CN101616690B

CN101616690B - Be used for the treatment of the method for IL-1 ss related diseases

Info

Publication number: CN101616690B
Application number: CN200780051536.4A
Authority: CN
Inventors: A·索林格; R·J·鲍尔; P·J·斯加侬; D·埃里瓦
Original assignee: Xoma US LLC
Current assignee: Xoma US LLC
Priority date: 2006-12-20
Filing date: 2007-12-20
Publication date: 2015-11-25
Anticipated expiration: 2027-12-20
Also published as: CN101616690A; CA2839162A1; ZA200904660B

Abstract

The invention discloses the method for morbid state and disease, obesity, hyperglycemia, hyperinsulinemia and the type 1 diabetes being used for the treatment of and/or preventing type 2 diabetes mellitus, insulin resistance and be characterised in that insulin resistance, it comprises anti-il-i-beta antibody or its fragment of using effective dose to experimenter.

Description

Be used for the treatment of the method for IL-1 ss related diseases

The application requires the U.S. Provisional Application submitted to for 20th in December in 2006 number 60/871 according to 35U.S.C. § 119,046, in the U.S. Provisional Application number 60/908 that on March 27th, 2007 submits to, 389, with the U.S. Provisional Application submitted on April 10th, 2007 number 60/911, the interests of 033, the disclosure entirety of described patent is incorporated herein by reference.

Invention field

The present invention relates to and be used for the treatment of and/or prevent type 2 diabetes mellitus, obesity, hyperglycemia, hyperinsulinemia, type 1 diabetes, insulin resistance and be characterised in that the morbid state of insulin resistance and the method for disease.This kind of method may be used for treating to be suffered from type 2 diabetes mellitus, obesity, hyperglycemia, hyperinsulinemia, type 1 diabetes, insulin resistance and is characterised in that the morbid state of insulin resistance and the mammalian subject of disease, or prevents its generation in risk subjects.

Background of invention

The disclosure relates to and is used for the treatment of and/or prevents the type 2 diabetes mellitus in mammal, obesity, hyperglycemia, hyperinsulinemia, type 1 diabetes, insulin resistance and be characterised in that the morbid state of insulin resistance and the method for disease.This kind of method may be used for treating mammal (such as people) experimenter of morbid state and the disease suffered from type 2 diabetes mellitus, obesity, hyperglycemia, hyperinsulinemia, type 1 diabetes, insulin resistance and be characterised in that insulin resistance, or prevents its generation in risk subjects.

Diabetes are people's metabolic disorder (Foster, D.W., Harrison ' sPrinciplesofInternalMedicine of the prevalence rate in general groups with about 1%, 114th chapter, 661-678 page, the 10th edition, McGraw-Hill, NewYork).This disease itself is the Developmental and Metabolic Disorder of a series of hormone induction, and it finally causes involving, and several tract is (comprising eye, kidney, nerve and blood vessel), debilitating, serious long-term complications.On pathology, the feature of this disease is visible basement membrane injury under an electron microscope.Diabetes can be divided into 2 kinds of clinical syndromes, 1 type and type 2 diabetes mellitus.1 type or insulin dependent diabetes mellitus (IDDM) (IDDM), also referred to as juvenile form, are chronic autoimmune disease, it is characterized in that a large amount of forfeitures of the β cell producing insulin in islets of langerhans.Because these cells are destroyed progressively, so the amount of insulin of secretion reduces, when amount of insulin secretion is reduced under normal required blood glucose levels, finally cause the hyperglycemia glucose of unusual high levels (in the blood).Although unknown about the definite triggering factors of this immunne response, IDDM patient has the antibody of the protein of expressing in high-caliber anti-pancreatic β cell.But all patients not with these antibody high-caliber develop IDDM.

Type 1 diabetes shows extremely low or immeasurablel plasma insulin and the adjoint glucagon raised characteristically.No matter what the definite cause of disease is, most of 1 type patient has the circulating antibody for himself pancreatic cell, comprises the antibody of Cytoplasm for insulin, pancreatic islet cells and glutamate decarboxylase.Immunne response specifically for β cell (insulin producing cell) causes type 1 diabetes.Current therapy for type 1 diabetes patient is injection of insulin, also can comprise the change to meals, so that the hyperglycemia making to result from natural insulin to lack (it is derived from again β cell injury conversely) drops to minimum.Also meals can be changed with regard to using of insulin, to offset the hypoglycemic effect of this hormone.

When muscle, fat and hepatocyte normally cannot respond insulin, there is type 2 diabetes mellitus (also referred to as non-insulin-dependent diabetes mellitus (NIDDM), Adult Onset, adult).This reason that cannot respond (being called insulin resistance) may be the minimizing of insulin receptor number on these cells, or the dysfunction of intracellular signaling pathway, or both.β cell compensates this insulin resistance by increasing insulin input quantity at first.Along with the past of time, these cells became and cannot produce enough insulins to maintain normal glucose level, thus instruction advances to type 2 diabetes mellitus.Type 2 diabetes mellitus is caused by combining with acquired risk factor of heredity, comprises high fat diet, does not get enough athletic exercise and aging.Suffer from type 2 diabetes mellitus more than 90% in diabetes community, and this sickness rate continues to rise, becoming principal element people such as (, DiabeticMed.14:Sl-85,1997) Amos of whole world death, morbidity and health care consumption.

Type 2 diabetes mellitus is the complex disease being characterised in that glucose and lipid metabolism disorder.Usually there is the upset of many metabolizing parameters, comprise the increase on fasting plasma glucose level, free fatty acid levels and triglyceride levels, and the reduction of HDL/LDL ratio.As mentioned above, one of the diabetes main basic cause of disease is considered to peripheral tissues, the mainly increase of insulin resistance in muscle and fat.The cause of disease of type 2 diabetes mellitus not yet obtains abundant understanding.It is believed that, the insulin secretion (" beta cell failure ") that target tissue also occurs the opposing of insulin action to reduce both has occurred.Be responsible for insulin homeostasis main insulin response tissue be liver, wherein insulin stimulating Glycogen synthesis and suppress glyconeogenesis; Muscle, insulin-stimulated glucose picked-up wherein and glycogen stimulate glucose uptake and suppress steatolysis.Therefore, as the result of diabetes, in blood, there is the glucose of elevated levels, this cytotoxicity that glucose can be caused to mediate and morbidity subsequently (nephropathy, neuropathy, retinopathy etc.).The development strong correlation of insulin resistance and type 2 diabetes mellitus.

At present, there are various pharmacological method people such as (, DiabetesCare, 22 (9): 1568-1577,1999) Scheen being used for the treatment of type 2 diabetes mellitus.They play a role via different binding mode: 1) sulfonylurea (such as, glimepiride (glimepiride), Glisentide (glisentide), sulfonylureas (sulfonylurea), AY31637) substantially stimulates insulin secretion, 2) biguanides (such as, metformin (metformin)) is by promoting glucose utilization, reducing liver glucose production and reduce intestinal glucose to export and play a role, 3) Alpha-glucosidase inhibitor (such as, acarbose (acarbose), miglitol (miglitol)) slows down carbohydrate digestion and thus from the absorption of intestinal with reduce postprandial hyperglycemia, 4) thiazolidinediones (such as, troglitazone (troglitazone), pioglitazone (pioglitazone), rosiglitazone (rosiglitazone), glipizide (glipizide), Ba Gelie ketone (balaglitazone), RIVOGLITAZONE (rivoglitazone), netoglitazone (netoglitazone), troglitazone (troglitazone), englitazone (englitazone), AD5075, T174, YM268, R102380, NC2100, NIP223, NIP221, MK0767, ciglitazone (ciglitazone), adaglitazone, CLX0921, darglitazone (darglitazone), CP92768, BM152054) insulin action is strengthened, promote the glucose utilization in peripheral tissues thus, 5) glucagon-like peptide, comprises DPP4 inhibitor (such as, sitagliptin (sitagliptin)), with 6) insulin stimulating tissue glucose utilizes and suppression liver glucose exports.Above-mentioned pharmacological method can individually or with conjoint therapy utilize.But often kind of method has its limitation and untoward reaction.Along with the time goes over, most of type 2 diabetes mellitus experimenter can lose its reaction to these activating agents.Insulinize generally starts from after meals, exercise and oral drugs cannot control blood-glucose fully.The shortcoming of insulinize needs the probability of drug injection, hypoglycemia and body weight to increase.

IL-1 β is by many different cell types, comprises mononuclear cell and macrophage, the proinflammatory cytokine of secretion.When the part as inflammatory reaction discharges, IL-1 β causes a series of biological effect, this is mainly through inducing other inflammatory mediators to mediate, and other inflammatory mediators described are thyroliberin, PF4, PGE2 (PGE2), IL-6 and IL-8 such as.IL-1 β, by activating IL-1 receptor (it is present on nearly all cell type), induces the inflammatory effector of local and whole body.Il-1 (IL-1) cytokine family has been prompted to involve numerous disease state.IL-1 family member comprises IL-1 α, IL-1 β and IL-1Ra.Although the ability combined by itself and IL-1 receptor (IL-1R1 and IL-1R2) and being associated, each of these cytokines is different, is expressed and have different primary amino acid sequences by different genes.In addition, the physiologically active of these cytokines can be distinguished from each other.Disclose instruction IL-1 β and obviously involve the experiment in diabetes.

The people such as Maedler, JClinInvest (2002) 110:851-860 points out chronic hyperglycemia in type 2 diabetes mellitus may damage pancreas beta cell, cause impaired insulin secretion, and point out that IL-1 β is the proinflammatory cytokine played a role in the self-immunprocess of type 1 diabetes, and β cell function can be suppressed.Especially, they examine following hypothesis: IL-1 β may mediate the illeffects of high glucose level.Treat diabetic animal with phlorhizin (phlorizin) and make Normalization of plasma glucose, and stop β cellular expression IL-1 β.It is said that this involves pathogenetic inflammatory process of glucose toxicity in type 2 diabetes mellitus (glucotoxicity), and IL-1 β/NF-kB pathway is accredited as preserves the amount of β cell and the target of function in this disease by them.

The people such as Donath, JMolmed (2003) 81:455-470 points out the clear meaning of IL-1 β in the apoptosis pathway (this causes insufficient insulin and diabetes) of pancreaticβ-cell death, and proposes the anti-inflammatory treatment method designing to block beta cell apoptosis in 1 type and type 2 diabetes mellitus.

WO2004/002512 relates to IL-1 receptor antagonist (IL-1Ra) and/or pyrrolidine dithiocarbamate (PDTC) is used for the treatment of or prevents the purposes of type 2 diabetes mellitus.But, the administration frequencies (injection in every 24 hours once) of advising is applied for the therapeutic of IL-Ra polypeptide in type 2 diabetes mellitus treatment, the problem of patient compliance may be caused, thus reduce effectiveness and/or the demand of restriction to it of this form of therapy.Therefore, still need the effective ways being used for the treatment of type 2 diabetes mellitus, those methods particularly not needing every day and inject.

The people such as Larsen, NewEnglandJournalofMedicine (2007) 356:1517-1526 describes the purposes that restructuring IL-1 receptor antagonist (IL-1Ra, Antril (Synergen) (anakinra)) is used for the treatment of type 2 diabetes mellitus.But every day, the administration of 1 100mg Antril (Synergen) continued the problem that 13 weeks may cause patient compliance, thus reduce effectiveness and/or the demand of restriction to it of this form of therapy.Therefore, still need the effective ways for the treatment of type 2 diabetes mellitus, particularly do not need the Therapeutic Method that frequent (such as, every day) injects.

US2005/0256197 and US2005/0152850 relates to for promoting that experimenter (such as, suffer from the experimenter of diabetes) in the method for Metabolism control (such as glucose), it comprises, particularly by using antiinflammatory, such as anti-inflammatory ketorolac gargarism (ketorolacoralrinse), reduce the IL-1 β level in experimenter's gum seam liquid, thus reduce circulation TNF level.

Obesity is the chronic disease of high prevalence, it is not only relevant with social discrimination, also relevant with numerous medical problem with the life-span reduced, described medical problem comprises bad mental development, dermatological conditions such as infects, varicosis, motion intolerance, diabetes, insulin resistance, hypertension, hypercholesterolemia and the coronary heart disease (people such as Rissanen, BritishMedicalJournal, 301:835-837,1990).In laboratory animal and people, fat of insulin resistance and diabetes height correlation.In fact, fat and insulin resistance (the wherein general adjoint hyperinsulinemia of the latter or hyperglycemia, or both) is the mark of type 2 diabetes mellitus.In addition, type 2 diabetes mellitus is relevant to 2 or 4 times of risks of coronary artery disease.Although have studied many decades to these serious health problems, the cause of disease that is fat and insulin resistance is still unknown.

Insulin resistance is relevant with disease to numerous morbid state, and is present in the non diabetic individuals of about 30-40%.These morbid states and disease include but not limited to, prediabetes and metabolism syndrome (also referred to as insulin resistance syndrome).Prediabetes is characterised in that glucose tolerance reduces the abnormal glucose tolerance state of (IGT) or impaired fasting plasma glucose (IFG).Having prediabetic patient is insulin resistant, and is in and is in progress in the excessive risk of dominant type 2 diabetes mellitus in the future.Metabolism syndrome is relevant cluster character, it includes but not limited to, hyperinsulinemia, abnormal glucose tolerance, obesity, fatty distribution again are to abdominal part or upper body compartment, hypertension, abnormal fibrinolysis (dysfibrinolysis) and dyslipidemia (being characterised in that high triglyceride, low HDL-cholesterol and small and dense LDL granule).Insulin resistance links together with all these character, and prompting Metabolic syndrome insulin resistance of seeking peace is closely related each other.The powerful risk factor making a definite diagnosis the atherosclerosis (causing heart attack, apoplexy and peripheral blood vessel) being generation type 2 diabetes mellitus and acceleration in the future of metabolism syndrome.Inflammatory cytokine, comprises IL-1, has been presented at transmitting inflammation in fatty tissue, and this seems the insulin resistance (people such as Trayhurn, Br.J.Nutr.92:347-355,2004 that relate to adipose cell; Wisse, J.Am.Soc.Nephrol.15:2792-2800,2004; Fantuzzi, J.AllergyClin.Immunol.115:911-919,2005; Matsuzawa, FEBSLett.580:2917-2921,2006; The people such as Greenberg, EurJ.Clin.Invest.32Suppl.3:24-34,2002; The people such as Jager, Endocrinology148:241-251,2007).Adipose cell is depot lipid and the cell of point resinosis therbligs (adipokine) (that is, a subgroup of cytokine), and is the key component of fatty tissue.Macrophage, as the main producers of inflammatory cell and inflammatory cytokine (IL-1, TNF-α and IL-6), be also present in fatty tissue, particularly relevant to the obesity inflammation fatty tissue (people such as Kern, Diabetes52:1779-1785,2003).Previous known TNF-α and IL-6 makes adipose cell to insulin stimulating desensitization (that is, insulin resistance).

Because to treat relevant problem at present, need the new therapy for the treatment of type 2 diabetes mellitus and other diseases indication such as those indications disclosed herein, to replace or to supplement existing medical procedures.The invention provides the method being used for the treatment of type 2 diabetes mellitus.In addition, the present invention is also provided for treatment obesity, hyperglycemia, hyperinsulinemia, type 1 diabetes, insulin resistance and is characterised in that the morbid state of insulin resistance and the method for disease.Method disclosed herein comprises such as, uses anti-il-i-beta antibody or its fragment.With antibody, particularly show the antibody of high-affinity, the method for direct targeting IL-1 beta ligands, provides and exceedes other potential Therapeutic Method, such as IL-1 beta receptor antagonist (such as, IL-1Ra, Antril (Synergen), ) advantage.To the challenge of the therapy based on IL-1 receptor antagonist be need this kind of therapeutic agent occupy a large amount of receptors, and this is a difficult task, because these receptors are expressed widely removing (Dinarello on exo-erythrocytic all cells, Curr.Opin.Pharmacol.4:378-385,2004).At most of immunologically mediated disease, such as, in disease disclosed herein, IL-1 β cytokine amount measurable or relevant to activating cell in body fluid is relatively low.Therefore, the method that treats and/or prevents of direct targeting IL-1 beta ligands is good strategy, particularly when using the IL-1 β antibody with high-affinity.

Summary of the invention

The disclosure relates to and is used for the treatment of and/or prevents the type 2 diabetes mellitus in mammal, obesity, hyperglycemia, hyperinsulinemia, insulin generation minimizing, type 1 diabetes, insulin resistance and/or be characterised in that the morbid state of insulin resistance and the method for disease.This kind of method may be used for treating to be suffered from following disease or has the mammal of the risk suffering from following disease (such as, people) experimenter: diabetes, obesity, hyperglycemia, hyperinsulinemia, insulin generate minimizing, insulin resistance and/or be characterised in that morbid state and the disease of insulin resistance.The method also may be used for preventing type 2 diabetes mellitus, type 1 diabetes, obesity, hyperglycemia, hyperinsulinemia, insulin generate minimizing, insulin resistance and are characterised in that the morbid state of insulin resistance and the generation of disease in risk subjects.

In one aspect, the present invention relates to treatment in people and be selected from following disease or the method for disease: type 2 diabetes mellitus, hyperglycemia, hyperinsulinemia, obesity, insulin generate minimizing, type 1 diabetes and insulin resistance, and the method comprises uses anti-il-i-beta antibody or its fragment to people.In a preferred embodiment, disease or disease are selected from type 2 diabetes mellitus, hyperglycemia, hyperinsulinemia, obesity, insulin generation minimizing and insulin resistance.Preferably, disease or disease are type 2 diabetes mellitus, obesity, insulin generation minimizing or insulin resistance.More preferably, disease or disease are type 2 diabetes mellitus, obesity or insulin resistance.Most preferably, disease or disease are type 2 diabetes mellitus.In one embodiment, the method does not increase cardiovascular disease or disease.In certain embodiments, antibody or fragment are used for the treatment of two or more above-mentioned disease or the diseases in same patient (such as, people experimenter).

In yet another aspect, the invention provides by using anti-il-i-beta antibody or fragment and be used for the treatment of or prevent disease or the method for disease, wherein disease or disease are prediabetes, dyslipidemia, hyperlipemia, hypertension, metabolism syndrome or illness behavior (sicknessbehavior).In another one, the method reduces or prevents to be selected from the following complication relevant to type 2 diabetes mellitus or disease in people: retinopathy, renal failure, cardiovascular disease and wound healing, and the method comprises uses anti-il-i-beta antibody or its fragment to people.In certain embodiments, antibody or fragment are used for the treatment of two or more above-mentioned disease or the diseases in same patient (such as, people experimenter).In another embodiment, antibody or fragment are used for the treatment of the renal failure (such as, kidney diaseases) of the disease that may be due to beyond type 2 diabetes mellitus.In another one embodiment, antibody or fragment are used for the level reducing CRP in the experimenter of the c reactive protein (CRP) of display elevated levels.

In yet another aspect, the invention provides in treatment or prevent the IL-1 β antibody that uses in disease as disclosed herein or disease or its antibody fragment.In further at one, the invention provides IL-1 β antibody or its antibody fragment, using for being selected from following disease or disease in treatment or prevention: type 2 diabetes mellitus, obesity, insulin generate and reduce and insulin resistance.In another one, the invention provides IL-1 β antibody or its antibody fragment, for using in treatment or prevention type 2 diabetes mellitus.

In yet another aspect, the invention provides the pharmaceutical composition comprising IL-1 β antibody or its antibody fragment and the optionally pharmaceutically acceptable excipient of at least one, in treatment or prevent to use in disease as disclosed herein or disease.In further at one, the invention provides the pharmaceutical composition comprising IL-1 β antibody or its antibody fragment and the optionally pharmaceutically acceptable excipient of at least one, using for being selected from following disease or disease in treatment or prevention: type 2 diabetes mellitus, obesity, insulin generate and reduce and insulin resistance.In another one, the invention provides the pharmaceutical composition comprising IL-1 β antibody or its antibody fragment and the optionally pharmaceutically acceptable excipient of at least one, for using in treatment or prevention type 2 diabetes mellitus.

The anti-il-i-beta antibody used in the method for the invention or antibody fragment are generally combined with IL-1 β with high-affinity.In preferred embodiments, antibody or antibody fragment are combined with IL-1 β, dissociation constant be about 10nM or less, about 5nM or less, about 1nM or less, about 500pM or less, about 250pM or less, about 100pM or less, about 50pM or less or about 25pM or less.In particularly preferred embodiments, antibody or antibody fragment are combined with people IL-1 β, dissociation constant be about 100pM or less, about 50pM or less, about 10pM or less, about 5pM or less, about 3pM or less, about 1pM or less, about 0.75pM or less, about 0.5pM or less, about 0.3pM or less, about 0.2pM or less or about 0.1pM or less.In particularly preferred embodiments, antibody or antibody fragment are combined with people IL-1 β, and dissociation constant is about 10pM or less.

In another aspect of the present invention, anti-il-i-beta antibody or antibody fragment are neutralizing antibodies.In yet another aspect, anti-il-i-beta antibody or antibody fragment are combined with IL-1 β epi-position, thus make the antibody of combination or fragment substantially allow IL-1 β and IL-1 receptor I (IL-1RI) to combine.In yet another aspect, anti-il-i-beta antibody or antibody fragment are combined with IL-1 β, but substantially do not stop the IL-1 β of combination and IL-1 receptor I (IL-1RI) to combine.In yet another aspect, the combination of antibody or antibody fragment and IL-1 α, IL-1R or IL-1Ra can not detect.In another aspect of the present invention, the epi-position that antibody or antibody fragment comprise in sequence ESVDPKNYPKKKMEKRFVFNKIE (SEQIDNO:1) is combined.In another aspect of the present invention, the epi-position that antibody or antibody fragment comprise Glu64 in IL-1 β is combined.In another aspect of the present invention, antibody or antibody fragment are combined with the amino acid/11-34 of IL-1 β N-terminal.Preferably, antibody or antibody fragment are people's through engineering approaches, humanized or people.

In yet another aspect, the invention provides treatment and show any above-mentioned disease or disease (such as, type 1 diabetes, type 2 diabetes mellitus, hyperglycemia, hyperinsulinemia, obesity, insulin generate reduce, insulin resistance) symptom or have the method for people of the risk developing any above-mentioned disease or disease, the method is included in one or more dosage and uses anti-il-i-beta antibody or its fragment to people.

In another aspect of the present invention, be provided for treatment in people and be selected from following disease or the method for disease: type 1 diabetes, type 2 diabetes mellitus, hyperglycemia, hyperinsulinemia, obesity, insulin generate and reduce and insulin resistance, the method comprises uses anti-il-i-beta antibody or its fragment to people, wherein after the predose using IL-1 β antibody or antibody fragment, uses one or more subsequent dose.In one embodiment, 2 or more subsequent dose are used after the predose of administration of antibodies or antibody fragment.In another embodiment, use one or more subsequent dose after the predose of administration of antibodies or antibody fragment, wherein said one or more subsequent dose is approximately equal to or less than predose in amount.In another embodiment, use one or more subsequent dose after the predose of administration of antibodies or antibody fragment, wherein at least one subsequent dose exceedes predose in amount.

In one embodiment, the antibody of 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more or 11 an or more subsequent dose is used.In another embodiment, using of each of predose and one or more subsequent dose is separated from each other, interval at least about 2 weeks, at least about 3 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months or at least about 12 months.

In another embodiment, antibody or fragment are used with one or more dosage, and described dosage is 5mg/kg or less antibody or fragment, 3mg/kg or less antibody or fragment, 2mg/kg or less antibody or fragment, 1mg/kg or less antibody or fragment, 0.75mg/kg or less antibody or fragment, 0.5mg/kg or less antibody or fragment, 0.3mg/kg or less antibody or fragment, 0.1mg/kg or less antibody or fragment or 0.03mg/kg or less antibody or fragment.Preferably, in each above-mentioned embodiment, antibody or fragment are used with one or more dosage, and described dosage is at least 0.01mg/kg antibody or fragment, at least 0.03mg/kg antibody or fragment, at least 0.05mg/kg antibody or fragment or at least 0.09mg/kg antibody or fragment.Above-mentioned dosage refers to mg (antibody or fragment)/kg (weight of individuality to be treated).

In another embodiment, predose and one or more subsequent dose of antibody or fragment are about 10mg/kg antibody for about 0.01mg/kg-separately, about 0.05-is about 5mg/kg antibody, about 0.05mg/kg-is about 3mg/kg antibody, about 0.1mg/kg-is about 3mg/kg antibody, about 0.1mg/kg-is about 1mg/kg antibody, about 0.1mg/kg-is about 0.5mg/kg antibody, about 0.3mg/kg-is about 5mg/kg antibody, about 0.3mg/kg-is about 3mg/kg antibody, about 0.3mg/kg-is about 1mg/kg antibody, about 0.5mg/kg-is about 5mg/kg antibody, about 0.5mg/kg-is about 3mg/kg antibody, about 0.5mg/kg-is about 1mg/kg antibody, about 1mg/kg-is about 5mg/kg antibody, or about 1mg/kg-is about 3mg/kg antibody.In certain embodiments, the antibody of 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more or 11 an or more subsequent dose is used.Above-mentioned dosage refers to mg (antibody or fragment)/kg (weight of individuality to be treated).If mention dosage hereinafter, so this applies equally.

In yet another aspect, the invention provides treatment in people and be selected from following disease or the method for disease: type 1 diabetes, type 2 diabetes mellitus, hyperglycemia, hyperinsulinemia, obesity, insulin generate and reduce and insulin resistance, the method comprises to the anti-il-i-beta antibody of people's administering therapeutic effective dose or its fragment, mode is the predose of about 5mg/kg or less antibody or fragment, in amount, be approximately equal to or less than the antibody of this predose or the subsequent dose of fragment with multiple, wherein subsequent dose is separated by the interval at least 2 weeks.

In yet another aspect, what the invention provides treatment people is selected from following disease or the method for disease: type 1 diabetes, type 2 diabetes mellitus, hyperglycemia, hyperinsulinemia, obesity, insulin generate and reduce and insulin resistance, the method comprises to the anti-il-i-beta antibody of people's administering therapeutic effective dose or its fragment, mode is the predose of about 3mg/kg or less antibody or fragment, in amount, be approximately equal to or less than the antibody of this predose or the subsequent dose of fragment with multiple, wherein subsequent dose is separated by the interval at least 2 weeks.

In yet another aspect, what the invention provides treatment people is selected from following disease or the method for disease: type 1 diabetes, type 2 diabetes mellitus, hyperglycemia, hyperinsulinemia, obesity, insulin generate and reduce and insulin resistance, the method comprises to the anti-il-i-beta antibody of people's administering therapeutic effective dose or its fragment, mode is the predose of about 1mg/kg or less antibody or fragment, in amount, be approximately equal to or less than the antibody of this predose or the subsequent dose of fragment with multiple, wherein subsequent dose is separated by the interval at least 2 weeks.

In yet another aspect, what the invention provides treatment people is selected from following disease or the method for disease: type 1 diabetes, type 2 diabetes mellitus, hyperglycemia, hyperinsulinemia, obesity, insulin generate and reduce and insulin resistance, the method comprises to the anti-il-i-beta antibody of people's administering therapeutic effective dose or its fragment, mode is the predose of about 0.5mg/kg or less antibody or fragment, in amount, be approximately equal to or less than the antibody of this predose or the subsequent dose of fragment with multiple, wherein subsequent dose is separated by the interval at least 2 weeks.

In yet another aspect, what the invention provides treatment people is selected from following disease or the method for disease: type 1 diabetes, type 2 diabetes mellitus, hyperglycemia, hyperinsulinemia, obesity, insulin generate and reduce and insulin resistance, the method comprises to the anti-il-i-beta antibody of people's administering therapeutic effective dose or its fragment, mode is the predose of about 0.3mg/kg or less antibody or fragment, in amount, be approximately equal to or less than the antibody of this predose or the subsequent dose of fragment with multiple, wherein subsequent dose is separated by the interval at least 2 weeks.

