraries expressed on phage or other binding molecules by capture uptake, a method that involves immobilizing candidate binding molecules on a solid support. Fv fragments are demonstrated on the phage surface by associating a chain expressed as a phage protein fusion (e.g., with M13 gene III) with the complementary strand expressed as a soluble fragment. It is considered that the phage may be a filamentous phage such as one of the class I phages: fd, M13, fl, Ifl, Ike, ZJ / Z, Ffe and one of the class II phages Xf, Pfl and Pf3. The phage may be M1 or fd or a derivative thereof.

Once the initial human Vl and Vh segments are selected, "mix and match" experiments, in which different pairs of the initially selected Vl and Vh segments are screened for target binding, are performed to select Vl./ pair combinations. Vh favorites. Additionally, to further improve antibody quality, the Vl and Vh segments of the preferred VL / VH pair (s) may be randomly mutated, preferably within any of the Vh CDR1, CDR2 or CDR3 regions. and / or VL, in a process analogous to the in vivo somatic mutation process responsible for antibody affinity maturation during a natural immune response. This in vitro affinity maturation can be accomplished by amplifying V1 and Vh regions using PCR primers complementary to Vh CDR1, CDR2 and CDR3 or VL CDR1, CDR2 and CDR3, respectively, which primers were "boosted" with a mixture. randomization of the four nucleotide bases at certain positions such that the resulting PCR products encode the V1 and Vh segments in which the random mutations were introduced into the Vh and / or VL CDR3 regions. These randomly mutated V1 and Vh segments can be screened again for binding to the target antigen.

Following screening and isolation of a specific target antibody from a recombinant immunoglobulin demo library, the nucleic acid encoding the selected antibody can be recovered from the demo pack (for example from the phage genome) and subcloned into other expression vectors by standard recombinant DNA techniques. If desired, the nucleic acid may be further engineered to create other forms of antibody of the invention as described below. To express a recombinant human antibody isolated by screening a combinatorial library, the DNA encoding the antibody is cloned into a recombinant expression vector and introduced into a mammalian host cell as described herein.

It is contemplated that the phage display method may be performed on a bacterial mutant strain or host cell. A mutant strain is a host cell that has a genetic defect that causes the replicated DNA within it to mutate with respect to its precursor DNA. Exemplary mutant strains are NR9046mutD5 and NR9046 mut Ti.

It is also considered that the phage display method can be performed using an auxiliary phage. This is a phage that is used to infect cells containing a defective phage genome that works to complement the defect. The defective phage genome may be a phagemid or a phage with some function encoding the removed gene sequences. Examples of phage aids are M13K07, M13K07 gene III No. 3; and phage demonstrating or encoding a binding molecule fused to a capsid protein.

Antibodies are also generated by phage demonstration screening methods using the hierarchical double combinatorial method as disclosed in WO 92/01047 wherein an individual colony containing an H or L chain clone is used to infect a complete library of clones. which encode the other chain (L or H) and the specific binding member of the two chains is selected according to phage display techniques such as those described herein. These techniques are also disclosed in Marks et al. (Bio / Technology, 10: 779-783, 1992).

Methods for demonstrating peptides on the surface of yeast and microbial cells were also used to identify antigen specific antibodies. See, for example, U.S. Patent No. 5,666,658. Antibody libraries may be linked to yeast proteins such as agglutinin, effectively mimicking the demonstration of antibody cell surface by B cells in the immune system.

In addition to phage display methods, antibodies can be isolated using ribosomal mRNA display methods 10 and microbial cell display methods. Polypeptide selection using the ribosome demonstration is described in Hanes et al., (Proc. Natl Acad Sci USA, 94: 4937-4942, 1997) and Pat. No. 5,643,768 and 5,658,754 issued to Kawasaki. Ribosome demonstration is also useful for rapid large scale mutational analysis of antibodies. The selective mutagenesis method also provides a method of producing antibodies with enhanced activities that can be selected using ribosomal demonstration techniques.

IL-I binding antibodies (e.g., IL-1β) and fragments may comprise one or more moieties that do not bind IL-20 1 β but instead are responsible for other functions, such as circulating half-life. , direct cytotoxic effect, detectable labeling or activation of receptor endogenous complement cascade or endogenous cellular cytotoxicity. The antibodies or fragments may comprise all or a portion of the constant region and may be of any isotype, including IgA (e.g., IgAl or IgA2), IgD, IgE, IgG (e.g. IgG1, IgG2, IgG3 or IgG4) or IgM . In addition to or instead of comprising a constant region, antigen binding compounds of the invention may include an epitope label, a recovery receptor epitope, a label portion for diagnostic or purification purposes or a cytotoxic portion such as a radionuclide or toxin.

The constant region (when present) of the present antibodies and fragments can be of type γΐ, y2, γ3, γ4, u, β2 or δ or ε, preferably of type γ, more preferably of type y, whereas the constant part of A human light chain may be of type κ or λ (which includes subtypes λ \, λ2 and λ3) but is preferably of type κ.

Variants also include antibodies or fragments comprising a modified Fc region, wherein the modified Fc region comprises at least one amino acid modification relative to a wild-type Fc region. The variant Fc region may be designed relative to a comparable molecule comprising the wild-type Fc region so as to bind Fc receptors with greater or lesser affinity.

For example, IL-1β binding antibodies and fragments present may comprise a modified Fc region. The Fc region refers to naturally occurring or synthetic polypeptides homologous to the IgG C-terminal domain that is produced in digestion with IgG papain. IgG Fe has a molecular weight of approximately 50 kD. In the present antibodies and fragments, an entire Fc region may be used or only a half-life enhancing portion. Furthermore, many modifications to the amino acid sequence are acceptable, since native activity is not always necessary or desired.

The Fc region may be mutated, if desired, to inhibit its ability to complement complement and bind the high affinity Fc receptor. For murine IgG Fe, substitution of Ala residues in place 25 of Glu 318, Lys 320 and Lys 322 makes the protein unable to direct ADCC. Glu substitution in place of Leu 235 inhibits the protein's ability to bind the high affinity Fc receptor. Various mutations for human IgG are also known (see, for example, Morrison et al., 1994, The Immunologist 2: 119 124 and Brekke et al., 1994, The Immunologist 2: 125). In some embodiments, the present antibodies or fragments are provided with a modified Fc region where a naturally occurring Fc region is modified to increase the half-life of the antibody or fragment in a biological environment, for example, the serum half or a half life as measured by an in vitro assay. Methods for altering the original form of an Fc region of an IgG are also described in U.S. Patent No. 6,998,253.

In certain embodiments, it may be desirable to modify the antibody or fragment to increase serum half-life, for example by adding molecules such as PEG or other water-soluble polymers, including polysaccharide polymers, to antibody fragments to increase serum half life. This can also be achieved, for example, by incorporating a salvage receptor binding epitope into the antibody fragment (e.g., by mutating the appropriate region in the antibody fragment or incorporating the epitope into a peptide label which is then fused to the antibody fragment at the end or in the medium, for example by DNA or peptide synthesis) (see International Publication No. WO96 / 32478). Recovery receptor binding epitope refers to an Fc region epitope of an IgG molecule (e.g., IgGi, IgG2, IgG3 or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.

A salvage receptor binding epitope may include a region in which any one or more amino acid residues of one or two loops of an Fc domain are transferred to an analogous position of the antibody fragment. Even more preferably, three or more residues from one or two loops of the Fc domain are transferred. Even more preferred, the epitope is taken from the CH2 domain of the Fc region (e.g., from an IgG) and transferred to the CHI, CH3 or Vh region or more than one such region of the antibody. Alternatively, the epitope is taken from the CH2 domain of the Fc region and transferred to the C1 region or region V1 or both of the antibody fragment. See also international applications WO 97/34631 and WO 96/32478 describing Fc variants and their interaction with the recovery receptor.

5 Mutation of residues within receptor binding sites

Fc may result in altered effector function, such as altered ADCC or CDC activity or altered half life. Potential mutations include insertion, deletion or substitution of one or more residues, including alanine substitution, conservative substitution, non-conservative substitution or substitution with a corresponding amino acid residue at the same position as a different IgG subclass (for example substituting an IgG1 residue with a corresponding IgG2 residue at that position). For example it has been reported that mutating serine at amino acid position 241 in IgG4 to proline (found at that position in IgG1 and 15 IgG2) led to the production of a homogeneous antibody as well as prolonged serum half-life and improving tissue distribution compared to Original chimeric IgG4. (Angal et al, Mol Immunol. 30: 105-8, 1993).

Antibody fragments are portions of a full-size intact antibody, such as an antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab ', F (ab') 2 and Fv fragments; diabodies; linear antibodies; single chain antibody molecules (e.g. scFv); multispecific antibody fragments such as bispecific, trypecific and multispecific antibodies (e.g., diabodies, triacibodies, tetribodies); 25 minibodies; recombinant chelating antibodies; tribodies or bi-bodies; intrabodies; nanobodies; small modular immunopharmaceuticals (SMIPs), adnectins, binding domain immunoglobulin fusion proteins; camelized antibodies; Vex-containing antibodies; and any other polypeptides formed from antibody fragments. The present invention includes IL-1β antibody binding fragments which comprise any of the foregoing heavy or light chain sequences and which bind IL-1β. The term fragments as used herein refers to any 3 or more contiguous amino acids (e.g., 4 or more, 5 or more 6 or more, 8 or more or even 10 or more contiguous amino acids) of the antibody and encompasses Fab fragments, Fab ', F (ab') 2 and F (v) or the individual light or heavy chain variable region or portion thereof. IL-Iβ binding fragments include, for example, Fab, Fab ', F (ab') 2, Fv and scFv. These fragments lack the Fc fragment of an intact antibody, clear faster from circulation, and may have less nonspecific tissue binding than an intact antibody. See Wahl et al. (1983), J. Nucl. Med. 24: 316-25. These fragments can be produced from intact antibodies using well known methods, for example by proteolytic cleavage with enzymes such as papain (to produce Fab fragments) or pepsin (to produce F (ab ') 2 fragments).

In vivo and cell-based assays are well described in the art for use in determining IL-1β binding to IL-I Type I receptor (IL-1R1), including assays that determine the presence of molecules (such as antibodies, antagonists or other inhibitors) that bind IL-1β or IL-1R1. (see for example Evans et al. (1995), J. Biol. Chem. 270: 11477-11483; Vigers et al. (2000), J. Biol. Chem. 275: 36927-36933; Yanofsky et al. (1996), Proc. Natl. Acad. Sci. USA 93: 7381-7386; Fredericks et al. (2004), Protein Eng. Des. Sci. 17: 95-106; Slack et al. J. Biol Chem 268: 2513-2524; Smith et al. (2003) Immunity 18: 87-96; Vigers et 25 al. (1997) Nature 386: 190-194; Ruggiero et al. (1997) J. Immunol 158: 3881-3887 Guo et al. (1995) J. Biol Chem. 270: 27562-27568 Svenson et al. (1995) Bur J. Immunol 25: 2842-2850; Arend et al. (1994), J. Immunol. 153: 4766-4774). Recombinant Type I IL-I receptor, including Type 1 human IL-I receptor, for such assays is readily available from a variety of commercial sources (see for example R&D Systems, SIGMA). The IL-I Type I receptor may also be expressed from a construct or expression vector introduced into an appropriate host cell using standard molecular biology and transfection techniques known in the art. The expressed IL-I Type I receptor may then be isolated and purified for use in binding assays or alternatively used directly in a cell associated form.

For example, binding of IL-1β to IL-I Type I receptor can be determined by immobilizing an IL-1β binding antibody, IO by contacting IL-1β with the immobilized antibody and determining whether IL-1β β was bound to the antibody and contacting a soluble form of IL-IR1 with the bound IL-1β / antibody complex and determining whether soluble IL-IR1 was bound to the complex. The protocol may also include contacting soluble IL-IR1 with immobilized antibody prior to contact with IL-1 β to confirm that soluble IL-15 IR1 does not bind to immobilized antibody. This protocol can be performed using a Biacore® instrument for kinetic analysis of binding interactions. Such a protocol may also be used to determine whether an antibody or other molecule permits or blocks binding of IL-1β to the Type I IL-1 receptor.

For other IL-1 / IL-1R1 binding assays, permission

or blockade of IL-1β binding to IL-I Type I receptor can be determined by comparing IL-1β binding to IL-IR1 in the presence or absence of IL-1β antibodies or IL-1β binding fragments thereof. . Blockade is identified in the assay reading as a designated 25 IL-1β reduction to IL-I Type I receptor in the presence of anti-IL-β antibodies or IL-1β binding fragments of these when compared to a sample containing the corresponding buffer or diluent but not an IL-1 β antibody or IL-β binding fragment thereof. The assay reading may be qualitatively visualized as indicating the presence or absence of blockage or may be quantitatively visualized as indicating a percent or fold reduction in binding due to the presence of antibody or fragment.

Alternatively or additionally, when an IL-1β binding antibody or IL-Iβ binding fragment substantially blocks IL-1β binding to IL-IRI, IL-1β binding to IL-IR1 is reduced by at least at least 10 times, alternatively at least about 20 times, alternatively at least about 50 times, alternatively at least about 100 times, alternatively at least about 1000 times, alternatively at least about 10,000 times or more compared to bonding same IL-1β and IL-IR1 concentrations in the absence of antibody or fragment. As another example, when an IL-1β binding antibody or IL-1β binding fragment substantially permits binding of IL-1β to IL-IR1, binding of IL-1β to IL-IR1 is at least about 90%, alternatively at least about 95%, alternatively at least about 99%, alternatively at least about 99.9%, alternatively at least about 99.99%, alternatively at least about 99.999 %, alternatively at least about 99.9999%, alternatively substantially identical to binding of the same concentrations of IL-Iβ and IL-IR1 in the absence of antibody or fragment.

The present invention may in certain embodiments encompass IL-Iβ binding antibodies or IL-Iβ binding fragments that bind to the same or substantially the same epitope as one or more of the exemplary antibodies described herein. Alternatively or in addition, IL-Iβ binding antibodies or IL-1β binding fragments compete with the binding of an antibody having AB7 variable region sequences, described in US application number 111472813 (sequences shown below). Alternatively or additionally, the present invention encompasses IL-1β-binding antibodies and fragments that bind to an epitope contained in the amino acid sequence ESVDPKNYPKKKMEKRFVFNKIF (SEQ ID NO: 1), an epitope to which antibodies are designated AB5 and AB7 (US application number 111472813) bind. As considered herein, it can be readily determined whether an IL-1β binding antibody or fragment binds to the same or substantially the same epitope as one or more of the exemplary antibodies, such as for example the antibody designated AB7, using any one of several methods known in the art.

For example, key amino acid (epitope) residues linked by an IL-1β binding antibody or fragment can be determined using a peptide array, such as for example a PepSpot® peptide array (JPT Peptide Technologies, Berlin, Germany), in which a scan of twelve amino acid peptides spanning the entire IL-1 β amino acid sequence, each peptide overlapping by 11 amino acids to the previous one, is synthesized directly on a membrane. The membrane carrying the peptides is then probed with the antibody for which epitope binding information is sought, for example at a concentration of 2 μg / ml, for 2 hours at room temperature. Antibody binding to membrane bound peptides can be detected using a goat anti-human (or mouse, where appropriate) conjugated to secondary HRP antibody, followed by enhanced chemiluminescence (ECL). The peptide spot (s) corresponding to the particular amino acid residues or sequences of the mature IL-1 β protein and which are positive for antibody binding are indicative of the epitope bound by the particular antibody.

Alternatively or in addition, antibody competition experiments may be performed and such assays are well known in the art. For example, to determine if an antibody or fragment binds to an epitope contained in a peptide sequence comprising the amino acids ESVDPKNYPKKKMEKRFVFNKIE (SEQ ID NO: 1), which corresponds to residues 83 to 105 of the mature IL-Iβ protein, An antibody of unknown specificity may be compared with any of the exemplary antibodies (e.g., AB7) of the present invention which are known to bind an epitope contained within this sequence. Binding competition assays can be performed, for example, using a Biacore instrument for kinetic analysis of binding interactions or by ELISA. In such an assay, antibody of unknown epitope specificity is evaluated for its ability to compete for binding against known comparator antibody (e.g., AB7). Competition for binding to a particular epitope is determined by a reduction in binding to the IL-1β epitope of at least about 50% or at least about 70% or at least about 80%) or at least about 90%. or at least about 95% or at least about 99% or about 15 100%) with respect to the known comparator antibody (e.g., AB7) and is indicative of binding to substantially the same epitope.

In view of the identification in this disclosure of IL-1β binding regions in exemplary antibodies and / or epitopes recognized by the disclosed antibodies, it is considered that additional antibodies having similar binding or therapeutic or diagnostic utility characteristics may be generated that compare to the embodiments of this disclosure.