In yet another aspect, what the invention provides treatment people is selected from following disease or the method for disease: type 1 diabetes, type 2 diabetes mellitus, hyperglycemia, hyperinsulinemia, obesity, insulin generate and reduce and insulin resistance, the method comprises to the anti-il-i-beta antibody of people's administering therapeutic effective dose or its fragment, mode is the predose of about 0.1mg/kg or less antibody or fragment, in amount, be approximately equal to or less than the antibody of this predose or the subsequent dose of fragment with multiple, wherein subsequent dose is separated by the interval at least 2 weeks.

Preferably, carry out in the above-mentioned embodiment used at antibody or fragment in the mode of predose and multiple subsequent dose, the dosage of antibody or fragment is at least 0.01mg/kg antibody or fragment, at least 0.03mg/kg antibody or fragment, at least 0.05mg/kg antibody or fragment or at least 0.09mg/kg antibody or fragment.

In another aspect of the present invention, antibody or fragment are used with fixed dosage, have nothing to do with dosage/subject weight ratio.In one embodiment, antibody or fragment are used with one or more fixed dosage, and described fixed dosage is 1000mg or less antibody or fragment, 750mg or less antibody or fragment, 500mg or less antibody or fragment, 250mg or less antibody or fragment, 100mg or less antibody or fragment or about 25mg or less antibody or fragment.In another embodiment, antibody or fragment are used with one or more fixed dosage, and described fixed dosage is at least about 1mg antibody or fragment, at least about 5mg antibody or fragment or at least about 10mg antibody or fragment.

In certain embodiments, fixed dosage is that about 1mg-is about 10mg, about 1mg-is about 25mg, about 10mg-is about 25mg, about 10mg-is about 50mg, about 10mg-is about 100mg, about 25mg-is about 50mg, about 25mg-is about 100mg, about 50mg-is about 100mg, about 50mg-is about 150mg, about 100mg-is about 150mg, about 100mg-is about 200mg, about 150mg-is about 200mg, about 150mg-is about 250mg, about 200mg-is about 250mg, about 200mg-is about 300mg, about 250mg-is about 300mg, about 250mg-is about 500mg, about 300mg-is about 400mg, about 400mg-is about 500mg, about 400mg-is about 600mg, about 500mg-is about 750mg, about 600mg-is about 750mg, about 700mg-is about 800mg, about 750mg-is about 1000mg.In a preferred embodiment, fixed dosage is selected from about 1mg-and is about 10mg, about 1mg-and is about 25mg, about 10mg-and is about 25mg, about 10mg-and is about 100mg, about 25mg-and is about 50mg, about 50mg-and is about 100mg, about 100mg-and is about 150mg, about 150mg-and is about 200mg, about 200mg-and is about 250mg.

In yet another aspect, what the invention provides treatment people is selected from following disease or the method for disease: type 1 diabetes, type 2 diabetes mellitus, hyperglycemia, hyperinsulinemia, fat, insulin generates and reduces and insulin resistance, the method comprises to the anti-il-i-beta antibody of people's administering therapeutic effective dose or its fragment, one or more subsequent dose is used after the predose of wherein administration of antibodies or antibody fragment, and wherein in the therapeutic process with described predose and one or more subsequent dose, for the time period being greater than about 1 week and being less than about 6 months between administered twice, described antibody or the antibody fragment plasma concentration in people is allowed to be reduced to the level lower than about 0.1ug/mL.In one embodiment, for being greater than about 1 week and being less than 5 months between administered twice, time period of about 4 months, about 3 months, about 2 months, about 1 month, about 3 week or about 2 weeks, the plasma concentration of described antibody or antibody fragment is allowed to the level be reduced to lower than about 0.07ug/mL, about 0.05ug/mL, about 0.03ug/mL or about 0.01ug/mL.In one embodiment, these blood plasma values refer to from utilizing the value obtained the individuality of antibody of the present invention or fragment treatment.In one embodiment, described individuality can be the patient suffering from one of disease mentioned below such as type 2 diabetes mellitus.

The present invention considers that the anti-il-i-beta antibody that uses according to context of methods or fragment can be used with any above-mentioned dosage, dosing interval between subsequent applications number of times and administered twice, and any disclosed dosage, dosing interval between subsequent applications number of times and administered twice can in alternative combination with one another, with adjustment for the treatment of benefit.In certain embodiments, one or more subsequent dose is approximately equal to or less than first dosage used in amount.In another embodiment, one or more subsequent dose is about greater than first dosage used in amount.Preferably, anti-il-i-beta antibody or fragment are used by subcutaneous, intramuscular or intravenous injection.The present invention considers, for each dosage of antibody or fragment, can use in one or more site.

In yet another aspect, the invention provides and use anti-il-i-beta antibody or antibody fragment treatment or prevent the disease of people or the method for disease, wherein disease or disease are type 2 diabetes mellitus, and wherein the dosage of antibody or fragment is enough to the improvement of at least 0.5, at least 1.0, at least 1.5, at least 2.0, at least 2.5 or at least 3.0 percentage points realizing glycated hemoglobin.In one embodiment, these parameter values refer to from utilizing the value obtained the individuality of antibody of the present invention or fragment treatment.In one embodiment, this individuality can be the patient suffering from one of disease mentioned below such as type 2 diabetes mellitus.

In a preferred embodiment, the improvement of this glycated hemoglobin is enough to the administration guide of the therapeutic agent met for ratifying treatment type 2 diabetes mellitus.Test method for Measuring hemoglobin A1c is well-known in the art.The present invention considers, the administration being enough to realize antibody that glycated hemoglobin improves or fragment can comprise any above-mentioned dosage, dosing interval between subsequent applications number of times and administered twice, and any combination of the dosage of antibody described herein or fragment, subsequent applications number of times and the dosing interval between using.In addition, glycated hemoglobin improvement can after one or more dosage of initial application antibody or fragment at least about 1 month, about 2 months, about 3 months, about 4 months or about 5 months and preferably about 6 months or longer, the time point of about 7 months or longer, about 8 months or longer, about 9 months or longer, about 10 months or longer, about 11 months or longer or about 12 months or longer occurs.

Be used for the treatment of or prevent type 2 diabetes mellitus said method another in, the method is enough to realize at least one following change: the decline of fasting blood glucose level, the decline of insulin resistance, the minimizing of hyperinsulinemia, the improvement of glucose tolerance, C reacts the minimizing of peptide (CRP), insulin generates the minimizing of increase and hyperglycemia, to the minimizing of diabetes medicament demand, the reduction of BMI, the change of glucose/insulin C peptide AUC, the reduction of urine glucose level, the minimizing of acute phase reactant, the improvement of the minimizing of serum lipids and the lipodogramme with regard to cardiovascular risk.Well-known in the art for measuring the test method of any above-mentioned change.In addition, the present invention also considers, one of above-mentioned change can after one or more dosage of initial application antibody or fragment at least about 1 month, about 2 months, about 3 months, about 4 months or about 5 months and preferably occur at least about on the time point of 6 months or longer, about 7 months or longer, about 8 months or longer, about 9 months or longer, about 10 months or longer, about 11 months or longer or about 12 months or longer.

In another aspect of the present invention, method provided herein reduces or prevention is selected from the following complication relevant to type 2 diabetes mellitus or disease: retinopathy, renal failure, cardiovascular disease and wound healing, the method comprises uses anti-il-i-beta antibody or its fragment to people.In one embodiment, complication or disease are cardiovascular disease, and wherein said cardiovascular disease is atherosclerosis or peripheral blood vessel.In another embodiment, complication or disease are wound healings, and wherein said wound healing disease is diabetic ulcer.In yet another aspect, the method is prevented or is postponed whole end stage renal disease or diabetic neuropathy.In one embodiment, anti-il-i-beta antibody or fragment and carry out combined administration for other treatments of medically generally acknowledging of at least one of this disease, disease or complication.In another embodiment, reduce or interrupt described other treatments of medically generally acknowledging of at least one for this disease, disease or complication, and with the treatment of constant dosage regimen maintenance anti-il-i-beta antibody or fragment.In another embodiment, reduce or interrupt described other treatments of medically generally acknowledging of at least one for this disease, disease or complication, and reducing the treatment with anti-il-i-beta antibody or fragment.In another embodiment, reduce or interrupt described other treatments of medically generally acknowledging of at least one for this disease, disease or complication, and increasing the treatment with anti-il-i-beta antibody or fragment.In another one embodiment, maintain described other treatments of medically generally acknowledging of at least one for this disease, disease or complication, and reduce or interrupt the treatment with anti-il-i-beta antibody or fragment.In another one embodiment, reduce or interrupt described other treatments of medically generally acknowledging of at least one for this disease, disease or complication and the treatment by anti-il-i-beta antibody or fragment.

In another aspect of the present invention, be provided in experimenter the method for the amount reducing c reactive protein, the method comprises uses anti-il-i-beta antibody or its fragment to experimenter.In one embodiment, antibody or antibody fragment are used with one or more dosage, and described dosage is 1mg/kg or less antibody or fragment, 0.75mg/kg or less antibody or fragment, 0.5mg/kg or less antibody or fragment, 0.3mg/kg or less antibody or fragment, 0.1mg/kg or less antibody or fragment or 0.03mg/kg or less antibody or fragment.Preferably, antibody or fragment are used with one or more dosage, and described dosage is at least 0.01mg/kg antibody or fragment, at least 0.03mg/kg antibody or fragment, at least 0.05mg/kg antibody or fragment or at least 0.09mg/kg antibody or fragment.In another embodiment, do not rely on dosage/subject weight ratio, antibody or fragment are used with one or more fixed dosage, and described fixed dosage is 500mg or less antibody or fragment, 250mg or less antibody or fragment, 100mg or less antibody or fragment or about 25mg or less antibody or fragment.Preferably, antibody or fragment are used with one or more fixed dosage, and described fixed dosage is at least about 1mg antibody or fragment, at least about 5mg antibody or fragment or at least about 10mg antibody or fragment.In another embodiment, antibody or antibody fragment are combined with IL-1 β, dissociation constant be about 500pM or less, 250pM or less, about 100pM or less, about 50pM or less or about 25pM or less, about 10pM or less, about 5pM or less, about 3pM or less, about 1pM or less, about 0.75pM or less, about 0.5pM or less, about 0.3pM or less, about 0.2pM or less or about 0.1pM or less.In another embodiment, after using predose for use one or more subsequent dose (they each other by least about 2 weeks, at least about 3 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months or at least about 12 months interval and separate).In another embodiment, be provided in experimenter the described method of the amount reducing c reactive protein, wherein experimenter suffers from kidney diaseases (such as, chronic kidney diseases, renal failure).In another embodiment, experimenter suffers from type 2 diabetes mellitus, type 1 diabetes, obesity, hyperglycemia, hyperinsulinemia, insulin generation minimizing, insulin resistance and is characterised in that morbid state and the disease of insulin resistance.In another embodiment, experimenter suffers from disease or disease---prediabetes, dyslipidemia, hyperlipemia, hypertension, metabolism syndrome or illness behavior.Above-mentioned dosage refers to mg (antibody or fragment)/kg (weight of individuality to be treated).There is using of the antibody of above-mentioned dissociation constant or fragment to carry out according to any above-mentioned dosage and dosing interval (when using 2 or more dosage).

In yet another aspect, the method provided herein Therapeutic Method other with at least one is combined, and described other Therapeutic Method comprises the pharmaceutical composition used at least one and comprise the activating agent except IL-1 β antibody or fragment.In another one, the inventive method stops or postpones the demand to the other Therapeutic Method of at least one, and described other Therapeutic Method comprises the pharmaceutical composition used at least one and comprise the activating agent except IL-1 β antibody or fragment.In another one, the inventive method is reduced by least a kind of amount of other Therapeutic Method, frequency or persistent period, and described other Therapeutic Method comprises the pharmaceutical composition used at least one and comprise the activating agent except IL-1 β antibody or fragment.In one embodiment, the pharmaceutical composition of activating agent that described at least one comprises except IL-1 β antibody or fragment is selected from: sulfonylurea, meglitinides, biguanides, Alpha-glucosidase inhibitor class, thiazolidinediones, glucagon-like peptide and insulin.In another embodiment, activating agent is sulfonylurea.In another embodiment, activating agent is meglitinide (meglitinide) class.In another embodiment, activating agent is biguanides.In another embodiment, activating agent is Alpha-glucosidase inhibitor.In another embodiment, activating agent is thiazolidinediones.In another embodiment, activating agent is glucagon-like peptide.In another embodiment, activating agent is insulin.In another embodiment, the pharmaceutical composition that described at least one comprises activating agent comprises 2 kinds of activating agents.In one embodiment, these 2 kinds of activating agents are sulfonylurea and biguanides.In another embodiment, these 2 kinds of activating agents are thiazolidinediones and biguanides.In another one embodiment, maintain the treatment with this at least one activating agent.In another embodiment, reduce or interrupt with the treatment of this at least one activating agent, and with the treatment of constant dosage regimen maintenance anti-il-i-beta antibody or fragment.In another embodiment, reduce or interrupt the treatment with this at least one activating agent, and reducing the treatment with anti-il-i-beta antibody or fragment.In another embodiment, reduce or interrupt the treatment with this at least one activating agent, and increasing the treatment with anti-il-i-beta antibody or fragment.In another one embodiment, maintain the treatment with this at least one activating agent, and reduce or interrupt the treatment with anti-il-i-beta antibody or fragment.In another one embodiment, reduce or interrupt with the treatment of this at least one activating agent and the treatment by anti-il-i-beta antibody or fragment.

In yet another aspect, what the invention provides treatment people is selected from following disease or the method for disease: type 1 diabetes, type 2 diabetes mellitus, hyperglycemia, hyperinsulinemia, fat, insulin generates and reduces and insulin resistance, the method comprises to the anti-il-i-beta antibody of people's administering therapeutic effective dose or its fragment, one or more subsequent dose is used after the predose of wherein administration of antibodies or antibody fragment, wherein in the therapeutic process with described predose and one or more subsequent dose, described antibody or the antibody fragment plasma concentration in people maintains following level: at least about 0.03ug/mL, at least about 0.05ug/mL, at least about 0.08ug/mL, at least about 0.1ug/mL, at least about 0.15ug/mL, at least about 0.2ug/mL, at least about 0.25ug/mL, at least about 0.3ug/mL, at least about 0.4ug/mL, at least about 0.5ug/mL, at least about 0.6ug/mL, at least about 0.8ug/mL, at least about 1ug/mL, at least about 1.5ug/mL, at least about 2ug/mL, at least about 3ug/mL, at least about 4ug/mL, or at least about 5ug/mL.In one embodiment, these blood plasma value pointers are to the value obtained with the individuality according to antibody of the present invention or fragment treatment.In one embodiment, described individuality can be the patient suffering from one of disease mentioned below such as type 2 diabetes mellitus.

In yet another aspect, what the invention provides treatment people is selected from following disease or the method for disease: type 1 diabetes, type 2 diabetes mellitus, hyperglycemia, hyperinsulinemia, fat, insulin generates and reduces and insulin resistance, the method comprises to the anti-il-i-beta antibody of people's administering therapeutic effective dose or its fragment, wherein use the production that anti-il-i-beta antibody or its fragment cause being selected from one or more following gene outcomes to people to reduce: leptin (leptin), phylaxin (resistin), Visfatin (visfatin), RANTES, IL-6, MCP-1, PAI-1, acylation stimulating protein matter, SAA3, Pentraxin-3, macrophage migration inhibition factor, IL-1RA, IL-12, IL-8 and TNF-α.In one embodiment, use the production that anti-il-i-beta antibody or its fragment cause being selected from one or more following gene outcomes to people to reduce: leptin, phylaxin and Visfatin.In another embodiment, use the production that anti-il-i-beta antibody or its fragment cause being selected from one or more following gene outcomes to people to reduce: MCP-1, RANTES, IL-6, TNF-α and Pentraxin-3.In another one embodiment, the production of one or more gene outcomes described reduces from fatty tissue.In another one embodiment, in human blood, detect described minimizing.

In yet another aspect, what the invention provides treatment people is selected from following disease or the method for disease: type 1 diabetes, type 2 diabetes mellitus, hyperglycemia, hyperinsulinemia, obesity, insulin generate and reduce and insulin resistance, the method comprises to the anti-il-i-beta antibody of people's administering therapeutic effective dose or its fragment, wherein uses the increase that anti-il-i-beta antibody or its fragment cause adiponectin production to people.In one embodiment, the increase of described adiponectin production is from fatty tissue.In another one embodiment, in human blood, detect described increase.

In yet another aspect, what the invention provides treatment people is selected from following disease or the method for disease: type 1 diabetes, type 2 diabetes mellitus, hyperglycemia, hyperinsulinemia, obesity, insulin generate and reduce and insulin resistance, the method comprises to the anti-il-i-beta antibody of people's administering therapeutic effective dose or its fragment, wherein in the people's whole blood IL-1 β inhibition test measuring the beta induced IL-8 production of IL-1, antibody or its fragment have the IC lower than IL-1 beta receptor antagonist ₅₀.In one embodiment, in the people's whole blood IL-1 β inhibition test measuring the beta induced IL-8 production of IL-1, antibody or fragment have the IC lower than IL-1 beta receptor antagonist ₅₀about 90%, 80%, 70%, 60%, 50% IC ₅₀.In a further embodiment, in the people's whole blood IL-1 β inhibition test measuring the beta induced IL-8 production of IL-1, antibody or fragment have the IC lower than IL-1 beta receptor antagonist ₅₀about 40%, 30%, 20%, 10% IC ₅₀.In a preferred embodiment, in the people's whole blood IL-1 β inhibition test measuring the beta induced IL-8 production of IL-1, antibody or fragment have the IC lower than IL-1 beta receptor antagonist ₅₀about 8%, 5%, 4%, 3%, 2%, 1% IC ₅₀.In one embodiment, IL-1 beta receptor antagonist be Antril (Synergen) (namely ).

In yet another aspect, what the invention provides treatment people is selected from following disease or the method for disease: type 1 diabetes, type 2 diabetes mellitus, hyperglycemia, hyperinsulinemia, obesity, insulin generate and reduce and insulin resistance, the method comprises to the anti-il-i-beta antibody of people's administering therapeutic effective dose or its fragment, wherein use the people such as Economides, NatureMed, the test that 9:47-52 (2003) (being incorporated herein by reference) describes, compare with control antibodies, antibody or its fragment suppress IL-1 β to stimulate in mice IL-6 release in body.In one embodiment, compare with control antibodies, the IL-6 release that antibody or fragment stimulate for IL-1 β in mice provides in the body at least about 10%, 20%, 30%, 40%, 50% and suppresses.In a further embodiment, compare with control antibodies, the IL-6 release that antibody or fragment stimulate for IL-1 β in mice provides in the body at least about 60%, 70%, 80%, 90%, 95% and suppresses.In one embodiment, control antibodies is Isotype control antibodies.

In yet another aspect, what the invention provides treatment people is selected from following disease or the method for disease: type 1 diabetes, type 2 diabetes mellitus, hyperglycemia, hyperinsulinemia, obesity, insulin generate and reduce and insulin resistance, the method comprises to the anti-il-i-beta antibody of people's administering therapeutic effective dose or its fragment, wherein, with do not use comparing of antibody, the cytokine production that antibody or its fragment suppress people's whole blood mesocuticle staphylococcus (Staphylococcusepidermidis) to induce.In one embodiment, and to compare, antibody or fragment for the cytokine production that people's whole blood mesocuticle staphylococcus is induced provide high at least about 10%, 20%, 30%, 40% or 50% suppression.In a further embodiment, and to compare, antibody or fragment for the cytokine production that people's whole blood mesocuticle staphylococcus is induced provide high at least about 60%, 70%, 80%, 90%, 95% suppression.In one embodiment, the cytokine of suppression is IL-1 β, IL-1a, IL-6, IL-8, IL-1Ra, TNF α or IFN γ.

In yet another aspect, the invention discloses anti-il-i-beta antibody or the purposes of its fragment in preparation compositions, described compositions is used for the treatment of type 1 diabetes, type 2 diabetes mellitus, hyperglycemia, hyperinsulinemia, obesity, insulin generation minimizing and insulin resistance, wherein, in the people's whole blood IL-1 β inhibition test measuring the beta induced IL-8 production of IL-1, described antibody or its fragment have the IC lower than IL-1 beta receptor antagonist ₅₀.In one embodiment, IL-1 beta receptor antagonist be Antril (Synergen) (namely ).

In another aspect of the present invention, consider IL-1 β antibody or binding fragment is preparing the purposes in medicine, described medicine is used for the treatment of or prevents disease as disclosed herein or disease.In this type of purposes any, medicine can be coordinated with the treatment of use second activating agent.In another embodiment of the invention, consider the purposes of synergistic combination for the preparation of medicine of antibody of the present invention, described medicine is used for the treatment of to demonstrate disease as disclosed herein or the risk of disease or the patient of symptom occurs, and its Chinese medicine is coordinated mutually with the treatment of use the second activating agent.In another one related embodiment, provide such compositions, wherein the second activating agent is another antibody, somatomedin, cytokine or insulin.Consider the embodiment of any such use, the IL-1 beta binding antibodies in its Chinese medicine or the amount of fragment are for effectively reducing on the dosage of the second active agent dose reached needed for curative effect.

In another aspect of the present invention, product is provided, it comprises container, in container, comprises compositions and the product description of anti-il-i-beta antibody or its fragment, and described description comprises people's administration of antibodies or the explanation of fragment for the treatment of to needs about said method according to the present invention.In one embodiment, container comprises pharmaceutically acceptable carrier, excipient or diluent further.In a related embodiment, the compositions in container comprises the second activating agent further.In another one related embodiment, provide such compositions, wherein the second activating agent is another kind of antibody, somatomedin, cytokine or insulin.

The present invention also considers test kit.In one embodiment, test kit comprises the treatment be packaged in container such as bottle or bottle or anti-il-i-beta antibody or the fragment of preventing effective dose, and comprise the label adhered to container or together with container package further, described label describes the content of container and provides and to be used for the treatment of about container contents said method according to the present invention or to prevent disease or the instruction of disease and/or explanation.In one embodiment, container comprises pharmaceutically acceptable carrier, excipient or diluent further.In a related embodiment, container comprises the second activating agent further.In another one related embodiment, the second activating agent comprises another kind of antibody, somatomedin, cytokine or insulin.

In one embodiment, product, test kit or medicine are used for the treatment of or prevent disease or the disease of people, and described disease or disease are selected from type 1 diabetes, type 2 diabetes mellitus, hyperglycemia, hyperinsulinemia, obesity, insulin generation minimizing and insulin resistance.In a preferred embodiment, disease or disease are selected from type 2 diabetes mellitus, obesity and insulin resistance.In another embodiment, the explanation on the product description of product and the label of test kit comprises the explanation using antibody or fragment according to any above-mentioned dosage, subsequent applications number of times and any combination of using a dosing interval and dosage described herein, subsequent applications number of times and using a dosing interval.In another one embodiment, the container of test kit or product is prefilled syringe.

Be to be understood that when this description mentions that utilization has certain character (such as Kd value or IC ₅₀value) antibody or the Therapeutic Method of its fragment time, this also means and relates to this kind of antibody or its fragment for the preparation of the purposes in the medicine of these methods.In addition, the antibody or its fragment with these character are also contained in the present invention, and comprise the pharmaceutical composition of these antibody or its fragment, in the Therapeutic Method hereafter discussed.

Accompanying drawing is sketched

Fig. 1 show called after AB7 antibody and result in vitro in IL-1 β Inhibition test, the IL-8 that described result relates to IL-1 induction produces.

The result of the antibody that Fig. 2 shows called after AB5 and AB7 in vivo in IL-1 β Inhibition test, described result relates to the IL-6 release that IL-1 stimulates.

The result (this result relate to IL-6 release that IL-1 stimulate) of the antibody that Fig. 2 B shows called after AB7 in vivo in IL-1 β Inhibition test, and the suppression (figure A) compared people IL-1 β and the suppression (figure B) to mice IL-1 β.

The serum-concentration after 0.1,1 or 10mg/kg anti-il-i-beta antibody is used in Fig. 3 display in rats.

Fig. 4 is presented in machin (Cynomolgusmonkey) serum-concentration after using 0.3 or 3mg/kg anti-il-i-beta antibody.

Fig. 5 mould build 0.1,0.3,1 or 5 of 3mg/kg monthly plasma concentration profile curves of anti-il-i-beta antibody in machin after dosage in.

The form of Fig. 6 shows the minimizing of cytokine production that caused by anti-il-i-beta antibody treatment, the induction of people's whole blood mesocuticle staphylococcus.

The pharmacokinetics of AB7 in people after 0.01mg/kg antibody dosage is used in the curve chart display of Fig. 7.

The curve chart of Fig. 8 is presented in people after the AB7 using 0.01mg/kg dosage the effect reducing CRP level.

The curve chart of Fig. 9 is presented in people after the AB7 using 0.03mg/kg dosage the effect reducing CRP level.

The curve chart display of Figure 10 from 0.01 and 0.03mg/kg dosage group placebo in the level of CRP.

Figure 11 mould is built in people and is used the impact on CRP level after the antibody (having similar quality with AB7) of various dosage with the interval of 28 days.

Figure 12 is presented at the effect of AB7 in the Mice model of obesity of the meals induction of type 2 diabetes mellitus.

Detailed Description Of The Invention

IL-1 β is the proinflammatory cytokine secreted by multiple different cell type (comprising mononuclear cell and macrophage).When the part as inflammatory reaction discharges, IL-1 β causes a series of biological effect, this is mainly through inducing other inflammatory mediators to mediate, and other inflammatory mediators described are thyroliberin, PF4, PGE2 (PGE2), IL-6 and IL-8 such as.IL-1 β by activating IL-1 receptor (it is present on nearly all cell type), induction local with the inflammatory effector of whole body.

Il-1 (IL-1) family of cytokine has been prompted to involve several morbid state, such as rheumatoid arthritis (RA), osteoarthritis, Crohn disease, ulcerative colitis (UC), septic shock, chronic obstructive pulmonary disease (COPD), asthma, graft versus host disease, atherosclerosis, adult T cell leukemia, multiple myeloma, multiple sclerosis, apoplexy and Alzheimer.IL-1 family member comprises IL-1 α, IL-1 β and IL-1Ra.Although the ability combined by itself and IL-1 receptor (IL-1R1 and IL-1R2) and being associated, each of these cytokines is all different, is expressed and have different primary amino acid sequences by different genes.In addition, the physiologically active of these cytokines can be distinguished from each other.

The compound destroying IL-1 receptor signal is studied as the therapeutic agent of disease such as some above-mentioned disease for the treatment of IL-1 mediation.These compounds comprise restructuring IL-1Ra (AmgenInc., ThousandOaks, CA), the IL-1 β antibody of IL-1 receptor " trap " peptide (RegeneronInc., Tarrytown, NY) and animal derived and restructuring IL-1 β antibody and fragment thereof.

As mentioned above, IL-1 receptor antagonist (IL-1Ra) polypeptide has been proposed for treatment type 2 diabetes mellitus (WO2004/002512), but still needs to treat the effective ways of type 2 diabetes mellitus, particularly do not need every day reinjected those.Based on IL-1 receptor antagonist treatment faced by another challenge be need this kind of therapeutic agent occupy a large amount of receptor, this is a difficult task, because these receptors are expressed widely removing (Dinarello on exo-erythrocytic all cells, Curr.Opin.Pharmacol.4:378-385,2004).In most of immune-mediated disease such as disease disclosed herein, the amount of IL-1 β cytokine measurable or relevant to activating cell in body fluid is relatively low.Therefore, the method that treats and/or prevents of direct targeting IL-1 beta ligands is good strategy, particularly when using the IL-1 β antibody with high-affinity.

The invention provides and use that IL-1 β specific antibody or its fragment treat and/or prevent type 2 diabetes mellitus in mammal, type 1 diabetes, obesity, hyperglycemia, hyperinsulinemia, insulin generate reduce, insulin resistance and/or be characterised in that the morbid state of insulin resistance and the method for disease and Related product.

As shown in Examples below 1, than IL-Ra (such as, be surprised to find this kind of antibody (such as, having high-affinity) can be ) much strong IL-1 approach restrainer, and as shown in Examples below 9, provide with than other drug (such as recombinate IL-1Ra) necessary dosage and/or the lower dosage of frequency of administration and/or lower frequency of administration to reach the chance of curative effect.

Utilize this kind of method of IL-1 β antibody or fragment can comprise treatment as described herein suffer from type 2 diabetes mellitus, type 1 diabetes, obesity, hyperglycemia, hyperinsulinemia, insulin generation minimizing (decreasedinsulinproduction), hypoinsulinemia, insulin resistance and/or be characterised in that the morbid state of insulin resistance and the experimenter of disease.The method can also comprise prevention type 2 diabetes mellitus, type 1 diabetes, obesity, hyperglycemia, hyperinsulinemia, insulin generate reduce, insulin resistance and/or be characterised in that the morbid state of insulin resistance and the generation of disease, or prevent its generation in risk subjects.As used herein, hyperinsulinemia refers to the relative high blood insulin caused by insulin resistance.

Antibody, humanized antibody and people's engineered antibody

IL-1 of the present invention (such as, IL-1 β) binding antibody can provide with following form: polyclonal antibody, monoclonal antibody (mAbs), recombinant antibodies, chimeric antibody, CDR grafted antibody, human antibody, single-chain antibody and/or bi-specific antibody, and by fragment that known technology provides, comprise its variant and derivant, described technology includes but not limited to enzymatic cutting, peptide symthesis or recombinant technique.

Antibody generally comprises 2 heavy chain polypeptides and 2 light chain polypeptides, but also considers have the single domain antibody of 1 heavy chain and 1 light chain and lack the heavy chain antibody of light chain.Based on the aminoacid sequence of heavy chain constant domain, there is the heavy chain of 5 types, be called α, δ, ε, γ and μ.These dissimilar heavy chains produce 5 antibody-likes respectively, and IgA (comprises IgA ₁and IgA ₂), IgD, IgE, IgG and IgM, comprise 4 IgG subclass, i.e. IgG ₁, IgG ₂, IgG ₃and IgG ₄.Based on the aminoacid sequence of constant domain, also there is the light chain of 2 types, be called kappa (κ) or lambda (λ).Full length antibody comprises constant domain and variable domains.Constant domain is without the need to being present in the Fab of antibody.The Fab of antibody disclosed herein can comprise Fab, Fab ', F (ab ') ₂with F (v) antibody fragment.As discussed in more detail, comprise can in conjunction with the antibody fragment of IL-1 β and antigen-binding polypeptides for IL-1 β binding fragment.

The heavy chain of antibody and sequence of light chain, or its Fab, include the variable region with 3 complementary determining regions (CDRs) and non-CDR framework region (FRs).Except as otherwise noted, as used herein, term " heavy chain " and " light chain " mean variable region of heavy chain and variable region of light chain respectively.Heavy chain CDRs is referred to herein as CDR-H1, CDR-H2 and CDR-H3.Light chain CDRs is referred to herein as CDR-L1, CDR-L2 and CDR-L3.Variable region in antibody sequence and CDRs can be identified by following: (i) is according to the general rule developed in this area, or the data base in relative for sequence known variable district compares by (ii).For the identification of the method in these regions at Kontermann and Dubel, editor, AntibodyEngineering, Springer, NewYork, NY, 2001, with people such as Dinarello, CurrentProtocolsinImmunology, JohnWileyandSonsInc., Hoboken, NJ, is described in 2000.Antibody sequence database is described in following, and can by " TheKabatman " data base of following acquisition: www.bioinf.org.uk/abs (by A.C.Martin at theDepartmentofBiochemistry & MolecularBiologyUniversityCollegeLondon, London, England safeguards), with the VBASE2 of www.vbase2.org, as people such as Retter, Nucl.AcidsRes., describe in 33 (Databaseissue): D671-D674 (2005)." Kabatman " database website also comprises the universal experience rule for the identification of CDRs.Except as otherwise noted, as used herein, term " CDR " is as people such as Kabat, and SequencesofImmunologicalInterest, the 5th edition, U.S.DepartmentofHealthandHumanServices, defines in 1991.

Polyclonal antibody produces in animal preferably by repeatedly subcutaneous (sc) of related antigen and adjuvant or intraperitoneal (ip) injection.The antibody response improved can make related antigen and protein-conjugate obtain by using difunctionality agent or derivating agent; described protein is immunogenic in the species treating immunity; such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin or soybean trypsin inhibitor, described difunctionality agent or derivating agent are such as maleimidobenzoyl sulfosuccinimide ester (being puted together by cysteine residues), N-hydroxy-succinamide (passing through lysine residue), glutaraldehyde, succinic anhydrides or other reagent known in the art.

Animal carries out immunity inoculation for antigen, immunogenic conjugate or derivant, mode is that such as 100 μ g or 5 μ g protein or conjugate (respectively for rabbit or mice) are combined with the Freund's complete adjuvant of 3 volumes, and at multiple this solution of site intradermal injection.After 1 month, animal by multiple site subcutaneous injection, is used in 1/5 in Freund's complete adjuvant and strengthens to the peptide of { mark (1/10) } original vol or conjugate.After booster injection during 7-14 days, animal carries out taking a blood sample and measures serum with regard to antibody titer.Animal booster immunization is until titre reaches platform.Preferably, animal same antigen but strengthen from different proteins and/or by the conjugate that different cross-linking agent is puted together.Conjugate can also be prepared as protein fusions in recombinant cell culture thing.In addition, assemble reagent such as Alumen and be suitable for enhancing immunne response.

Monoclonal antibody refers to derive from the antibody of a group antibody of homogenizing substantially.Monoclonal antibody is generally high degree of specificity, and can for single antigen site, different from routine (polyclone) antibody preparation generally comprised for the different antibodies of different determinant (epi-position).Except its specificity, monoclonal antibody or favourable, because they are synthesized by homogenous culture, is not polluted by other immunoglobulins with not homospecificity and feature.

Treat that the monoclonal antibody used according to the present invention can be passed through first by people such as Kohler, (Nature, 256:495-7,1975) hybridoma method described is prepared, maybe can pass through recombinant DNA method (see such as, U.S. Patent number 4,816,567) be prepared.Monoclonal antibody can also use the people such as such as Clackson, the people such as (Nature352:624-628,1991) and Marks, and the technology described in (J.MolBiol.222:581-597,1991), is separated from phage antibody library.

In hybridoma method, mice or other suitable host animal such as hamster or stump-tailed macaque are as described herein carries out immunity, maybe can produce the lymphocyte of the antibody be combined with the protein specific being used for immunity to cause generation.Alternately, lymphocyte can carry out immunity in vitro.Suitable fusion agent such as Polyethylene Glycol is used to make lymphocyte and myeloma cell fusion subsequently, to form hybridoma (Goding, MonoclonalAntibodies:PrinciplesandPractice, 59-103 page (AcademicPress, 1986)).

Inoculated by the hybridoma prepared thus and grow in suitable culture medium, described culture medium preferably comprises the Parent Myeloma Cell growth that suppression is not merged or one or more materials of surviving.Such as, if Parent Myeloma Cell lacks hypoxanthine-guaninephosphoribosyl transferase (HGPRT or HPRT), culture medium so for hybridoma generally will comprise hypoxanthine, aminopterin and thymidine (HAT culture medium), and described material stops the growth of HGPRT deficient cells.

Preferred myeloma cell is such cell, it can merge effectively, support selected by antibody producing cells stably high level production antibody, and responsive to culture medium.Human myeloma and mouse-human heteromyeloma (heteromyeloma) cell line are also described, for generation of human monoclonal antibodies (Kozbor, J.Immunol, 133:3001 (1984); The people such as Brodeur, MonoclonalAntibodyProductionTechniquesandApplications, 51-63 page (MarcelDekker, Inc., NewYork, 1987)).Exemplary murine myeloma system comprises can from SalkInstituteCellDistributionCenter, SanDiego, that Calif.USA obtains, derived from those of MOP-21 and M.C-11 mouse tumor, and can from American type culture collection, SP-2 or the X63-Ag8-653 cell that Rockville, Md.USA obtain.

With regard to the production of the monoclonal antibody of antigen, analyze the culture medium that wherein hybridoma is growing.Preferably, the binding specificity of the monoclonal antibody of being produced by hybridoma measures by immunoprecipitation or by external in conjunction with determination test, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).The binding affinity of monoclonal antibody such as can be analyzed people such as (, Anal.Biochem., 107:220 (1980)) Munson by Scatchard and measure.

Identify produce there is the hybridoma of the antibody of required specificity, affinity and/or activity after, these clones can carry out sub-clone by limiting dilution operation, and undertaken growing (Goding by standard method, MonoclonalAntibodies:PrinciplesandPractice, 59-103 page (AcademicPress, 1986)).Suitable culture medium for this purposes comprises such as DMEM or RPMI-1640 culture medium.In addition, hybridoma can grow as the ascites tumour in animal in vivo.The monoclonal antibody of being secreted by sub-clone suitably can be separated by conventional immune globulins purification process from culture medium, ascites fluid or serum, and described operational example is as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatograph.

Further consider that antibody of the present invention can use by less Fab that is well-known with this area and antibody described herein.

IL-1 (such as, the IL-1 β) binding antibody comprising 2 total length heavy chains and 2 full-length light chains is contained in the present invention.Alternately, IL-beta binding antibodies can be retain the construct for the binding activities of IL-1 β, such as single-chain antibody or " miniature " antibody.This kind of construct can be prepared by methods known in the art, single-chain antibody clone and the assembling of such as PCR mediation are used at expression in escherichia coli (as AntibodyEngineering, Thepracticalapproachseries, J.McCafferty, H.R.Hoogenboom, andD.J.Chiswell, editor, OxfordUniversityPress, describes in 1996).In the construct of this type, weight and the light chain variable part of antibody molecule carry out pcr amplification by cDNA.The amplicon obtained can such as be assembled by linker DNA subsequently in second PCR step, the elastin matter joint that described linker DNA coding aminoacid Gly and Ser forms.This joint allows Weight variable and chain moiety to fold by this way, and which makes antibody binding pocket regenerate, and with usually suitable with parent's total length dimerization immunoglobulin molecules affinity conjugated antigen.

IL-1 of the present invention (such as, IL-1 β) binding antibody and fragment comprise the variant of exemplary antibodies disclosed herein, fragment and sequence.Variant comprises and comprises one or more amino acid sequence substitutions, the peptide of disappearance and/or interpolation and polypeptide, and it has the epi-position binding affinity identical or substantially the same with sequence with one or more exemplary antibodies disclosed herein, fragment and specificity.Therefore, variant comprises the one or more amino acid sequence substitutions, the peptide of disappearance and/or interpolation and the polypeptide that comprise exemplary antibodies disclosed herein, fragment and sequence, wherein this kind of displacement, disappearance and/or the affinity and specific material change that add not cause epi-position to combine.Such as, the variant of antibody or fragment can be caused by the one or more change for antibody or fragment, and the antibody of wherein this change or fragment have the epi-position binding affinity identical or substantially the same with homing sequence and specificity.Variant can be naturally occurring, such as allele variant or splice variant, can be maybe artificial constructed.Variant can be prepared by the corresponding nucleic molecule of the described variant of coding.The variant of antibody of the present invention and IL-1 β binding fragment can have change in light and/or heavy chain amino acid sequence, and described change can be maybe can transforming native sequences in vitro by using recombinant DNA technology and introduce of natural generation.Naturally occurring variant comprises " somatic cell " variant, and it produces in vivo in the production process of the antibody response of anti-exogenous antigen in corresponding embryonal system (germline) nucleotide sequence.

The variant of IL-1 (such as, IL-1 β) binding antibody and binding fragment also can be prepared by induced-mutation technique.Such as, amino acid change can be introduced randomly in whole antibody coding region, and the variant obtained can just screen for the binding affinity of IL-1 β or other character.Alternately, amino acid change can be introduced in region selected by IL-1 β antibody, such as, in light and/or heavy chain CDRs, and/or introduces in framework region, and the antibody obtained can just screen with the combination of IL-1 β or some other activity.Amino acid change is included in the one or more amino acid replacements in CDR, changes from single amino acid differences to introducing multiple aminoacid in given CDR such as CDR3.In another approach, each residue in CDR can by assessing with alanine substitution at least one residue in this CDR to the contribution that IL-1 β combines.The people such as Lewis (1995), Mol.Immunol.32:1065-72.For the combination with IL-1 β and the residue of non-optimal can be changed subsequently, to determine the sequence more optimized.The present invention is also contained by inserting aminoacid to increase the size of CDR such as CDR3 and the variant that produces.Such as, long 9 aminoacid of most of light chain CDR3 sequence.The sequence of light chain being shorter than 9 residues in antibody by inserting suitable aminoacid to increase the length of this CDR, can optimize the combination with IL-1 β.

Variant can also pass through gently or " the chain reorganization " of heavy chain is prepared.The people such as Marks (1992), Biotechnology10:779-83.Wall scroll light (or heavy) chain can with the libraries Combinatorial heavily with (or light) chain storehouse (repertoire), and the colony obtained with regard to required activity such as with IL-1 β be incorporated into row filter.Compared with possible the situation used when not only having comprised heavy chain storehouse but also comprised the library in light chain storehouse, this more different heavy chains (or light chain) sample allowing screening and wall scroll light chain (or heavy chain) to combine.

IL-1 of the present invention (such as, IL-1 β) binding antibody and fragment contain the derivant of exemplary antibodies disclosed herein, fragment and sequence.Derivant comprises the polypeptide or peptide or its variant, fragment or derivant that carry out chemical modification.Example comprises one or more polymer, and such as water-soluble polymer, N connection or O join the covalent attachment of carbohydrate, sugar, phosphoric acid and/or other this kind of molecule.In the type or position of the molecule of attachment, derivant is modified in the mode being different from naturally occurring or initial peptide or polypeptide.Derivant also comprises the disappearance of the natural one or more chemical groups be present on peptide or polypeptide.

IL-1 beta binding antibodies of the present invention and fragment can be bispecifics.Bi-specific antibody or fragment can have several configuration.Such as, bi-specific antibody can similar single antibody (or antibody fragment), but have 2 different antigen binding sites (variable region).Bi-specific antibody can pass through the chemical technology (people (1981) such as Kranz, Proc.Natl.Acad.Sci.USA, 78:5807), by " polyoma " (polydoma) technology (U.S. Patent number 4,474,893) or by recombinant DNA technology produce.Bi-specific antibody of the present invention can have the binding specificity at least 2 kinds of different epi-positions, and wherein at least one is the epi-position of IL-1 β.IL-1 beta binding antibodies and fragment also can be assorted antibody (heteroantibody).Assorted antibody is two or more antibody or antibody binding fragment (Fab) of linking together, and often kind of antibody or fragment have not homospecificity.

Walk around monoclonal antibody produce, can consider for IL-1 of the present invention (such as, IL-1 β) binding antibody and fragment for the preparation of the technology of the recombinant DNA form of the antigen binding regions of antibody molecule.By DNA clone in bacterial expression system.Be suitable for the example putting into practice this kind of technology of the present invention, use the bacteriophage k vector system with targeting sequencing, described targeting sequencing causes the Fab protein of expression to move to periplasmic space (between bacterial cell membrane and cell wall) or secretion.A large amount of functional Fab fragment can be produced fast, and be incorporated into row filter with regard to itself and IL-1 β.This kind of IL-1 β bonding agent (having specific Fab fragment for IL-1 beta polypeptides) is especially included in IL-1 beta binding antibodies of the present invention and fragment.

IL-1 of the present invention (such as, IL-1 β) binding antibody and fragment can be humanization or people's engineered antibody.As used herein, humanized antibody or its Fab are recombinant polypeptides, and it comprises from the part of the antigen binding site of non-human antibody and the framework of people's antibody and/or the part of constant region.People's engineered antibody or antibody fragment are by revising (such as in specific location, disappearance, insert or displacement) aminoacid and by transform inhuman (such as, mice) antibody, wherein said amino acid whose amendment causes reducing or eliminating any in people of this modified antibodies and detects immunogenicity.

Humanized antibody comprises chimeric antibody and CDR grafted antibody.Chimeric antibody is the antibody comprising the non-human antibody variable region be connected with human constant region.Therefore, in chimeric antibody, variable region major part is inhuman, and constant region is people.Chimeric antibody and for the preparation of its method people such as Morrison, Proc.Natl.Acad.SclUSA, 81:6841-6855 (1984), the people such as Boulianne, Nature, 312:643-646 (1984), and described in the open WO86/01533 of PCT application.Although they can immunogenicity more less than mouse monoclonal antibody, the using of chimeric antibody is replied (HAMA) with the human anti-mouse antibody of the nonhuman portions for antibody and is associated.Chimeric antibody also can by by from have suitable antigen binding specificity amouse antibody molecule gene with from have suitable bioactive human antibody molecules gene splicing together with produce, described biological activity such as activate people's complement and mediation ADCC ability.The people such as Morrison (1984), Proc.Natl.Acad.Scl, 81:6851; The people such as Neuberger (1984), Nature, 312:604.An example Shi Jiang Fc district replaces with the Fc district of different isotype.

CDR grafted antibody is the antibody comprising the CDRs from inhuman " donor " antibody be connected with the framework region from people's " receptor " antibody.Usually, CDR grafted antibody comprises human antibody sequence more more than chimeric antibody, because they comprise constant-region sequences from people's antibody and variable region (framework) sequence.Therefore, such as, CDR of the present invention transplants humanized antibody can comprise heavy chain, described heavy chain comprise from people's antibody framework region (such as, FR-1, FR-2 or FR-3 of people's antibody) continuous amino acid sequence (such as, about 5 or more, 10 or more or even 15 or more continuous amino acid residues), or optionally, the great majority of the whole framework region of people's antibody or all.CDR grafted antibody and for the preparation of its method people such as Jones, Nature, 321:522-525 (1986), the people such as Riechmann, Nature, 332:323-327 (1988), and the people such as Verhoeyen, Science, 239:1534-1536 (1988)) in described.May be used for the method for generating humanized antibodies also at U.S. Patent number 4,816,567,5,721,367,5,837,243 and 6,180, described in 377.CDR grafted antibody is considered to comparatively can not induce for non-human antibody's immunoreation partly than chimeric antibody.But the frame sequence having reported from donor antibody is needed for the binding affinity of donor antibody and/or specificity, infers this is because these frame sequences affects folding of the antigen-binding portion thereof of donor antibody.Therefore, when donor non-human CDR sequences be transplanted to irrelevant people build in sequence time, in some cases, relative to initial non-human donor antibody, the CDR grafted antibody obtained may show the forfeiture of binding affinity.See such as, the people such as Riechmann, Nature, 332:323-327 (1988), and the people such as Verhoeyen, Science, 239:1534-1536 (1988).

People's engineered antibody comprises such as " inlaying " antibody and use HUMANENGINEERING ^tMantibody prepared by technology (United States Patent (USP) 5,869,619).HUMANENGINEERING ^tMtechnology is obtained commercially, and relate to and change non-human antibody or antibody fragment, such as mice or chimeric antibody or antibody fragment, mode is that the aminoacid sequence of antagonist carries out special change, to produce the immunogenicity in people with minimizing but still to retain the modified antibodies of binding property needed for original non-human antibody.Usually, this technology relates to the amino acid residue of inhuman (such as, mice) antibody is categorized as " low-risk ", " medium risk " or " excessive risk " residue.Classification use overall risk/return calculating is carried out, and this calculating assessment will affect the risk of the folding of gained antibody and/or antigen-binding matter relative to displacement, carry out the expection benefit (immunogenicity such as, in people) that this is specifically replaced.Therefore, low-risk position is expection is favourable position in the displacement at this place, because expect that this will reduce immunogenicity and not appreciable impact antigen-binding matter.Medium risk position is that the displacement of expection at this place can reduce immunogenicity but have the larger position that may affect protein folding and/or antigen combination.Excessive risk position comprises the residue that most probable participates in correctly folding or antigen combines.Usually, the low-risk position employment residue in non-human antibody is replaced, and excessive risk position is seldom replaced, and sometimes, but be not without restriction, carry out humanization displacement in medium risk position.The position in non-human antibody's variable region sequences with proline is categorized as at least medium risk position usually.

Can be selected by following at the particular person amino acid residue of the given low or medium risk position of inhuman (such as, mice) antibody sequence displacement: make the aminoacid sequence from the variable region of non-human antibody and respective regions comparison that is specific or joint owner's antibody sequence.According to this comparison, the corresponding residue in human antibody sequence can be replaced at the amino acid residue of the low of non human antibody sequences or medium risk position.For the preparation of the technology of people's engineered proteins people such as Studnicka, ProteinEngineering, 7:805-814 (1994), United States Patent (USP) 5,766,886,5,770,196,5,821,123 and 5,869,619 and the open WO93/11794 of PCT application in be described in more detail.

" inlaying " antibody is inhuman or humanization (such as, chimeric or CDR grafted antibody) antibody, and it is through transforming to replace the amino acid residue that some is exposed to solvent, to reduce its immunogenicity further or to strengthen its function.Because infer that the surface residue of chimeric antibody comparatively can not affect correct antibody and folds but comparatively may cause immunoreation, so inlaying of chimeric antibody can comprise such as, be exposed to the residue of solvent in the non-human framework regions of qualification chimeric antibody, and replace at least one in them with the respective surfaces residue from people's framework region.Inlay and can have been come by any suitable engineering, comprise above-mentioned HUMANENGINEERING ^tMthe use of technology.

In a distinct methods, binding affinity can be recovered by making CDR grafted antibody " remove humanization ".Go humanization to comprise and make the residue of the framework region from donor antibody get back to CDR grafted antibody, thus recover correct folding.Similar " removing humanization " can be reached by following: (i) comprises the part of " donor " framework region at " receptor " antibody, or the part (the donor CDRs together with transplanting) of " donor " antibody framework is transplanted in receptor antibody by (ii).

About antibody, humanized antibody, people's engineered antibody and the further discussion for its method prepared, see KontermannandDubel, editor, AntibodyEngineering, Springer, NewYork, NY, 2001.

Exemplary humanized or people's engineered antibody comprises IgG, IgM, IgE, IgA and IgD antibody.Antibody of the present invention can be any type (IgG, IgA, IgM, IgE, IgD etc.) or isotype, and can comprise κ or lambda light chain.Such as, the fragment that people's antibody can comprise IgG heavy chain or determine, such as at least one isotype, IgG1, IgG2, IgG3 or IgG4.As further example, antibody of the present invention or fragment can comprise IgG1 heavy chain and IgG1 light chain.

Antibody of the present invention and fragment can be that people's antibody is (such as in conjunction with IL-1 beta polypeptides and by the antibody of following nucleic acid sequence encoding, described nucleotide sequence is the natural somatic cell variant of people's germ-line immunoglobulin nucleotide sequence), and fragment, synthesis variant, derivant and fusions.This kind of antibody can be produced by any method known in the art, and such as, by using transgene mammal (such as, transgenic mice), wherein natural in this mammalian chromosome immunoglobulin storehouse is replaced by people V gene.This kind of mammal shows the VDJ restructuring and somatic hypermutation that realize people's germline antibody gene in the normal fashion, thus produces the high-affinity antibody with total man's sequence.

People's antibody for target protein also can use transgenic animal to produce, and described transgenic animal do not have endogenous immunoglobulin and produce, and comprise human immunoglobulin gene's seat through transformation.Such as, WO98/24893 discloses the transgenic animal with people Ig locus, and wherein due to the inactivation of endogenous heavy and light chain gene seat, this animal does not produce functional endo immunoglobulin.WO91/00906 also discloses the transgenic non-primate mammal host that can produce for immunogenic immunne response, and wherein antibody has primate constant region and/or variable region, and the wherein replaced or inactivation of endogenous immunoglobulin encoding gene seat.WO96/30498 and U.S. Patent number 6,091,001 disclose and use Cre/Lox system to modify immunoglobulin loci in mammal, such as to replace all or part of of constant or variable region, thus form the antibody molecule modified.WO94/02602 discloses the non-human mammal host of endogenous Ig loci and the functional human Ig loci with deactivation.U.S. Patent number 5,939,598 disclose the method preparing transgenic mice, and wherein this mice lacks endogenous heavy chain, and express the exogenous immunoglobulin genes seat comprising one or more xenogenesis constant region.Also see, U.S. Patent number 6,114,598,6,657,103 and 6,833,268.

Use above-described transgenic animal, the immunne response for selected antigen molecule can be produced, antibody producing cells can be taken out from animal, and for generation of secreting the hybridoma of human monoclonal antibodies.Immunization scheme, adjuvant etc. are known in the art, and can use in the immunity inoculation of such as transgenic mice, as described in WO96/33735.This publication disclose the monoclonal antibody for various antigen molecule, described antigen molecule comprises IL-6, IL-8, TNFa, people CD4, L selection albumen, gp39 and tetanus toxin.Monoclonal antibody can with regard to suppress or in and the biological activity of respective egg white matter or the ability of physiological effect test.WO96/33735 discloses the monoclonal antibody for IL-8, the neutrophil cell function of its immunocyte derived from the transgenic mice with IL-8 immunity, blocking-up IL-8 induction.To the antigen for immunize transgenic animals, there is specific human monoclonal antibodies and be also disclosed in WO96/34096 and U.S. Patent Application No. 20030194404; And in U.S. Patent Application No. 20030031667.

Other transgenic animal for the preparation of monoclonal antibody are included in U.S. Patent number 5,770,429 and the people (Nat.Biotechnol.14:845-851,1996) such as Fishwild in the MedarexHuMAb-that describes it comprises the gene order of weight from the encoding human antibody not resetting human immunoglobulin gene and light chain.To HuMAb- immunity make it possible to produce for the human monoclonal antibodies of target protein.

In addition, the people such as Ishida (CloningStemCells.4:91-102,2002) describe TransChromoMouse (TCMOUSE ^tM), it comprises people's region of DNA section of megabasse size, and mixes complete human normal immunoglobulin (hIg) locus.TCMOUSE ^tMthere is complete various storehouse of hIg, comprise all subclass (IgG1-G4) of IgGs.With various human antigen immunity inoculation TCMOUSE ^tMproduce the antibody response comprising people's antibody.

Also see people such as Jakobovits, Proc.Natl.Acad.Sci.USA, 90:2551 (1993); The people such as Jakobovits, Nature, 362:255-258 (1993); The people such as Bruggermann, YearinImmunol., 7:33 (1993); With U.S. Patent number 5,591,669, U.S. Patent number 5,589,369, U.S. Patent number 5,545,807; With U.S. Patent Publication No. 20020199213.U.S. Patent Publication No. 20030092125 describes the method for the required epi-position of immunne response deflection for making animal.People's antibody also can be produced by the B cell of Activated in Vitro (see U.S. Patent number 5,567,610 and 5,229,275).

People's antibody also can be produced by the in-vitro screening of antibody display libraries.See the people such as Hoogenboom (1991), J.Mol.Biol.227:381; With people (1991) such as Marks, J.Mol.Biol.222:581.The various phage display library comprising antibody is described, and can easily prepare.Library can comprise various human antibody sequence, such as human Fab, Fv and scFv fragment, can screen these sequences for suitable target.Phage display library can comprise peptide or the protein of non-antibody, can screen this peptide or protein to identify the selective binding agent of IL-1 β.