Antigen binding fragments of an antibody include fragments that retain the ability to specifically bind to an antigen, generally by retaining the antigen binding portion of the antibody. It is well established that the antigen binding function of an antibody can be performed by fragments of a life-size antibody. Examples of antigen binding moieties include (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) an F (ab ') 2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region;

(iii) an Fd fragment which is the VH and CHI domains; (iv) an Fv fragment that is the VL and VH domains of a single antibody branch, (v) a dAb fragment (Ward et al., (1989) Nature 341: 544-546), which is a VH domain ; and (vi) an isolated complementarity determining region (CDR). Single chain antibodies are also encompassed within the term antigen binding portion of an antibody. IL-1β binding antibodies and fragments of the present invention also encompass monovalent or multivalent or monomeric or multimeric (e.g. tetrameric) CDR-derived binding domains, with or without a backbone (e.g., protein or carbohydrate backbone) .

The present IL-1β binding antibodies or fragments may be part of a larger immunoadhesion molecule formed by covalent or non-covalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of the streptavidin nucleus to make a tetrameric scFv molecule (Kipriyanov, SM, et al. (1995) Human Antibody and Hybridomas 6: 93-101) and the use of a cysteine residue , a marker peptide and a C-terminal polystidine label for making divalent and biotinylated scFv molecules (Kipriyanov, SM, et al. (1994) Mol. Immunol. 31: 1047-1058). Antibodies and fragments comprising immunoadhesion molecules may be obtained using standard recombinant DNA techniques as described herein. Preferred antigen binding moieties are whole domains or full domain pairs.

IL-1β binding antibodies and fragments of the present invention also encompass domain (dAb) antibody fragments (Ward et al., Nature 341: 544-546, 1989) consisting of a VH domain. The IL-1β binding antibodies and fragments of the present invention also encompass diabodies, which are bivalent antibodies wherein the Vh and V1 domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing. between the two domains in the same chain, thereby forcing the domains to pair with the complementary domains of another chain and creating two antigen binding sites (see for example, EP 404,097; WO 93111161; Holliger et al. , Proc. Natl. Acad. Sci. USA 90: 6444-6448, 1993 and Poljak et al., Structure 2: 1121-1123, 1994). Diabodies can be bispecific or monospecific.

IL-1β binding antibodies and fragments of the present

The invention also encompasses single chain antibody (scFv) fragments that bind IL-1β. An scFv comprises an antibody heavy chain variable region (Vh) operably linked to an antibody light chain variable region (V1) wherein the heavy chain variable region and the light chain variable region, together or individually, form one another. a binding site that binds IL-Iβ. An scFv may comprise an amino terminal end Vh region and a carboxy terminal end V1 region. Alternatively, scFv may comprise an amino terminal end V1 region and a carboxy terminal end Vh region. In addition, although the two domains of the Fv fragment, V1 and VH, are encoded by separate genes, they can be joined using recombinant methods by a synthetic linker that enables them to be made as a single protein chain in which the V1 regions. and Vh form a pair to form monovalent molecules (known as single chain Fv (scFv); see for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad Sci USA 85: 5879-5883).

An scFv may optionally further comprise a polypeptide linker between the heavy chain variable region and the light chain variable region. Such polypeptide linkers generally comprise from 1 to 50 amino acids, alternatively from 3 to 12 amino acids, alternatively

2 amino acids. An example of a linker peptide for ligating heavy and light chains into an scFv comprises the 5 amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 2). Other examples comprise one or 5 more tandem repeats of this sequence (for example, a polypeptide comprising two to four Gly-Gly-Gly-Gly-Ser repeats (SEQ ID NO: 2) to create linkers.

IL-1β binding antibodies and fragments of the present invention also encompass heavy chain antibodies (HCAb). The 10 exceptions to the H2L2 framework of conventional antibodies occur in some immunoglobulin isotypes found in camelids (camels, dromedaries and llamas; Hamers-Casterman et al., 1993 Nature 363: 446; Nguyen et al., 1998 J. Mol. Biol 275: 413), wobbegong sharks (Nuttall et al., Mol Immunol. 38: 313-26, 2001), lambaru sharks (Greenberg et al., 15 Nature 374: 168-73, 1995; Roux et al., 1998 Proc. Nat. Acad. Sci. USA 95: 11804) and the painted ratfish (Nguyen, et al., "Heavy-chain antibodies in Camelidae; a case of evolutionary innovation," 2002 Immunogenetics 54 (1): 39- 47). These antibodies may apparently form antigen binding regions using only heavy chain variable regions, wherein these functional antibodies are heavy chain only dimers (alluded to as "heavy chain antibodies" or "HCAbs"). Accordingly, some embodiments of the present IL-1β binding antibodies and fragments may be heavy chain antibodies that specifically bind IL-Iβ. For example, heavy chain antibodies that are a class of 25 IgG and devoid of light chains are produced by animals of the genus Camelidae which include camels, dromedaries and llamas (Hamers-Casterman et al., Nature 363: 446-448 (1993)). . HCAbs have a molecular weight of about 95 kDa rather than the molecular weight of about 160 kDa of conventional IgG antibodies. Their binding domains consist only of heavy chain variable domains, often alluded to as VHh to distinguish them from conventional Vh. Muildermans et al., J. Mol. Recognit. 12: 131-140 (1999). The variable domain of heavy chain antibodies is sometimes alluded to as a nanobody (Cortez-Retamozo et al, Cancer 5 Research 64: 2853-57, 2004). A nanobody library can be generated from an immunized dromedary as described in Conrath et al. (Antimicrob Agents Chemother 45: 2807-12, 2001) or using recombinant methods.

Since the first constant domain (CHi) is missing (removed during mRNA processing due to loss of a union consensus signal), the variable domain (VHh) is immediately followed by the hinge region, the Ch2 and Cro domains ( Nguyen et al., Mot. Immunol. 36: 515-524 (1999); Woolven et al., Immunogenetics 50: 98-101 (1999)). Camelid VHh VHh reportedly recombines with the hinge-containing IgG2 and IgG3 constant regions, the CH2 and CH3 domains, and lack a CH1 domain (Hamers-Casterman et al., Supra). For example, llama IgG1 is a conventional antibody isotype (H2L2) wherein Vh recombines with a hinge-containing constant region, the CHI, CH2, and CH3 domains, while llama IgG2 and IgG3 are chain-only isotypes. which lack CH1 domains and do not contain any light chains.

Although HCAbs are light chain-free, they have an antigen binding repertoire. The mechanism of genetic generation of HCAbs is reviewed in Nguyen et al. Adv. Immunol 79: 261-296 (2001) and 25 Nguyen et al., Immunogenetics 54: 39-47 (2002). Sharks, including lambaru shark, demonstrate unique monomeric V domains containing similar antigen receptor. Irving et al., J. Immunol. Methods 248: 31-45 (2001); Roux et al., Proc. Natl. Acad. Know. USA 95: 11804 (1998).

VHhS comprise small intact antigen binding fragments (e.g. fragments that are about 15 kDa, 118 to 136 residues). Camelid VHh domains have been found to bind to the high affinity antigen (Desmyter et al., J. Biol. Chem. 276: 26285-90, 2001), with Vhi affinities typically in the nanomolar range and comparable to those of Fab fragments. and scFv. VffilS are highly soluble and more stable than the corresponding derivatives of scFv and Fab fragments. Vh fragments were relatively difficult to produce in soluble form, but improvement in solubility and specific binding can be obtained when matrix residues are changed to be more stable. same as VHh (See, for example, Reichman et al, J Immunol Methods 1999, 231: 25-38). VHhS carry amino acid substitutions that make them more hydrophilic and prevent prolonged interaction with BiP (immunoglobulin heavy chain binding protein), which normally binds to the H chain in the Endoplasmic Reticulum (ER) during folding and assembly until it is displaced by the L chain. Because of the increased hydrophilicity of the VHhS, ER secretion is improved.

Functional VhhS can be obtained by proteolytic cleavage of HCAb from an immunized camelid, by direct cloning of B cell VHh genes from an immunized camelid that results in recombinant VHhS or from naive or synthetic libraries. VffilS with the desired antigen specificity can also be obtained by the phage demonstration methodology. Using VffilS for phage demonstration is much simpler and more efficient compared to Fabs or scFvs, as only one domain needs to be cloned and expressed to obtain a functional antigen binding fragment. Muildermans, Biotechnol. 74: 277-302 (2001); Ghahroudi et al, FEBS Lett. 414: 521-526 (1997); and van der Linden et al, J. Biotechnol. 80: 261-270 (2000). Methods for generating antibodies having camelid heavy chain are also described in US Patent Publication No. 20050136049 and 20050037421. Ribosome demonstration methods can be used to identify and isolate scFv and / or VHh molecules having binding activity and affinity. desired. Irving et al., J. Immunol. Methods 248: 31-45 (2001). Ribosome demonstration and selection has the potential to generate and demonstrate large libraries (1014).

Other embodiments provide VHh-like molecules generated by the camelization process by modifying non-Camelidae VHs, such as human VhhS, to improve their solubility and prevent non-specific binding. This is achieved by replacing residues 10 on the VLs side of VHs with residues equal to VHh, thereby mimicking the most soluble VHh fragments. Camelized Vh fragments, particularly those based on the human matrix, are expected to exhibit a greatly reduced immune response when administered to a patient in vivo and, consequently, are expected to have significant advantages for therapeutic applications. Davies et al., FEBS Lett. 339: 285-290 (1994); Davies et al., Protein Eng. 9: 531-537 (1996); Tanha et al., J. Biol. Chem. 276: 24774-24780 (2001); and Riechmann etal, Immunol. Methods 231: 25-38 (1999).

A wide variety of expression systems are available for the production of IL-1β fragments including Fab, scFv and VhhS fragments. For example, expression systems of both prokaryotic and eukaryotic origin may be used for the large scale production of antibody fragments and antibody fusion proteins. Particularly advantageous are expression systems that allow the secretion of large amounts of antibody fragments into the culture medium.

The production of bispecific Fab-scFv ("bi-body") and Fab- (scFv) (2) trypecific Fab ("tric body") is described in Schoonjans et al. (J Immunol. 165: 7050-57, 2000) and Willems et al. (J Chromatogr B Analytical Technol Biomed Life Sci. 786: 161-76, 2003). For bi-bodies or tribodies, an scFv molecule is fused to one or both of the VL-CL (L) and VH-CH1 (Fd) chains, for example, to produce a tribody two scFvs are fused to the C-terminus of Fab while in a bibody. a scFv is fused to the C-terminus of Fab. A "minibody" consisting of scFv fused to CH3 via a peptide linker (without hinge) or via an IgG hinge has been described in Olafsen, et al., Protein Eng Des I know. April 2004; 17 (4): 315-23.

Intrabodies are single-chain antibodies that demonstrate intracellular expression and can manipulate intracellular protein function 10 (Biocca, et al., EMBO J. 9: 101-108, 1990; Colby et al., Proc Natl Acad Sci USA. 101: 17616-21, 2004). Intrabodies, which comprise cell signal sequences that retain the antibody construct in intracellular regions, may be produced as described in Mhashilkar et al (EMBO J 14: 1542-51, 1995) and Wheeler et al. (FASEB J. 17: 1733-5. 2003). Transbodies are cell-permeable antibodies in which a protein transduction domain (PTD) is fused to single chain variable fragment (scFv) antibodies Heng et al., (Med Hypotheses. 64: 1105-8, 2005).

IL-1β binding antibodies and fragments of the present invention also encompass antibodies that are SMIPs or protein-specific immunoglobulin fusion protein binding domain. These constructs are single stranded polypeptides comprising antigen binding domains fused to the immunoglobulin domains required to perform antibody effector functions. See for example, W003 / 041600, U.S. Patent Publication 20030133939 and US Patent Publication 20030118592.

IL-1β binding antibodies and fragments of the present invention also encompass immunoadhesins. One or more CDRs may be incorporated into a covalent or non-covalently molecule to make it an immunoadhesin. An immunoadhesin may incorporate the CDR (s) as part of a larger polypeptide chain, may covalently link the CDR (s) to another polypeptide chain or may incorporate the non-covalently incorporated CDR (s). . The CDRs disclosed herein allow immunoadhesin to specifically bind to IL-1β.

IL-1β binding antibodies and fragments of the present

The invention also encompasses antibody mimics comprising one or more IL-1β binding moieties constructed on an organic or molecular backbone (such as a protein or carbohydrate backbone). Proteins having relatively defined three-dimensional structures, 10 commonly referred to as protein backbones, can be used as reagents for the design of antibody imitations. These skeletons typically contain one or more regions that are amenable to specific or random sequence variation and such sequence randomization is often performed to produce protein libraries from which desired products can be selected. For example, an antibody imitation may comprise a non-chimeric binding polypeptide having a domain equal to the skeleton-containing immunoglobulin having two or more solvent-exposed handles containing a CDR other than a precursor antibody inserted into each of the handles and exhibiting binding activity. selective binding against a ligand binding by the precursor antibody. Non-immunoglobulin protein skeletons have been proposed to obtain proteins with novel binding properties. (Tramontano et al, J. Mol. Recognit. 7: 9, 1994; McConnell and Hoess, J. Mol. Biol. 250: 460, 1995). Other proteins were tested as arrays and were used to demonstrate random residues on alpha helical surfaces (Nord et al., Nat. Biotechnol. 15: 772, 1997; Nord et al., Protein Eng. 8: 601, 1995). between alpha helices in alpha helix beams (Ku and Schultz, Proc. Natl. Acad. Sci. USA 92: 6552, 1995) and disulfide-bridged loop handles, such as those of small protease inhibitors (Markland et al. , Biochemistry 35: 8045, 1996; Markland et al, Bioehemistry 35: 8058, 1996; Rottgen and Collins, Gene 164: 243, 1995; Wang et al., J. Biol. Chem. 270: 12250, 1995). Methods for using skeletons for antibody imitations are disclosed in US Patent 5,770,380 and US Patent Publications 5 2004/0171116, 2004/0266993 and 2005/0038229.

Preferred IL-Iβ antibodies or antibody fragments for use according to the invention generally bind to high affinity human IL-1β (e.g. as determined with BIACORE) such as for example with a constant equilibrium binding dissociation (K0) 10 for IL-1β of about 10 nM or less, about 5 nM or less, about 1 nM or less, about 500 pM or less or more preferably about 250 pM or less. less, about 100 pM or less, about 50 pM or less, about 25 pM or less, about 10 pM or less, about 5 pM or less, about 3 pM or less, about 1 pM or less about 0.75 pM 15 or less, about 0.5 pM or less or about 0.3 pM or less.

Antibodies or fragments of the present invention, for example, may bind to IL-1β with an IC50 of about 10 nM or less, about 5 nM or less, about 2 nM or less, about 1 nM or less, about 0.75 nM or less, about 0.5 nM or less, about 0.4 nM or less, about 0.3 nM or less, or about 0.2 nM or less, as determined by enzyme linked immunosorbent assay (ELISA). Preferably, the antibody or antibody fragment of the present invention does not cross-react with any target other than IL-1. For example, the present antibodies and fragments may bind to IL-1β but not detectably bind to IL-1a or are at least about 100-fold (e.g. at least about 150-fold, at least about 200 times or at least about 250 times) greater selectivity in their IL-Iβ binding over their IL-I binding a. Antibodies or fragments used according to the invention, in certain embodiments, may inhibit IL-1β-induced expression of serum IL-6 in an animal by at least 50% (e.g. at least 60% at least 70% or at least 80%) when compared to serum IL-6 level in an IL-1β-stimulated animal that was not administered with an antibody or fragment of the invention. The antibodies may bind IL-1β but allow or substantially permit binding of IL-1β ligand bound to the IL-I Type I receptor (IL-1 RI). Unlike many known IL-1β binding antibodies that block or substantially interfere with IL-1β binding to IL-I RI, antibodies designated AB5 and AB7 (US Application No. 111472813) 10 selectively bind to the IL-1β ligand. IL-1 β, but allow binding of IL-IRL-bound IL-1β ligand For example, the antibody designated AB7 binds to an IL-1 β epitope but still allows binding of IL-1 β to bind to IL In certain embodiments, the antibody may decrease the interaction affinity of bound IL-1 β to bind to IL-IRI. Accordingly, the invention provides, in a related aspect, the use of an IL-1β binding antibody or IL-1β binding antibody fragment which has at least one of the above-mentioned characteristics. Any of the foregoing antibodies, antibody fragments or polypeptides of the invention may be humanized or human engineered as described herein.