For the preparation of the development of the technology of recombinant human antibody gene bank and the development for coded antibody fragment being illustrated in the technology on filobactivirus surface, the means of directly preparation people antibody are provided.The form that the antibody produced by phage technology is with Fab---being generally Fv or Fab fragment---produces in antibacterial, and therefore lacks effector function.Effector function can be introduced by one of 2 kinds of strategies: these fragments can be transformed into complete antibody for expressing in mammalian cell, or is transformed into that have can the bispecific antibody fragment of the second binding site of trigger effect subfunction.

The present invention considers the method for generation of target-specific antibody or its antigen-binding portion thereof, and it comprises the steps: to synthesize people's antibody library in phage, obtain antibody by the phage of target protein or its part screening library, separating and combining target by phage.Such as, a kind of method for the preparation of the antibody library used in display technique of bacteriophage comprises the steps: the non-human animal comprising human immunoglobulin gene's seat with target antigen or the immunity of its antigenic portions, to produce immunne response, from the animal of immunity inoculation, extract antibody producing cells; Isolation of RNA from the cell extracted, by RNA reverse transcription to produce cDNA, uses primer amplification cDNA, and is inserted in Vector for Phage Display by cDNA, thus antibody is expressed in phage.Restructuring target-specific antibody of the present invention can obtain by this way.

Phage display method passes through at filobactivirus displaying on surface antibody library, and subsequently by selecting phage with the combination of selected antigen, thus simulation immunoselection.A kind of this kind of technology describes in WO99/10494, which depict and uses this kind of method Separated pin to the high-affinity of MPL and msk receptor and functional antagonism antibody.Antibody of the present invention can by screening restructuring combinatorial antibody library, and preferably scFv phage display library is separated, and wherein said library can use the people V prepared by the lymphocytic mRNA of derived from human _land V _hprepared by cDNA.For the preparation of with screening this kind of library method be known in the art.See such as, U.S. Patent number 5,969,108.Exist and be obtained commercially test kit (such as, PharmaciaRecombinantPhageAntibodySystem, catalog number (Cat.No.) 27-9400-01 for generation of phage display library; And StratageneSurfZAP.TM.phagedisplaykit, catalog number (Cat.No.) 240612).Also exist additive method and reagent may be used for producing and screening antibodies display libraries (see such as, people's U.S. Patent numbers such as Ladner 5,223,409; The people PCT publication number WO92/18619 such as Kang; The people PCT publication number WO91/17271 such as Dower; The people PCT publication number WO92/20791 such as Winter; The people PCT publication number WO92/15679 such as Markland; The people PCT publication number WO93/01288 such as Breitling; The people PCT publication number WO92/01047 such as McCafferty; The people PCT publication number WO92/09690 such as Garrard; The people such as Fuchs (1991) Bio/Technology9:1370-1372; The people such as Hay (1992) Hum.Antibod.Hybridomas3:81-85; The people such as Huse (1989) Science246:1275-1281; The people such as McCafferty, Nature (1990) 348:552-554; The people such as Griffiths (1993) EMBOJ12:725-734; The people such as Hawkins (1992) J.Mol.Biol.226:889-896; The people such as Clackson (1991) Nature352:624-628; The people such as Gram (1992) Proc.Natl.Acad.Sci.USA89:3576-3580; The people such as Garrad (1991) Bio/Technology9:1373-1377; The people such as Hoogenboom (1991) NucAcidRes19:4133-4137; With people (1991) Proc.Natl.Acad.Sci.USA88:7978-7982 such as Barbas.

In one embodiment, in order to be separated the target antigen human antibodies specific with required feature, screening people V _hand V _llibrary, to select having required specific antibody fragment.The antibody library used in this approach is preferably as herein and preparation as described in this area and the scFv library of the screening (people such as McCafferty, PCT publication number WO92/01047, the people such as McCafferty, (Nature348:552-554,1990); With people such as Griffiths, (EMBOJ12:725-734,1993).ScFv antibody library preferably uses target protein to screen as antigen.

Alternately, the Fd fragment (V of antibody _h-C _h1) and light chain (V _l-C _l) cloned respectively by PCR, and recombinate at random in combination phage display library, can just select with the combination of specific antigen subsequently.These Fab fragments are expressed on phage surface, namely with its gene physical connection of coding.Therefore, combined the common selection selecting Fab can cause Fab coded sequence by antigen, can increase this sequence subsequently.Combine by number wheel antigen and increase again (program being called elutriation), can enrichment and the final Fab be separated for antigen-specific.

In 1994, describe the method for antibody humanization being called " pathfinder selection ".The strength of display technique of bacteriophage is used for the humanization (see people such as Jespers, L.S., Bio/Technology12,899-903 (1994)) of mouse monoclonal antibody by pathfinder selection.For this reason, the Fd fragment of mouse monoclonal antibody can combine with people's light chain libraries shows, and the hybridization Fab library obtained can be selected with antigen subsequently.Mice Fd fragment thus provide the template of pathfinder selection.Subsequently, selected people's light chain and people Fd frag-ment libraries is made to combine.Total man Fab is produced to the selection in obtained library.

Multiple method for obtaining people's antibody from phage display library has obtained describing (see such as, the people such as Hoogenboom, J.Mol.Biol., 227:381 (1991); The people such as Marks, J.Mol.Biol, 222:581-597 (1991); U.S. Patent number 5,565,332 and 5,573,905; Clackson, T., and Wells, J.A., TIBTECH12,173-184 (1994)).Especially, powerful has been become (see Burton, D.R., with BarbasIII, C.F., Adv.Immunol.57,191-280 (1994) to the external selection of the antibody derived from phage display library and evolving; The people such as Winter, G., Annu.Rev.Immunol.12,433-455 (1994); U.S. Patent Publication No. 20020004215 and WO92/01047; U.S. Patent Publication No. 20030190317; And U.S. Patent number 6,054,287 and 5,877,293.

Watkins, " ScreeningofPhage-ExpressedAntibodyLibrariesbyCaptureLift; " MethodsinMolecularBiology, AntibodyPhageDisplay:MethodsandProtocols178:187-193 (2002), with on March in 2003 6 disclosed in U.S. Patent Publication No. 20030044772 describe by the method for CaptureLift for the antibody library or other binding molecules that screen phage expression, CaptureLift a kind ofly relates to the method be fixed on by candidate biding molecules on solid phase carrier.

Fv fragment is illustrated on phage surface in the following way: a chain of expressing as phage protein fusions (such as, having M13 gene III) is combined with the complementary strand as solvable fragment expression.One of consider that phage can be filobactivirus, such as one of I class phage: fd, M13, f1, If1, lke, ZJ/Z, Ff, and II class phage: Xf, Pf1 and Pf3.Phage can be M13 or fd or derivatives thereof.

Once have selected initial people V _land V _hsection, can perform " mixing and coupling " experiment, to select preferred V _l/ V _hto combination, wherein screen the V of initial selection with regard to the combination of target _land V _hthe difference of section is right.In addition, in order to improve the quality of antibody further, can be similar in innate immunity process be responsible for antibody affinity maturation body in somatic mutation process program in; The preferred V of random mutation _l/ V _hright V _land V _hsection, preferably at V _hand/or V _lcDR1, CDR2 or CDR3 district any one in suddenly change.This external affinity maturation can by using PCR primer amplification V _land V _hdistrict has come, described PCR primer respectively with V _hcDR1, CDR2 and CDR3, or V _lcDR1, CDR2 and CDR3 are complementary, and described primer is the random mixture of 4 kinds of nucleotide bases at some position admixture, thus makes produced PCR primer coding random mutation to introduce V _hand/or V _lv in CDR3 district _land V _hsection.The V of these random mutations _land V _hsection can just screen with the combination of target antigen again.

From recombination immunoglobulin display libraries screening be separated target-specific antibody after, the nucleic acid of the selected antibody of coding can from displaying bag (displaypackage) (such as, from phage genome) middle recovery, and be subcloned in other expression vectors by standard recombinant dna technology.When needing, nucleic acid can process further, to produce other antibody formations of the present invention, as mentioned below.In order to express the recombinant human antibody be separated by screening combinatorial library, by the DNA clone of encoding antibody in recombinant expression carrier, and introduce in mammalian host cell, as described herein.

Consider that phage display method can carry out in the mutator of antibacterial or host cell.Mutator is the host cell with genetic defect, and it causes the DNA copied to undergo mutation relative to parent DNA within it.Example mutator strain is NR9046mutD5 and NR9046mutT1.

Also consider that phage display method can use helper phage to carry out.This comprises the genomic cell of defective phage and function is to make up the phage of this defect for infecting.Defective phage genome can be the wherein removed phasmid of some function coding gene order or phage.The example of helper phage is M13K07, M13K07 gene III numbering 3; With the phage of binding molecule of showing or coding and capsid protein merge.

Antibody also can produce via phage display screening technique, wherein use layering double combinations method (hierarchicaldualcombinatorialapproach) disclosed in WO92/01047, wherein comprise single bacterium colony of H or L chain clone for infecting a complete library, described library is made up of the clone of another chain (L or H) of encoding, and the double-stranded specific binding members obtained is selected according to display technique of bacteriophage such as described herein those.This technology is also disclosed in the people such as Marks, in (Bio/Technology, 10:779-783,1992).

For the method for displayed polypeptide on yeast and microbial cell surface also for the identification of antigen-specific antibodies.See such as, U.S. Patent number 6,699,658.Antibody library can adhere to yeast protein such as agglutinin, effectively to simulate in immune system antibody via the cell surface display of B cell.

Except phage display method, antibody can use ribosome mRNA methods of exhibiting to be separated with microbial cell display method.Use the polypeptide that carries out of ribosomal display to select people such as Hanes, (Proc.NatlAcadSciUSA, 94:4937-4942,1997) and authorize the U.S. Patent number 5,643,768 and 5,658 of Kawasaki, described in 754.Ribosomal display is also for the extensive fast mutation analysis of antibody.Selectivity method of mutagenesis also provides the method producing and have and improve active antibody, and described antibody can use ribosomal display technology to select.

IL-1 (such as IL-1 β) binding antibody and fragment can comprise such a or multiple part, it is not in conjunction with IL-1 β but be responsible for other functions, such as circulating half-life, directly cytotoxic effect, detectable label or activate endogenous complement cascade or the endogenous cell toxicity of receiver.Antibody or fragment can comprise all or part of constant region, and can have any isotype, comprise IgA (such as, IgA1 or IgA2), IgD, IgE, IgG (such as IgG1, IgG2, IgG3 or IgG4) or IgM.Except or replace comprising constant region, antigen-binding compound of the present invention can comprise epitope tag, scavenger receptor (salvagereceptor) epi-position, for diagnosing or the label segment of purification applications or cytotoxic moieties such as radionuclide or toxin.

The constant region (when it is present) of antibody of the present invention and fragment can be γ 1, γ 2, γ 3, γ 4, μ, β 2 or δ or ε type, preferred γ type, more preferably y type, and the constant portion of people's light chain can be that (it comprises λ to κ or λ type ₁, λ ₂and λ ₃hypotype), but preferred κ type.

Variant also comprises the antibody or fragment that comprise modified Fc district, wherein modified Fc district comprise relative to wild type Fc district at least one is amino acid modified.Variant Fc district can design relative to the compared molecule comprising wild type Fc district, so that with more or less affinity in conjunction with Fc receptor.

Such as, IL-1 beta binding antibodies of the present invention and fragment can comprise modified Fc district.Fc district refers to and polypeptide that is following IgGC terminal domains homology, naturally occurring or that synthesize, and described domain produces after the papain digestion of IgG.IgGFc has the molecular weight of about 50kD.In antibody of the present invention and fragment, whole Fc district can be used, or only the half-life strengthens part.In addition, the many modifications in aminoacid sequence are acceptable, because natural activity is all not required in all cases or expects.

When needing, Fc district can suddenly change, to suppress its complement-fixing and with the ability of high-affinity in conjunction with Fc receptor.Protein is made to instruct ADCC for muroid IgGFc, Ala residue substitutions Glu318, Lys320 and Lys322.Glu replaces Leu235 Profilin matter with the ability of high-affinity in conjunction with Fc receptor.Various sudden changes about human IgG are known (see such as, the people such as Morrison, the people such as 1994, TheImmunologist2:119124 and Brekke, 1994, TheImmunologist2:125).

In certain embodiments, provide the antibody of the present invention or fragment with modified Fc district, wherein naturally occurring Fc district carries out modifying to increase antibody or the half-life of fragment in biotic environment, such as serum half-life or half-life of being measured by vitro tests.For changing the method for the original form in IgGFc district at U.S. Patent number 6,998, described in 253.

In certain embodiments, may wish that modified antibodies or fragment are to increase its serum half-life, such as, add molecule such as PEG or other water-soluble polymers to antibody fragment, comprise polysaccharide polymer, to increase the half-life.This also can such as by mixing in antibody fragment (such as by scavenger receptor in conjunction with epi-position, by the sudden change of appropriate area in antibody fragment, or by this epi-position being mixed (such as by means of DNA or peptide symthesis) in peptide tag, subsequently peptide tag is merged in arbitrary end of antibody fragment or centre) reach (see international publication number WO96/32478).Scavenger receptor refers to IgG molecule (such as, the IgG of serum half-life in the responsible body increasing IgG molecule in conjunction with epi-position ₁, IgG ₂, IgG ₃or IgG ₄) epi-position in Fc district.

Scavenger receptor can comprise such region in conjunction with epi-position, and any one or more amino acid residues wherein from 1 or 2 ring of Fc domain are transferred in the similar position of antibody fragment.More preferably, 3 of 1 or 2 ring from Fc domain or more amino acid residue is shifted.More preferably, epi-position takes from the CH2 domain of Fc district (such as, the Fc district of IgG), and is transferred to CH1, CH3 or V of antibody _hdistrict or more than on this kind of region.Alternately, epi-position takes from the CH2 domain in Fc district, and is transferred to the C of antibody fragment _ldistrict or V _ldistrict or both.Also see international application WO97/34631 and WO96/32478, which depict Fc variant and the interaction with scavenger receptor thereof.

The sudden change of the residue in Fc receptor binding site can cause the effector function changed, ADCC or CDC such as changed is active, or the half-life changed.Potential sudden change comprises the insertion of one or more residue, disappearance or displacement, comprise with alanine substitution, conservative substitution, non-conservative displacement or the corresponding amino acid residue replacement (such as, IgG1 residue is used in the corresponding IgG2 residue replacement of that position) with the same position place from different I gG subclass.Such as, report and to have compared with original chimeric IgG4, the mutant serine of the amino acid position 241 in IgG4 is made to become proline (residue that this position in IgG1 and IgG2 finds) to cause the generation of homogeneity antibody, and the tissue distribution of the serum half-life extended and the improvement (people such as Angal, MolImmunol.30:105-8,1993).

Antibody fragment is the part of intact full length antibody, the antigen binding domain of such as complete antibody or variable region.The example of antibody fragment comprise Fab, Fab ', F (ab ') ₂with Fv fragment; Double-chain antibody (diabody); Linear antibodies; Single-chain antibody molecules (such as, scFv); Multispecific antibody fragments is bispecific, tri-specific and multi-specificity antibody (such as, double-chain antibody, three chain antibodies (triabody), four chain antibodies (tetrabody)) such as; Miniantibody (minibody); Chelating recombinant antibody (chelatingrecombinantantibody); Three bodies (tribody) or binary (bibody); Intracellular antibody; Nano antibody; Little module immune drug (smallmodularimmunopharmaceuticals, SMIP), adnectins, binding structural domain domain-immunoglobulin fusion proteins; Camel source antibody (camelizedantibody); Containing V _hHantibody; With any other polypeptide formed by antibody fragment.

The present invention includes and comprise any aforementioned heavy or sequence of light chain and in conjunction with the IL-1 beta binding antibodies fragment of IL-1 β.As used herein, term fragment any 3 that refer to antibody or more continuous amino acid (such as, 4 or more, 5 or more, 6 or more, 8 or more or even 10 or more continuous amino acids), and contain Fab, Fab ', F (ab ') ₂with F (v) fragment or single light or variable region of heavy chain or its part.IL-1 β binding fragment comprise such as Fab, Fab ', F (ab ') ₂, Fv and scFv.These fragments lack the Fc fragment of complete antibody, are eliminated more quickly from circulation, and can have the nonspecific tissue combination more less than complete antibody.See the people such as Wahl (1983), J.Nucl.Med., 24:316-25.These fragments can use well-known method to be produced by complete antibody, such as, pass through with enzyme such as papain (to produce Fab fragment) or pepsin (to produce F (ab ') ₂fragment) proteolysis cutting.

For measuring the external of the combination of IL-1 β and IL-1 receptor type I (IL-1R1) and being that this area fully describes based on the determination test of cell, be included in the test carrying out under the molecule (such as antibody, antagonist or other inhibitor) be combined with IL-1 β or IL-1R1 exists measuring.(see people such as such as Evans, (1995), J.Biol.Chem.270:11477-11483; The people such as Vigers, (2000), J.Biol.Chem.275:36927-36933; The people such as Yanofsky, (1996), Proc.Natl.Acad.Sci.USA93:7381-7386; The people such as Fredericks, (2004), ProteinEng.Des.SeI.17:95-106; The people such as Slack, (1993), J.Biol.Chem.268:2513-2524; The people such as Smith, (2003), Immunity18:87-96; The people such as Vigers, (1997), Nature386:190-194; The people such as Ruggiero, (1997), J.Immunol.158:3881-3887; The people such as Guo, (1995), J.Biol.Chem.270:27562-27568; The people such as Svenson, (1995), Eur.J.Immunol.25:2842-2850; The people such as Arend, (1994), J.Immunol.153:4766-4774).For the restructuring IL-1 receptor type I (comprising people IL-1 receptor type I) of this kind of determination test, easily can obtain from various commercial source (see such as R & DSystems, SIGMA).IL-1 receptor type I also can from the expression construct used in standard molecular biology known in the art and rotaring dyeing technology introducing Suitable host cells or vector expression.The IL-1 receptor type I expressed can be separated subsequently and purification uses in measuring in combination, or alternately directly uses with cell associated form.

Such as, IL-1 β can be measured by following with the combination of IL-1 receptor type I: fixing IL-1 beta binding antibodies, IL-1 β is contacted with sessile antibody and measure IL-1 β whether with antibodies, the IL-1RI of soluble form is contacted with the IL-1 β/antibody complex of combination, and measures solvable IL-1RI and whether be combined with complex.The program makes solvable IL-1RI contact with sessile antibody before also can being included in and contacting with IL-1 β, to confirm that solvable IL-1RI is not combined with sessile antibody.This scheme can to use the dynamic analysis for binding interactions instrument carries out.This kind of scheme also may be used for measuring antibody or whether other molecules allow or block the combination of IL-1 β and IL-1 receptor type I.

For other IL-1 β/IL-1RI in conjunction with determination test, to the permission of IL-1 β and the combination of IL-1 receptor type I or block and can be determined by following: compare the combination of comparing IL-1 β and IL-1RI under IL-1 β antibody or its IL-1 β binding fragment presence or absence.Block in the reading of this test and be accredited as: to comprise corresponding buffer agent or diluent but the control sample not comprising IL-1 β antibody or its IL-1 β binding fragment compares, the appointment that IL-1 β is combined with IL-1 receptor type I under anti-il-i-beta antibody or its IL-1 β binding fragment exist reduces.This test reads the presence or absence that can be rendered as instruction qualitatively and block, or can be rendered as quantitatively instruction due to antibody or fragment existence caused by the percentage ratio that reduces of combination or multiple.

Alternately or additionally, when IL-1 beta binding antibodies or IL-1 β binding fragment substantially block IL-1 β and IL-1RI in conjunction with time, compare with the combination of IL-1 β and IL-1RI of the same concentrations when there is not antibody or fragment, the combination of IL-1 β and IL-1RI be reduced by least 10 times, alternately at least about 20 times, alternately at least about 50 times, alternately at least about 100 times, alternately at least about 1000 times, alternately at least about 10000 times or more.As another example, when IL-1 beta binding antibodies or IL-1 β binding fragment substantially allow IL-1 β and IL-1RI in conjunction with time, the combination of IL-1 β and IL-1RI be the combination of IL-1 β with IL-1RI of the same concentrations when there is not antibody or fragment at least about 90%, alternately at least about 95%, alternately at least about 99%, alternately at least about 99.9%, alternately at least about 99.99%, alternately at least about 99.999%, alternately at least about 99.9999%, alternately substantially the same.

In certain embodiments, the present invention can comprise IL-1 beta binding antibodies or IL-1 β binding fragment, and itself and one or more exemplary antibodies disclosed herein are in conjunction with identical epi-position or substantially the same epi-position.Alternately or additionally, this IL-1 beta binding antibodies or IL-1 β binding fragment are combined with the antibody competition with the AB7 variable region sequences (sequence is hereafter showing) described in U. S. application number 11/472813.Alternately or additionally, the present invention includes IL-1 beta binding antibodies and fragment, the epi-position comprised in its binding amino acid sequence ESVDPKNYPKKKMEKRFVFNKIE (SEQIDNO:1), the epi-position that the antibody of called after AB5 and AB7 (U. S. application number 11/472813) combines.As used herein considered, can use any one in several known method in this area easily measure IL-1 beta binding antibodies or fragment whether with one or more exemplary antibodies, the epi-position that such as antibodies of called after AB7 is identical or substantially the same epi-position.

Such as, the critical amino acid residues (epi-position) combined by IL-1 beta binding antibodies or fragment can use peptide array to measure, such as PepSpot ^tMpeptide array (JPTPeptideTechnologies, Berlin, Germany), wherein on film, directly a series of 12 amino acid whose scanning peptides of whole IL-1 beta amino acids sequence are crossed in synthesis, often kind of peptide 11 aminoacid overlapping with front a kind of peptide.The film of carry peptides at room temperature such as detects 2 hours with the concentration of 2 μ g/ml with antibody subsequently, to obtain epi-position combining information for described antibody.Goat anti human that the combination of antibody and film binding peptide can use secondary HRP to put together (or time suitably, mice) antibody, use enhanced chemical luminescence (ECL) to detect subsequently.Antibodies is had to the peptide point of just marking corresponding to the particular amino acid residue of ripe IL-1 beta protein or sequence, indicate the epi-position combined by this specific antibodies.

Alternately or additionally, antibody competition experiment can be performed, and this kind of determination test is well-known in the art.Such as, the epi-position whether comprised in following peptide sequence to measure antibody or fragment is combined, described peptide sequence comprises the aminoacid ESVDPKNYPKKKMEKRFVFNKIE (SEQIDNO:1) corresponding to the residue 83-105 of ripe IL-1 beta protein, the any exemplary antibodies of the present invention (such as, AB7) of specific for the unknown antibody and the known epi-position in conjunction with comprising in this sequence can be compared.The dynamic analysis for binding interactions such as can be used in conjunction with competition assay instrument or undertaken by ELISA.In this kind measures, the antibody of unknown epitope specificity is assessed with the known ability comparing antibody (such as, AB7) competition binding with regard to it.Known antibodies is passed through (such as with the competition binding of defined epitope, AB7) 50% is reduced by least about with the combination of this IL-1 β epi-position, or at least about 70% or at least about 80% or at least about 90% or at least about 95% or determine at least about 99% or about 100%, and can indicate and combine with substantially the same epi-position.

In view of the epi-position that the IL-1 β calmodulin binding domain CaM and/or these open antibody that identify exemplary antibodies in the disclosure identify, can consider to produce and have similar in conjunction with characteristic sum treatment or other antibody of diagnostic uses, it is the embodiment parallel with embodiment of the present disclosure.

The Fab of antibody comprises the fragment retaining the ability (the general antigen-binding portion thereof by retaining antibody) be combined with antigenic specificity.The antigen combined function fully having established antibody can be realized by the fragment of full length antibody.The example of antigen-binding portion thereof comprises (i) Fab fragment, and it is the monovalent fragment be made up of VL, VH, CL and CH1 domain; (ii) F (ab ') ₂fragment, it is be included in the bivalent fragment of hinge region place by 2 Fab fragments of disulfide bridge connects; (iii) Fd fragment, it is VH and CH1 domain; (iv) Fv fragment, it is VL and the VH domain of antibody single armed, (v) dAb fragment (people such as Ward, (1989) Nature341:544-546), and it is VH domain; (vi) complementary determining region (CDR) be separated.Single-chain antibody is also contained in the antigen-binding portion thereof of term antibody.IL-1 beta binding antibodies of the present invention and fragment also contain unit price or multivalence or monomer or polymer (such as, the tetramer), contain or do not contain the derivative binding structural domain of the CDR of support (such as, protein or carbohydrate support).

IL-1 beta binding antibodies of the present invention or fragment can be the parts of larger immunoadhesin molecule, are formed with other protein one or more or being covalently or non-covalently combined of peptide by antibody or antibody moiety.The example of this kind of immunoadhesin molecule comprises use Streptavidin core space to form tetramer scFv molecule (Kipriyanov, S.M. people (1995) HumanAntibodiesandHybridomas6:93-101 is waited), with use cysteine residues, labelling peptide and C-terminal polyhistidine tag to form bivalence and biotinylated scFv molecule people (1994) Mol.Immunol.31:1047-1058 such as () Kipriyanov, S.M..The immunoadhesin molecule comprising antibody and fragment can use standard recombinant dna technology to obtain, as described herein.Preferred antigen-binding portion thereof is complete domain or complete domain pair.

IL-1 beta binding antibodies of the present invention and fragment also contain by V _hdomain antibodies (dAb) fragment people such as (, Nature341:544-546,1989) Ward of domain composition.IL-1 beta binding antibodies of the present invention and fragment also contain double-chain antibody, and it is bivalent antibody, wherein V _hand V _ldomain is expressed on wall scroll polypeptide chain, but the joint used is too short and do not allow to match between in same chain 2 domains, thus forces the complementary domain of these domains and another chain to match, produce 2 antigen binding sites (see such as, EP404,097; WO93/11161; The people such as Holliger, Proc.Natl.Acad.Sci.USA90:6444-6448,1993, and the people such as Poljak, Structure2:1121-1123,1994).Double-chain antibody can be bispecific or monospecific.

IL-1 beta binding antibodies of the present invention and fragment also contain the single chain antibody fragments (scFv) be combined with IL-1 β.ScFv comprises and antibody chain variable region (V _l) antibody heavy chain variable region (V that is operably connected _h), wherein variable region of heavy chain forms together with variable region of light chain or respectively the binding site in conjunction with IL-1 β.ScFv can be included in the V at amino terminal place _hdistrict and the V at carboxyl terminal place _ldistrict.Alternately, scFv can be included in the V at amino terminal place _ldistrict and the V at carboxyl terminal place _hdistrict.In addition, although 2 of Fv fragment domain V _land V _hby the gene code separated, but they can use recombination method pass through synthetic linker and connect, and described synthetic linker makes them can be prepared to wall scroll polypeptide chain, wherein V _land V _hdistrict's pairing (is called scFv (scFv) to form monovalent molecule; See people (1988) Science242:423-426 such as such as Bird; With people (1988) Proc.Natl.Acad.Sci.USA85:5879-5883 such as Huston).

ScFv optionally can be included in the peptide linker between variable region of heavy chain and variable region of light chain further.This peptide species joint generally comprises 1-50 aminoacid, alternately 3-12 aminoacid, alternately 2 aminoacid.Example for the joint peptide connecting the heavy and light chain in scFv comprises 5 aminoacid sequence Gly-Gly-Gly-Gly-Ser (SEQIDNO:2).Other examples comprise one or more tandem sequence repeats (such as, comprising 2-4 the polypeptide repeated of Gly-Gly-Gly-Gly-Ser (SEQIDNO:2)) of this sequence, to produce joint.