A variety of IL-I antibodies (eg IL-1 β) and

fragments known in the art may be used according to the methods provided herein, including for example described antibodies or derivatives using methods described in the following patents and patent applications: US 4,935,343, US 2003/0026806, US 2003/0124617, WO 25 2006/081139, WO 03/034984, WO 95/01997, WO 02/16436, WO 03/010282, WO 03/073982, WO 2004/072116, WO 2004/067568, EP 0 267 611 BI, EP 0 364 778 Bl and US Application No. 11/472813. As a non-limiting example, antibodies AB5 and AB7 (US Application No. 11/472813, W02007 / 002261) may be used in accordance with the invention. The variable region sequences of AB5 and AB7 are as follows:

AB5

Light chain

DIQMTQTTSSLSASLGDRVTISCRASODISNYLSWYOOKPDGTVKLLIYYTSKLHS GVPSRFSGSGSGTDYSLTISNLEOEDIATYFCLOGKMLPWTFGGGTKLEIK (SEQ ID NO: 3)

The underlined sequences represent (left to right) CDR1, 2, and 3.

Heavy chain

OVTLKESGPGILKPSOTLSLTCSFSGFSLSTSGMGVGWIROPSGKGLEWLAHIWWD GDESYNPSLKTOLTISKDTSRNOVFLKITSVDTVDTATYFCARNRYDPPWFVDWG QGTLVTVSS (SEQ ID NO: 4)

The underlined sequences represent (left to right) CDR1, 2, and 3.

AB7

Light chain

DIOMTOSTSSLSASVGDRVTITCRASODISNYLSWYOOKPGKAVKLLIYYTSKLHS GVPSRFSGSGSGTDYTLTISSLOOEDFATYFCLOGKMLPWTFGQGTKLEIK (SEQ ID NO: 5)

The underlined sequences represent (from left to right)

right) CDR1, 2 and 3.

Heavy chain

OVOLOESGPGLVKPSOTLSLTCSFSGFSLSTSGMGVGWIROPSGKGLEWLAHIWW DGDESYNPSLKSRLTISKDTSKNOVSLKITSVTAADTAVYFCARNRYDPPWFVDW GQGTLVTVSS (SEQ ID NO: 6)

The underlined sequences represent (left to right) CDR1, 2, and 3.

Antibodies and antibody fragments described herein may be prepared by any suitable method. Suitable methods for preparing such antibodies and antibody fragments are known in the art. Other methods for preparing antibodies and antibody fragments are as described herein as part of the invention. The antibody, antibody fragment or polypeptide of the invention as described herein may be isolated or purified to any degree. As used herein, an isolated compound is a compound that has been removed from its natural environment. A purified compound is a compound that has been increased in purity such that the compound exists in a form that is purer than it exists (i) in its natural environment or (ii) when initially synthesized and / or amplified under 10 laboratory conditions. where "purity" is a relative term and does not necessarily mean "absolute purity."

Pharmaceutical Compositions

IL-I-binding antibodies (e.g., IL-1β) and antibody fragments for use in accordance with the present invention may be formulated into compositions, especially pharmaceutical compositions, for use in the methods herein. Such compositions comprise a therapeutically or prophylactically effective amount of an IL-1β binding antibody or antibody fragment of the invention in admixture with a suitable carrier, for example a pharmaceutically acceptable agent. Typically, IL-1β binding antibodies and antibody fragments of the invention are sufficiently purified for administration to an animal prior to formulation into a pharmaceutical composition.

Pharmaceutically acceptable agents include fillers, excipients, diluents, antioxidants, preservatives, colorants, flavoring and diluents, emulsifying agents, suspending agents, solvents, fillers, bulking agents, buffers, release vehicles, tonicity agents, co-agents. solvents, wetting agents, complexing agents, buffering agents, antimicrobials and surfactants.

Neutral buffered saline or albumin mixed saline are exemplary appropriate carriers. Pharmaceutical compositions may include antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween, pluronics or polyethylene glycol (PEG). Also by way of example, suitable tonicity enhancing agents include alkali metal halides (preferably sodium or potassium chloride), mannitol, sorbitol and the like. Suitable preservatives include benzalkonium chloride, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid and others. Hydrogen peroxide can also be used as a preservative. Suitable cosolvents include glycerine, propylene glycol and PEG. Suitable complexing agents include caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin. Suitable surfactants or wetting agents include sorbitan esters, polysorbates such as polysorbate 80, tromethamine, lecithin, cholesterol, tiloxapal and the like. The buffers may be conventional buffers such as acetate, borate, citrate, phosphate, bicarbonate or Tris-HCl. Acetate buffer may be around pH 4 to 5.5 and Tris buffer may be around pH 7 to 8.5. Additional pharmaceutical agents are disclosed in Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed., Mack Publishing Company, 1990.

The composition may be in liquid form or in a freeze-dried or freeze-dried form and may include one or more lyoprotectants, excipients, surfactants, high molecular weight structural additives and / or bulking agents (see for example US Patent 6,685,940 , 6,566,329 and 6,372,716). In one embodiment, a lyoprotectant is included, which is a non-reducing sugar such as sucrose, lactose or trehalose. The amount of lyoprotectant generally included is such that upon reconstitution the resulting formulation will be isotonic, although hypertonic or slightly hypotonic formulations may also be suitable. In addition, the amount of lyoprotectant should be sufficient to prevent an unacceptable amount of protein degradation and / or aggregation in lyophilization. Exemplary lyoprotectant concentrations for sugars (e.g. sucrose, lactose, trehalose) in the pre-lyophilized formulation are from about 10 10 mM to about 400 mM. In another embodiment, a surfactant is included, such as for example nonionic surfactants and ionic surfactants such as polysorbates (e.g. polysorbate 20, polysorbate 80); poloxamers (for example poloxamer 188); poly (ethylene glycol) phenyl ethers (e.g. Triton); sodium dodecyl sulfate (SDS); sodium lauryl sulfate; octyl sodium glycoside; lauryl, myristyl, linoleyl or stearyl sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl sarcosine; linoleyl-, myristyl- or cetyl betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl- or isostearamidopropyl betaine (e.g. lauroamidopropyl); myristamidopropyl-, palmidopropyl- or isostearamidopropyl dimethylamine; sodium methyl cocoyl taurate or disodium methylphenyl taurate; and the MONAQUAT® series (Mona Industries, Inc., Paterson, N.J.), polyethyl glycol, polypropyl glycol and ethylene propylene glycol copolymers (e.g. Pluronics, PF68 etc). Exemplary amounts of surfactant that may be present in the pre-lyophilized formulation are about 0.001 to 0.5%. High molecular weight structural additives (e.g. fillers, binders) may include for example acacia, albumin, alginic acid, (dibasic) calcium phosphate, cellulose, carboxymethylcellulose, sodium carboxymethylcellulose,

hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, dextran, dextrin, dextrates, sucrose, tilose, pregelatinized starch, calcium sulfate, amylose, glycine, bentonite, maltose, sorbitol, ethylcellulose, disodium disodium phosphate phosphate, disodium, polyvinyl alcohol, gelatin, glucose, guar gum, liquid glucose, compressible sugar, magnesium alumina silicate, maltodextrin, polyethylene oxide, polymethacrylates, povidone, sodium alginate, tragacanth, microcrystalline cellulose, starch and zein. Exemplary concentrations of high molecular weight structural additives are from 0.1% to 10% by weight. In other embodiments, a bulking agent (e.g., mannitol, glycine) may be included.

The compositions may be suitable for parenteral administration. Exemplary compositions are suitable for injection or infusion into an animal by any route available to the skilled artisan, such as intraarticular, subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intralesional routes. , intrarectal, transdermal, oral and inhaled. A parenteral formulation will typically be a sterile, isotonic, pyrogen-free solution, optionally containing pharmaceutically acceptable preservatives.

Examples of non-aqueous solvents are propylene glycol,

polyethylene glycol, vegetable oils such as olive oil and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose and others. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and others. See generally, Remington's Pharmaceutical Science, 16th Ed., Mack Eds., 1980, which is incorporated herein by reference.

The pharmaceutical compositions described herein may be formulated for controlled or prolonged release in a manner that provides local sustained release concentration (e.g., bolus, depot effect) and / or increased product stability or half-life in a particular local environment. The invention considers that in certain embodiments such compositions may include a significantly larger amount of antibody or fragment at the initial depot, while the effective amount of antibody or fragment actually released and available at any time point is in accordance with the disclosure herein. in a much lower amount than the initial deposit. Compositions may include formulating IL-1β binding antibodies, antibody fragments, nucleic acids or vectors of the invention with particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc., as well as agents such as a matrix biodegradable, injectable microspheres, microcapsular particles, microcapsules, bioerodible particle beads, liposomes and implantable delivery devices that provide controlled or prolonged release of the active agent which can then be released as a depot injection. Techniques for formulating such sustained or controlled release media are known and a variety of polymers have been developed and used for controlled release and drug release. Such polymers are typically biodegradable and biocompatible. Polymeric hydrogels, including those formed by complexation of enantiomeric polymer or polypeptide segments and hydrogels with temperature or pH sensitive properties, may be desirable to provide drug deposition effect because of the mild and aqueous conditions involved in entrapping bioactive protein agents. (e.g. antibodies). See, for example, the description of controlled release porous polymer microparticles for the release of pharmaceutical compositions in PCT Application Publication WO 93/15722.

Suitable materials for this purpose include

polylactides (see, for example, US Patent 3,773,919), poly (α-hydroxycarboxylic acids) polymers such as poly-D - (-) - 3-hydroxybutyric acid (EP 133,988A), acid copolymers Ethyl L-Glutamic and Gamma L-Glutamate (Sidman et al., Biopolymers, 22: 547-556 (1983)), 2- (10-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater. Res ., 15: 167-277 (1981) and Langer, Chem. Tech., 12: 98-105 (1982)), ethylene vinyl acetate or poly-D (-) - 3-hydroxybutyric acid. Other biodegradable polymers include poly (lactones), poly (acetals), poly (orthoesters) and poly (orthocarbonates). Sustained release compositions may also include liposomes, which may be prepared by any of a number of methods known in the art (see, for example, Eppstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-92 (1985)). The charger itself or its degradation products must be non-toxic to the target tissue and should not further aggravate the condition. This can be determined by routine screening in animal models of the target disorder or, if such models are unavailable, in normal animals.

Microencapsulation of recombinant proteins for prolonged release was successfully performed with human growth hormone (rhGH), interferon- (rhIFN-), interleukin-2 and MN rgpl20. Johnson 25 et al., Nat. Med., 2: 795-799 (1996); Yasuda, Biomed. Ther., 27: 1221-1223 (1993); Hora et al., Bio / Technology. 8: 755-758 (1990); Cleland, “Design and Production of Single Immunization Vaccines Using Polylactide Poliglycolide Microsphere Systems,” in Vaccine Design: The Subunit and Adjuvant Method, Powell and Newman, eds, (Plenum Press: New York, 1995), pp. 439-462; WO 97/03692, WO 96140072, WO 96/07399; and Pat. No. 5,654,010. The extended release formulations of these proteins were developed using the poly-lactic-co-glycolic acid (PLGA) polymer due to its biocompatibility and wide range of biodegradable properties. The 5 degradation products of PLGA, lactic and glycolic acids can be rapidly cleared within the human body. In addition, the degradability of this polymer may be dependent on its molecular weight and composition. Lewis, “Controlled release of active bioagents from lactidelglycolide polymer,” in: M. Chasm and R. Langer (Eds.), Biodegradable 10 Drug Delivery Systems (Mareei Dekker: New York, 1990), pp. 1-41. Additional examples of sustained release compositions include, for example, EP 58,481 A, Pat. No. 3,887,699, EP 158,277A, Canadian Patent Nos. 1176565, U. Sidman et al., Biopolymers 22, 547 [1983], R. Langer et al., Chem. Tech. 12, 98 [1982], Sinha et al., J. Control. 15 Release 90, 261 [2003], Zhu et al., Nat. Biotechnol. 18, 24 [2000] and Dai et al., Colloids SurfB Biointerfaces 41, 117 [2005].

Bioadhesive polymers are also considered for use in or with the compositions of the present invention. Bioadhesives are naturally occurring, synthetic materials capable of adhering to biological substrates for extended periods of time. For example, Carbopol and polycarbophil are both synthetic cross-linked derivatives of poly (acrylic acid). Naturally occurring substance-based bioadhesive release systems include for example hyaluronic acid, also known as hyaluronan. Hyaluronic acid is a naturally occurring mucopolysaccharide consisting of residues of D-glucuronic acid and N-acetyl-D-glucosamine. Hyaluronic acid is found in the matrix of vertebrate extracellular tissue, including connective tissues, as well as in synovial fluid and the vitreous and aqueous humor of the eye. Esterified derivatives of hyaluronic acid have been used to produce release microspheres that are bioeompatible and biodegradable (see for example, Cortivo et al., Biomaterials (1991) 12: 727-730; European Publication No. 517.565; International Publication N WO 96129998; Thum et al., J. Controlled King. (1994) 29: 133-141). Exemplary hyaluronic acid-containing compositions of the present invention comprise a hyaluronic acid ester polymer in an amount from approximately 0.1% to about 40% (w / w) of an IL-1β binding antibody or polymer fragment of hyaluronic acid.

Both biodegradable and non-biodegradable polymeric matrices may be used to release compositions according to the invention and such polymeric matrices may comprise natural or synthetic polymers. Biodegradable matrices are preferred. The time period in which the release occurs is based on polymer selection. Typically, release over a period of a few hours to three to twelve months is more desirable. Exemplary synthetic polymers that can be used to form the biodegradable release system include: lactic acid and glycolic acid polymers, polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyvinyl alcohols, polyvinyl ethers, polyvinyl ethers, Polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyaldehydes, polyurethanes and copolymers thereof, poly (butyric acid), poly (valeric acid), alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, acrylic and methacrylic ester polymers, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethyl cellulose triacetate, cellulose sulfate sodium salt, poly (methyl methacrylate), poly (ethyl methacrylate), poly (butyl methacrylate), poly (isobutyl methacrylate), poly (hexyl methacrylate), poly ( isodecyl methacrylate), poly (lauryl methacrylate), poly (phenyl methacrylate), poly (methyl acrylate), poly (isopropyl aerylate), poly (isobutyl acrylate), poly (octadecyl aerylate), polyethylene, polypropylene poly (ethylene glycol), poly (ethylene oxide), poly (ethylene terephthalate), poly (vinyl alcohols), polyvinyl acetate, polyvinyl chloride, polystyrene and polyvinylpyrrolidone. Exemplary natural polymers include alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example alkyl, alkylene, hydroxylations, oxidations and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these materials degrade by enzymatic hydrolysis or exposure to water in vivo, surface or volumetric erosion. The polymer optionally is in the form of a hydrogel (see for example WO 04/009664, WO 05/087201, Sawhney, et al., Macromolecules, 1993, 26, 581-587), which can absorb up to about 90%. of its weight in water and furthermore is optionally cross-linked with multivalent ions or other polymers.

Delivery systems also include non-polymeric systems which are lipid including sterols such as cholesterol, cholesterol esters and neutral fatty acids or fats such as mono-di- and triglycerides; hydrogel release systems; silastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and 25 others. Specific examples include, but are not limited to: (a) erosional systems wherein the product is contained in a form within a matrix such as that described in U.S. Pat. No. 4,452,775, 4,675,189 and 5,736,152 and (b) diffusional systems wherein a product permeates at a controlled rate from a polymer as described in U.S. Pats. No. 3,854,480, 5,133,974 and 5,407,686. Liposomes containing the product may be prepared by known methods, such as for example (DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-3692 (1985); Hwang et al., Natl. Acad Sci USA, 77: 4030-4034 (1980); EP 52,322; EP 5 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Patent Application 83-118008; US Pat. 045 and 4,544,545, and EP 102,324).

A pharmaceutical composition comprising an IL-Iβ binding antibody or fragment may be formulated for inhalation, such as for example as a dry powder. Inhalation solutions may also be formulated in a liquefied propellant for aerosol release. In another formulation, the solutions may be nebulized. Additional pharmaceutical composition for pulmonary administration includes those described, for example, in PCT Application Publication WO 94/20069, which discloses pulmonary release of chemically modified proteins. For pulmonary release, the particle size should be adequate for release to the distal lung. For example, the particle size may be from 1 μηι to 5 μηι; however, larger particles may be used, for example, if each particle is reasonably porous.

Certain formulations containing IL-Iβ 20 binding antibodies or antibody fragments may be administered orally. Formulations administered in this manner may be formulated with or without those carriers commonly used in the combination of solid dosage forms such as tablets and capsules. For example, a capsule may be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and presystemic is minimized. Additional agents may be included to facilitate absorption of a selective binding agent. Diluents, flavorings, low melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents and binders may also be used.

Another preparation may involve an effective amount of an IL-IB binding antibody or fragment in a mixture with non-toxic excipients that are suitable for tablet manufacture. By dissolving the tablets in sterile water or another suitable vehicle, solutions may be prepared in unit dose form. Suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose or calcium phosphate; or binding agents such as starch, gelatin or acacia; or lubricating agents 10 such as magnesium stearate, stearic acid or talc.

Suitable and / or preferred pharmaceutical formulations may be determined in view of the present disclosure and general knowledge of the formulation technology, depending upon the intended route of administration, release format and desired dosage. Regardless of the mode of administration, an effective dose may be calculated according to the patient's body weight, surface area or organ size. Another refinement of the calculations to determine the appropriate dosage for treatment involving each of the formulations described herein are routinely made in the art and are within the scope of tasks routinely performed in the art. Appropriate dosages can be ascertained through the use of appropriate dose response data.

Additional formulations will be apparent in light of the present disclosure, including formulations involving IL-1β binding antibodies and fragments in combination with one or more other therapeutic agents. For example, in some formulations, an IL-1β binding antibody, antibody fragment, nucleic acid or vector of the invention is formulated with a second inhibitor of an IL-1 signaling pathway. Representative secondary inhibitors include, but are not limited to, antibodies, antibody fragments, peptides, polypeptides, compounds, nucleic acids, vectors and pharmaceutical compositions, such as, for example, those described in US 6899878, US 2003022869, US 20060094663, US20050186615, US20030166069, W0 / 04022718, W0 / 05084696, WO / 05019259. For example, a composition may comprise an IL-1β binding antibody, antibody fragment, nucleic acid or vector of the invention in combination with an IL-1β binding antibody, fragment or a nucleic acid or vector encoding a such antibody or fragment.

The pharmaceutical compositions may comprise IL-1β binding antibodies or fragments in combination with other active agents. Such combinations are those useful for their intended purpose. The combinations which are part of this invention may be IL-1β antibodies and fragments, such as for example those described herein and at least one additional agent selected from the lists below. The active agents presented below are for illustrative purposes and not intended to be limiting. The combination may also include more than one additional agent, for example two or three additional agents if the combination is such that the composition formed may perform its intended function.

The invention further provides that pharmaceutical compositions comprising one or more other active agents may be administered separately from IL-1β binding antibodies or fragments and such separate administrations may be performed at the same or different time points, such as for example. on the same or different days. Administration of the other active agents may be in accordance with standard medical practices known in the art or administration may be modified (e.g., longer intervals, lower dosages, delayed onset) when used in conjunction with administration of antigen-binding antibodies. IL-1β or fragments as disclosed herein.

Active agents or combinations with the present antibodies or fragments include a non-steroidal anti-inflammatory drug (NSAID) such as aspirin, ibuprofen and other propionic acid derivatives (alminoprofen, benoxaprofen, bucolic acid, carprofen, fenbufen, fenoprofen, fluprofen, flurbiprofen, indoprofen , ketoprofen, miroprofen, naproxen, oxaprozin, pirprofen, pranoprofen, suprofen, thiaprofenic acid and thioxaprofen), acetic acid derivatives (indomethacin, acemetacin, alclofenac, clidanac, diclofenac, fenclofenac, fencofacazole, fenclofenac, fenclofenac, , sulindac, thiopinac, tolmetine, zidometacin and zomepirac), fenamic acid derivatives (flufenamic acid, meclofenamic acid, mefenamic acid, niflumic acid and tolfenamic acid), biphenylcarboxylic acid derivatives (diflunisal and flufenisal), oxicans (isoxicam, poxicam, suxicam, poxicam and tenoxican), salicylates (acetyl salicylic acid sulfasalazine) and pyrazolones (apazone, bezpiperilon, feprazone, mofebutazone, oxyphenbutazone, phenylbutazone). Other combinations include cyclooxygenase-2 (COX-2) inhibitors. Other active agents for the combination include steroids such as prednisolone, prednisone, methylprednisolone, betamethasone, dexamethasone or hydrocortisone. A combination may be especially advantageous as long as one or more steroid side effects can be reduced or even eliminated by decreasing the required steroid dose when treating patients in combination with the present antibodies and fragments.

Alternatively or in addition, therapeutic treatment with at least one or more additional active agents may be used which may act through different modes of action: 1) sulfonylureas (e.g. chlorpropamide, tolazamide, acetoexamide, tolbutamide, glyburide, glimepiride, glipizide) and / or meglitinides (e.g. repaglinide, nateglinide) which essentially stimulate insulin secretion; 2) biguanides (eg, metformin) act by promoting glucose utilization, reducing hepatic glucose production and decreasing intestinal glucose production; 3) alpha-glucosidase inhibitor (e.g. acarbose, miglitol) decreases carbohydrate digestion and consequently absorption from the intestine and reduces postprandial hyperglycemia; 4) thiazolidinediones (eg troglitazone, pioglitazone, rosiglitazone, glipizide, balaglitazone, rivoglitazone, netoglitazone, troglitazone, englitazone, AD 5075, T 174, YM 268, R 102380, NC 2100, NIP 223, NIP761, NIP 221 , adaglitazone, CLX 0921, darglitazone, CP 92768, BM 152054) which enhance the action of insulin, thereby promoting the utilization of glucose in peripheral tissues; 5) glucagon equivalent peptides including DPP4 inhibitors (e.g. sitagliptin); and 6) insulin, which stimulates tissue glucose utilization and inhibits hepatic glucose production. Glucagon Equivalent Peptide-1 (GLP-1), DPP-IV resistant analogs (incretin mimetics), DPP-IV inhibitors, insulin, insulin analogs, PPAR gamma agonists, double acting PPAR agonists, agonists or GLP-1 analogs, PTP1B inhibitors, SGLT inhibitors, insulin secretagogues, RXR agonists, glycogen synthase kinase-3 inhibitors, insulin sensitizers, immunomodulators, beta-3 adrenergic receptor agonists, Pan-PPAR agonists 1beta-HSDI inhibitors, amylin analogues, biguanides, alpha-glucosidase inhibitors, meglitinides, thiazolidine diones, sulfonylureas and others may also be used as other active agent (s) (see for example Nathan, 2006, N. Engl. J. Med. 355: 2477-2480; Kahn et al., 2006, N. Engl. J. Med. 355: 2427-2443). In another embodiment, the active agent may be an HMG Co-A reductase inhibitor (eg, statins).

It is further contemplated that an anti-IL-ββ antibody or fragment administered to a patient according to the invention may be administered in combination with treatment with at least one additional active agent, such as for example any of the above-mentioned active agents. In one embodiment, treatment with at least one active agent is maintained. In another embodiment, treatment with at least one active agent is reduced or discontinued (for example, when the patient is stable), while treatment with the anti-IL-ββ antibody or fragment is maintained in one. constant dosing regimen. In another embodiment, treatment with at least one active agent is reduced or discontinued (e.g., when the patient is stable) and treatment with the anti-IL-ββ antibody or fragment is reduced (e.g., dose lower, less frequent dosing 10, shorter treatment regimen). In another embodiment, treatment with the at least one active agent is reduced or discontinued (e.g., when the patient is stable) and treatment with the anti-IL-ββ antibody or fragment is increased (e.g., dose higher, more frequent dosage, longer treatment regimen). In another embodiment, treatment with at least one active agent is maintained and treatment with the anti-IL-β antibody or fragment is reduced or discontinued (eg, lower dose, less frequent dosage, regimen). of shorter treatment). In another embodiment, treatment with at least one active agent and treatment with anti-IL-ββ antibody or fragment are reduced or discontinued (eg, lower dose, less frequent dosage, treatment regimen). shorter).

The pharmaceutical compositions used in the invention may include a therapeutically effective amount or a prophylactically effective amount of the IL-1β binding antibodies or fragments. A therapeutically effective amount refers to an amount effective at the dosages and for periods of time necessary to obtain the desired therapeutic result. A therapeutically effective amount of the antibody or antibody moiety may vary according to factors such as the disease state, age, sex and weight of the individual and the ability of the antibody or antibody moiety to elicit a desired response in the individual. A therapeutically effective amount is also one wherein any toxic or deleterious effects of the antibody or antibody moiety are outweighed by the therapeutically beneficial effects. A prophylactically effective amount refers to an amount effective, at dosages and for periods of time required, to obtain the desired prophylactic result.

A therapeutically or prophylactically effective amount of a pharmaceutical composition comprising an IL-1β 10 binding antibody or fragment will depend, for example, on therapeutic purposes such as the indication for which the composition is being used, the route of administration and of the patient's condition. The pharmaceutical compositions are administered in a therapeutically or prophylactically effective amount to treat an IL-1 related condition. A "therapeutic or prophylactically effective amount" of an IL-1β binding antibody or fragment of the invention is that amount that can treat or prevent one or more symptoms of an IL-I-related disease in a patient, such as disclosed here.

Usage Methods

Anti-IL-1 β antibodies in an effective amount may be

used in the present invention for the treatment and / or prevention of Type 1 diabetes, Type 2 diabetes, obesity, hyperglycemia, hyperinsulinemia, insulin resistance, and disease states and conditions characterized by insulin resistance, such methods may be used to treat a patient. mammal 25 (e.g., human) suffering from Type 1 diabetes, Type 2 diabetes, obesity, hyperglycemia, hyperinsulinemia, insulin resistance, and disease conditions and conditions characterized by insulin resistance or to prevent its occurrence in a patient at risk.

The terms "prevention", "prevent", "prevent", "suppress", "suppress", "suppress", "inhibit" and "inhibit" as used herein refer to a course of action (such as administering a compound or pharmaceutical composition) initiated in a manner (for example, prior to the onset of a clinical symptom of a disease state or condition) so as to temporarily or permanently prevent, suppress or reduce the onset of a clinical manifestation of the state or condition of disease. Such prevention, suppression or reduction need not be absolute to be useful.

The terms "treating", "treating" and "treating" as used herein refer to a course of action (such as the administration of a compound or pharmaceutical composition) initiated after the onset of a clinical symptom of a condition or condition. disease in order to eliminate, reduce, suppress or ameliorate, temporarily or permanently, a clinical manifestation or progression of the disease state or condition. Such treatment need not be absolute to be useful.

The term "in need of treatment" as used herein refers to a judgment made by a healthcare professional that a patient requires or will benefit from treatment. This judgment is based on a variety of factors that are within the domain of a health professional's expertise, but include the knowledge that the patient is ill or ill as a result of a condition that is treatable by a method. or compound of the disclosure.

The term "in need of prevention" as used herein refers to a judgment made by a healthcare professional that a patient requires or will benefit from prevention. This judgment is made on the basis of a variety of factors that are within the skill of the healthcare professional, but include the knowledge that the patient will be ill or may become ill as a result of a condition that is preventable. a method or compound of the disclosure.

The term "therapeutically effective amount" as used herein refers to an amount of a compound (e.g., antibody), alone or as a part of a pharmaceutical composition, which is capable of having any detectable, positive effect on any symptom, aspect or characteristics of a disease state or condition when administered to a patient (e.g., as one or more doses), such effect need not be absolute to be beneficial.

The term "insulin resistance" as used herein refers to a condition where a normal amount of insulin is unable to produce a normal physiological or molecular response. In some cases, a hyperphysiological amount of endogenously produced or exogenously added insulin is capable of overcoming insulin resistance in whole or in part and producing a biological response.

Anti-IL-β antibodies or fragments may be administered to a human in an amount effective for the treatment and / or prevention of Type 1 diabetes, Type 2 diabetes, obesity, hyperglycemia, hyperinsulinemia, insulin resistance and / or conditions and conditions. disease conditions characterized by insulin resistance. Other diseases or conditions considered for treatment with anti-IL-β antibodies or fragments according to the present invention include pre-diabetes, dyslipidemia, hyperlipidemia, hypertension, metabolic syndrome and unhealthy behavior. The invention further provides methods of using such antibodies or fragments to decrease the incidence or severity or stabilize complications or conditions associated with Type 2 diabetes, such as for example retinopathy, renal failure, cardiovascular disease (e.g. atherosclerosis, vascular disease). peripheral) and wound healing (eg, diabetic ulcer).

In addition, the invention further contemplates the use of IL-1β antibodies and fragments as described herein to reduce the level of C-reactive protein (CRP) in a patient. CRP is an acute phase protein that is predominantly produced by hepatocytes under the influence of cytokines such as IL-1, IL-6 and tumor necrosis factor (TNF). Based on the 2007 electronic version of the UpToDate® Internal Medicine textbook, despite a lack of specificity for the cause of inflammation (eg, 5 infection, chronic kidney disease, auto-inflammatory disease, cancer), data from More than 30 epidemiological studies have shown a significant association between elevated CRP serum or plasma concentrations and the prevalence of underlying atherosclerosis, the risk of recurrent cardiovascular events among patients with the established disease, and the incidence of the first cardiovascular event among individuals in risk for atherosclerosis. In addition, the interaction of primary renal disease, resulting renal failure with its oxidative stress and postsynthetic protein modifications, dialysis with associated contaminants, and the effect of the dialysis membrane on serum proteins and associated infections 15 repeated access site entry and subsequent systemic infections lead these patients to an excessive burden of inflammatory stimuli. Since serum creatinine clearance levels fall as renal function worsens, there is a proportional increase in serum inflammatory mediators (eg, TNF, IL-6, IL-1 cytokines) as well as evidence of the body's attempt to combat this. situation with increased but inefficient production of IL-I RA and IL-10, anti-inflammatory mediators. This inflammatory state in chronic renal failure patients leads to atherosclerotic plaque instability due to direct outbreak of apoptosis of vascular smooth muscle cells. The consequence of elevated cytokine leads to one of two major mortalities in these patients - a marked increase in cardiovascular deaths from myocardial infarction and stroke. A direct illustration of this increased risk is observed by assessing the patient's CRP levels; when divided into quartiles of CRP values, the group with the highest CRP values has a 12-month mortality rate of approximately 35%. Thus, the present invention discloses the use of an IL-1β antibody or fragment as provided herein to reduce CRP levels in such patients (e.g., patients suffering from kidney disease). Reduction in CRP levels in a patient as described herein is a suitable means of achieving a corresponding proportional decrease in cardiovascular morbidity and mortality.

In one embodiment, the anti-IL-ββ antibody or fragment is administered to a patient with at least one of the aforementioned diseases, conditions or complications and the patient also receives at least one other medically accepted treatment (e.g., medication, medicine, therapeutic product, active agent) for the disease, condition or complication. In another embodiment, the at least one other medically accepted treatment for the disease, condition or complication is reduced or discontinued (for example, when the patient is stable), while treatment with the anti-IL-ββ antibody or The fragment is maintained at a constant dosage regimen. In another embodiment, the at least one other medically accepted treatment for the disease, condition or complication is reduced or discontinued (e.g., when the patient is stable) and treatment with the anti-IL-ββ antibody or fragment is reduced (eg lower dose, less frequent dosage, shorter treatment regimen). In another embodiment, the at least one other medically accepted treatment for the disease, condition or complication is reduced or discontinued (e.g., when the patient is stable) and treatment with the anti-IL-1 β antibody or fragment. is increased (eg higher dose, more frequent dosage, longer treatment regimen). In another embodiment, at least one other medically accepted treatment for the disease, condition or complication is maintained and treatment with the anti-IL-β antibody or fragment is reduced or discontinued (eg, lower dose, less frequent dosing, shorter treatment regimen). In yet another embodiment, the at least one other medically acceptable treatment for the disease, condition or complication and treatment with the anti-IL-β antibody or fragment is reduced or discontinued (eg, lower dose, lower dose). frequent, shorter treatment regimen).