IL-1 beta binding antibodies of the present invention and fragment also contain heavy chain antibody (HCAb).Hunchbacked class animal (camelids) (camel (camel), one-humped camel (dromedary) and yamma (llama); The people such as Hamers-Casterman, 1993Nature363:446; The people such as Nguyen, 1998J.Mol.Biol.275:413), blanket shark (people such as Nuttall, MolImmunol.38:313-26,2001), nurse shark (people such as Greenberg, Nature374:168-73,1995; The people such as Roux, 1998Proc.Nat.Acad.Sci.USA95:11804) and Ratfiss (people such as Nguyen, " Heavy-chainantibodiesinCamelidae; Acaseofevolutionaryinnovation, " 2002Immunogenetics54 (1): 39-47) some Immunoglobulin Isotype in find conventional antibody H ₂l ₂the exception of structure.These antibody seem only to use formation antigen binding regions, variable region of heavy chain, because these function antibodies are dimers (being called " heavy chain antibody " or " HCAbs ") of only heavy chain.Therefore, some embodiment of IL-1 beta binding antibodies of the present invention and fragment can be the heavy chain antibody with IL-1 β specific binding.Such as, IgG class and lack the heavy chain antibody of light chain and produced by camelidae (camelidae) animal, described cameloid comprises camel, one-humped camel and yamma (people such as Hamers-Casterman, Nature363:446-448 (1993)).HCAbs has the molecular weight of the about 160kDa of about 95kDa instead of conventional IgG antibody.Their binding structural domain is only made up of heavy-chain variable domains, is commonly referred to V _hHto make itself and conventional V _hdistinguish.The people such as Muyldermans, J.Mol.Recognit.12:131-140 (1999).What the variable domains of heavy chain antibody had is called nano antibody (people such as Cortez-Retamozo, CancerResearch64:2853-57,2004).Nano antibody library as people such as Conrath, can be produced by the one-humped camel of immunity inoculation described in (AntimicrobAgentsChemother45:2807-12,2001) or uses recombination method to produce.

Because there is not first constant domain (C _h1) (because the event of disappearance montage shared signal is fallen by montage in the mRNA course of processing), so variable domains (V _hH) be immediately hinge region, C afterwards _h2and C _h3domain (people such as Nguyen, Mol.Immunol.36:515-524 (1999); The people such as Woolven, Immunogenetics50:98-101 (1999)).Camelidae V _hHit was reported and to recombinate with IgG2 and IgG3 constant region, described IgG2 and IgG3 constant region comprises hinge, CH2 and CH3 domain and lack CH1 domain people such as (, the same quoted passage) Hamers-Casterman.Such as, yamma IgG1 is conventional (H ₂l ₂) antibody isotype, wherein V _hwith comprise hinge, the constant region of CH1, CH2 and CH3 domain recombinates, and yamma IgG2 and IgG3 is the isotype of only heavy chain, and it lacks CH1 domain and does not comprise light chain.

Although HCAbs lacks light chain, they have antigen in conjunction with storehouse (repereoire).The hereditary generation mechanism of HCAbs is people such as people Adv.Immunol79:261-296 (2001) and Nguyen such as Nguyen, and Immunogenetics54:39-47 is summarized in (2002).Shark, comprises nurse shark, shows the similar single monomer V domain containing antigen receptor.The people such as Irving, J.Immunol.Methods248:31-45 (2001); The people such as Roux, Proc.Natl.Acad.Sci.USA95:11804 (1998).

V _hHcomprise the little intact antigen binding fragment fragment of 15kDa, 118-136 residue (such as, about).Find camelidae V _hHdomain is combined with antigen (people such as Desmyter, J.BiolChem.276:26285-90,2001) with high-affinity, wherein V _hHaffinity, and can be suitable with the affinity of Fab and scFv fragment generally in nanomolar range.V _hHbe high soluble and more stable than the corresponding derivative of Fab and scFv fragment.V _hfragment is difficult to produce with soluble form relatively, but when Framework residues changes over more V _hHduring sample, the improvement of solubility and specific binding can be obtained.(see such as, the people such as Reichman, JImmunolMethods1999,231:25-38.).V _hHcarry and make it more hydrophilic and stop and BiP (immunoglobulin heavy chain binding protein matter) long-term interactional amino acid replacement, normally in folding and assembling process Bip will in endoplasmic reticulum (ER) with H chain combination, until it is replaced by L chain.Because V _hHthe hydrophilic increased, so improve from the secretion ER.

Functional V _hHthe HCAb of immune hunchbacked class animal can be cut by proteolysis, (cause the V that recombinates by the B cell from immune hunchbacked class animal _hH) or from library that is natural or synthesis Direct Cloning V _hHgene, and obtain.There is the V of required antigenic specificity _hHalso can be obtained by display technique of bacteriophage.Compare with Fabs or scFvs, in phage display, use V _hHmore simply and more effective, because only need clone and express 1 domain to obtain function Fab.Muyldermans, Biotechnol.74:277-302 (2001); The people such as Ghahroudi, FEBSLett.414:521-526 (1997); With people such as vanderLinden, J.Biotechnol.80:261-270 (2000).Method for generation of the antibody with camelidae heavy chain is also described in U.S. Patent Application No. 20050136049 and 20050037421.

Ribosomal display method may be used for identifying and is separated scFv and/or V with required binding activities and affinity _hHmolecule.The people such as Irving, J.Immunol.Methods248:31-45 (2001).Ribosomal display and selection have generation and presenting large library (10 ¹⁴) potentiality.

Other embodiments provide the V produced by hunchbacked source process _hHsample molecule, modifies non-camelidae V in this process _h, such as people V _hH, to improve its dissolubility and to stop non-specific binding.This is by using V _hHsample residue replaces V _hv _lresidue on side reaches, thus the V that simulation is more solvable _hHfragment.When being applied to patient in vivo, hunchbacked source V _hfragment, particularly based on those of people's framework, expection demonstrates the immunne response greatly reduced, and therefore expection has the remarkable advantage being used for the treatment of application.The people such as Davies, FEBSLett.339:285-290 (1994); The people such as Davies, ProteinEng.9:531-537 (1996); The people such as Tanha, J.Biol.Chem.276:24774-24780 (2001); With people such as Riechmann, Immunol.Methods231:25-38 (1999).

Extensively various expression system can be used for producing IL-1 β fragment, comprises Fab fragment, scFv and V _hH.Such as, the expression system of protokaryon and eukaryotic origin may be used for large-scale production antibody fragment and antibody fusion protein.Particularly advantageously expression system lot of antibodies fragment be secreted in culture medium is allowed.

The generation of bispecific Fab-scFv (" binary ") and tri-specific Fab-(scFv) (2) (" three bodies ") is at the people such as Schoonjans (JImmunol.165:7050-57,2000) and in the people (JChromatogrBAnalytTechnolBiomedLifeSci.786:161-76,2003) such as Willems described.For binary or three bodies, make scFv molecule and VL-CL (L) and VH-CH ₁(Fd) one or two in chain merges, and such as, in order to produce three bodies, the C-terminal of 2 scFvs and Fab is merged, and in binary, the C-terminal of 1 scFv and Fab is merged." miniantibody " that scFv forms is merged people such as Olafsen, ProteinEngDesSeI.2004Apr by via peptide linker (hinge-less) or via IgG hinge and CH3; Described in 17 (4): 315-23.

Intracellular antibody to be expressed in showed cell and can single-chain antibody (people such as Biocca, EMBOJ.9:101-108,1990 of manipulating cells internal protein function; The people such as Colby, ProcNatlAcadSciUSA.101:17616-21,2004).The intracellular antibody comprising following cell signal sequence can as the people such as Mhashilkar (EMBOJ14:1542-51,1995) and described in the people (FASEBJ.17:1733-5.2003) such as Wheeler produce, described cell signal sequence makes antibody construct be retained in intracellular space.Wearing membrane antibody (transbody) is can through the antibody of cell, wherein protein transduction domains (PTD) and single chain variable fragment (scFv) Antibody Fusion.The people such as Heng, (MedHypotheses.64:1105-8,2005).

IL-1 beta binding antibodies of the present invention and fragment also contain such antibody, and it is SMIPs or for the special binding structural domain domain-immunoglobulin fusion proteins of target protein.These constructs are the single chain polypeptides comprising the antigen-binding domains merged with immunoglobulin domains, and described immunoglobulin domains performs needed for antibody mediated effect subfunction.See such as, WO03/041600, U.S. Patent application 20030133939 and U.S. Patent application 20030118592.

IL-1 beta binding antibodies of the present invention and fragment also contain immunoadhesin.One or more CDRs can covalently or non-covalently mix in molecule, to become immunoadhesin.Immunoadhesin can mix the part of CDR (s) as larger polypeptide chain, CDR (s) and another polypeptide chain can be made covalently bound, or non-covalently can mix CDR (s).CDRs disclosed herein allows immunoadhesin and IL-1 β specific binding.

IL-1 beta binding antibodies of the present invention and fragment also contain antibody analog, and it is included in the organic or upper one or more IL-1 β bound fractions built of molecular scaffold (such as protein or carbohydrate support).There is the three dimensional structure relatively limited, the protein being commonly referred to protein scaffolds, reagent can be used as and be used for designerantibodies analogies.These supports generally comprise one or more easy acceptance special or the region of random sequence variations, usually to this type of sequence randomization to produce protein library, by the product wherein can selecting expectation.Such as, antibody analog can comprise the chimeric NIg Binding peptide with immunoglobulin like domain, described polypeptide comprises and has the support that 2 or more solvents expose ring, described solvent exposes ring and contains the different CDR from parental antibody inserted in each ring, and demonstrates selective binding activity to the part that parental antibody combines.The protein scaffolds of NIg is proposed for obtaining the protein with new binding property.(people such as Tramontano, J.Mol.Recognit.7:9,1994; McConnell and Hoess, J.Mol.Biol.250:460,1995).Other protein are tested as framework, and in α helical surface (people such as Nord, Nat.Biotechnol.15:772,1997; The people such as Nord, ProteinEng.8:601,1995), on ring between the α spiral of α helical bundle (Ku and Schultz, Proc.Natl.Acad.Sci.USA92:6552,1995), with on the ring retrained by disulphide bridges, such as, on those rings of small protein enzyme inhibitor (people such as Markland, Biochemistry35:8045,1996; The people such as Markland, Biochemistry35:8058,1996; Rottgen and Collins, Gene164:243,1995; The people such as Wang, J.Biol.Chem.270:12250,1995) show randomized residue.Be disclosed in United States Patent (USP) 5,770 about method support being used for antibody analog, 380 and U.S. Patent application 2004/0171116,2004/0266993 and 2005/0038229 in.

The preferred IL-1 β antibody used according to the present invention or antibody fragment are generally combined with people IL-1 β (such as, as measured with BIACORE) with high-affinity, such as, have balance in conjunction with dissociation constant (K for IL-1 β _d) be about 10nM or less, about 5nM or less, about 1nM or less, about 500pM or less, more preferably from about 250pM or less, about 100pM or less, about 50pM or less, about 25pM or less, about 10pM or less, about 5pM or less, about 3pM or less, about 1pM or less, about 0.75pM or less, about 0.5pM or less or about 0.3pM or less.

As measured by enzyme-linked immunosorbent assay (ELISA), antibody of the present invention or fragment can such as with about 10nM or less, about 5nM or less, about 2nM or less, about 1nM or less, about 0.75nM or less, about 0.5nM or less, about 0.4nM or less, about 0.3nM or less or even about 0.2nM or less IC ₅₀be combined with IL-1 β.Preferably, antibody of the present invention or antibody fragment not with any target cross reaction except IL-1.Such as, antibody of the present invention and fragment can be combined with IL-1 β, but can not combine with detecting with IL-1 α, or relative to the combination of itself and IL-1 α, the combination of itself and IL-1 β has the high selectivity at least about 100 times (such as, at least about 150 times, at least about 200 times or even at least about 250 times).In certain embodiments, compare with the IL-6 in the animal stimulated at the IL-1 β not using antibody of the present invention or fragment, antibody used according to the invention or fragment can make the blood serum IL-6 that in animal, IL-1 is beta induced express the suppression being subject at least 50% (such as, at least 60%, at least 70% or even at least 80%).Antibody can in conjunction with IL-1 β, but allow or substantially allow the IL-1 beta ligands of combination and IL-1 receptor type I (IL-RI) to combine.Different from the many known IL-1 beta binding antibodies blocked or substantially disturb IL-1 β and IL-1RI to combine, the antibody of called after AB5 and AB7 (U. S. application number 11/472813) and IL-1 beta ligands selective binding, but allow the IL-1 beta ligands combined to be combined with IL-1RI.Such as, the antibody of called after AB7 is combined with IL-1 β epi-position, but still allows IL-1 β and the IL-1RI combined to combine.In certain embodiments, antibody can reduce the interactional affinity of IL-1 β and the IL-1RI of combination.Therefore, in a related aspect, the invention provides the purposes of the IL-1 beta binding antibodies with at least one above-mentioned feature or IL-1 beta binding antibodies fragment.As described herein, any afore mentioned antibodies of the present invention, antibody fragment or polypeptide can be humanized or people's through engineering approaches.

Various IL-1 known in the art (such as, IL-1 β) antibody and fragment can use according to method provided herein, comprise the antibody described in such as following patent and patent application or the antibody using the method described in following patent and patent application to obtain: US4,935,343; US2003/0026806; US2003/0124617; WO2006/081139; WO03/034984; WO95/01997; WO02/16436; WO03/010282; WO03/073982, WO2004/072116, WO2004/067568, EP0267611B1, EP0364778B1 and U. S. application number 11/472813.As non-limitative example, antibody A B5 and AB7 (U. S. application number 11/472813, WO2007/002261) can use according to the present invention.The variable region sequences of AB5 and AB7 is as follows:

AB5

Light chain

DIQMTQTTSSLSASLGDRVTISC RASQDISNYLSWYQQKPDGTVKLLIY YTSKLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFC LQGKMLPWTFGGGTKLEIK(SEQIDNO：3)

There is sequence description (from left to right) CDR1,2 and 3 of underscore.

Heavy chain

QVTLKESGPGILKPSQTLSLTCSFSGFSLS TSGMGVGWIRQPSGKGLEWLA HIWWDG DESYNPSLKTQLTISKDTSRNQVFLKITSVDTVDTATYFCAR NRYDPPWFVDWGQGTLVTVSS(SEQIDNO：4)

There is sequence description (from left to right) CDR1,2 and 3 of underscore.

AB7

Light chain

DIQMTQSTSSLSASVGDRVTITC RASQDISNYLSWYQQKPGKAVKLLIY YTSKLHSGVPSRFSGSGSGTDYTLTISSLQQEDFATYFC LQGKMLPWTFGQGTKLEIK(SEQIDNO：5)

There is sequence description (from left to right) CDR1,2 and 3 of underscore.

Heavy chain

QVQLQESGPGLVKPSQTLSLTCSFSGFSLS TSGMGVGWIRQPSGKGLEWLA HIWWD GDESYNPSLKSRLTISKDTSKNQVSLKITSVTAADTAVYFCAR NRYDPPWFVDWGQGTLVTVSS(SEQIDNO：6)

There is sequence description (from left to right) CDR1,2 and 3 of underscore.

Antibody described herein and antibody fragment can be prepared by any suitable method.Appropriate method for the preparation of this kind of antibody and antibody fragment is known in the art.For the preparation of the additive method of antibody and antibody fragment as described herein as part of the present invention.As described herein, antibody of the present invention, antibody fragment or polypeptide can isolated or purified extremely any degree.As used herein, the compound of separation is the compound taken out from its natural surroundings.The compound of purification is the compound increased in purity, thus with its (i) in its natural surroundings time or (ii) compare when at first synthesizing and/or increase in laboratory conditions, this compound exists with purer form, wherein " purity " is relative terms, not necessarily refers to " absolute purity ".

Pharmaceutical composition

IL-1 (such as, IL-1 β) binding antibody used according to the invention and antibody fragment can be formulated in compositions, particularly in pharmaceutical composition, for using in method herein.This kind of compositions comprises the treatment mixed with suitable carrier or the IL-1 beta binding antibodies of the present invention preventing effective dose or antibody fragment, and described carrier is pharmaceutically acceptable reagent such as.Usually, in order to use to animal, before being formulated in pharmaceutical composition, abundant purification IL-1 beta binding antibodies of the present invention and antibody fragment.

Pharmaceutically acceptable reagent comprises carrier, excipient, diluent, antioxidant, antiseptic, coloring agent, flavoring agent and diluent, emulsifying agent, suspending agent, solvent, filler, extender (bulkingagent), buffer agent, delivery vehicle, tonicity agents, cosolvent, wetting agent, chelating agent, buffer reagent, antimicrobial and surfactant.

Neutral buffered saline or the saline mixed with albumin are exemplary suitable carriers.Pharmaceutical composition can comprise antioxidant such as ascorbic acid; Low molecular weight polypeptide; Protein, such as serum albumin, gelatin or immunoglobulin; Hydrophilic polymer is polyvinylpyrrolidone such as; Aminoacid is glycine, glutamine, agedoite, arginine or lysine such as; Monosaccharide, disaccharide and other carbohydrates, comprise glucose, mannose or dextrin; Chelating agen is EDTA such as; Sugar alcohol such as mannitol or Sorbitol; Salt-forming counterion is sodium such as; And/or nonionic surfactant such as Tween, general stream Buddhist nun gram (pluronics) or Polyethylene Glycol (PEG).In addition, such as, suitable tension-elevating agent comprises alkali halide (preferred sodium chloride or potassium), mannitol, Sorbitol etc.Suitable antiseptic comprises benzalkonium chloride, thimerosal, phenethanol, methyl parahydroxybenzoate, propyl p-hydroxybenzoate, chlorhexidine, sorbic acid etc.Hydrogen peroxide also can be used as antiseptic.Suitable cosolvent comprises glycerol, propylene glycol and PEG.Suitable chelating agent comprises caffeine, polyvinylpyrrolidone, beta-schardinger dextrin-or HP-β-CD.Suitable surfactant or wetting agent comprise sorbitan ester, polysorbate such as polysorbate80, tromethane, lecithin, cholesterol, tyloxapal etc.Buffer agent can be Conventional buffering agents such as acetate, borate, citrate, phosphate, bicarbonate or Tris-HCl.Acetate buffer can be about pH4-5.5, and Tris buffer can be about pH7-8.5.Other pharmaceutical agents is at Remington ' sPharmaceuticalSciences, and the 18th edition, A.R.Gennaro, editor, MackPublishingCompany, sets forth in 1990.

Compositions can be liquid form or lyophilizing or freeze-dried, and one or more freezing protectants, excipient, surfactant, high molecular structural additives and/or extender can be comprised (see such as United States Patent (USP) 6,685,940,6,566,329 and 6,372,716).In one embodiment, comprise freezing protectant, it is non-reducing sugar such as sucrose, lactose or trehalose.The amount of the freezing protectant generally comprised makes preparation obtained after reconstitution to be isotonic, although height oozes or slightly hypotonic preparation also can be suitable.In addition, the amount of freezing protectant should be enough to stop the protein degraded of unacceptable amount and/or gathering after freeze drying.Before lyophilizing in preparation for sugar (such as, sucrose, lactose, trehalose) exemplary freezing protectant concentration be that about 10mM-is about 400mM.In another embodiment, comprise surfactant, such as nonionic surfactant and ionic surfactant, such as polysorbate (such as polysorbate20, polysorbate80); Poloxamer (such as PLURONICS F87); PEG phenyl ether (such as Triton); Sodium lauryl sulphate (SDS); Sodium lauryl sulfate; OG sodium; Lauryl-, myristyl-, sub-oil base-or stearyl-sulfobetaines; Lauryl-, myristyl-, sub-oil base-or stearyl-sarcosine; Sub-oil base-, myristyl-or cetyl-betanin; Lauramido-, Cocamidopropyl-, sub-oleoyl aminopropyl-, myristoyl aminopropyl-, palmitoylamino propyl group-or isostearoyl aminopropyl-betanin (such as lauramido); Myristoyl aminopropyl-, palmitoylamino propyl group-or isostearoyl aminopropyl-dimethylamine; Sodium methyl cocoyl taurate or methyl oil base-taurine disodium; And MONAQUAT ^tMthe copolymer of series (MonaIndustries, Inc., Paterson, NJ.), Polyethylene Glycol, polypropylene glycol and ethylene glycol and propylene glycol (such as general stream Buddhist nun gram, PF68 etc.).The Exemplary amounts that may reside in the surfactant in pre-lyophilization formulation is about 0.001-0.5%.High molecular structural additives (such as filler, binding agent) such as Radix Acaciae senegalis can be comprised, albumin, alginic acid, calcium phosphate (secondary), cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose, hydroxyethyl-cellulose, hydroxypropyl cellulose, hydroxypropyl emthylcellulose, microcrystalline Cellulose, glucosan, dextrin, dextrates, sucrose, tylose, pre-gelatinized starch, calcium sulfate, amylose, glycine, bentonite, maltose, Sorbitol, ethyl cellulose, sodium hydrogen phosphate, disodium hydrogen phosphate, disodium pyrosulfite, polyvinyl alcohol, gelatin, glucose, guar gum, liquid glucose, sompressible sugar, aluminium-magnesium silicate, maltodextrin, polyethylene glycol oxide, polymethacrylates, polyvidone, sodium alginate, Tragacanth microcrystalline Cellulose, starch and zein.The exemplary concentration of high molecular structural additives is 0.1 % by weight-10 % by weight.In other embodiments, extender (such as, mannitol, glycine) can be comprised.

Compositions can be suitable for parenteral administration.Exemplary composition be adapted to pass through technical staff can the injection of any approach or be infused in animal body, such as, in intraarticular, subcutaneous, intravenous, intramuscular, intraperitoneal, brain in (in brain essence), Intraventricular, intramuscular, ophthalmic, intra-arterial, damage, internal rectum, percutaneous, per os and inhalation route.Parenteral administration generally by be aseptic, without pyrogen, isotonic aqueous solution, optionally comprise pharmaceutically acceptable antiseptic.

The example of nonaqueous solvent comprises propylene glycol, Polyethylene Glycol, vegetable oil as olive oil and injectable organic ester such as ethyl oleate.Aqueous carrier comprises water, alcohol/aqueous solution, emulsion or suspension, comprises saline and buffer medium.Parenteral vehicles comprises sodium chloride solution, woods grignard glucose, glucose and sodium chloride, Lactated Ringer'S Solution or fixed oil.Intravenous vehicles comprises liquid and supplementary, electrolyte replenisher, such as, based on those of woods grignard glucose, etc.Also antiseptic and other additives can be there are, such as, antimicrobial, antioxidant, chelating agen, noble gas etc.Generally see, the Remington ' sPharmaceuticalScience be incorporated herein by reference, the 16th edition, Mack edits, and 1980.

Pharmaceutical composition described herein can be prepared for controlling by this way or continual delivery, described mode provides the sustained release of product (such as, bolus injection (bolus), reservoir (depot) effect) of local concentration and/or the stability of increase or half-life in specific portion environment.The present invention considers, in certain embodiments, this kind of compositions can comprise remarkable more substantial antibody or fragment in initial stock, and according to present disclosure upper release in fact at any time and the effective dose of available antibody or fragment is the amount more much lower than initial stock.Compositions can comprise IL-1 beta binding antibodies of the present invention, antibody fragment, nucleic acid or carrier and polymerizable compound (such as polylactic acid, polyglycolic acid etc.) particle product and reagent such as biodegradable substrate, Injectable microspheres body, microcapsule granule, microcapsule, biology can lose the preparation separating granule pearl, liposome and implantable delivery apparatus (providing control or the sustained release of activating agent), and said preparation can be sent with the form of reservoir devices injection subsequently.Continue or control the technology of delivery means to be known for preparing this kind, and various polymer has been developed and for medicine Co ntrolled release and send.This kind of polymer is generally biodegradable and biocompatible.Polyalcohol hydrogel, comprise by the complexation of enantiomer polymer or polypeptide section and the polyalcohol hydrogel formed, and there is the hydrogel of temperature or pH sensitive natur, due to the gentle aqueous conditions related to when catching biological activity protein reagent (such as antibody), and may for providing desired by drug depot effect.See such as, for the description of the Co ntrolled release porous polymer particle of delivering drugs compositions in the open WO93/15722 of PCT application.

Suitable material for this purposes comprises polylactic acid (see such as, United States Patent (USP) 3, 773, 919), poly-(a-hydroxy carboxylic acid) polymer, such as poly-D-(-)-3-hydroxybutyric acid (EP133, 988A), copolymer (the people such as Sidman of Pidolidone and γ ethyl-L-glutamate ester, Biopolymers, 22:547-556 (1983)), poly-(2-hydroxyethyl methacrylate) (people such as Langer, 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 biological degradable polymer comprises poly-(lactone), poly-(acetal), poly-(ortho esters) and poly-(orthocarbonic ester).Sustained-release composition can also comprise liposome, it can be prepared by any one in several methods known in the art (see such as, the people such as Eppstein, Proc.Natl.Acad.Sci.USA, 82:3688-92 (1985)).Carrier self or its catabolite should be nontoxic in target tissue, and disease should do not made to worsen further.This can be measured by the routine screening in the animal model of target disease, if or this kind of unavailable words of model, measure in intact animal.

Microencapsulation for the recombinant protein of sustained release has used human growth hormone (rhGH), interferon-(rhIFN-), interleukin-2 and MNrgp120 successfully to carry out.The people such as Johnson, Nat.Med., 2:795-799 (1996); Yasuda, Biomed.Ther., 27:1221-1223 (1993); The people such as Hora, Bio/Technologv.8:755-758 (1990); Cleland, " DesignandProductionofSingleImmunizationVaccinesUsingPoly lactidePolyglycolideMicrosphereSystems; " inVaccineDesign:TheSubunitandAdjuvantApproach, PowellandNewman, editor, (PlenumPress:NewYork, 1995), 439-462 page; WO97/03692, WO96/40072, WO96/07399; With U.S. Patent number 5,654,010.The extended release preparation of these protein uses polylactide co-glycolide (PLGA) polymer (the biodegradable character due to its biocompatibility and broad range) to develop.The catabolite of PLGA---lactic acid and glycolic can be removed fast in human body.In addition, the degree of degradation of this polymer can depend on its molecular weight and composition.Lewis, " Controlledreleaseofbioactiveagentsfromlactide/glycolidep olymer; " in:M.ChasinandR.Langer (editor), BiodegradablePolymersasDrugDeliverySystems (MarcelDekker:NewYork, 1990), 1-41 page.The other example of sustained-release composition comprises such as EP58,481A, U.S. Patent number 3,887,699, EP158,277A, the people such as Canadian Patent numbers 1176565, U.Sidman, Biopolymers22, the people such as 547 [1983], R.Langer, Chem.Tech.12,98 [1982], the people such as Sinha, J.Control.Release90,261 [2003], the people such as Zhu, Nat.Biotechnol.18,24 [2000], and the people such as Dai, ColloidsSurfBBiointerfaces41,117 [2005].