In preferred methods of treating or preventing the aforementioned diseases or conditions (e.g., Type 1 diabetes, Type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, insulin resistance) the anti-IL-ββ antibody or fragment thereof is administered to the human patient. according to the dose numbers, amounts per dose and / or dosing intervals mentioned above. Alternatively, the anti-IL-ββ antibody or fragment may be administered as one or more starting doses of the aforementioned amounts which are lower than one or more subsequent dose amounts. By promoting the starting dose (s) in a lower amount, the efficacy and / or tolerability of the treatment may be enhanced. For example, in a non-limiting embodiment of the invention, one or more starting doses (e.g., 1, 2, 3, 4, 5) of an amount of antibody or fragment <1 mg / kg (e.g., <0 , 9 mg / kg, <0.8 mg / kg, <0.7 mg / kg, <50.6 mg / kg, <0.5 mg / kg, <0.4 mg / kg, <0.3 mg / kg, <02 mg / kg, <0.1 mg / kg, <0.05 mg / kg, <0.03 mg / kg, <0.01 mg / kg) may be administered, followed by one or more subsequent doses in an amount greater than the starting dose (s) (eg> 0.01 mg / kg,> 0.03 mg / kg,> 0.1 mg / kg,>

0.3 mg / kg> 0.5 mg / kg,> 0.6 mg / kg,> 0.7 mg / kg,> 0.8 mg / kg,> 0.9 mg / kg,

> 1.0 mg / kg>> 1.5 mg / kg> 2 mg / kg> 2.5 mg / kg> 3 mg / kg> 3.5 mg / kg>

4 mg / kg,> 4.5 mg / kg,> 5 mg / kg). The invention considers that each antibody dose or fragment may be administered at one or more sites. Methods of treating or preventing a disease or condition according to the present invention may use a predetermined or "routine" program for administration of the antibody or fragment. As used herein a routine schedule refers to a predetermined designated period of time between dose administrations. The routine program may cover time periods that are identical or differ in length as long as the program is predetermined. Any particular combination would be covered by the routine program as long as it was determined ahead of time when the appropriate program involves administration on a particular day.

The invention further considers that IL-1β antibodies or fragments used according to the methods provided herein may be administered in conjunction with more traditional methods of treatment and pharmaceutical compositions (e.g. active agents). Such compositions may include, for example, DPP-IV inhibitors, insulin, insulin analogs, PPAR gamma agonists, double-acting PPAR agonists, GLP-1 agonists or analogs, PTP1B inhibitors, SGLT inhibitors, insulin, RXR agonists, glycogen synthase kinase-3 inhibitors, insulin sensitizers, immunomodulators, beta-3 adrenergic receptor agonists, Pan-PPAR agonists,

Ibeta-HSD 1, amylin analogues, biguanides, alpha glycosidase inhibitors, meglitinides, thiazolidinediones, sulfonylureas and others (see for example Nathan, 2006, N. Engl. J. Med. 355: 2477-2480; Kahn et al , 2006, N. Engl. J. Med. 355: 2427-2443). In certain embodiments, antibodies and fragments used in accordance with the invention may prevent or delay the need for additional treatment methods or pharmaceutical compositions. In other embodiments, antibodies or fragments may reduce the amount, frequency or duration of the treatment method or additional pharmaceutical compositions. Alternatively, methods of treating or preventing a disease or condition according to the present invention may use a program for administering the antibody or fragment that is based on the presence of disease symptoms and / or changes in any of the assessments herein ( eg HbA lc, fasting blood sugar levels, OGTT, glucose / insulin C-peptide AUC, diabetes medication use, insulin sensitivity, serum cytokine levels, CRP levels, quality of life measurements, improvement of BMI) as a means to determine when to administer one or more subsequent doses. Similarly, this method can be used as a means of determining whether a subsequent dose should be increased or decreased based on the effect of a previous dose.

Diagnosis of such diseases or conditions in patients or alternatively the risk of developing such diseases or conditions may be in accordance with standard medical practices known in the art. Following administration of an anti-IL-ββ antibody or fragment, clinical evaluations for a preventive treatment or effect on the aforementioned diseases and conditions are well known in the art and may be used as a means to monitor the effectiveness of the methods of the invention. .

For example, the response to Type 2 diabetes treatment may

be evaluated based on a primary efficacy endpoint of hemoglobin HbAlc Alc improvement, see for example Reynolds et al., BMJ, 333 (7568): 586-589, 2006). Improvements in HbAlc that are indicative of therapeutic efficacy may vary depending on the initial baseline measurements in a patient, with a larger decrease often corresponding to a higher initial reference and a smaller decrease often corresponding to a lower initial reference. In one aspect of the invention, the method should result in an HbAlc decrease of at least about 0.5% (for example, at least about 0.5%, at least about 1%, at least about 1%). 5%, at least about 2%, at least about 2.5%, at least about 3%, at least about 3.5%, at least about 4% or more) compared to pre-dose.

5 One or more of the following secondary endpoints also

can be determined to assess treatment efficacy, such as for example fasting blood sugar levels (eg glucose) (eg decrease to <130, <125, <120, <115, <110 , <105, <100; alternatively> 20%,> 30%,> 40%,> 50%,> 60%),> 10 70%,> 80%),> 90%,> 95% decrease compared to (pre-dose levels), 120-minute oral glucose tolerance test (OGTT) (eg <200, <190, <180, <170, <160, <150, <140), C-peptide AUC glucose / insulin (eg> 25%,> 50%,> 60%,> 70%,> 80%,> 90%,> 100% increase in pretreatment), reduction in diabetes medication (eg eg insulin, oral hypoglycemic agent), improvement in insulin sensitivity, serum cytokine levels (eg normalization), CRP levels (eg decrease of> 0.2,> 0.4,> 0.6, > 0.8,> 1.0,> 1.4,> 1.8,> 2.2,> 2.6, > 3.0 mg / L, alternatively a decrease of> 20%,> 30%,> 40%,

> 50%>,> 60%),> 70%,> 80%,> 90%,> 95% of pretreatment), quality of life measurements, BMI improvement (1% reduction, 3%, 5 %), pharmacokinetics and others (Saudek, et al., JAMA, 295: 1688-97, 2006; Pfutzner et al., Diabetes Technol Ther. 8: 28-36, 2006; Norberg, et al., J Intem Med. 260: 263-71, 2006).

Similarly, efficacy evaluation for other diseases or conditions may use one or more of the above-mentioned and / or other known endpoints in the art. For example, the effect on hyperglycemia can be assessed by measuring fasting blood sugar levels (ie glucose), the effect on hyperinsulinemia can be assessed by measuring insulin levels and / or peptide levels. C, the effect on obesity can be assessed by measuring weight and / or EMI and the effect on insulin resistance can be assessed by the OGTT.

Alternatively or in addition, patients treated according to the invention may experience a decrease in the patient's blood triglyceride level of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% 'or more of the pretreatment level. Alternatively or in addition, patients treated according to the invention may experience a decrease in free fatty acid level of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45. %, 50%, 10 55%, 60%) or more of the pretreatment level.

EXAMPLES

The following examples are intended merely to further illustrate the practice of the present invention, but should not be construed as limiting its scope in any way. The disclosures of all patents and scientific literature cited herein are hereby expressly incorporated in their entirety by reference.

Example 1

IL-1β inhibition using a high affinity IL-Iβ antibody in an in vitro cell-based assay, with IL-I induced production of IL-8 as a reading

The inhibitory effect of IL-1β specific antibody was compared with a non-IL-1 pathway antibody inhibitor, Kineret® (anakinra), which is a recombinant IL-I receptor antagonist. Fresh, heparinized peripheral blood was collected from healthy donors. 180 μΐ of 25 whole blood were plated in a 96 well plate and incubated with various concentrations of antibody AB7 (US Application No. 11/472813, WO 2007/002261) and 100 pM rhlL-β. For samples treated with Kineret®, Kineret® and rhIL-Ιβ they were combined 1: 1 before mixing with blood. Samples were incubated for 6 hours at 37 ° C with 5% CO2. Whole blood cells were then lysed with 50 μΐ of 12.5% Triton X-100. Interleukin-8 (IL-8) concentration in purified lysates was assayed by ELISA (Quantikine human IL-8 ELISA kit, R&D Systems) according to the manufacturer's instructions. IL-58 concentrations in AB 7 and Kineret® treated samples were compared with a control sample treated with anti-KLH control. The results are shown in Fig. 1 and summarized in Table 6. IC50 is the antibody concentration required to inhibit 50%) of IL-8 released by IL-1 β stimulation.

Table 1

IC50 (PM) AB 7 1.9 pM Kineret * 53.4 pM These results demonstrate the in vitro potency of AB7 as

measured by inhibition of IL-Iβ-stimulated release of IL-8. These results showing higher potency compared to Kineret® indicate that the antibody will have IL-1β inhibitory efficacy in vivo.

Example 2

In vivo inhibition of human IL-1 β biological activity using IL-1 β-specific antibodies as measured by impact on IL-1 β stimulated release of IL-6

To confirm the in vivo efficacy of AB7, its ability to block the biological activity of human IL-1 β was tested in 20 mice. Details of the assay are described in Economides et al., Nature Med., 9: 47-52 (2003). In summary, male C57 / B16 mice (Jackson Laboratory Bar Harbor, Maine) were injected intraperitoneally with titrated doses of AB7, another IL-1β antibody, AB5 or a control antibody. Twenty-four hours after antibody injection, the 25 mice were injected subcutaneously with recombinant human IL-1β (rhIL-beta) (from PeproTech Inc., Rocky Hill, NJ) at a dose of

1 μg / kg. Two hours after rhIL-Ιβ injection (peak IL-6 response time), mice were sacrificed and blood was collected and processed for serum. Serum IL-6 levels were assayed by ELISA (BD Pharmingen, Franklin Lakes, NJ) according to the manufacturer's protocol. Percent inhibition was calculated from the ratio of IL-65 detected in experimental animal serum to IL-6 detected in control animal serum (multiplied by 100).

The results are shown in Figure 2. The ability to inhibit IL-1β in vivo activity was assessed as a function of serum IL-1β-stimulated IL-6 levels. As illustrated by Figure 2, the 10 antibodies AB7 and AB5 were effective to inhibit in vivo activity of human IL-1β. These results also show that a single injection of AB7 or AB5 may block systemic action for IL-1β stimulation and that such antibodies are useful for inhibition of IL-1β activity in vivo.

A similar experiment was conducted to further demonstrate the ability of AB7 to neutralize mouse IL-1β in vivo to support the use of this antibody in mouse models of the disease. AB7 has been found to have an affinity for human IL-1β that is -10,000 times greater than the affinity for mouse IL-1β and an in vitro potency in assay D10.G4.1 which is -1,000 times greater than than that 20 for mouse IL-Iβ. In the IL-6 reading C57BL16 mouse model, mice were injected with AB7 (3 or 300 μg) or PBS i.p. 24 hours before an s.c. injection of human IL-1β (Figure 2B, panel A) or mouse (Figure 2B, panel B) (20 ng). Blood was drained 2 hours later and serum samples were analyzed for IL-6 levels by ELISA. These data show the maximum suppression of IL-6 levels (~ 75%) induced by human IL-1β at 3 μg (panel A), while submaximal suppression of IL-6 levels (~ 50%>) by mouse IL-1 β was demonstrated with 300 μg (panel B). These results are compatible with the observation of much higher affinity and in vitro potency of the AB7 antibody to human IL-1 β when compared to mouse IL-1 β. In addition, the data indicate that this antibody can be used for in vivo mouse disease models with a higher appropriate dose than would be needed for treatment of human patients, where the antibody has much higher affinity and potency. In the case of other IL-1β antibodies, such as for example the antibodies disclosed and / or cited herein, which do not exhibit such a significantly higher affinity property and in vitro potency for human IL-1β as compared to IL-1β. 1 β mouse, similar higher 10 doses in mouse models may not be necessary. Example 3

Pharmacokinetics of an anti-IL-ββ antibody following single intravenous or subcutaneous administration to rats

To examine the pharmacokinetic profile, an IL-1β antibody designated AB7 was administered to adult male rats as an intravenous (IV) bolus in the tail vein at doses of 0.1, 1.0 or 10 mg / kg.

1, 2 and 3 respectively) or a subcutaneous dose (SC) between the shoulder blades to be

1.0 mg / kg (Group 4). Blood samples were collected by cannulation in the jugular vein or retro-orbital bell at the times specified for 20 to 91 days after dosing. Blood samples were centrifuged to obtain serum. Samples were analyzed for anti-IL-ββ antibody concentration using an alkaline phosphatase based ELISA assay as follows.

IL-1 β (Preprotech) was diluted to 0.5 μg / ml in PBS and 50 μΐ 25 of this solution was added to the Nunc-Immuno Maxisorp (VWR) microtiter plate wells and incubated overnight at 2 to 8 ° C. The antigen solution was removed and 200 μΐ blocking buffer [1% bovine serum albumin (BSA) in IX PBS containing 0.05% Tween 20] was added to all wells and incubated for 1 hour at room temperature. After blocking, the reservoirs were washed three times with wash buffer (IX PBS containing 0.05% Tween 20). Standards, samples and controls were diluted in sample diluent (25% Rat Serum in IX PBS containing 1% BSA and 0.05% Tween 20).

Anti-IL-ββ antibody standard solutions were prepared as two-fold serial dilutions from 2000 to 0.24 ng / ml. Each replicate and dilution of the standards, samples and controls (50 μΐ) were transferred to the blocked microtiter plates and incubated for 1 hour at 37 ° C. After incubation, the reservoirs were washed 3 times with wash buffer. Alkaline phosphatase-conjugated goat anti-human IgG 10 (H + L) antibody (Southern Biotech Associates Inc, Birmingham, AL) was diluted 1/1000 in conjugate diluent (1% BSA in IX PBS containing 0.05% Tween 20). Fifty μΐ of diluted conjugate was added to all reservoirs except for WHITE reservoirs, which received only 50 μΐ of 15 conjugate diluent. The plates were incubated for 1 hour at 37 ° C and then all wells were washed 3 times with wash buffer and 3 times with deionized water. The p-nitrophenyl phosphate substrate (1 mg / ml in 10%) of diethanolamine buffer, pH 9.8) was added to all reservoirs and color development was allowed to process for 1 hour at room temperature after 50 ° C. μΐ of 1 N NaOH was added to stop the reaction. Absorbance at 405 nm was determined using a SPECTRAmax M2 Plate Reader (Molecular Devices, Menlo Park, CA) and a standard curve was then plotted as A405 versus ng / mL antibody standard. A regression analysis was performed and concentrations were determined for samples and controls by interpolation from the standard curve. The limit of quantification was 40 ng / ml.

As shown in Figure 3, serum concentrations declined bi-exponentially between dose groups IV. A compartmental analysis was performed on individual animal data and the resulting pharmacokinetic parameters were averaged for each dose group excluding those animals in which an RAHA response was generated. Serum anti-IL-β antibody levels declined with a mean alpha phase half-life of 0.189 ± 0.094 to 0.429 ± 0.043 days (4.54 to 10.3 hours) and a beta phase half-life of 9.68 ± 0.70 to 14.5 ± 1.7 days. Among rats receiving a subcutaneous dose of 1 mg / kg serum levels increased to a peak of 4.26 ± 0.065 μg / ml over 2 to 3 days and declined with a half life of 2.59 ± 0.25.

Example 4

Pharmacokinetics of an anti-IL-ββ antibody following single intravenous dose administration to cynomolgus monkeys

Adult male and female cynomolgus monkeys received the anti-IL-Ιβ antibody designated AB7 as a single intravenous (IV) bolus injection at doses of 0.3, 3.0 or 30 mg / kg. Blood samples 15 were collected from animals before dosing, 5 minutes, 4 and 8 hours after dosing on Day I and Days 2, 4, 8, 11, 15, 22, 29, 43 and 56. Samples were analyzed. for anti-IL-ββ antibody concentration using an alkaline phosphatase-based ELISA assay as follows.

The IL-Iβ solution was diluted to 0.5 μg / ml in PBS and 50 μΐ 20 of this solution was added to the Nunc-Immuno Maxisorp (VWR) microtiter plate wells and incubated overnight at 2 to 8 ° C. The antigen solution was removed and 200 μΐ blocking buffer [1% bovine serum albumin (BSA) in IX PBS containing 0.05% Tween 20] was added to all wells and then incubated for 1 to 4 hours at 25 ° C. room temperature. After blocking, the wells of each plate were washed three times with wash buffer (IX PBS containing 0.05% Tween 20). Standards, samples and controls were diluted in sample diluent (2% Normal Cinomolgo Serum (NCS) in IX PBS containing 1% BSA and 0.05% Tween 20). Anti-IL-β standard solutions were prepared as serial two-fold dilutions of 8000 ng / ml. Each replicate and dilution of the standards, samples and controls (50 μΐ) were transferred to the blocked microtiter plates and incubated for 1 hour at 37 ° C. After primary incubation, the wells were washed 35 times with wash buffer and 50 μΐ biotinylated rhIL-lbeta was added to all wells. The plates were then incubated for 1 hour at 37 ° C. The wells were washed 3 times with wash buffer and a tertiary incubation with fifty μΐ diluted alkaline phosphatase-conjugated streptavidin were added to all wells except 10 for the WHITE reservoirs, which received only 50 μΐ diluent. The plates were incubated for 30 minutes at 37 ° C and then all wells were washed 3 times with wash buffer and 3 times with deionized water. The p-nitrophenyl phosphate substrate (1 mg / ml in 10% diethanolamine buffer, pH 9.8) was added to all reservoirs. The color development was allowed to process in the dark for 1 hour at room temperature, after 50 μΐ of 1 N NaOH was added to stop the reaction. Absorbance at 405 nm was determined for all reservoirs using a SPECTRAmax M2 Plate Reader (Molecular Devices, Menlo Park, CA). A standard curve was then plotted as A40S 20 versus ng / ml anti-IL-ββ standard. A 4-parameter regression analysis was performed and concentrations were determined for samples and controls by interpolation from the standard curve. The limit of quantification was 40 ng / ml.

For the single dose groups of 0.3 and 3 mg / kg, serum anti-IL-Ιβ antibody levels declined with an average alpha phase half-life of 9.40 ± 2.00 hours, followed by a half-life. of 13.3 ± beta phase

1.0 days (Figure 5). In cinomolgus monkeys given a single 30 mg / kg IV injection, serum antibody levels declined more rapidly, with an alpha phase half-life of 10.9 ± 3.2 hours, followed by a beta phase half-life of 7.54 ± 1.79 days. Modeling of plasma concentration-time profiles of doses of 0.1, 0.3, 1 and 10 mg / kg administered at five monthly intervals was also performed and is shown in Figure 5.

Example 5

Effect of anti-IL-β antibodies on a human islet cell assay system

As an in vitro model, human islet cells are isolated and then treated with high glucose levels to mimic the Type 2 diabetic environment. Anti-IL-ββ antibodies can be used in the islet cell system to examine the effect. on beta cell function (release of insulin in response to glucose), beta cell proliferation and apoptosis.

Islets are isolated from the pancreas of multiple human organ donors with no history of diabetes or metabolic disease as described (Linetsky et al., Diabetes 46: 1120-1123, 1997; Oberholzer et al., Transplantation 69: 1115-1123, 2000 ; Ricordi et al., Diabetes 37: 413-420, 1988, Maedler et al., Proc. Natl. Acad. Sci. USA 101: 8138-8143, 2004; WO 2004/0002512). The islets are then cultured in extracellular matrix coated plates derived from bovine comeal endothelial cells (Novamed Ltd, Jerusalem), allowing the cells to bind to the plates and preserving their functional integrity. Islets are grown in CMRL 1066 medium containing 100 U / ml penicillin, 100 μg / ml streptomycin and 10% fetal bovine serum (GIBCO, Gaithersburg, MD). To stimulate insulin secretion, the culture medium is replaced with culture medium supplemented with 5 μΐ or 33 mM glucose, with or without the addition of fatty acid.

To measure insulin release in response to glucose, islet cells are washed and preincubated for 30 minutes in Krebs-Ringer Bicarbonate (KRB) buffer containing 3.3 mM glucose and 0.5% BSA. KRB is then replaced with 3.3 mM KRB glucose for 1 hour, which is then followed by an additional 1 hour in 16.7 mM KRB glucose. Islet cells are extracted with 0.18 M HCl in 70% ethanol for determination of insulin content using a human insulin RIA kit (Cl S Biointemational, Gif-sur-Yvette, France). Beta cell apoptosis can be measured by a variety of methods. For example, cells are double stained by standard terminal deoxynucleotidyl transferase (TUNEL) mediated dUTP cut-off labeling techniques and also to insulin. In parallel, apoptosis is also confirmed by detection of caspase 3 activation or Fas expression as described (see for example, WO 2004/002512; Maedler et al., 2004, ibid.

Example 6

Effect of anti-IL-ββ antibodies on a rat pseudoislet cell assay system

Alternatively or in addition to the human islet cell model, rat pseudoislet cells can be used as an in vitro model to evaluate the effects of anti-IL-ββ antibodies. For example, pseudoislets can be prepared and tested as described in US 20 20060094714. The pancreas of four Sprague Dawley rats are divided into small pieces, rinsed three times with Hanks-Hepes buffer and digested with collagenase (Liberase, 0.25). mg / ml, Roche Diagnostic Corp., Indianapolis, Ind., USA) at 37 ° C on a water bath shaker for 10 minutes. The tissue of the digested pancreas is then rinsed three times with 25 50 ml of Hanks-Hepes buffer to remove collagenase and the tissue pellet is then filtered through a 250 micron filter. The filtrate is mixed with 16 ml of 27% Ficoll (Sigma, St. Louis, Mo., USA) w / v in Hanks-Hepes buffer and then centrifuged on a Ficoll gradient (23%, 20.5%). and 11%, respectively; 8 ml of each concentration) at 1,600 rpm for 10 minutes at room temperature. Pancreatic islets are concentrated at the interphase between 11% and 20.5% and between 20.5% and 23% depending on the size of the islets. The islets are collected from the two interphase, rinsed twice with 5 calcium-free Hanks-Hepes buffer and then suspended in 5 ml of antibody-free Hanks-Hepes buffer containing 1 mM EDTA and incubated for 8 minutes at room temperature. environment. Trypsin and DNAse I are added to the islet suspension to a final concentration of 25 μg / ml and 2 μg / ml, respectively, and the suspension is incubated with shaking at 30 ° C for 10 minutes. Digestion with 10 trypsin is interrupted by the addition of 40 ml RPMI 1640 (GIBCO Life Technologies, Invitrogen, Carlsbad, Calif.) With 10% FBS. Trypsin digested islet cells are then filtered through a 63 micron nylon filter (PGC Scientific, Frederick, Md.) To remove large cell groups. Dispersed islet cells are then washed, counted and seeded in 96-well bottom plates (2,500 cells per well). The dispersed islet cell suspension is then centrifuged at

1,000 rpm for 5 minutes. The Hanks-Hepes buffer is removed and replaced with 200 μΐ RPMI 1640 medium containing 10% FBS, 1% Penicillin-Streptomycin and 2 mM L-glutamine. The 96 well plates are then centrifuged at 1,000 rpm for 5 minutes to collect the scattered islet cells concentrated at the bottom of the plate forming pseudoislets. These pseudo-islets are then grown overnight in a 37 ° C cell culture incubator with 5% CO 2 and then used for the assays.

Example 7

Effect of an IL-Iβ antibody on insulin sensitivity in an animal model

The in vivo efficacy of an IL-1β antibody as an insulin sensitizing agent can be measured as the activity of the insulin and glucose lowering antibody in a dietary model of insulin resistance. Male Sprague-Dawley rats are placed on a high fat, high carbohydrate diet containing 60% fructose, 10% lard and 0.06% magnesium at 6 weeks of age. Two days after starting the diet, rats are randomized into different groups based on antibody dose levels (ranging from 0.1 to 5 mg / kg body weight), route of administration (subcutaneous, intravenous or intraperitoneal routes). ) and frequency of administration (daily to bi-weekly). Control groups receive either buffer (vehicle) or an irrelevant antibody only. The 10 food and fluid intakes are measured each day and a peer feeding protocol is used to ensure food intake between the 3 groups. After 5 weeks, serum glucose, insulin and triglyceride levels are obtained in the semi-fasting state (the night before blood drainage, the animals are given a restricted amount of food) and the blood is drained in the morning. Following. The protocol is continued for an additional 9 weeks in which the glucose tolerance test (OGTT) is performed on conscious animals in the fasting state by blood sampling for glucose and insulin measurements after oral administration of a glucose load (100 mg / 100 grams of 20 body weight). Serum glucose and triglyceride levels are measured by spectrophotometric methods and insulin levels are measured by radioimmunoassay (Linco, St. Louis, MO).

Example 8

IL-1β antibodies for treatment in an animal model of Psammomys obesus DE T2D

The therapeutic efficacy of an IL-1 β antibody to prevent the decline in β cell mass observed in patients with Type 2 diabetes is evaluated in the Psammomys obesus gerbil, which shows insulin resistance and develops diet-induced obesity-related diabetes initially. associated with hyperinsulinemia and gradually progressing to severe hyperglycemia accompanied by a transient increase in beta cell proliferative activity and a prolonged increase in beta cell death rate with disruption of islet architecture (Donath et al., 5). Diabetes 48: 738-744, 1999). To determine the effect of IL-Iβ antibody on hyperglycemia-induced beta-cell apoptosis and impaired proliferation in the pancreatic islets of Psammomys obesus during diabetes development, the antibody is administered to diabetes-prone animals (switched to a high-energy diet). at multiple dose levels ranging from 0.1 to 10 5 mg / kg body weight by subcutaneous, intravenous or intraperitoneal routes, with repeated antibody administrations at intervals ranging from daily to weekly. Animal control groups are kept on a low energy diet or switched to a high energy diet and treated with buffer (vehicle) only or an irrelevant antibody. 15 Animal subgroups are sacrificed on days 4, 7, 14, 21 and 28, after which blood is collected and used to determine plasma glucose, insulin and triglycerides. The pancreas is also removed, with one portion frozen at -70 ° C for subsequent determination of insulin content and the remaining portion fixed in 10% phosphate-buffered formalin, paraffin embedded and sectioned for Fas analysis, IL-1 β and insulin expression and beta cell proliferation and apoptosis. Such analyzes will allow the determination of prevention or delay in the onset of diabetes, protection of hyperglycemia-induced beta-cell apoptosis, impaired proliferation and decreased β-cell mass and normalization of pancreatic insulin content.

Example 9

Use of an IL-1 β antibody in the treatment of T2D in humans

The IL-1β antibodies or fragments described herein may be administered to a human patient according to the invention for the therapeutic treatment and / or prevention of Type 2 diabetes. Specifically, in one example, an IL-1β antibody having the properties (AB7, described above) is used for the therapeutic treatment of patients demonstrating signs and symptoms of Type 2 diabetes. More specifically, the safety and efficacy of an IL-1 β antibody for Type 5 diabetes are demonstrated in a or more human clinical studies, including for example a test of the following design.

A double-blind placebo-controlled human clinical trial is performed in patients with Type 2 diabetes. Patients who meet the inclusion criteria for this study according to the American Diabetes Association (ADA) diagnostic criteria for T2D:

- Fasting blood glucose concentration> 126 mg / dL (>

7.0 mmol / L) (should be measured within 28 days before Day 0)

OR

- Symptoms of hyperglycaemia (eg thirst, polyuria, weight loss, visual shuffling) and a plasma glucose value

200 mg / dL (> 11.1 mmol / L) (should be measured within 28 days before Day 0),

and with an HbAlc> 7.5% and <12% (standard DCCT), are listed in the study sequentially by study group and within each group are randomly assigned to receive IL-IB antibody or placebo. To minimize risk to patients, safety and tolerability are reviewed at each dose level before escalating to the next dose level. Treatment groups and patient numbers for the study are shown in the following table:

Placebo Antibody Group Via # Patients Dose # Patients 1 IV or SC 5 0.01 mg / kg 1 2 IVor SC 5 0.03 mg / kg 1 3 IVor SC 5 0.1 mg / kg 1 IV or SC 5 0.3 mg / kg 1 S IV or SC 5 1.0 mg / kg 1 6 IV or SC 5 3.0 mg / kg 1 On Day 1 of the study, the antibody or placebo is administered subcutaneously or via a rate intravenous infusion. constant 30 minutes. Safety assessments, including record of adverse events, physical examinations, vital signs, clinical laboratory tests (eg, blood chemistry, hematology, urinalysis), plasma cytokine 5 levels, and electrocardiograms (ECGs) are conducted using standard practices. standard medical devices known in the art. Blood samples are collected at pre-dose administration and multiple time periods (eg, days) after administration to evaluate HbAlc, lipid profile including free fatty acids, HDL and LDL cholesterol, 10 IL antibody levels. -1 β (pharmacokinetics), anti-IL-Ιβ antibody responses, cytokine levels (eg IL-1 β, IL-6, TNFα), CRP, sodium, potassium, creatinine, AST, ALT, and hematogram. Assays may also be performed for other cytokines and lymphokines, such as for example those described herein. Additional blood samples may be collected later than 15 days initially planned in those cases when levels of the administered IL-1β antibody did not fall below the detection limit. Study evaluations are conducted at specified posttreatment times.

Clinical monitoring of treatment for Type 2 diabetes is based on a primary efficacy endpoint of improvement in hemoglobin Alc 20 (HbAlc, see for example Reynolds et al., BUT, 333 (7568): 586-589, 2006), Improvement in HbAlc level is indicative of therapeutic efficacy of anti-IL-Ιβ treatment and should generally result in a decrease of at least about 0.5% or more. One or more of the following secondary endpoints are also determined to evaluate the effectiveness of treatment for Type 2 diabetes, such as for example fasting blood sugar levels (e.g. glucose) (e.g. <130, < 120), 120-minute oral glucose tolerance test (OGTT), glucose / insulin C-peptide AUC (eg> 50%,> 60% increase), reduction in diabetes medication (eg insulin, agent hypoglycemia), improvement in insulin sensitivity, serum cytokine levels (eg normalization), CRP levels, quality of life measurements, BMI improvement (1%, 3%, 5% reduction), pharmacokinetics and others. (Saudek, et al., JAMA, 295: 1688-97, 2006; Pfiitzner et al., Diabetes Technol Ther. 8: 28-36, 5 2006; Norberg, et al., J Intem Med. 260: 263-71 , 2006). Additional sample lipid profile analyzes include the following tests performed in accordance with

according to accepted standard methods known in the art.

Test Method Lipoprotein Electrophoresis Gel Electrophoresis Serum Apolipoprotein AI (apoA-I) Nephelometry Apolipoprotein A-II (apo A-II) Nephelometry Serum Apolipoprotein B-48 (apo B-48) ELISA Serum Apolipoprotein B-100 (apo B-100 ) Nephelometry Serum Apolipoprotein Cs (apo Cs) Immunoturbidimetry for Apo CII and ApoCIII Serum Apolipoprotein E (apo E) Nephelometry Serum Apolipoprotein J (apo J) ELISA Serum Amyloid A Nephelometry Plasma Free Fatty Acid (FFA) Colorimetry Glycerol Plasma Glycerol o Serum LCAT Serum LCAT Cholesteryl Ester Transfer Protein Serum ELISA (CETP) Serum Hepatic Lipase (HL) Fluorometry Serum Paraoxonase I (PON1) UV / Colorimetry Pharmacokinetic Analysis

Samples are obtained for pharmacokinetic analysis on days 10 0, 1, 2, 3, 4, 7, 9 ± 1, 11 ± 1, 14 ± 1, 21 ± 2, 28 ± 2, 42 ± 3 and 56 ± 3. Preliminary analysis of AB7 pharmacokinetics in Type 2 diabetes patients receiving a single IV dose of 0.01 mg / kg showed serum concentration - time profiles with a 22 - day terminal half - life, clearance of 2.9 ml / day. / kg and central compartment volume of distribution of 50 ml / kg, 15 very similar to serum volume (Figure 7).

Blood Glucose and HbAlc Analysis

Samples are obtained and blood glucose measured on days 0, 7, 14 ± 1, 21 ± 2, 28 ± 2, 42 ± 3 and 56 ± 3, as well as on the day of screening. The evaluation of these samples for a decrease in blood glucose levels is shown below for samples from the first two dose groups (upper data line for each patient). Samples are obtained and HbAle measured on days 0, 28 ± 2, 42 ± 3 and 56 ± 3, as well as on the day of screening. The evaluation of these samples for a decrease in HbAle levels is shown below for samples from the first two dose groups (lower data line for each patient).