Bioadhesive polymer is also expected for using in the present compositions or using together with compositions of the present invention.Bioadhesive polymer be can with the synthesis adhered to for a long time at the bottom of bio-based and naturally occurring material.Such as, carbomer and polycarbophil are the synthesizing cross-linked derivants of poly-(acrylic acid).Bioadhesive delivery systems based on natural occuring article matter comprises such as hyaluronic acid, also referred to as hyaluronan.The naturally occurring mucopolysaccharide that hyaluronic acid is made up of D-glucuronic acid and N-acetyl-GLUCOSAMINE residue.Hyaluronic acid is present in vertebrate ECT substrate, is included in connective tissue, and in the vitreous humor and aqueous humor of synovial fluid and eye.Hyaluronic esterification derivative is used for using in sending for generation of microsphere, described microsphere be bio-compatible and biodegradable (see such as, the people such as Cortivo, Biomaterials (1991) 12:727-730; European publication number 517,565; International publication number WO96/29998; The people such as Ilium, J.ControlledReI. (1994) 29:133-141).The amount that exemplary composition containing hyaluronic acid of the present invention is about 0.1% to about 40% (w/w) with the ratio of IL-1 beta binding antibodies or fragment and hyaluronic acid polymer comprises hyaluronic acid ester polymer.

Biodegradable and biological non-degradable polymeric matrices may be used for according to delivering compositions of the present invention, and this kind of polymeric matrices can comprise natural or synthetic polymer.Biodegradable substrate is preferred.The time period of release generation depends on the selection of polymer.Usually, go through a few hours to wish most to the 3-12 release of individual month.The Exemplary synthetic polymers that may be used for being formed biodegradable delivery system comprises: the polymer of lactic acid and glycolic, polyamide, Merlon, polyolefin, poly alkylene glycol, polyalkylene oxide, polyalkylene terephthalates ester, polyvinyl alcohol, polyvinylether, polyvinyl ester, polyvinyl halides, polyvinylpyrrolidone, PGA, polysiloxanes, polyanhydride, polyurethanes and copolymer thereof, poly-(butanoic acid), poly-(valeric acid), alkylcellulose, hydroxy alkyl cellulose, cellulose ether, cellulose esters, NC Nitroncellulose, the polymer of acrylic acid and methacrylate, methylcellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl emthylcellulose, hydroxy butyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate-butyrate, Cellacefate, carboxyethyl cellulose, cellulose triacetate, sulfate cellulose sodium salt, poly-(methyl methacrylate), poly-(ethyl methacrylate), poly-(butyl methacrylate), poly-(isobutyl methacrylate), poly-(N-Hexyl methacrylate), poly-(isodecyl methacrylate), poly-(lauryl methacrylate), poly-(phenyl methacrylate), poly-(acrylic acid methyl ester .), poly-(isopropyl acrylate), poly-(Isobutyl 2-propenoate), poly-(octadecyl acrylate), polyethylene, polypropylene, PEG, poly-(oxirane), poly-(PETP), poly-(vinyl alcohol), polyvinyl acetate, polrvinyl chloride, polystyrene and polyvinylpyrrolidone.Exemplary natural polymer comprises alginate and other polysaccharide, comprise glucosan and cellulose, collagen, its chemical derivative (replacement of chemical group, interpolation, such as alkyl, thiazolinyl, hydroxylating, oxidation and undertaken by those skilled in the art's routine other modify), albumin and other hydrophilic protein, zein and other prolamin and hydrophobic protein, copolymer and composition thereof.Generally speaking, these materials are by enzymatic hydrolysis in vivo or be exposed to water, degraded by surface or bulk erosion (Bulkerosion).Polymer is optionally that the form of hydrogel is (see such as WO04/009664, WO05/087201, the people such as Sawhney, Macromolecules, 1993,26,581-587), it can absorb the water up to about 90% its weight, and further optionally with multivalent ion or other crosslinked polymers.

Delivery system also comprises following non-polymer systems, and it is lipid, comprises sterin such as cholesterol, cholesteryl ester and fatty acid or neutral fat such as glycerol list, two and three esters; Hydrogel delivery system; Silicone rubber system; Based on the system of peptide; Wax coating; Use the compressed tablets of traditional binders and excipient; The implants of partial fusion; Deng.Object lesson includes but not limited to: (a) be the dissolution systems that is included with form in the substrate of product wherein, such as U.S. Patent number 4,452,775,4,675,189 and 5,736, describe in 152 those, and (b) wherein diffusion system of infiltrating from polymer with speed control of product, such as at U.S. Patent number 3,854,480,5,133,974 and 5,407, describe in 686.The liposome comprising product can be prepared by known method, such as (DE3,218,121; The people such as Epstein, Proc.Natl.Acad.Sci.USA, 82:3688-3692 (1985); The people such as Hwang, Proc.Natl.Acad.Sci.USA, 77:4030-4034 (1980); EP52,322; EP36,676; EP88,046; EP143,949; EP142,641; Japanese patent application 83-118008; U.S. Patent number 4,485,045 and 4,544,545; And EP102,324).

The pharmaceutical composition comprising IL-1 beta binding antibodies or fragment can be prepared for sucking, such as, as dry powder.Inhalation solution also can carry out preparing for Aerosol delivery in liquefied propellant.In another one embodiment, solution can carry out atomization.The other pharmaceutical composition used for lung comprises those that such as describe in PCT patent application WO94/20069, described in patent document discloses chemically modified protein matter lung send.Send for lung, particle size should be suitable for being delivered to far-end lung.Such as, particle size can be 1 μm-5 μm; But, can larger particles be used, if such as each granule is many holes.

Some preparation comprising IL-1 beta binding antibodies or draw together body piece section can carry out Orally administered.The preparation used by this way can with or need not prepare by carrier as follows, described carrier is conventional in the preparation of solid dosage forms such as Tablet and Capsula.Such as, capsule can be designed as in the gastrointestinal tract in bioavailability can be made to maximize and before circulation minimum degradation point on the active part of delivery formulations.Other reagent can be comprised, to promote the absorption of selective binding agent.Diluent, flavoring agent, low melt wax, vegetable oil, lubricant, suspending agent, tablet disintegrant and binding agent can also be adopted.

Another kind of preparation can relate to and the IL-1 beta binding antibodies of the effective dose of non-toxic mixed with excipients or fragment, and described excipient is suitable for manufacturing tablet.By making tablet dissolved in sterilized water or another kind of suitable vehicle, solution can be prepared with unit dosage forms.Suitable excipient includes but not limited to, inert diluent is calcium carbonate, sodium carbonate or sodium bicarbonate, lactose or calcium phosphate such as; Or bonding agent such as starch, gelatin or Radix Acaciae senegalis; Or lubricant such as magnesium stearate, stearic acid or Talcum.

Suitable and/or preferred pharmaceutical preparation can be determined based on the general knowledge of present disclosure and preparation technique, and this depends on expection route of administration, delivery form and required dosage.Have nothing to do with method of application, effective dose can calculate according to weight in patients, body surface area or organ size.The appropriate dosage for treatment becoming more meticulous further to determine to relate to often kind of preparation as herein described is calculated to these, is conventional in the art, and belongs to the category of this area conventional practice.Suitable dosage can be determined by using suitable dose response data.

According to present disclosure, other preparation will be apparent, comprise the preparation of IL-1 beta binding antibodies and the fragment related to one or more combination with other therapeutic agents.Such as, in some preparation, IL-1 beta binding antibodies of the present invention, antibody fragment, nucleic acid or carrier are prepared together with the second inhibitor of IL-1 signal pathway.Representative second inhibitor includes but not limited to, antibody, antibody fragment, peptide, polypeptide, compound, nucleic acid, carrier and pharmaceutical composition, such as, describe in US6899878, US2003022869, US20060094663, US20050186615, US20030166069, WO/04022718, WO/05084696, WO/05019259 those.Such as, compositions can comprise and the IL-1 beta binding antibodies of the present invention of the nucleic acid of IL-1 beta binding antibodies, fragment or encode this kind of antibody or fragment or carrier combinations, antibody fragment, nucleic acid or carrier.

Pharmaceutical composition can comprise the IL-1 beta binding antibodies or fragment that combine with other activating agents.This kind of combination is for the useful combination of its desired use.This type of combination as a part of the present invention can be IL-1 β antibody and fragment, such as described herein those, and be selected from the other activating agent of following at least one.The activating agent of hereafter setting forth is illustrative, and not intended to be is construed as limiting.This combination can also comprise and exceed a kind of other activating agent, such as 2 kinds or 3 kinds of other activating agents, as long as this combination can make formed compositions can perform its expectation function.

The present invention further considers, comprise one or more other activating agents pharmaceutical composition can with IL-1 beta binding antibodies or fragment separate administration, and this kind of separate administration can be carried out on same time point or different time points, such as on the same day or not on the same day.When with IL-1 beta binding antibodies or fragment use be combined time, using of these other activating agents can be carried out according to SMP known in the art, maybe can this be used and is modified (such as, longer interval, more low dose of, postpone initial), such as disclosed herein.

Activating agent or comprise nonsteroidal antiinflammatory drug (NSAID) with the combination of antibody of the present invention or fragment, such as aspirin (aspirin), ibuprofen (ibuprofen) and other propanoic derivatives (alminoprofen (alminoprofen), benoxaprofen (benoxaprofen), bucloxic acid (bucloxicacid), carprofen (carprofen), fenbufen (fenbufen), fenoprofen (fenoprofen), fluprofen (fluprofen), flurbiprofen (flurbiprofen), indoprofen (indoprofen), ketoprofen (ketoprofen), miroprofen (miroprofen), naproxen (naproxen), oxaprozin (oxaprozin), pirprofen (pirprofen), pranoprofen (pranoprofen), suprofen (suprofen), tiaprofenic acid (tiaprofenicacid) is with tioxaprofen (tioxaprofen)), acetogenin (indomethacin (indomethacin), acemetacin (acemetacin), alclofenac (alclofenac), clidanac (clidanac), diclofenac (diclofenac), fenclofenac (fenclofenac), fenclozic acid (fenclozicacid), fentiazac (fentiazac), furofenac (fuirofenac), ibufenac (ibufenac), Isoxepac (isoxepac), oxpinac, sulindac (sulindac), tiopinac (tiopinac), tolmetin (tolmetin), zidometacin (zidometacin) and zomepirac (zomepirac)), fragrant that acid (fenamicacid) derivant (flufenamic acid (flufenamicacid), meclofenamic acid (meclofenamicacid), mefenamic acid (mefenamicacid), niflumic acid (niflumicacid) and tolfenamic acid (tolfenamicacid)), biphenyl carboxylic acid's derivant (diflunisal (diflunisal) and flufenisal (flufenisal)), thiazole amide (oxicams) (isoxicam (isoxicam), piroxicam (piroxicam), sudoxicam (sudoxicam) and tenoxicam (tenoxican)), salicylic acid (aspirin (acetylsalicylicacid), sulfasalazine (sulfasalazine)) and pyrazoline ketone (azapropazone (apazone), bezpiperylon, feprazone (feprazone), mofebutazone (mofebutazone), oxyphenbutazone (oxyphenbutazone), Phenylbutazone (phenylbutazone)).Other combinations comprise cyclo-oxygenase-2 (COX-2) inhibitor.Other activating agents for combining comprise steroid, such as prednisolone (prednisolone), prednisone (prednisone), methylprednisolone (methylprednisolone), betamethasone (betamethasone), dexamethasone (dexamethasone) or hydrocortisone (hydrocortisone).This kind of combination can be particularly advantageous, because when treating patient with antibody of the present invention and fragment combination, by reducing required steroid dosage gradually, can reduce or even eliminate one or more side effect of steroid.

Alternately or additionally, the treatment of associating at least one or multiple other activating agent can be used, wherein said other activating agent can be played a role by different binding mode: 1) sulfonylurea that stimulates insulin secretion of substance (such as, chlorpropamide (chlorpropamide), tolazamide (tolazamide), acetohexamide (acetohexamide), tolbutamide (tolbutamide), glibenclamide (glyburide), glimepiride, glipizide) and/or meglitinides (such as repaglinide (repaglinide), Nateglinide (nateglinide)), 2) by the biguanides (such as, metformin) promoting glucose utilization, reduce Hepatic glucose production and reduce intestinal glucose to export and play a role, 3) carbohydrate digestion is slowed down and thus from the absorption of intestinal and the Alpha-glucosidase inhibitor (such as, acarbose, miglitol) reducing postprandial hyperglycemia, 4) strengthen insulin action thus promote the thiazolidinediones (such as, troglitazone, pioglitazone, rosiglitazone, glipizide, Ba Gelie ketone, RIVOGLITAZONE, netoglitazone, troglitazone, englitazone, AD5075, T174, YM268, R102380, NC2100, NIP223, NIP221, MK0767, ciglitazone, adaglitazone, CLX0921, darglitazone, CP92768, BM152054) of the glucose utilization in peripheral tissues thus, 5) glucagon-like peptide, comprises DPP4 inhibitor (such as, sitagliptin), with 6) stimulate the insulin organized glucose utilization and suppress hepatic glucose to export.Glucagon-like-peptide-1 (GLP-1), DPP-IV-resistant analogs (incretin analogies), DPP-IV inhibitor, insulin, insulin analog, PPAR gamma agonist, dual-acting PPAR agonists, GLP-1 agonist or analog, PTP1B inhibitor, SGLT inhibitor, insulin secretagogue element, rxr agonist, glycogen synthase kinase-3 inhibitors, insulin sensitisers, immunomodulator, β-3 3 adrenergic receptor agonists, Pan-PPAR agonist, 11 beta-HSD 1 inhibitors, amylin analogue, biguanides, Alpha-glucosidase inhibitor, meglitinides, thiazolidinediones, sulfonylureas etc. also can be used as described other activating agent (see such as, Nathan, 2006, N.Engl.J.Med.355:2477-2480, the people such as Kahn, 2006, N.Engl.J.Med.355:2427-2443).In another one embodiment, activating agent can be HMGCo-A reductase inhibitor (such as, Statins (statins)).

Further consider the anti-il-i-beta antibody used to experimenter according to the present invention or fragment can with the other activating agent of at least one, such as the treatment of any above-mentioned activating agent is combined uses.In one embodiment, the treatment with this at least one activating agent is maintained.In another embodiment, reduce or interrupt (such as, when experimenter stablizes) with treatment of this at least one activating agent, and with the treatment of constant dosage regimen maintenance anti-il-i-beta antibody or fragment.In another embodiment, reduce or interrupt (such as, when experimenter stablizes) with the treatment of this at least one activating agent, and reduce (such as, compared with low dosage, lower frequency administration, shorter therapeutic scheme) treatment by anti-il-i-beta antibody or fragment.In another embodiment, reduce or interrupt (such as, when experimenter stablizes) with the treatment of this at least one activating agent, and increase (such as, higher dosage, upper frequency administration, longer therapeutic scheme) treatment by anti-il-i-beta antibody or fragment.In another one embodiment, maintain the treatment with this at least one activating agent, and reduce or interrupt the treatment that (such as, compared with low dosage, lower frequency administration, shorter therapeutic scheme) uses anti-il-i-beta antibody or fragment.In another one embodiment, reduce or interrupt (such as, compared with low dosage, lower frequency administration, shorter therapeutic scheme) with the treatment of this at least one activating agent and the treatment by anti-il-i-beta antibody or fragment.

The pharmaceutical composition used in the present invention can comprise IL-1 beta binding antibodies or the fragment for the treatment of effective dose or prevention effective dose.Treatment effective dose refers to effectively to reach with required dosage and time the amount expecting therapeutic outcome.The treatment effective dose of antibody or antibody moiety can become according to following factor, such as individual morbid state, age, sex and weight, and antibody or antibody moiety cause required ability of replying in individuality.Treatment effective dose is also wherein treat favourable effect to exceed any toxicity of antibody or antibody moiety or the amount of ill-effect.Prevention effective dose refers to effectively to reach with required dosage and time the amount expecting prevention result.

The treatment or the prevention effective dose that comprise the pharmaceutical composition of IL-1 beta binding antibodies or fragment such as will depend on therapeutic goal, the situation of such as, indication used for compositions, route of administration and experimenter and becoming.Pharmaceutical composition is used with treatment or prevention effective dose, to treat IL-1 associated conditions." treatment or the prevention effective dose " of IL-1 beta binding antibodies of the present invention or fragment is the amount can treating or prevent one or more symptoms (as disclosed herein) of IL-1 relevant disease in experimenter.

Using method

The anti-il-i-beta antibody of effective dose can be used for the treatment of and/or prevent type 1 diabetes, type 2 diabetes mellitus, obesity, hyperglycemia, hyperinsulinemia, insulin resistance in the present invention and be characterised in that morbid state and the disease of insulin resistance.This kind of method may be used for treating the mammalian subject of morbid state and the disease suffered from type 2 diabetes mellitus, type 1 diabetes, obesity, hyperglycemia, hyperinsulinemia, insulin resistance and be characterised in that insulin resistance (such as, people), or in risk subjects, prevent its generation.

As used herein, the mechanism (such as administered compound or pharmaceutical composition) that mode that the clinical manifestation that term " prevention ", " prevention ", " checking ", " preventing " and " suppression " refer to temporarily or for good and all to prevent, check or reduce morbid state or disease occurs (such as, before the clinical manifestation of morbid state or disease occurs) starts.This kind prevents, check or reduce not necessarily definitely useful.

As used herein, term " treatment " refers to the mechanism (such as administered compound or pharmaceutical composition) started after the onset of clinical symptoms of morbid state or disease, temporarily or for good and all to eliminate, to reduce, to check or to improve clinical manifestation or the process of morbid state or disease.This kind for the treatment of is not necessarily definitely useful.

As used herein, term " needs to treat " patients needing treatment referring to be made by Senior Nurse (caregiver) or the judgement that will benefit from treat.This judgement is made based on various factors, these factors in the expertise of Senior Nurse, but comprise about patient because by method of the present disclosure or compounds for treating disease sick or will be sick knowledge.

As used herein, term " needs to prevent " the needs of patients prevention referring to be made by Senior Nurse or the judgement will benefited from prevention.This judgement is made based on various factors, these factors in the expertise of Senior Nurse, but comprise about patient because by method of the present disclosure or compound prevention disease will sick or may be sick knowledge.

As used herein, term " treatment effective dose " refers to compound, separately or as the part of pharmaceutical composition, when being applied to patient (such as, with one or more dosage) time, this amount can have the amount of any detectable positive effect to any symptom of morbid state or disease, aspect or feature.Described effect is not necessarily definitely useful.

As used herein, term " insulin resistance " refers to that the insulin of wherein normal amount cannot produce the disease of normal physiological or Molecular responses.In some cases, the insulin of the supraphysiologic amount that endogenous generation or external source are added can overcome insulin resistance in whole or in part, produces biological answer-reply.

Anti-il-i-beta antibody or fragment can be applied to people with effective dose, are used for the treatment of and/or prevent type 2 diabetes mellitus, type 1 diabetes, obesity, hyperglycemia, hyperinsulinemia, insulin resistance and be characterised in that morbid state and the disease of insulin resistance.Consider with anti-il-i-beta antibody according to the present invention or fragment treatment other diseases or disease comprise prediabetes, dyslipidemia, hyperlipemia, hypertension, metabolism syndrome or illness behavior.The present invention further considers the incidence rate or the seriousness that use this kind of antibody or fragment to reduce the complication relevant to type 2 diabetes mellitus or disease, or stablize the complication relevant to type 2 diabetes mellitus or disease, described complication or disease are, such as retinopathy, renal failure, cardiovascular disease (such as, atherosclerosis, peripheral blood vessel) and wound healing (such as, diabetic ulcer),

In addition, the present invention also considers that IL-1 β antibody as described herein and fragment reduce the purposes of c reactive protein (CRP) level in experimenter.CRP is primarily of the acute phase protein that hepatocyte produces under the impact of cytokine, described cytokine such as IL-1, IL-6 and tumor necrosis factor (TNF).Based on the internal medicine textbook of 2007 electronic versions although lack to inflammation reason (such as, infection, chronic kidney diseases, self inflammatory diseases, cancer) specificity, but confirmed the serum that raises or plasma C RP concentration and potential atherosclerotic prevalence rate from more than the data of 30 epidemiological studies, in the patient with built vertical disease, recurred the risk of cardiovascular event and the generation significant correlation of cardiovascular event first in Atherosclerosis Risk individuality.In addition, modify after Primary renal disease, the renal failure caused thus and oxidative stress thereof and protein synthesis, with contaminants associated dialysis and dialyzer on the impact of serum proteins and to enter relevant infection and systemic infection subsequently, these the inflammatory stimulus load causing patient excessive that influences each other at entry site repeatedly.When serum creatinine removes level along with the decreased renal function worsened, there is serum inflammatory mediator (such as, cytokine TNF, IL-6, IL-1) proportional rising, and body is attempted producing with (but invalid) IL-1RA and IL-10 (Anti-inflammatory mediator) increased resisting the sign of this state.Due to the direct triggering of vascular smooth muscle cell apoptosis, this inflammatory conditions in patients with chronic renal failure causes the unstability of atheromatous plaque.The consequence that cytokine raises causes one of 2 kinds of topmost death in these patients---from the remarkable increase of the cardiovascular death of myocardial infarction and apoplexy.The direct illustration of the danger of this increase can be seen by assess patient CRP level; When being divided into the quartile of CRP value, the group with the highest CRP value has 12 months mortality rates of about 35%.Therefore, the invention discloses the purposes that IL-1 β antibody as provided herein or fragment reduce the CRP level in this kind of patient (such as, suffering from the experimenter of kidney diaseases).As described herein, in experimenter, reduce the suitable means that CRP level is the corresponding proportional reduction realizing cardiovascular morbidity and mortality rate.

In one embodiment, anti-il-i-beta antibody or fragment are applied to the experimenter with the above-mentioned disease of at least one, disease or complication, and experimenter also accept at least one other for this disease, disease or complication medically generally acknowledge treatment (such as, medication, medicine, therapeutic agent, activating agent).In another embodiment, reduce or interrupt (such as, when experimenter stablizes) other the treatment of medically generally acknowledging for this disease, disease or complication of this at least one, and with the treatment of constant dosage regimen maintenance anti-il-i-beta antibody or fragment.In another embodiment, reduce or interrupt (such as, when experimenter stablizes) this at least one other for this disease, disease or complication medically generally acknowledge treatment, and reduce (such as, more low dosage, more low frequency administration, shorter therapeutic scheme) treatment by anti-il-i-beta antibody or fragment.In another embodiment, reduce or interrupt (such as, when experimenter stablizes) this at least one other for this disease, disease or complication medically generally acknowledge treatment, and increase (such as, more high dose, higher frequency administration, longer therapeutic scheme) treatment by anti-il-i-beta antibody or fragment.In another one embodiment, maintain this at least one other for this disease, disease or complication medically generally acknowledge treatment, and reduce or interrupt (such as, more low dosage, more low frequency administration, shorter therapeutic scheme) treatment by anti-il-i-beta antibody or fragment.In another one embodiment, reduce or interrupt (such as, more low dosage, more low frequency administration, shorter therapeutic scheme) other the treatment of medically generally acknowledging for this disease, disease or complication of this at least one, and with the treatment of anti-il-i-beta antibody or fragment.

Treating or preventing above-mentioned disease or disease (such as, type 1 diabetes, type 2 diabetes mellitus, hyperglycemia, hyperinsulinemia, obesity, insulin resistance) method for optimizing in, according to amount and/or the dosing interval of above-mentioned number of doses, every dosage, anti-il-i-beta antibody or its fragment are applied to people experimenter.Alternately, anti-il-i-beta antibody or fragment can be used with the predose of the above-mentioned amount of one or more tool, and the amount of this predose is lower than the amount of one or more subsequent dose.By providing predose with relatively low amount, effectiveness and/or the toleration for the treatment of can be strengthened.Such as, in a non-limiting embodiments of the present invention, can use≤1mg/kg (such as, ≤ 0.9mg/kg, ≤ 0.8mg/kg, ≤ 0.7mg/kg, ≤ 0.6mg/kg, ≤ 0.5mg/kg, ≤ 0.4mg/kg, ≤ 0.3mg/kg, ≤ 0.2mg/kg, ≤ 0.1mg/kg, ≤ 0.05mg/kg, ≤ 0.03mg/kg, ≤ 0.01mg/kg) antibody or fragment amount one or more predoses (such as, 1, 2, 3, 4, 5), subsequently to exceed the amount of predose (such as, >=0.01mg/kg, >=0.03mg/kg, >=0.1mg/kg, >=0.3mg/kg, >=0.5mg/kg, >=0.6mg/kg, >=0.7mg/kg, >=0.8mg/kg, >=0.9mg/kg, >=1.0mg/kg, >=1.5mg/kg, >=2mg/kg, >=2.5mg/kg, >=3mg/kg, >=3.5mg/kg, >=4mg/kg, >=4.5mg/kg, >=5mg/kg) use one or more subsequent dose.The present invention considers that each dosage of antibody or fragment can be used in one or more site.

According to the present invention's treatment or to prevent disease or the method for disease can use predetermined or " conventional " antibody or fragment time of application table.As used herein, routine schedule refers to fixed time section predetermined between two doses is used.Routine schedule can be encompassed in the length identical or different time period, as long as this timetable is predetermined.This routine schedule can cover any particular combination, as long as it is determine in advance that this suitable timetable relates to using of one day.

The present invention further considers, the IL-1 β antibody used according to method provided herein or fragment can combine with multiple conventional treatments and pharmaceutical composition (such as, activating agent) to be used.This kind of compositions can comprise such as, DPP-IV inhibitor, insulin, insulin analog, PPAR gamma agonist, dual-acting PPAR agonists, GLP-1 agonist or analog, PTP1B inhibitor, SGLT inhibitor, insulin short point of element, rxr agonist, glycogen synthase kinase-3 inhibitors, insulin sensitisers, immunomodulator, β-3 3 adrenergic receptor agonists, Pan-PPAR agonist, 11 beta-HSD 1 inhibitors, amylin analogue, biguanides, Alpha-glucosidase inhibitor, meglitinides, thiazolidinediones, sulfonylureas etc. are (see such as Nathan, 2006, N.Engl.J.Med.355:2477-2480, the people such as Kahn, 2006, N.Engl.J.Med.355:2427-2443).In certain embodiments, the antibody used according to the present invention and fragment can stop or postpone the needs of Therapeutic Method to other or pharmaceutical composition.In other embodiments, antibody or fragment can reduce the amount of other Therapeutic Method or pharmaceutical composition, frequency or persistent period.

Alternately, according to the present invention's treatment or to prevent disease or the method for disease can use such antibody or fragment time of application table, the basis of described timetable is as the means determining when to use one or more subsequent dose by the change of the existence of disease symptoms and/or any one evaluation of estimate herein (improvement of such as, the use of HbA1c, fasting blood glucose level, OGTT, glucose/insulin C-peptide AUC, diabetes medicament, insulin sensitivity, serum cytokines, CRP level, the measuring of quality of life, BMI).Similarly, this method can be used as a kind of means, with the effect based on preceding dosage, determines whether should increase or reduce with post dose.

Can according to SMP known in the art, diagnose this kind of disease or disease in patients or alternately develop the risk of this kind of disease or disease.After using anti-il-i-beta antibody or fragment, be well-known in the art to above-mentioned disease or the treatment of disease or the clinical assessment of preventive effect, and the instrument of the effectiveness of monitoring the inventive method can be used as.

Such as, (HbA1c is assessed to the primary efficacy endpoint (primaryefficacyendpoint) that the response of type 2 diabetes mellitus treatment can improve based on glycated hemoglobin, see people such as such as Reynolds, BMJ, 333 (7568): 586-589,2006).The HbA1c of instruction curative effect improves and can become because of the initial baseline measures in patient, and wherein larger reduction corresponds to higher initial baseline usually, and less reduction corresponds to lower initial baseline usually.In one aspect of the invention, compare with level before administration, the HbA1c that the inventive method should cause at least about 0.5% (such as, 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) reduces.