Group at the dose of 0.01 mg / kg

Patient Screening Day 0 Day 7 Day 14 Day 21 Day 28 Day 42 Day 56 Labs Labs Labs Labs Labs Labs Labs 5 200.00 247.00 212.00 179.00 213.00 242.00 235.00 200.00 8, 40 8.60 8.50 7.10 9.30 1 211.00 232.00 235.00 236.00 199.00 221.00 252.00 204.00 9.30 9.60 9.50 9.10 9.80 2 229.00 131.00 160.00 191.00 193.00 224.00 204.00 207.00 9.00 8.80 8.40 8.60 9.00 11 290.00 283.00 300.00 177.00 308.00 278.00 292.00 302.00 11.80 11.60 11.40 11.20 11.80 3 175.00 158.00 175.00 154.00 154.00 162 .00 183.00 170.00 8.20 8.20 7.70 7.80 8.10 4 238.00 255.00 270.00 275.00 289.00 278.00 255.00 245.00 8, 50 9.40 10.50 10.60 10.40 Group at the dose of 0.03 mg / kg

Patient Screening Day 10 Day 7 Day 14 Day 21 Day 28 Day 42 Day 56 Labs Labs Labs Labs Labs Labs Labs 6 222.00 164.00 148.00 166.00 162.00 145.00 207.00 8.40 8, 40 8.20 8.40 7 208.00 109.00 101.00 120.00 81.00 108.00 113.00 8.00 7.50 6.70 6.40 8 364.00 287.00 289, 00 260.00 237.00 12.40 12.00 9 204.00 128.00 124.00 117.00 112.00 113.00 7.90 7.50 7.10 12 275.00 235.00 250, 00 126.00 168.00 9.90 10.30 10 332.00 398.00 243.00 187.00 220.00 11.50 11.50 These data indicate the lower limits of and a dose of IL-1β antibody as provided herein, useful for achieving a therapeutic effect (e.g., decrease in glucose and / or HbAlc levels) in a patient following single antibody administration. C-Reactive Protein Analysis

C-reactive protein (CRP), which is released by the liver in response to various stress triggers, including IL-6, produced in response to IL-1, was also measured in serum at the same time points 5 as the PK samples. A PKIPD model was developed that incorporated a two-compartment model for serum antibody level and a concentration-dependent indirect antibody response over the CRP production rate, with a linear CRP elimination rate. Following a single IV dose of 0.01 mg / kg antibody in patients with Type 2 diabetes, 10 CRP declined within 7 to 10 days to 66 ± 22% from 100% pre-dose based on adjustments model (Figure 8). Following a single IV dose of 0.03 mg / kg antibody in patients with Type 2 diabetes, CRP declined within 7 to 10 days to 40 ± 12% over 100% of the pre-dose (Figure 9). Data for placebo controls are shown in Figure 15. Based on these data and model projections, the following predicted maintained CRP levels of monthly antibody injections are approximate to be 40% to 0.03 mg / kg. 16.5% at 0.1 mg / kg, 6.2% at 0.3 mg / kg, 1.9% at 1 mg / kg and 0.66% at 3 mg / kg per month (Figure 11 ). These data indicate that an IL-1β antibody as provided herein may be administered as infrequently as one month or longer to achieve a therapeutic effect (e.g., decrease in CRP levels) in a single patient following antibody administration.

Based on the results obtained from the first clinical studies, additional clinical tests are performed. Such assays may include one or more of the above dosages as well as or alternatively one or more other IL-1β antibody dosages, longer treatment and / or observation period and larger numbers of patients per group (at least about 10, 50, 100, 200, 300, 400, 500, 750, 1000) according to the invention. In addition, these and other studies can also be used to determine the length of time required to achieve a desired therapeutic benefit based on a change in a specific parameter (eg decrease in blood sugar, decrease in HbAlc, decrease in CRP). as well as the duration of the desired therapeutic benefit based on a change in a specific parameter (e.g., decrease in blood sugar, decrease in HbAlc, decrease in CRP) before additional dosages are administered.

Example 10

Effect of IL-Iβ Antibody on Adipocyte Function and Insulin Resistance

An in vitro assay using cultured adipocytes may be used to demonstrate a reduction (eg blocking) of IL-1β-induced insulin resistance by the use of anti-IL-ββ antibodies. The pre-adipocyte cell line 3T3-L1 obtained from ATCC (# CL-173) is grown in 15 7% CO2 and 37 ° C in DMEM, 25 mM glucose and 10% calf serum and induced to differentiate into adipocytes. In summary, 2 days after confluence, the medium is switched to DMEM, 25 mM glucose and 10% FCS supplemented with isobutyl methyl xanthine (0.25 mM), dexamethasone (0.25 μΜ), insulin (5 μg / ml). and pioglitazone (10 μΜ). The medium is removed after 2 20 days and replaced with DMEM, 25 mM glucose and 10% insulin supplemented FCS (5 μg / ml) and pioglitazone (10 μΜ) for 2 days. Then the cells are fed every 2 days with DMEM, 25 mM glucose and 10% FCS. 3T3-L1 adipocytes are used 8 to 15 days after the start of the differentiation protocol.

Human pre-adipocytes (Biopredic International, Rennes,

France) are grown in 5% CO2 and 37 ° C in Ham DMEM F12 containing 15 mM HEPES, 2 mM L-glutamine, 5% FCS, 1% antimycotic solution, ECGS / H-2, hEGF- 5 and HC-500 of the supplement packet pre-adipocyte growth medium (Promocell, Heidelberg, Germany). Adipocyte differentiation is induced after confluence by changing the medium by Ham DMEM F12 15 mM HEPES, 2 mM L-glutamine and 3% FCS supplemented with biotin (33 μΜ), insulin (100 nM), pantothenate ( 17 μΜ), isobutyl methylxanthine (0.2 mM), dexamethasone (1 μΜ), and 5 rosiglitazone (10 μΜ). The medium is removed after 3 days and replaced with Ham F12 containing 15 mM HEPES, 2 mM L-glutamine and 10% biotin-supplemented FCS (33 μΜ), insulin (100 nM), pantothenate (17 μΜ) and dexamethasone (1 μΜ). Then the cells are fed every 2 days with the same medium. Human adipocytes are used 15 days after the start of the differentiation protocol. Human preadipocytes may also be obtained from alternative sources, such as for example XA15Al and XM18B1 cell lines (Lonza, Allendale, NJ).

The role of IL-1β in inducing insulin resistance (decreased insulin sensitivity) in cultured adipocytes is shown 15 by incubating adipocytes with IL-1β (eg 20 ng / ml, 48 hours) and then incubating with different adipocytes. insulin concentrations (eg 0.5 nM, 100 nM; 20 min), followed by measuring glucose transport after the addition of 2- [H] deoxyglucose. Insulin resistance is determined as a reduction in glucose uptake and the effect of an anti-IL-20 1 β antibody on reducing (eg blocking) insulin resistance is easily measured in this adipocyte cell culture system.

The role of IL-Iβ in direct stimulation of adipokine and cytokine production by adipocytes (eg, leptin, resistin, visfatin, IL-6, MCP-I (CCL2), RANTES, PM-1, 25-acylation stimulating protein , SAA3, Pentraxin-3, macrophage migration inhibition factor, IL-IRA, IL-12, IL-8, IL-6, TNF-α) is determined by adipocyte culture in the absence or presence of different concentrations of IL -1 β for different time dimensions as described above and measuring adipocyte and cytokine levels in conditioned culture medium, usually by ELISA or other commonly used methods. In addition, the effects of treating IL-1β-stimulated adipocyte cultures with an anti-IL-

1 β neutralizer in adipokine induction and / or insulin resistance related cytokine secretion are measured. Similarly, the effects of treating with an anti-IL-Ιβ antibody on insulin-sensitizing adipokine suppression, adiponectin, are measured. To indicate whether anti-IL-ββ antibody treatment neutralizes the effects of endogenously produced IL-I derived from immune / inflammatory cells (eg macrophages) during adipose tissue inflammation, different numbers of human macrophages (monocyte derivatives or various monocytic cell lines) are cultured with the adipocyte cultures described above in the absence or presence of anti-IL-1β antibody and adipokine and cytokine modulation are measured. In addition, modulatory effects on adipokine and cytokine secretion following in vivo treatment of a patient with an anti-IL-ββ antibody are measured in the circulation (eg serum, plasma) as a means to demonstrate efficacy.

Example 11

Inhibition of cytokine production in human whole blood by an IL-Ιβ antibody

Measuring blood cytokines during an illness or treating an illness can be helpful in determining disease severity or response to therapy. Usually cytokine levels are measured in serum, but this method does not necessarily measure total cytokines. Many cytokines may be inside (intracellular) cells. In addition, a cell's ability to produce a cytokine may be more useful information than the circulating cytokine level.

A method of stimulating whole blood was used to determine cytokine production and the effect of treating with an anti-IL-1β antibody. Blood was drained from patients into the sterile heparinized tubes and then 250 μΐ of whole blood was added to each adjusted 4 ml orange Corning sterile cryotube as follows:

Control Series

All tubes were pre-filled with 550 μΐ RPMI. To tube 1 (control), 200 μΐ RPMI was added and to tubes 2 to 10, 100 μΐ additional RPMI were added. To each of tubes 2 to 10, 100 μΐ dilutions of an anti-IL-Ιβ (AB7) antibody was added.

Test Series

A similar series of antibody dilutions were adjusted as detailed above.

All tubes were well mixed using a swirl of seconds. Control series Al-10 tubes then received an additional 100 μΐ RPMI, vortexed 10 seconds, the screw cap firmly attached and the tubes placed in the incubator. To heat-killed Staphilococcus epidermidis Bl 10 test tubes at 10 ° C (final stock concentration of 1: 1000 resulting in a bacterial: white blood cell ratio of 10: 1) were added, the tubes were then swirled. for 10 seconds, capped and incubated at 37 ° C. After 24 hours of incubation, the cultures were all lysed with Triton X (0.5% final) to release cell contents and the lysates were frozen. Following lysis of whole blood cultures, thawed freeze cycles and cytokine levels are measured by standard cytokine ELISA assays for human TNFα, IL-6, IFNγ, IL-8, IL-Ia, IL-IRa and IL-β (R&D Systems, Minneapolis, MN).

Cytokines measured in control series tubes, which contain only sterile culture medium and antibody (where indicated), reflect the spontaneous level of stimulation. In healthy patients, very low levels of various cytokines are found when measured after 24 hours of incubation. In patients with untreated diseases, the levels may be higher. The test tube series additionally contained a defined amount of heat-killed Staphilococcus epidermidis, which stimulates the production of various cytokines. If treatment with the anti-IL-ββ antibody is effective, this will be reflected by reduced cytokine production.

5 As shown in Figure 6, the high-level anti-IL-anticorpoβ antibody

AB7 affinity was very effective in inhibiting IL-1β production in human blood. In an average of three human samples, the antibody inhibited Staphilococcus epidermidis-induced IL-1 β production by 50% to 0.1 pM and 75% to 3 pM. At 100 pM, the inhibition was 100%. Interferon gamma 10 (IFNγ) was induced by Staphilococcus epidermidis and AB7 reduced IFNγ induced by Staphilococcus epidermidis by 75% to 100 pM.

Example 12

Effect of anti-IL-Ιβ antibody on diabetes in the non-obese diabetic mouse (NOD)

To demonstrate the effectiveness of an anti-IL-1 β antibody in a

mouse diabetes model, 3- to 4-week-old female NOD mice (Jackson Laboratories, Bar Harbor, ME) are obtained and housed in a nursery under pathogen-free conditions. Multiple doses of anti-IL-ββ antibody (eg, 3 to 600 μg) are diluted in a suitable vehicle (eg, PBS) and administered to pre-diabetic female NOD starting mice no later than 6 weeks of age. , using different pathways (e.g., intraperitoneal, subcutaneously, intravenously) at defined intervals (e.g. weekly, biweekly, monthly). Blood glucose is monitored using a glucose meter (Encore Glucometer; Bayer, Elkhart, IN) at weekly intervals, starting at 10 weeks of age. Mice with blood glucose levels of 200 mg / dl on two consecutive occasions are considered diabetic (onset of diabetes is usually seen around 15 to 20 weeks of age and the incidence usually reaches a maximum of around 90% in 30 weeks old). Data are calculated as the percentage of animals remaining free of diabetes during the course of the experiment. Differences between curves are tested using the log rank test, which compares distributions over the entire observation period.

5 In another NOD mouse model, the effectiveness of the

anti-IL-1β antibody is demonstrated in a cyclophosphamide (CY) accelerated disease model (Reddy et al., Histochem J. 33: 317-327, 2001; Cailleau et al., Diabetes 46: 937-940, 1997 ; Reddy et al., Histochem J., 34: 1-12, 2002; Harada et al., Diabetologia 27: 604-606, 1984; Nicoletti, et al., Eur J Immunol 24: 1843-7, 1994). Male (or female) 4- to 8-week-old non-diabetic NOD mice are obtained (Jackson Laboratories, Bar Harbor, ME) and housed in a nursery under pathogen-free conditions. Mice are injected with a single dose of CY (Sigma) at 200 mg / kg and are treated at various accelerated intervals due to the accelerated nature of the model (eg once a week, twice a week) for two to three weeks. with or without multiple doses of anti-IL-1β antibodies (eg 3 μg, 30 μg, 150 μg, 600 μg) or isotype control diluted in a suitable vehicle (eg PBS) using different routes of administration (eg intraperitoneal, subcutaneously, intravenously). Urine glucose (glycosuria) levels are monitored three times a week and blood glucose levels are monitored once a week using a blood glucose meter starting the day before CY injection. Urine glucose levels> 20 mmol / L on two consecutive occasions are considered diabetic and the reduction in urine glucose levels by an anti-IL-1 β antibody is a measure of effectiveness.

In another model, the efficacy of an anti-IL-1β antibody is evaluated in a model of pancreatic islet transplantation diabetes reoccurrence (non-transplant rejection) (Mellgren et al., Diabetologia 29: 670-2, 1986 Sandberg, et al., Clin Exp Immunol 108: 314-7, 1997). Non-diabetic 4- to 8-week-old female NOD mice are obtained (Jackson Laboratories, Bar Harbor, ME) and housed in a nursery under pathogen-free conditions. Pancreatic islets are prepared from 5 to 6 week old non-diabetic male and 5 female NOD mice prior to observed leukocytic infiltration and transplanted under the renal capsule of female spontaneous diabetic NOD mice (15 to 20 week old) (400 at 450 islets / mouse). Transient normoglycemia occurs shortly after transplantation and hyperglycemia usually recurs approximately 6 days after transplantation. Mice are 10 treated with or without various doses of anti-lL-Ιβ (eg 3 μg, 30 μg, 150 μg, 600 μg) or diluted isotype control antibodies in a suitable vehicle (eg, PBS) using different routes of administration (eg intraperitoneal, subcutaneously, intravenously). Blood glucose levels are monitored before and after transplantation once or twice 15 times a week using a glucometer, and mice with levels> 200 mg / dl on two consecutive occasions are considered diabetic and reduced blood glucose levels. by an anti-IL-1 β antibody are a measure of effectiveness.

Example 13

Treatment of the low-dose streptozotocin-induced diabetes model in C57BLIK mice.

To demonstrate the efficacy of an anti-IL-1 β antibody in a multiple low dose streptozotocin (STZ) induced hyperglycemia and diabetes mellitus model (Sandberg, et al., Biochem Biophys Res 25 Commun 202: 543-548, 1994 ; Reddy, et al., Ann NY Acad Sci 1079: 109-113, 2006), 4- to 8-week-old C57BL / K mice are obtained (Jackson Laboratories, Bar Harbor, ME) and housed in a nursery under exempt conditions. of pathogen. Mice receive five daily injections of STZ (40 mg / kg) in this accelerated model and undergo accelerated treatment (starting one day prior to STZ injection) at various intervals (eg once, twice or three times per day). week) for one to three weeks with or without multiple doses of anti-IL-1β antibody (eg 3 μg, 30 μg, 150 μg, 600 μg) or isotype 5 control antibody diluted in a suitable vehicle (eg , PBS) using different routes of administration (e.g., intraperitoneal, subcutaneously, intravenously). Blood glucose levels are monitored once a week using a blood glucose meter starting the day before STZ injection. Blood glucose levels> 200 mg / dl on two consecutive occasions 10 are considered diabetic and the reduction in blood glucose levels by an anti-IL-1 β antibody is a measure of effectiveness.

Example 14

Treatment in the Type 2 Diabetes Diet-Induced Obesity Model

The efficacy of an anti-IL-1β antibody was tested in the Type 2 diabetes diet-induced obesity (DIO) model 15. In this model, mice fed a high-fat diet become obese over a period of several weeks and they exhibit impaired glucose tolerance and impaired insulin secretion when inoculated with a glucose bolus injection, 6 week-old male C57BL / 6 mice were fed a normal diet (ND, Teklad, 5 kcal% of fat) or high fat diet, Surwit High Sucrose (HFD, Research Diets # D12331, 58 kcal% fat). Antibody dosing was started one day earlier. Test anti-IL-1β antibody (AB7) and isotype control human IgG2 antibody were administered by intraperitoneal injection (i.p.). The 25 antibodies were dosed twice a week for 4 weeks. Body weight was also recorded twice a week. After 4 weeks, the mice underwent a glucose tolerance test (GTT). In GTT, mice are fasted overnight and then injected i.p. with 1 g / kg glucose. Blood glucose is measured from tail cuts at 0, 15, 30, 60, 90 and 120 minutes after injection using a FreeStile glucometer. Figure 12 shows that mice fed a high fat diet for 4 weeks had impaired glucose tolerance compared to mice on the normal diet (Figure I). Administration of a test IL-1β antibody protected the HFD mice from impaired glucose tolerance. At 60 minutes during GTT, performance of mice dosed with 1 mg / kg IL-1 β antibody was significantly better than mice receiving control IgG2 antibody (*, p <0.05). Notably, positive results in this mouse model were observed even if the AB7 antibody had a much lower affinity (-10,000 fold) and in vitro potency for mouse IL-1β as compared to human IL-1β as described in Example 2 above.

All references, including publications, patent applications and patents, cited herein are hereby incorporated by reference to the same degree as if each reference were individually and specifically indicated to be incorporated by reference and set forth herein in their entirety.

The use of the terms "one" and "one" and "the" and "a" and the like in the context of describing the invention (especially in the context of the following claims) shall be construed to encompass both the singular and the plural, the unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" shall be construed as unlimited terms (ie 25 meaning "including, but not limited to"), unless otherwise noted. Whenever an unlimited term is used to describe a feature or element of the invention, it is specifically contemplated that a limited term may be used in place of the unlimited term without departing from the spirit and scope of the invention. The recitation of value ranges here is merely intended to serve as a summary method of individually referring to each separate value falling within the range, unless otherwise indicated herein and each separate value is incorporated into the descriptive report as if even individually reported here. All 5 methods described herein may be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all of the exemplary or exemplary languages (e.g. "as") provided herein is intended merely to better illustrate the invention and does not propose a limitation on the scope of the invention unless otherwise claimed. No language in the descriptive report should be interpreted as indicating any unclaimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors embodying the invention. Variations of these preferred embodiments may be apparent to those skilled in the art in reading the foregoing description. The inventors expect skilled artisans to use such variations as appropriate and the inventors intend the invention to be practiced otherwise as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter reported in the appended claims as permitted by applicable law. Further, any combination of the elements described above in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. I LIST OF SEQUENCE <110> XOMA Technology Ltd. <120> "METHODS FOR THE TREATMENT OF IL-I-BETA-RELATED DISEASES" <1 30> 117791-100 <140> PCT / US07 / 88411 <14 1> 2007- 12-20 <150> US 60 / 871,046 <151> 2006-12-20 <150> US 60 / 908,389 <151> 2007-03-27 <150> US 60 / 911,033 <151> 2007-04-10 <160 > 6 <17 0> PatentIn version 3.5 <210> 1 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> IL-I-BETA Epitope <4 00> 1 Glu Ser Val Asp Pro Lys Asn Tyr Pro Lys Lys Met Glu Lys Arg 10 15

Phe Val Phe Asn Lys Ile Glu 20

<210> 2 <211> 5 <212> PRT

<213> Artificial Sequence <220>

<223> Linker Peptide <4 00> 2

Gly Gly Gly Gly Ser 1 5

<210> 3 <211> 107 <212> PRT

<213> Artificial Sequence <220>

<223> AB5-LC <400> 3

Asp Ile Gln Met Thr Gln Thr Thr Be Being Read Be Ala Be Read Gly 1 5 10 15 Asp Arg Val Thr Ile Be Cys Arg Wing Be Gln Asp Ile Be Asn Tyr 20 2 5 30 Read Be Trp Tyr Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Lys Leu His Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Wing Thr Tyr Phe Cys Leu Gln Gly Lys Met Leu Pro Trp 85 90 95 Thr Phe Gly Gly Gly Leu Glu Thr Lys Ile Lys 100 105 <210> 4

<211> 120

<212> PRT

<213> Artificial Sequence <220>

<223> AB5-HC

<400> 4 Gln Val Thr Read Lys Glu Be Gly Pro Gly Ile Read Lys Lys Pro Be Gln 1 5 10 15 Thr Read Le Be Read Thr Cys Be Phe Be Gly Phe Be Read Thr Be 20 25 30 Gly Met Gly Val Gly Trp Ile Arg Gln Pro Gly Lys Gly Leu Glu 35 40 45 Trp Leu Wing His His Ile Trp Asp Gly Asp Glu Ser Tyr Asn Pro Ser 50 55 60 Leu Lys Thr Gln Leu Thr Ile Ser Lys Asp Thr Be Val Asn Gln Val 65 70 75 80 Phe Leu Lys Ile Thr Be Val Asp Thr Val Asp Thr Wing Tyr Thr 85 85 95 Cys Wing Arg Asn Arg Tyr Asp Pro Pro Pp Val Asp Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 5

<211> 107

<212> PRT

<213> Artificial Sequence <220>

<223> AB7-LC

<4 00> 5

Asp Ile Gln Met Thr Gln Be Thr Be Be Read Be Wing Be Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Wing Cys Be Gln Asp Ile Be Wing Asn Tyr 20 25 30 Read Le Be Trp Tyr Gln Lys Pro Gly Lys Wing Val Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Be Lys Leu His Gly Val Pro Be Arg Phe Ser Gly 50 55 60 Be Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Be Leu Gln 65 70 75 80 Glu Asp Phe Wing Thr Tyr Phe Cys Leu Gln Gly Lys Met Leu Pro Trp 85 90 95 Thr Phe Gly Gln Le Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 6

<211> 120

<212> PRT

<213> Artificial Sequence <220>

<223> AB7-HC

<4 00> 6

Gln Val Gln Leu Gln Glu Be Gly Pro Gly Leu Val Lys Pro Be Gln 1 5 10 15 Thr Leu Be Leu Thr Cys Be Phe Be Gly Phe Be Leu Thr Thr Be 20 25 30 Gly Met Gly Val Gly Trp Ile Arg Gln Pro Be Gly Lys Gly Leu Glu 35 40 45 Trp Leu Wing His Ile Trp Asp Gly Asp Glu Ser Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Ser Leu Lys Ile Thr Be Val Thr Wing Asp Thr Wing Val Tyr Phe 85 90 95 Cys Arg Wing Asn Arg Tyr Asp Pro Pro Pp Val Asp Trp Gly Gln 100 105 110 Gly Thr Read Val Val Val Ser Ser 115 120

Claims (77)

Method for treating in a human being a disease or condition selected from the group consisting of Type 2 diabetes, insulin resistance, decreased insulin production, hyperglycemia, hyperinsulinemia, Type 2 diabetes, and obesity, characterized by the fact that it comprises administering an anti-IL-β antibody or human fragment thereof. Method according to claim 1, characterized in that the disease or condition is selected from the group consisting of Type 2 diabetes, insulin resistance, decreased insulin production, hyperglycemia, hyperinsulinemia and obesity. Method according to claim 2, characterized in that the disease or condition is selected from the group consisting of Type 2 diabetes, insulin resistance and obesity. Method according to claim 1, characterized in that the disease or condition is Type 2 diabetes. Method according to claim 2, characterized in that the disease or condition is hyperglycemia. Method according to claim 2, characterized in that the disease or condition is hyperinsulinemia. Method according to claim 3, characterized in that the disease or condition is obesity. Method according to claim 3, characterized in that the disease or condition is insulin resistance. Method according to claim 3, characterized in that the disease or condition is Type 2 diabetes. A method according to claims 1 to 9, characterized in that the administration of an initial dose of antibody or antibody fragment is followed by the administration of one or more subsequent doses. Method according to claim 10, characterized in that the administration of an initial dose of the antibody or antibody fragment is followed by the administration of two or more subsequent doses. Method according to Claims 1 to 9, characterized in that the administration of an initial dose of antibody or antibody fragment is followed by the administration of one or more subsequent doses, and wherein said one or more doses are administered. Subsequent doses are in an amount that is approximately the same or less than the initial dose. Method according to claims 1 to 9, characterized in that the administration of an initial dose of the antibody or antibody fragment is followed by the administration of one or more subsequent doses, and wherein at least one of the subsequent doses. It is in an amount that is more than the initial dose. Method according to claims 1 to 13, characterized in that the antibody or antibody fragment binds to human IL-1β with a dissociation constant of about 250 pM or less. A method according to claim 14, characterized in that the antibody or antibody fragment binds to human IL-1β with a dissociation constant of about 10 pM or less. Method according to claim 15, characterized in that the antibody or antibody fragment binds to human IL-1β with a dissociation constant of about 1 pM or less. Method according to claim 16, characterized in that the antibody or antibody fragment binds to human IL-1β with a dissociation constant of about 0.5 pM or less. A method according to claims 10 to 17, characterized in that the starting dose and each or more subsequent doses are separated from each other by an interval of at least about 2 weeks. A method according to claim 18, characterized in that the starting dose and each or more subsequent doses are separated from each other by an interval of at least about 1 month. Method according to claim 19, characterized in that the starting dose and each or more subsequent doses are separated from each other by an interval of at least about 3 months. Method according to claim 20, characterized in that the starting dose and each or more subsequent doses are separated from each other by an interval of at least about 6 months. Method according to claim 21, characterized in that the starting dose and each or more subsequent doses are separated from each other by an interval of at least about 12 months. Method according to claims 1 to 22, characterized in that the antibody or antibody fragment is administered at one or more doses of 3 mg / kg or less of antibody or fragment. A method according to claim 23, characterized in that the antibody or antibody fragment is administered in one or more doses of 1 mg / kg or less of antibody or fragment. A method according to claim 24, characterized in that the antibody or antibody fragment is administered in one or more doses of 0.5 mg / kg or less of antibody or fragment. A method according to claim 25, characterized in that the antibody or antibody fragment is administered in one or more doses of 0.1 mg / kg or less of antibody or fragment. The method of claim 26, wherein the antibody or antibody fragment is administered in one or more doses of 0.03 mg / kg or less of antibody or fragment. Method according to claim 27, characterized in that the antibody or antibody fragment is administered in one or more doses of 0.01 mg / kg or less of antibody or fragment. A method according to claims 23 to 28, characterized in that at one or more doses it is at least 0.01 mg / kg antibody or fragment. Method according to Claims 1 to 29, characterized in that the anti-IL-1β antibody or antibody fragment is a neutralizing antibody. A method according to claims 1 to 29, characterized in that the anti-IL-1β antibody or antibody fragment binds an IL-1β epitope such that the substantially bound antibody or fragment allows IL-binding. -1 β to IL-I receptor 1 (IL-1R1). A method according to claims 1 to 29, characterized in that the antibody or antibody fragment does not detectably bind IL-I to, IL-IR or IL-IRa. A method according to claims 1 to 29, characterized in that the antibody or antibody fragment binds an epitope contained in the sequence ESVDPKNYPKKKMEKRFVFNKIE. A method according to claims 1 to 29, characterized in that the antibody or antibody fragment binds to an epitope incorporating IL-1β Glu64. A method according to claims 1 to 29, characterized in that the antibody or antibody fragment binds to the N-terminal amino acids 1 to 34 of IL-1 β. A method according to claims 1 to 35, characterized in that the antibody or antibody fragment is human, engineered or humanized. A method according to claims 1 to 35, characterized in that the antibody or antibody fragment is human. A method according to claims 1 to 37, characterized in that the anti-IL-1β antibody or fragment is administered by subcutaneous, intravenous or intramuscular injection. A method according to claims 1 to 22, characterized in that the antibody or fragment is administered as a fixed dose, independent of a dose by weight ratio of the patient. The method of claim 39, wherein the antibody or fragment is administered in one or more doses of 500 mg or less of antibody or fragment. The method of claim 40, wherein the antibody or fragment is administered in one or more doses of 250 mg or less of antibody or fragment. A method according to claim 41, characterized in that the antibody or fragment is administered in one or more doses of 100 mg or less of antibody or fragment. A method according to claim 42, wherein the antibody or fragment is administered in one or more doses of 25 mg or less of antibody or fragment. A method according to claim 39, characterized in that the antibody or fragment is administered in one or more doses of at least 1 mg of antibody or fragment. The method of claim 39, wherein the antibody or fragment is administered in one or more doses of from about 10 mg to about 100 mg of antibody or fragment. A method according to claims 9 to 45, characterized in that a dose of antibody or fragment is sufficient to obtain at least 0.5 percentage point improvement in hemoglobin A1c. A method according to claim 46, characterized in that the dose of antibody or fragment is sufficient to obtain at least 1 percentage point improvement in hemoglobin Alc. A method according to claim 47, characterized in that the dose of antibody or fragment is sufficient to obtain at least 2 percentage points improvement in hemoglobin Alc. A method according to claim 48, characterized in that the dose of antibody or fragment is sufficient to obtain at least 3 percentage points improvement in hemoglobin Alc. A method according to claims 9 to 49, characterized in that said method is sufficient to obtain at least one of the following modifications: reduction in fasting blood sugar, decrease in insulin resistance, reduction in hyperinsulinemia, improvement in glucose tolerance, reduction in reactive peptide (CRP), reduction in hyperglycemia, reduction in need for diabetes medication, reduction in BMI, change in glucose / insulin C-peptide AUC, reduction in acute phase reagents , decrease in serum lipids with improvement in lipid profile. A method according to claims 9 to 45, characterized in that it reduces or prevents a complication or condition associated with Type 2 diabetes selected from the group consisting of retinopathy, renal failure, cardiovascular disease, and wound healing. a method comprising administering an anti-IL-1β antibody or fragment thereof to a human. Method according to claim 51, characterized in that the complication or condition is cardiovascular disease and wherein said cardiovascular disease is atherosclerosis or peripheral vascular disease. The method according to claim 51, characterized in that the complication or condition is wound healing and wherein said wound healing condition is diabetic ulcer. A method according to any one of claims 1 to 53, characterized in that said method is in conjunction with at least one additional treatment method, said further treatment method comprising administering at least one pharmaceutical composition comprising an agent. active other than an IL-1 β antibody or fragment. A method according to any one of claims 1 to 53, characterized in that said method prevents or delays the need for at least one further treatment method, said further treatment method comprising administering at least one pharmaceutical composition which comprises at least one pharmaceutical composition. comprises an active agent other than an IL-Iβ antibody or fragment. A method according to claims 1 to 53, characterized in that said method reduces the amount, frequency or duration of at least one further treatment method, said further treatment method comprising administering at least one pharmaceutical composition. which comprises an active agent other than an IL-1β antibody or fragment. A method according to claims 54 to 56, characterized in that said at least one pharmaceutical composition comprising an active agent other than an IL-1β antibody or fragment is selected from the group consisting of a sulfonylurea, a meglitinide, a biguanide, an alpha-glucosidase inhibitor, a thiazolidinedione, a glucagon and insulin equivalent peptide. A method according to claim 57, characterized in that said active agent is a sulfonylurea. A method according to claim 57, characterized in that said active agent is a meglitinide. A method according to claim 57, characterized in that said active agent is a biguanide. A method according to claim 57, characterized in that said active agent is an alpha glycosidase inhibitor. A method according to claim 57, characterized in that said active agent is a thiazolidinedione. A method according to claim 57, characterized in that said active agent is a glucagon equivalent peptide. A method according to claim 57, characterized in that said active agent is insulin. A method according to claims 1 to 53, characterized in that the administration of an initial dose of the antibody or antibody fragment is followed by the administration of one or more subsequent doses, and wherein the plasma concentration of said antibody. or human antibody fragment is maintained at a level of at least about 0.03 µg / ml during a course of treatment with said initial dose and one or more subsequent doses. A method according to claim 65, wherein the plasma concentration of said antibody or antibody fragment is maintained at a level of at least about 0.1 µg / ml during the course of treatment. A method according to claim 66, characterized in that the plasma concentration of said antibody or antibody fragment is maintained at a level of at least about 0.3 μg / ml during the course of treatment. Method according to claims 1 to 67, characterized in that the administration of the anti-IL-1β antibody or fragment thereof to humans results in a decrease in the production of one or more gene products selected from the group. consisting of leptin, resistin, visfatin, RANTES, IL-6, MCP-1, PAI-1, acylation stimulating protein, SAA3, Pentraxin-3, macrophage migration inhibiting factor, IL-1RA, IL-12, IL-8 and TNF-a. The method of claim 68, wherein administration of the anti-IL-1β antibody or fragment thereof to humans results in a decrease in the production of one or more gene products selected from the group consisting of leptin, resistin and visfatin. The method of claim 68, wherein administration of the anti-IL-1β antibody or fragment thereof to humans results in a decrease in the production of one or more gene products selected from the group consisting of MCP-1, RANTES, IL-6, TNF-α, and Pentraxin-3. A method according to claim 68, characterized in that said decrease in the production of one or more gene products is from adipose tissue. Method according to claims 1 to 67, characterized in that administration of the anti-IL-1β antibody or fragment thereof to humans results in an increase in adiponectin production. 73. The method of claim 72, wherein said increase in adiponectin production is from adipose tissue. A method according to claims 68 and 72, characterized in that said decrease or increase is detected in human blood. A method according to claims 1 to 74, characterized in that the antibody or fragment thereof has a lower IC50 than an IL-1β receptor antagonist in a whole blood IL-β inhibition assay. measuring human IL-10 induced production of IL-8. 76. Use of an anti-IL-1β antibody or fragment thereof that has a lower IC50 than an IL-1β receptor antagonist, characterized in that it is in a whole blood IL-β inhibition assay. human being measuring IL-Iβ-induced production of IL-8 in the manufacture of a composition for use in the treatment of Type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin production, and insulin resistance. The method according to claim 75 or the use according to claim 76, characterized in that the IL-1β receptor antagonist is anakinear.