Also one or more following secondary endpoints can be measured, so that effect of assessment treatment, such as fasting glucose (such as, glucose) level (such as, compare with level before administration, be reduced to≤130 ,≤125 ,≤120 ,≤115 ,≤110 ,≤105 ,≤100, alternately, > 20% is reduced, > 30%, > 40%, > 50%, > 60%, > 70%, > 80%, > 90%, > 95%), 120 minutes oral glucose tolerance tests (OGTT) (such as ,≤200, ≤ 190, ≤ 180, ≤ 170, ≤ 160, ≤ 150, ≤ 140), glucose/insulin C-peptide AUC (such as, increases > 25% than before treatment, > 50%, > 60%, > 70%, > 80%, > 90%, > 100%), diabetes medicament (such as, insulin, oral hypoglycemic) minimizing, the improvement of insulin sensitivity, serum cytokines (such as, normalization), CRP level (such as, reduces >=0.2 than before treatment, >=0.4, >=0.6, >=0.8, >=1.0, >=1.4, >=1.8, >=2.2, >=2.6, >=3.0mg/L, alternately reduce >=20%, >=30%, >=40%, >=50%, >=60%, >=70%, >=80%, >=90%, >=95%), the measuring of quality of life, BMI improve the (people such as Saudek such as (reducing 1%, 3%, 5%), pharmacokinetics, JAMA, 295:1688-97,2006, the people such as Pfutzner, DiabetesTechnolTher.8:28-36,2006, the people such as Norberg, JInternMed.260:263-71,2006).

Similarly, the assessment about effect of other diseases or disease can use one or more above-mentioned terminals and/or other terminals known in the art.Such as, can by measuring fasting glucose (such as on the impact of hyperglycemia, glucose) level assesses, can be assessed by measurement insulin level and/or C-peptide level the impact of hyperinsulinemia, can be assessed by measurement weight and/or/BMI the impact of obesity, and can be assessed by OGTT the impact of insulin resistance.

Alternately, or additionally, the experimenter according to the present invention's treatment can experience triglyceride levels in experimenter's blood relative to level at least 5% before treatment, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, the minimizing of 70% or more.Alternately, or additionally, the experimenter according to the present invention's treatment can experience free fatty acid levels relative to level at least 5% before treatment, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, the minimizing of 60% or more.

Embodiment

Following embodiment is only intended to for illustrating practice of the present invention further, but should not be construed as and limit its scope by any way.The all patents wherein quoted and the disclosure of scientific literature are incorporated herein by reference in this entirety especially.

Embodiment 1

In vitro based on using high-affinity IL-1 β antibody suppression IL-1 β in the determination test of cell, the IL-8 wherein reading IL-1 induction produces

The depression effect of IL-1 β specific antibody and the non-antibody inhibitor of IL-1 approach (Antril (Synergen)) compares, described in it is restructuring IL-1 receptor antagonist.Fresh, Heparinised peripheral blood is collected from healthy donors.180 μ l whole bloods are added 96 orifice plates, and carries out incubation with the antibody A B7 of various concentration (U. S. application number 11/472813, WO2007/002261) and 100pMrhIL-1 β.For the sample of process, before mixing with blood, 1: 1 mixing is carried out with rhIL-1 β.Sample at 37 DEG C, 5%CO ₂lower incubation 6 hours.Use 50 μ l2.5%TritonX-100 cracking complete blood cell subsequently.Interleukin-8 (IL-8) concentration in cleared lysate is assessed according to the description of manufacturer by ELISA (Quantikine people IL-8ELISA test kit, R & DSystems).AB7 and control sample handled by IL-8 concentration in processing sample contrasts with anti-KLH compares.The results are described in Fig. 1, and be summarized in table 6.IC ₅₀be suppression 50% by IL-1 β stimulate the IL-8 that causes discharge needed for antibody concentration.

Table 1

Measured by the suppression of the IL-8 release by stimulating IL-1 β, these results confirm the vitro efficacy of AB7.These results demonstrate with the larger effect compared, indicates this antibody will have IL-1 β in body and suppresses effect.

Embodiment 2

Measured by the impact of the IL-6 release by stimulating IL-1 β, use IL-1 β specific antibody to suppress the biological activity of people IL-1 β in vivo

In order to confirm AB7 body in effect, in mice, test the bioactive ability that AB7 blocks people IL-1 β.The details measured is people such as Economides, and NatureMed., 9:47-52 are described in (2003).In brief, male C57/B16 mice (JacksonLaboratoryBarHarbor, Maine) carries out peritoneal injection by the AB7 of titration dosage, another kind of IL-1 β antibody A B5 or control antibodies.Latter 24 hours of antibody injection, mice carries out subcutaneous injection with the dosage of 1 μ g/kg recombined human IL-1 β (rhIL-1 β) (from PeproTechInc., RockyHill, NJ).RhIL-1 β injects latter 2 hours (peak IL-6 response time), puts to death mice, and collects blood and process to obtain serum.IL-6 is measured according to the scheme of manufacturer by ELISA (BDPharmingen, FranklinLakes, NJ).Percentage ratio is suppressed to be undertaken calculating (being multiplied by 100) by the IL-6 detected in laboratory animal serum and the ratio of the IL-6 detected in control animal serum.

The results are shown in Fig. 2.The function of the IL-6 level suppressing the ability of IL-1 β activity in vivo to stimulate as IL-1 β in serum is assessed.As illustrational by Fig. 2, AB7 and AB5 antibody is effectively for suppressing the activity in vivo of people IL-1 β.The single injection that these results also show AB7 or AB5 can block the systemic effect stimulated IL-1 β, and these antibody are useful for IL-1 'beta ' activity in suppression body.

Perform similar experiment, to confirm during AB7 in vivo further and the ability of mice IL-1 β, to support the use of this antibody in mouse disease model.After measured, AB7 for people IL-1 β have be compared to mice IL-1 β large ~ affinity of 10,000 times, and the vitro efficacy in D10.G4.1 measures be compared to mice IL-1 β large ~ 1,000 times.In the C57BL/6 mouse model with IL-6 reading, at subcutaneous injection people (Fig. 2 B, figure A) or first 24 hours of mice (Fig. 2 B, figure B) IL-1 β (20ng), mice AB7 (3 or 300ug) or PBS vehicle control carry out peritoneal injection.Blood is extracted after 2 hours, and via ELISA with regard to IL-6 horizontal analysis blood serum sample.These data are presented at the maximum suppression (~ 75%) (figure A) of 3 μ g to the beta induced IL-6 level of people IL-1, and show secondary maximum suppression (~ 50%) (scheming B) to the beta induced IL-6 level of mice IL-1 with 300 μ g.The observation that these results have much higher affinity and vitro efficacy with AB7 antibody on human IL-1 β comparison mice IL-1 β is consistent.In addition, these data show that this antibody can with the dosage suitably higher than the dosage needed for treatment people experimenter in disease model in Mice Body, and in human body, antibody has much better affinity and effect.Other IL-1 β antibody of---having significantly higher affinity and vitro efficacy to people IL-1 β than mice IL-1 β---when not showing this kind of character (such as herein open the and/or antibody quoted), in mouse model, similar higher dosage may be optional.

Embodiment 3

To use in single dose intravenous to rat or the pharmacokinetics of anti-il-i-beta antibody after subcutaneous dosage

In order to check pharmacokinetic property, the IL-1 β antibody of called after AB7 is administered to the tail intravenous of Adult male rats with 0.1,1.0 or the dosage (being respectively group 1,2 and 3) of 10mg/kg with intravenous (IV) form of injecting, or uses with 1.0mg/kg (group 4) subcutaneous between scapula (SC).At the appointed time collect blood sample via jugular vein intubate or retro-orbital sinus (retro-orbitalsinus), to being no more than after administration 91 days.Make blood sample centrifugal to obtain serum.Following use analyzes sample based on the ELISA determination test of alkali phosphatase with regard to anti-il-i-beta antibody concentration.

Make IL-1 β (Preprotech) in PBS, be diluted to 0.5 μ g/mL, and this solution of 50 μ L is added in the hole of Nunc-ImmunoMaxisorp microtitration plate (VWR), and be incubated overnight at 2-8 DEG C.Remove antigenic solution, and to porose in add 200 μ L Block buffer [1% bovine serum albumin (BSA) in the 1XPBS comprising 0.05%Tween20], and at room temperature incubation 1 hour.After closing, hole lavation buffer solution (comprising the 1XPBS of 0.05%Tween20) washs 3 times.Make standard, sample and dilute impinging upon in sample diluting liquid (25% rat blood serum in the 1XPBS comprising 1%BSA and 0.05%Tween20).Anti-il-i-beta antibody standard solution is prepared as the series 2 times of dilutions from 2000 to 0.24ng/mL.Each repetition of standard, sample and contrast and dilution (50 μ L) are transferred to closed microtitration plate, and at 37 DEG C incubation 1 hour.After incubation, hole lavation buffer solution washs 3 times.Goat anti human IgG (H+L) antibody (SouthernBiotechAssociatesInc that alkali phosphatase is puted together, Birmingham, AL) dilution 1/1000 in Conjugate Diluent (1%BSA in the 1XPBS comprising 0.05%Tween20).During the institute added except BLANK hole by the conjugate of 50 μ L dilutions is porose, BLANK hole only accepts 50 μ L Conjugate Diluent.Make dull and stereotyped at 37 DEG C incubation 1 hour, then porose lavation buffer solution washs 3 times and with deionized water wash 3 times.Substrate para-nitro-pheneye phosphate (at 10% diethanolamine buffer, the 1mg/mL in pH9.8) is added institute porose in, and allow colour developing at room temperature to carry out 1 hour, add 50 μ L1NNaOH with cessation reaction after this.SPECTRAmaxM2PlateReader (MolecularDevices, MenloPark, CA) is used to be determined at the absorbance at 405nm place, and subsequently with A ₄₀₅to ng/mL antibody standard, drawing standard curve.Perform regression analysis, and by pushing away the concentration determining sample and contrast in standard curve.Quantitative limit is 40ng/mL.

As shown in Figure 3, serum-concentration is two in IV dosage group declines exponentially.Compartment analysis (compartmentalanalysis) is performed to the data of single animal, and for each dosage group, after getting rid of those animals producing RAHA response, asks the meansigma methods of gained pharmacokinetic parameter.The serum levels of anti-il-i-beta antibody declines, and the average alpha phase half-life is 0.189 ± 0.094 to 0.429 ± 0.043 day (4.54 to 10.3 hours), and the β phase half-life is 9.68 ± 0.70 to 14.5 ± 1.7 days.In the rat of AB7 accepting 1mg/kg subcutaneous dosage, serum levels increased to the peak of 4.26 ± 0.065 μ g/mL by 2-3 days, and declined with the half-life of 2.59 ± 0.25 days.

Embodiment 4

The pharmacokinetics of anti-il-i-beta antibody after dosage in single dose intravenous is used to machin

With intravenous (IV) single bolus injection form, the anti-il-i-beta antibody of the called after AB7 of bull and female cynomolgus monkeys acceptance 0.3,3.0 or 30mg/kg dosage.At the 1st day before administration, 5 minutes upon administration, 4 and 8 hours, and the 2nd, 4,8,11,15,22,29,43 and 56 day time, from animal, collect blood sample.Following use analyzes sample based on the ELISA determination test of alkali phosphatase with regard to anti-il-i-beta antibody concentration.

Make IL-1 β solution in PBS, be diluted to 0.5 μ g/mL, and this solution of 50 μ L is added in the hole of Nunc-ImmunoMaxisorp microtitration plate (VWR), and be incubated overnight at 2-8 DEG C.Remove antigenic solution, and to porose in add 200 μ L Block buffer [1% bovine serum albumin (BSA) in the 1XPBS comprising 0.05%Tween20], and at room temperature incubation 1-4 hour subsequently.After closing, the hole lavation buffer solution of every block plate washs 3 times at (1XPBS comprises 0.05%Tween20).Make standard, sample and dilute impinging upon in diluents (in the 1XPBS comprising 1%BSA and 0.05%Tween20 2% normal machin serum (NCS)).Anti-il-i-beta standard solution is prepared as the 2 times of dilutions of the series from 8000ng/mL.Each repetition of standard, sample and contrast and dilution (50 μ L) are transferred to closed microtitration plate, and at 37 DEG C incubation 1 hour.This first time incubation after, hole lavation buffer solution washs 3 times, and to institute porose in add the biotinylated rhIL-1 β of 50 μ L.Flat board is incubation 1 hour at 37 DEG C subsequently.Hole lavation buffer solution washs 3 times, by except BLANK hole porose in add the Streptavidin puted together of alkali phosphatase that 50 μ L dilute, carry out third time incubation, wherein BLANK hole only accepts the diluent of 50 μ L.Make dull and stereotyped at 37 DEG C incubation 30 minutes, then porose lavation buffer solution washs 3 times and with deionized water wash 3 times.Substrate para-nitro-pheneye phosphate (at 10% diethanolamine buffer, the 1mg/mL in pH9.8) is added institute porose in.Allow colour developing at room temperature to carry out 1 hour in the dark, after this, add 50 μ L1NNaOH with cessation reaction.Use SPECTRAmaxM2PlateReader (MolecularDevices, MenloPark, CA) for the porose absorbance being determined at 405nm place.Subsequently with A ₄₀₅to ng/mL anti-il-i-beta standard, drawing standard curve.Perform 4 parametric regression analyses, and by pushing away the concentration determining sample and contrast in standard curve.Quantitative limit is 40ng/mL.

For single dose 0.3 and 3mg/kg group, serum anti-il-i-beta antibody horizontal declines, and the average alpha phase half-life is 9.40 ± 2.00 hours, is the β phase half-life (Fig. 5) of 13.3 ± 1.0 days subsequently.In the machin that the single IV accepting 30mg/kg injects, the serum levels of antibody declines more quickly, and the α phase half-life is 10.9 ± 3.2 hours, is the β phase half-life of 7.54 ± 1.79 days subsequently.Also carry out the modeling of plasma concentration v. time curve to 5 with use every January 0.1,0.3,1 and 10mg/kg dosage, and be shown in Fig. 5.

Embodiment 5

The effect of anti-il-i-beta antibody in human pancreatic island cell Analytical system

As external model, be separated human pancreatic island cell, then process by high glucose level, to simulate type 2 diabetes mellitus environment.Anti-il-i-beta antibody can use in islet cells system, to check the impact on β cell function (insulin releasing of response glucose), Beta cell proliferation and apoptosis.

As described in (people such as Linetsky, Diabetes46:1120-1123,1997; The people such as Oberholzer, Transplantation69:1115-1123,2000; The people such as Ricordi, Diabetes37:413-420, the people such as 1988, Maedler, Proc.Natl.Acad.Sci.USA101:8138-8143,2004; WO2004/0002512), from the pancreas of multiple human organs donors of non-diabetic or metabolism medical history, islets of langerhans is separated.On the flat board of the extracellular matrix bag quilt derived from bovine corneal inner skin cell (NovamedLtd, Jerusalem), cultivate islets of langerhans subsequently, allow cell to adhere to dull and stereotyped, and preserve its functional completeness.Islets of langerhans is cultivated in the CMRL1066 culture medium comprising 100U/mL penicillin, 100ug/mL Streptavidin and 10% hyclone (GIBCO, Gaithersburg, MD).In order to stimulate insulin secretion, culture medium with to be supplemented with further 5,11 or 33mM glucose, interpolation or the culture medium of not adding fatty acid replace.

In order to measure the insulin releasing of response glucose, islet cells washs, and the middle precincubation of Ke-Lin Er Shi bicarbonate buffer (KRB) comprising 3.3mM glucose and 0.5%BSA 30 minutes.KRB replaces 1 hour by KRB3.3mM glucose subsequently, and this is other 1 hour in KRB16.7mM glucose subsequently.The 0.18MHCl be used in 70% ethanol extracts islet cells, measures insulin content for end user's Insulin RIA Kit (CISBiointernational, Gif-sur-Yvette, France).β apoptosis can be measured by various method.Such as, dUTP nick end labelling (TUNEL) technology that mediated by Typical end deoxynucleotidyl transferase of cell and carry out double staining with regard to insulin.Abreast, as described in (see such as, WO2004/002512; The people such as Maedler, 2004, the same quoted passage), can also be expressed by the activation of detection caspase 3 or Fas and verify apoptosis.

Embodiment 6

The effect of anti-il-i-beta antibody in rat artificial insular cell assay system

Alternately or except human pancreatic island cell model, rat artificial insular cell can be used as external model, to assess the effect of anti-il-i-beta antibody.Such as, artificial insular can be prepared and test as described in US20060094714.Pancreas from 4 SpragueDawley rats is divided into small pieces, with Hanks-Hepes buffer rinsing 3 times, and at 37 DEG C, in shaking bath, use collagenase (Liberase, 0.25mg/ml, RocheDiagnosticCorp., Indianapolis, Ind., USA) digest 10 minutes.The pancreatic tissue of digestion uses 50mlHanks-Hepes buffer rinsing 3 times subsequently, to remove collagenase, and makes to organize agglomerate to be filtered by 250 micron membrane filter.Filtrate and the 27%Ficoll of 16ml in Hanks-Hepes buffer (Sigma, St.Louis, Mo., USA) w/v are mixed, then (is respectively 23%, 20.5% and 11% in Ficoll gradient; Each concentration 8ml) under 1,600rpm and room temperature centrifugal 10 minutes.Depend on the size of islets of langerhans, islets of langerhans concentrates at 11% and 20.5% and interface between 20.5% and 23%.Collect islets of langerhans from 2 interfaces, with without calcium Hanks-Hepes buffer rinsing 2 times, be then suspended in comprise 1mMEDTA 5ml without in calcium Hanks-Hepes buffer, and at room temperature incubation 8 minutes.Trypsin and DNA enzymatic I are added in islets of langerhans suspension, is respectively the final concentration of 25ug/ml and 2ug/ml, and suspension is with vibration incubation 10 minutes at 30 DEG C.Trypsinization stops by adding the 40mlRPMI1640 (GIBCOLifeTechnologies, Invitrogen, Carlsbad, Calif.) with 10%FBS.The islet cells of trypsinization is filtered by 63 micrometer nylon filters (PGCScientific, Frederick, Md.), to remove maxicell aggregation subsequently.The islet cells of dispersion carries out washing subsequently, count and plant " at the bottom of V-arrangement " 96 orifice plate interior (2,500 cells/well).Make the islet cells suspension of dispersion subsequently under 1,000rpm centrifugal 5 minutes.Remove Hanks-Hepes buffer, and replace by the 200ulRPMI1640 culture medium comprising 10%FBS, 1% Pen .-Strep and 2mML-glutamine.Next, make 96 orifice plates under 1,000rpm centrifugal 5 minutes, to be collected in the concentrated dispersed islet cells cell in dull and stereotyped V-arrangement bottom, form artificial insular.These artificial insulars subsequently in cell culture incubator at 37 DEG C and 5%CO ₂lower overnight incubation, subsequently for measuring.

Embodiment 7

In animal model, IL-1 β antibody is on the impact of insulin sensitivity

IL-1 β antibody can be measured by the insulin of antibody and glucose lowering activity as effect in the body of insulin sensitizer in the meals model of insulin resistance.When 6 week age, be placed in by male Sprague-Dawley rat in higher fatty acid, high carbohydrate diet, described meals comprised 60% fructose, 10% Adeps Sus domestica and 0.06% magnesium.Meals start latter 2 days, based on antibody dosing level (0.1-5mg/kg body weight), route of administration (subcutaneous, intravenous or intraperitoneal routes) and frequency of administration (every day was to every 2 weeks), rat are divided into different groups at random.Matched group only accepts buffer (vehicle) or irrelevant antibody.Measure food and fluid absorption every day, and utilize feeding (pair-feedingprotocol), to guarantee that the same food between 3 groups is taken in.After 5 weeks, obtain the serum levels of glucose, insulin and triglyceride at half fasted conditions (draw blood last evening, animal gives the food of limit amount, and draws blood in ensuing morning).The program continues other 9 weeks, at this moment perform in conscious animal in half fasted conditions glucose tolerance test (OGTT), mode be Orally administered glucose load (100mg/100 gram of body weight) afterwards blood sampling be used for glucose and insulin measurement.The serum levels of glucose and triglyceride is measured by spectrophotography, and insulin level is measured by radioimmunoassay (Linco, St.Louis, MO).

Embodiment 8

IL-1 β antibody is used for the treatment of in fertile gerbil jird (Psammomysobesus) animal model of T2D

The therapeutic efficiency that IL-1 β antibody reduces for preventing the beta cell amount observed in type 2 diabetes mellitus patient, assess in fertile gerbil jird, described fertile gerbil jird shows insulin resistance and the fat relevant diabetes of development meals induction type, initial relevant to hyperinsulinemia, and advance to Severe hyperglycemia gradually, with the instantaneous rising of beta-cell proliferation activity and the long-term increase of beta cell mortality rate, and the destruction of the Pancreas Islet Structure (people such as Donath, Diabetes48:738-744,1999).In order to measure the impact impaired with propagation of the beta cell apoptosis of IL-1 β antibody on hyperglycemia induction in gerbil jird islets of langerhans fertile in diabetes de-velopment process, by subcutaneous, intravenous or intraperitoneal routes, antibody is applied to potential diabetes animal (being transformed into high-energy meals) with multiple dosage levels of 0.1-5mg/kg body weight, and wherein antibody uses to carry out repetition to interval weekly every day.Control animal group maintains low-yield meals, or is transformed into high-energy meals and only uses buffer (vehicle) or irrelevant antibody to process.The the 4th, 7,14,21 and 28 day time, putting to death animal subgroup, at this moment collecting blood and for measuring plasma glucose, insulin and triglyceride.Also take out pancreas, wherein part is freezing for measuring insulin content subsequently at-70 DEG C, and remainder is fixing in 10% PBF, embedding and section in paraffin, for analyzing Fas, IL-1 β and insulin expression, and beta-cell proliferation and apoptosis.This kind is analyzed and will be allowed to determine to the prevention of diabetes onset or delay action, to the beta cell apoptosis of hyperglycemia induction, breed the opposing protective effect that impaired and beta cell amount reduces, and the normalization of pancreatic insulin content.

Embodiment 9

IL-1 β antibody in people for T2D treatment purposes

IL-1 β antibody described herein or fragment can be applied to people patient according to the present invention, are used for the treatment of and/or prevent type 2 diabetes mellitus.Particularly, in one example in which, the IL-1 β antibody (AB7, above-described) with above-mentioned character is used for the treatment of the patient of the S&S of display type 2 diabetes mellitus.More specifically, IL-1 β antibody is used for the safety of type 2 diabetes mellitus and effectiveness is confirmed in one or more people's clinical research, comprises the test such as with following design.

People's clinical research of double blinding, placebo is performed in type 2 diabetes mellitus patient.T2D diagnostic criteria according to ADA (ADA):

-fasting plasma glucose concentration >=126mg/dL (>=7.0mmol/L) (must measure in 28 days before the 0th day)

Or

The symptom of-hyperglycemia (such as, thirsty, polyuria, lose weight, blurred vision) and accidentally/casual plasma glucose value >=200mg/dL (>=11.1mmol/L) (must measure in 28 days before the 0th day),

With there is HbA1c >=7.5% and≤12% (DCCT standard), meet the patient of the inclusive criteria of this research and enter anthology research in turn by seminar, in each group, random appointment accepts IL-1 β antibody or placebo.In order to make to drop to minimum to the danger of experimenter, before being increased to next dosage level, at each dosage level assessment safety and toleration.Be shown in following table for the processed group of this research and experimenter's number:

When research the 1st day, subcutaneous or via 30 minutes constant rate of speed intravenous infusion administration of antibodies or placebo.SMP known in the art is used to carry out safety evaluation, comprise the record of adverse events, physical examination, vital sign, clinical laboratory's mensuration (such as, hematochemistry, hematology, urinalysis), Plasma Cytokine Levels and electrocardiogram (ECGs).Before dosage is used and after application, multiple time period (such as, sky) collects blood sample, to assess HbA1c, lipodogramme (comprising free fatty, HDL and LDL-C), IL-1 β antibody horizontal (pharmacokinetics), anti-il-i-beta antibody response, cytokine (such as, IL-1 β, IL-6, TNF α) level, CRP, sodium, potassium, kreatinin, AST, ALT and hematogram.Mensuration can also be performed to other cytokines and lymphokine, such as described herein those.When the IL-1 β antibody horizontal used does not drop under detection limit, other blood sample can be collected on the date more late than initial design.Carry out research assessment after treatment at fixed time.

Based on the primary efficacy endpoint (HbA1c, see people such as such as Reynolds, BMJ, 333 (7568): 586-589,2006) that glycated hemoglobin improves, perform the clinical monitoring to type 2 diabetes mellitus treatment.The therapeutic effect that the improvement of HbA1c level indicates anti-IL-1b to treat, and generally should cause the reduction at least about 0.5% or more.Also one or more following secondary endpoints can be measured, to assess the therapeutic efficiency of type 2 diabetes mellitus, such as fasting glucose (such as, glucose) level is (such as, ≤ 130, ≤ 120), 120 minutes oral glucose tolerances test (OGTT), glucose/insulin C-peptide AUC (such as, >=50%, >=60% increases), diabetes medicament (such as, insulin, oral hypoglycemic) minimizing, the improvement of insulin sensitivity, serum cytokines (such as, normalization), CRP level, measuring of quality of life, BMI improves (reduces 1%, 3%, 5%), (the people such as Saudek such as pharmacokinetics, JAMA, 295:1688-97, 2006, the people such as Pfutzner, DiabetesTechnolTher.8:28-36,2006, the people such as Norberg, JInternMed.260:263-71,2006).Other sample lipodogramme analysis comprises the following test performed according to standard accepted method known in the art.

Test	Method
		Lipoprotein electrophoresis	Gel electrophoresis
Serum apolipoprotein A-I (apoA-I)	Turbidimetry
		Serum apolipoprotein A-II (apoA-II)	Turbidimetry
Apolipoprotein B-48 (apoB-48)	ELISA
		Apolipoprotein B-100 (apoB-100)	Turbidimetry
Serum Apoprotein Cs (apo Cs)	For the Immunoturbidimetric assay of Apo CII and ApoCIII
		Serum Apoprotein E (apo E)	Turbidimetry
Serum Apoprotein J (apo J)	ELISA
		Serum amyloid A	Turbidimetry
Blood plasma free fatty acid (FFA)	Colorimetry
		Plasma glycerol	Colorimetry
Serum LCAT	ELISA
		Serum cholesterol ester transferring protein (CETP)	ELISA
Serum liver esterase (HL)	Fluorimetry
		Serum paraoxonase 1 (PON1)	UV/ colorimetry

pharmacokinetic analysis

The the 0th, 1,2,3,4,7,9 ± 1,11 ± 1,14 ± 1,21 ± 2,28 ± 2,42 ± 3 and 56 ± 3 day time, obtain sample and be used for pharmacokinetic analysis.The preliminary analysis display plasma concentration v. time curve of AB7 pharmacokinetics in the type 2 diabetes mellitus experimenter of single IV dosage accepting 0.01mg/kg, wherein 22 days t1/2, the 2.9mL/ days/clearance rate of kg and central compartment (centralcompartment) volume of distribution (being very similar to serum volume) (Fig. 7) of 50mL/kg.

blood-glucose and HbA1c analyze

At the 0th, 7,14 ± 1,21 ± 2,28 ± 2,42 ± 3 and 56 ± 3 day and at screening day (screeningday), obtain sample and measure blood-glucose.For the sample from front 2 dosage groups, in these samples, the assessment of the minimizing of blood glucose levels is shown in hereafter (upper data lines of each experimenter).The the 0th, 28 ± 2,42 ± 3 and 56 ± 3 day and screening day, obtain sample and measure HbA1c.For the sample from front 2 dosage groups, in these samples, the assessment of the minimizing of HbA1c level is shown in hereafter (lower data lines of each experimenter).

0.01mg/kg dosage group

Experimenter	Screening	0th day laboratory	7th day laboratory	14th day laboratory	21st day laboratory	28th day laboratory	42nd day laboratory	56th day laboratory
									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

(Labs=laboratory)

0.03mg/kg dosage group

Experimenter	Screening	0th day laboratory	7th day laboratory	14th day laboratory	21st day laboratory	28th day laboratory	42nd day laboratory	56th day laboratory
									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 to point out after the single administration of antibody can in experimenter for reach curative effect (such as, the minimizing of glucose and/or HbA1c level), the lower bound of the dosage of IL-1 β antibody as provided herein.

c reactive protein is analyzed

By liver respond various stress trigger agent and discharge C reactive protein (CRP) (described stress trigger agent comprise response IL-1 produce IL-6), also measure in serum at the time point identical with PK sample.The concentration dependent indirect reaction in CRP generation speed and the linear clearance rate of CRP for antibody level of serum and antibody, develop PK/PD model, it mixes two-compartment model.In type 2 diabetes mellitus patient 0.01mg/kg antibody single IV dosage after, based on models fitting, in 7-10 days, drop to 66 ± 22% (Fig. 8) relative to 100%, CRP before dosage.In type 2 diabetes mellitus patient 0.03mg/kg antibody single IV dosage after, in 7-10 days, drop to 40 ± 12% (Fig. 9) relative to 100%, CRP before dosage.Data about placebo are also shown in Figure 10.Based on these data and model projection (modelprojections), after antibody is monthly injected the CRP level of maintenance of expection when the 0.03mg/kg/ month close to 40%, when the 0.1mg/kg/ month 16.5%, when the 0.3mg/kg/ month 6.2%, when the 1mg/kg/ month 1.9%, and when the 3mg/kg/ month 0.66% (Figure 11).These data show IL-1 β antibody as herein provided can be low to moderate one month or longer time frequency use, to reach curative effect (such as, the minimizing of CRP level) after antibody single administration in patients.

Based on the result obtaining first clinical research since then, perform other clinical trial.This kind of test can according to the present invention includes one or more above-mentioned dosage, and or alternately other IL-1 β antibody dosages one or more, the patient/group (at least about 10,50,100,200,300,400,500,750,1000) of longer treatment and/or observation period and more more number.In addition, these and other researchs also may be used for before using other dosage, based on special parameter change (such as, the minimizing of the minimizing of blood glucose, the minimizing of HbA1c, CRP) determine to reach the desired time required for treatment benefit, and the persistent period of the treatment benefit of this expectation is determined based on the change (such as, the minimizing of blood glucose, the minimizing of HbA1c, the minimizing of CRP) of special parameter.

Embodiment 10

IL-1 β antibody is on the impact of adipose cell function and insulin resistance

By using the external test test of the adipose cell cultivated, the reducing effect (such as, block) of anti-il-i-beta antibody to the beta induced insulin resistance of IL-1 can be illustrated.The 3T3-L1 PECTORAL LIMB SKELETON cell line deriving from ATCC (#CL-173) in DMEM, 25mM glucose and 10% calf serum at 7%CO ₂with 37 DEG C at grow, and be induced to differentiate into adipose cell.In brief, converge latter 2 days, culture medium is replaced with DMEM, 25mM glucose and 10%FCS that are supplemented with isobutyl methylxanthine (0.25mM), dexamethasone (0.25 μM), insulin (5 μ g/ml) and pioglitazone (10 μMs).Remove culture medium after 2 days, and replace with DMEM, 25mM glucose and 10%FCS that are supplemented with insulin (5 μ g/ml) and pioglitazone (10 μMs), cultivate 2 days.Cell is fed with DMEM, 25mM glucose and 10%FCS for every 2 days subsequently.Within 8-15 days after this differentiation scheme starts, use 3T3-L1 adipose cell.

People's PECTORAL LIMB SKELETON (BiopredicInternationl, Rennes, France) in the DMEMHam ' sF12 from Supplementpack PECTORAL LIMB SKELETON growth medium (Promocell, Heidelberg, Germany) at 5%CO ₂with 37 DEG C at grow, described DMEMHam ' sF12 comprises 15mMHEPES, 2mML-glutamine, 5%FCS, 1% antimycotic solution, ECGS/H-2, hEGF-5 and HC-500.After converging, by culture medium being replaced with DMEMHam ' sF12,15mMHEPES, 2mML-glutamine and 3%FCS, differentiation-inducing lipoblast, described culture media supplemented has biotin (33 μMs), insulin (100nM), pantothenate (17 μMs), isobutyl methylxanthine (0.2mM), dexamethasone (1 μM) and rosiglitazone (10 μMs).Culture medium is removed after 3 days, and replacing with the Ham ' sF12 comprising 15mMHEPES, 2mML-glutamine and 10%FCS, described Ham ' sF12 is supplemented with biotin (33 μMs), insulin (100nM), pantothenate (17 μMs) and dexamethasone (1 μM).Cell is fed by same medium for every 2 days subsequently.People's adipose cell can use for 15 days after this differentiation scheme starts.People's PECTORAL LIMB SKELETON also can derive from alternative source, such as cell line XA15A1 and XM18B1 (Lonza, Allendale, NJ).

The effect of IL-1 β induced insulin resistance (reduction insulin sensitivity) in the adipose cell cultivated is confirmed by following: make adipose cell and IL-1 β incubation (such as 20ng/mL, 48 hours), then with insulin incubation (such as, 0.5nM, 100nM of variable concentrations; 20 minutes), subsequently interpolation 2-[ ³h] measure glucose transport after deoxyglucose.Insulin resistance is determined as the minimizing of glucose uptake, and anti-il-i-beta antibody is reducing acting in this adipose cell cell culture system and can easily measure on (such as, blocking) insulin resistance.

IL-1 β directly stimulates adipose cell generation fat therbligs and cytokine (such as, leptin, phylaxin, Visfatin, IL-6, MCP-1 (CCL2), RANTES, PAI-1, acylation stimulating protein matter, SAA3, Pentraxin-3, macrophage migration inhibition factor, IL-1RA, IL-12, IL-8, IL-6, TNF-α) effect can be measured by following: under not the existing or exist of IL-1 β of variable concentrations, culture adipocytes different time length described above, and in conditioned medium, measure adipose cell and cytokine levels (usually via ELISA or other common methods).In addition, the impact of the fat therbligs that the adipocyte culture thing that measurement anti-il-i-beta neutralizing antibody process IL-1 β stimulates is correlated with on insulin resistance and/or cytokine secretion.Similarly, measurement fat therbligs---the impact of adiponectin of anti-il-i-beta antibody treatment on suppression sensitization insulin.In order to study anti-il-i-beta antibody treatment whether in and in fatty tissue inflammation process, be derived from immunity/inflammatory cell (such as, macrophage) the effect of IL-1 of endogenous generation, under not the existing or exist of anti-il-i-beta antibody, the human macrophage of different number (monocyte derived or various monocytic series) is cultivated together with above-described adipocyte culture thing, and measures the adjustment to fat therbligs and cytokine.In addition, with after processing experimenter in anti-il-i-beta antibody body, (such as, serum, blood plasma) measures the regulating action to fat therbligs and cytokine secretion in the circulating cycle, as the instrument confirming effect.

Embodiment 11

By the cytokine production in IL-1 β antibody suppression people whole blood

The cytokine measured in disease or treatment of diseases in blood may be used for determining Disease severity or the response to treatment.Usually, cytokine levels is measured in serum, but this method not necessarily measures total cytokine.Many cytokines can in cell interior (intracellular).In addition, the ability about cell cytokine production may be the information more useful than circulating cells factor level.

Use the method for stimulation of whole to determine cytokine production and the effect by anti-il-i-beta antibody treatment.Blood is drawn in aseptic heparinization pipe from patient, and is added by 250ul whole blood in the aseptic cryopreservation tube of Corning of the following orange lid of each 4mL arranged subsequently:

Control series

550ulRPMI is pre-installed in all pipes.To in pipe 1 (contrast), add 200ulRPMI, and in pipe 2-10, add the RPMI that 100ul is other.For each in pipe 2-10, add the 100ul dilution of anti-il-i-beta antibody (AB7).

Test series

Similar antibody dilution system of setting up as detailed above arranges.

Use 10 seconds vortexs that all pipes are fully mixed.Control series tubulation A1-10 accepts other 100ulRPMI subsequently, vortex 10 seconds, fastening screw turncap, and pipe is placed in incubator.To in test series pipe B1-10, add the heat-inactivated staphylococcus epidermidis of 100ul (final concentration of stock solution 1: 1000, causes the antibacterial of 10: 1: leukocyte than), make pipe vortex subsequently 10 seconds, cover lid and be placed in 37 DEG C of incubators.Incubation is after 24 hours, make the whole cracking of culture with TritonX (0.5% eventually), makes lysate freezing with release cells content.After Whole blood cultures cracking, freeze-thaw cycle is implemented to pipe, and with regard to human TNF alpha, IL-6, IFN γ, IL-8, IL-1 α, IL-1Ra and IL-1 β, by standard cytokine ELISA algoscopy, measure cytokine levels (R & DSystems, Minneapolis, MN).

Only comprising the cytokine measured in the control series tubulation of aseptic culture medium and antibody (in pointed pipe), reflect spontaneous irritation level.In health volunteer, when incubation was measured after 24 hours, find extremely low-level various cytokine.In the patient with non-disease therapy, level may be higher.Test series pipe comprises the hot deactivation staphylococcus epidermidis of ormal weight in addition, and this stimulates many cytokines to produce.If anti-il-i-beta antibody treatment is effective, so this will be reflected as the cytokine production of minimizing.

As shown in Figure 6, high-affinity anti-il-i-beta antibody A B7 is suppressing very effective in the IL-1 β production in human blood.In the meansigma methods of 3 parts of human samples, when 0.1pM, this antibody is produced the IL-1 β that induces via staphylococcus epidermidis and is caused 50% suppression, and 75% to suppress when 3pM.When 100pM, suppression is 100%.Interferon gamma (IFN γ) is via staphylococcus epidermidis induction, and AB7 makes when 100pM to reduce 75% via the IFN γ of staphylococcus epidermidis induction.

Embodiment 12

In non-obese diabetes (NOD) mice, anti-il-i-beta antibody is on the impact of diabetes

In order to confirm anti-il-i-beta antibody effect in diabetes mice model, obtaining female 3 to 4 NOD mice in age in weeks (JacksonLaboratories, BarHarbor, ME), and raising in cages in vivarium under pathogen free conditions.The anti-il-i-beta antibody of various dosage (such as, 3-600 μ g) be diluted in suitable vehicle (such as, PBS) in, and use in prediabetes female NOD mice earlier than starting 6 week age, wherein use different approaches (such as, intraperitoneal, subcutaneous, intravenous) at predetermined intervals (such as, weekly, every 2 weeks, monthly) use.During from 10 week age, use glucose meters (EncoreGlucometer with every weekly interval; Bayer, Elkhart, IN) monitor blood-glucose.The continuous mice 2 times with 200mg/dl blood glucose levels is regarded as (diabetes onset is observed when about 15-20 age in week usually, and sickness rate reaches maximum to during 30 week age usually, about 90%) of diabetes.Data are calculated as along with experimentation is still the percentage ratio of the animal of non-diabetic.Difference between curve uses log-rank inspection to test, the distribution in this inspection more whole observation period.

In another kind of NOD mouse model, in the disease model that cyclophosphamide (CY) accelerates, confirm effect (people such as Reddy, HistochemJ.33:317-327,2001 of anti-il-i-beta antibody; The people such as Cailleau, Diabetes46:937-940,1997; The people such as Reddy, HistochemJ., 34:1-12,2002; The people such as Harada, Diabetologia27:604-606,1984; The people such as Nicoletti, EurJImmunol24:1843-7,1994).Obtain ND 4 to 8 week age male (or female) NOD mice (JacksonLaboratories, BarHarbor, ME), and raise in cages in vivarium under pathogen free conditions.With the single dose injection mice of 200mg/Kg with CY (Sigma), and due to the acceleration character of model, with the interval of various acceleration (such as, 1 time weekly, weekly 2 times), use or do not use and coexist suitable vehicle (such as, PBS) in, the anti-il-i-beta antibody of the various dosage of dilution (such as, 3ug, 30ug, 150ug, 600 μ g) or Isotype control antibodies, use different administration approach (such as, intraperitoneal, subcutaneous, intravenous) process mice, 2-3 week altogether.Inject the previous day at CY, use glucose meters to monitor weekly 3 urine glucose (glycosuria) levels, and monitor 1 blood glucose levels weekly.Continuous 2 urine glucose level > 20mmol/L are considered as diabetes, and to urinate glucose level via the minimizing of anti-il-i-beta antibody be measuring of effect.

In another kind of model, effect of anti-il-i-beta antibody carries out assessing (people such as Mellgren, Diabetologia29:670-2,1986 in the diabetes recurrence model (Nonimplantation repulsion) of islet transplantation; The people such as Sandberg, ClinExpImmunol108:314-7,1997).Obtain ND 4 to 8 female NOD mice in age in weeks (JacksonLaboratories, BarHarbor, ME), and raise in cages in vivarium under pathogen free conditions.Before remarkable leukocyte infiltration, prepare islets of langerhans by the male and female NOD mice of non-diabetic in 5-6 age in week, and under being implanted in the scrotum of Spontaneous Diabetic (15-20 age in week) female NOD mice (400-450 islets of langerhans/mice).Instantaneous euglycemia occurs after the transfer soon, and hyperglycemia reappears usually after the transfer for about 6 days.Mice is used or not be used in suitable vehicle (such as, PBS) in, the anti-il-i-beta antibody of the various dosage of dilution (such as, 3ug, 30ug, 150ug, 600 μ g) or Isotype control antibodies process, wherein use different administration approach (such as, intraperitoneal, subcutaneous, intravenous).Before transplantation and after 1 time or 2 times use glucose meters monitoring blood glucose levels weekly, and the continuous mice 2 times with horizontal > 200mg/dl is considered as diabetes, and blood glucose levels is measuring of effect via the minimizing of anti-il-i-beta antibody.

Embodiment 13

The process of the diabetes model of low dosage streptozotocin induction in C57BL/K mice

In order to confirm the hyperglycemia that anti-il-i-beta antibody is induced at repeatedly low dosage streptozotocin (STZ) and insulitis diabetes model (people such as Sandberg, BiochemBiophysResCommun202:543-548,1994; The people such as Reddy, AnnNYAcadSci1079:109-113,2006) in effect, acquisition 4 to 8 C57BL/K mice (JacksonLaboratories in age in week, BarHarbor, ME), and raise in cages in vivarium under pathogen free conditions.Mice accepts 5 STZ daily doses (40mg/kg) in this acceleration model, and with various interval (such as, 1 time weekly, 2 times or 3 times) accept process (starting for first 1 day in STZ injection) altogether 1-3 week of accelerating, wherein use different administration approach (such as, intraperitoneal, subcutaneous, intravenous), use or be not applied in suitable vehicle (such as, the anti-il-i-beta antibody (such as, 3ug, 30ug, 150ug, 600 μ g) of the various dosage diluted PBS) or Isotype control antibodies.Within first 1 day, start in STZ injection, use glucose meters to monitor weekly 1 blood glucose levels.Continuous 2 blood glucose levels > 200mg/dl are considered as diabetes, and blood glucose levels is measuring of effect via the minimizing of anti-il-i-beta antibody.

Embodiment 14

Process in the obese model that the meals of type 2 diabetes mellitus are induced

Test in obesity (DIO) model that effect of anti-il-i-beta antibody is induced at the meals of type 2 diabetes mellitus.In this model, the mice fed with high fat content meals becomes fat through time several weeks, and when attacking by the mode of injecting glucose, they demonstrate glucose tolerance and reduce and impaired insulin secretion.C57BL/6 male mice, in 6 week age, feeds normal meals (ND, Teklad, 5 kilocalories of % fat) or Surwit ' s is higher fatty acid, high-sucrose meals (HFD, ResearchDiets#D12331,58 kilocalories of % fat).First 1 day starts antibody administration.Anti-il-i-beta Subject antibodies (AB7) and isotype controls human IgG2 antibody are used by intraperitoneal (i.p.) injection.Antibody Per-Hop behavior 2 times, totally 4 weeks.Body weight also records 2 times weekly.After 4 weeks, glucose tolerance test (GTT) is implemented to mice.In GTT, mice overnight fasting, and carry out peritoneal injection with 1g/kg glucose subsequently.Use FreeStyle glucose meters, after injection 0,15,30,60,90 and 120 minute time measure blood-glucose from tail otch.Figure 12 display is compared with the mice of making a living with normal meals, feed high fat diet the mice of totally 4 weeks there is glucose tolerance reduction (Fig. 1).Using of IL-1 β Subject antibodies provides the opposing protective effect that glucose tolerance reduces to HFD mice.In GTT process when 60 minutes, with the performance of the mice of 1mg/kgIL-1 β antibody administration be obviously better than having accepted IgG2 control antibodies mice ( ^*, p < 0.05).It should be noted that, although as described in Example 2, compare with people IL-1 β, AB7 antibody has much lower affinity (~ 10 for mice IL-1 β, 000 times) and vitro efficacy, but still positive findings is observed in this mouse model.

All references cited herein, comprises publication, patent application and patent and is hereby incorporated by, its degree and each list of references indivedual and particularly point out be incorporated herein by reference and in this article overall set forth identical.

Unless otherwise indicated herein or contradiction obvious with context, should be interpreted as containing odd number and plural number with similar referring to " the " with " an " describing the term " a " that in context of the present invention, (particularly in the context of following claim) uses.Except as otherwise noted, term " comprises ", " having ", " comprising " and " containing " should be interpreted as open term (that is, meaning " including but not limited to ").When no matter when opening term for describing feature of the present invention or element, all special consideration can use closed term to replace open term, and does not deviate from the spirit and scope of the present invention.Unless otherwise indicated herein, numerical range as herein described is only intended to serve as the stenography method one by one mentioning each independent numerical value be included within the scope of this, and each independent numerical value is integrated with in description, just as it is addressed separately in this article.Unless otherwise indicated herein or contradiction obvious with context, all methods described herein can be carried out with any suitable order.Except as otherwise noted, the use of any and all examples or exemplary language (such as, " such as ") provided herein, is only intended to better illustrate the present invention and does not cause restriction to scope of the present invention.It is necessary that the element that should be interpreted as indicating any failed call to protect without any language in description is that the present invention puts into practice.

The preferred embodiments of the invention are described in this article, comprise the present inventor known for performing best mode of the present invention.After reading aforementioned specification, the change of these preferred embodiments can be apparent for those skilled in the art.The present inventor expect technical staff can suitably time adopt this kind change, and the present inventor expect the present invention can with such as herein specifically describe outside mode put into practice.Therefore, as allowed by law by what be suitable for, the present invention includes all modifications and the equivalence of the theme described in appended claim.In addition, unless otherwise indicated herein or contradiction obvious with context, said elements with its any combination of likely variation pattern all fall into the present invention.

Claims

1. anti-il-i-beta antibody or its fragment are in the purposes be selected from the medicine of following disease or disease for the preparation for the treatment of people: the obesity that type 2 diabetes mellitus, insulin resistance, the type 2 diabetes mellitus hyperglycemia of being correlated with is relevant with type 2 diabetes mellitus,

Wherein said antibody or antibody fragment comprise variable region of light chain and variable region of heavy chain, and the aminoacid sequence of SEQIDNO:5 is contained in described variable region of light chain, and the aminoacid sequence of SEQIDNO:6 is contained in described variable region of heavy chain.

2. the purposes of claim 1, wherein said disease or disease are selected from type 2 diabetes mellitus, obesity that insulin resistance is relevant with type 2 diabetes mellitus.

3. the purposes of claim 1, wherein said disease or disease are type 2 diabetes mellitus.

4. the purposes of claim 1, wherein said disease or disease are insulin resistances.

5. the purposes of claim 1, wherein said disease or disease are the hyperglycemias that type 2 diabetes mellitus is relevant.

6. the purposes of claim 1, wherein said disease or disease are the obesities that type 2 diabetes mellitus is relevant.

7. the purposes of any one of claim 1-6, wherein, in described treatment, using for one or more subsequent dose after the using of predose of described antibody or antibody fragment.

8. the purposes of claim 7, the predose of wherein said antibody or antibody fragment is using of 2 or more subsequent dose after using.

9. the purposes of any one of claim 1-6, wherein, in described treatment, the predose of described antibody or antibody fragment uses rear using for one or more subsequent dose, and wherein said one or more subsequent dose is approximately equal to or less than described predose in amount.

10. the purposes of any one of claim 1-6, wherein, in described treatment, the using of predose of described antibody or antibody fragment is using of one or more subsequent dose afterwards, and wherein subsequent dose described at least one exceedes described predose in amount.

The purposes of 11. claim 7, wherein, in described treatment, described predose and each one or more subsequent dose are separated from each other interval at least 2 week.

The purposes of 12. claim 11, wherein said predose and each one or more subsequent dose are separated from each other interval at least 1 month.

The purposes of 13. claim 11, wherein said predose and each one or more subsequent dose are separated from each other interval at least 3 months.

The purposes of 14. claim 11, wherein said predose and each one or more subsequent dose are separated from each other interval at least 6 months.

The purposes of 15. claim 11, wherein said predose and each one or more subsequent dose are separated from each other interval at least 12 months.

The purposes of 16. claim 1, wherein, in described treatment, described antibody or antibody fragment are used with one or more 3mg/kg or less antibody or fragment dosage.

The purposes of 17. claim 16, wherein said antibody or antibody fragment are used with one or more 1mg/kg or less antibody or fragment dosage.

The purposes of 18. claim 17, wherein said antibody or antibody fragment are used with one or more 0.5mg/kg or less antibody or fragment dosage.

The purposes of 19. claim 18, wherein said antibody or antibody fragment are used with one or more 0.1mg/kg or less antibody or fragment dosage.

The purposes of 20. claim 19, wherein said antibody or antibody fragment are used with one or more 0.03mg/kg or less antibody or fragment dosage.

The purposes of 21. claim 20, wherein said antibody or antibody fragment are used with one or more 0.01mg/kg or less antibody or fragment dosage.

The purposes of any one of 22. claim 16-21, wherein said one or more dosage is at least 0.01mg/kg antibody or fragment.

The purposes of 23. claim 1, wherein said anti-il-i-beta antibody or fragment are used by subcutaneous, intravenous or intramuscular injection.

The purposes of 24. claim 1, wherein said antibody or fragment are used with fixed dosage, do not rely on dosage/subject weight ratio.

The purposes of 25. claim 24, wherein said antibody or fragment are used with one or more 500mg or less antibody or fragment dosage.

The purposes of 26. claim 25, wherein said antibody or fragment are used with one or more 250mg or less antibody or fragment dosage.

The purposes of 27. claim 26, wherein said antibody or fragment are used with one or more 100mg or less antibody or fragment dosage.

The purposes of 28. claim 27, wherein said antibody or fragment are used with one or more 25mg or less antibody or fragment dosage.

29. the purposes of any one of claim 24-28, wherein said antibody or fragment are used with the antibody of one or more at least 1mg or fragment dosage.

The purposes of 30. claim 24, wherein said antibody or fragment are used with the antibody of one or more 10mg-100mg or fragment dosage.

The purposes of 31. claim 7, wherein, in described treatment, the dosage of described antibody or fragment is enough to the improvement of at least 0.5 percentage point realizing glycated hemoglobin.

The purposes of 32. claim 31, the dosage of wherein said antibody or fragment is enough to the improvement of at least 1 percentage point realizing glycated hemoglobin.

The purposes of 33. claim 32, the dosage of wherein said antibody or fragment is enough to the improvement of at least 2 percentage points realizing glycated hemoglobin.

The purposes of 34. claim 33, the dosage of wherein said antibody or fragment is enough to the improvement of at least 3 percentage points realizing glycated hemoglobin.

The purposes of 35. claim 7, wherein said treatment realizes at least one following modification: the minimizing that the reduction of fasting blood glucose level, the decline of insulin resistance, hyperinsulinemia reduce, the improvement of glucose tolerance, C react peptide (CRP), hyperglycemia reduce, minimizing that diabetes medicament is needed, the reduction of BMI, the change of glucose/insulin C peptide AUC, the minimizing of acute phase reactant, the decline of blood fat and lipodogramme improvement.

The purposes of 36. claim 7, wherein said treatment reduces or prevention is selected from the following complication relevant to type 2 diabetes mellitus or disease: retinopathy, renal failure, cardiovascular disease and wound healing.

The purposes of 37. claim 36, wherein said complication or disease are cardiovascular disease, and wherein said cardiovascular disease is atherosclerosis or peripheral blood vessel.

The purposes of 38. claim 36, wherein said complication or disease are wound healings, and wherein said wound healing is diabetic ulcer.

The purposes of 39. claim 1, wherein, in described treatment, the needs used prevention or postpone the other treatment of at least one of IL-1 β antibody or fragment, described treatment in addition comprises the pharmaceutical composition used at least one and comprise the activating agent except IL-1 β antibody or fragment.

The purposes of 40. claim 1, wherein, in described treatment, the amount that the using of IL-1 β antibody or fragment is reduced by least a kind for the treatment of in addition, frequency or persistent period, described treatment in addition comprises the pharmaceutical composition used at least one and comprise the activating agent except IL-1 β antibody or fragment.

The purposes of 41. claim 1, wherein in described treatment, the using of predose of described antibody or antibody fragment is using of one or more subsequent dose afterwards, and wherein in the therapeutic process with described predose and one or more subsequent dose, described antibody or the plasma concentration of antibody fragment in described people maintain in the level of at least 0.03ug/mL.

The purposes of 42. claim 41, wherein in described therapeutic process, the plasma concentration of described antibody or antibody fragment maintains in the level of at least 0.1ug/mL.

The purposes of 43. claim 42, wherein in described therapeutic process, the plasma concentration of described antibody or antibody fragment maintains in the level of at least 0.3ug/mL.

The purposes of 44. claim 1, generation that described anti-il-i-beta antibody or its fragment cause being selected from one or more following gene outcomes reduces to use wherein to described people: leptin, phylaxin, Visfatin, RANTES, IL-6, MCP-1, PAI-1, acylation stimulating protein matter, SAA3, Pentraxin-3, macrophage migration inhibition factor, IL-1RA, IL-12, IL-8 and TNF-α.

The purposes of 45. claim 44, generation that described anti-il-i-beta antibody or its fragment cause being selected from one or more following gene outcomes reduces to use wherein to described people: leptin, phylaxin and Visfatin.

The purposes of 46. claim 44, generation that described anti-il-i-beta antibody or its fragment cause being selected from one or more following gene outcomes reduces to use wherein to described people: MCP-1, RANTES, IL-6, TNF-α and Pentraxin-3.

The purposes of 47. claim 44, the minimizing of wherein one or more gene outcomes production is from fatty tissue.

The purposes of 48. claim 1, the increase of using described anti-il-i-beta antibody or its fragment wherein to described people to cause adiponectin production.

The purposes of 49. claim 48, wherein the increase of adiponectin production is from fatty tissue.

The purposes of 50. claim 44 or 48, wherein detects described minimizing or increase in described human blood.

The purposes of 51. claim 1, wherein in the people's whole blood IL-1 β inhibition test measuring the beta induced IL-8 production of IL-1, described antibody or its fragment have the IC lower than IL-1 beta receptor antagonist ₅₀.

The purposes of 52. claim 51, wherein said IL-1 beta receptor antagonist is Antril (Synergen).

53. what be used for the treatment of people is selected from following disease or the medicine of disease: the obesity that type 2 diabetes mellitus, insulin resistance, the type 2 diabetes mellitus hyperglycemia of being correlated with is relevant with type 2 diabetes mellitus, wherein said pharmaceutical pack containing anti-il-i-beta antibody or its fragment, wherein said medicine as in any one of claim 1-52 define.