WO2011083477A1 - Immuno-tolerance formulations - Google Patents

Abstract

A method of inducing immune tolerance to a pancreatic beta cell in a diabetic or a pre-diabetic patient having antibodies directed against at least one beta cell antigen is provided. Moreover, compositions which comprise 500-100,000 pancreatic beta cells, for the induction of immune tolerance to a pancreatic beta cell in patients in need of such treatment, are also provided.

Description

IMMUNO-TOLERANCE FORMULATIONS

FIELD OF INVENTION

[001] A method of inducing immune tolerance to a pancreatic beta cell in a subject is provided.

BACKGROUND OF THE INVENTION [002] In response to the growing health burden of diabetes, the diabetes community has three choices: prevent diabetes; cure diabetes; and take better care of people with diabetes to prevent devastating complications. All three approaches are actively being pursued by the US Department of Health and Human Services. However, diabetes prevention and cure can change the lives of millions of patients that depend on multiple medications for their every day survival.

[003] In type 1 diabetes, the beta cells of the pancreas produce insufficient amount or no insulin, the hormone that allows glucose to enter body cells. Without adequate insulin, glucose builds up in the bloodstream instead of going into the cells. The body is unable to use this glucose for energy despite high levels in the bloodstream, leading to increased hunger. The high levels of glucose in the blood causes the patient to urinate frequently, which in turn causes excessive thirst. Within 5 to 10 years after diagnosis, the insulin-producing beta cells of the pancreas are completely destroyed, and no more insulin is produced.

[004] Type 1 diabetes can occur at any age, but it usually starts in people younger than 30. Symptoms are usually severe and occur rapidly. The exact cause of type 1 diabetes is not known. Type 1 diabetes accounts for 10% of all new cases of diabetes each year. New cases are less common among adults older than 20.

[005] At diagnosis, the immediate goals of treatment are to treat diabetic ketoacidosis (also called DKA) and high blood glucose levels. Because of the sudden onset and severity of symptoms in type 1 diabetes, treatment for newly diagnosed people may involve hospitalization. The long-term goals of treatment are to prolong life, reduce symptoms, and prevent diabetes-related complications such as blindness, kidney failure, and amputation of limbs. [006] Several approaches to "cure" diabetes were being pursued including: pancreas transplantation, islet cell transplantation, artificial pancreas development, and genetic manipulation of cells. Each of these approaches still has a lot of challenges, such as preventing immune rejection; finding an adequate number of insulin cells; keeping cells alive; and others.

SUMMARY OF THE INVENTION

[007] In one embodiment, the present invention provides a method of inducing a state of immune tolerance to a pancreatic beta cell in a subject in need thereof, comprising the step of administering to the subject a composition comprising an effective amount of beta cells, thereby inducing a state of immune tolerance to a pancreatic beta cell in a subject.

[008] In another embodiment, the present invention further provides a method of reducing type 1 diabetes risk in a subject in need thereof, comprising the step of administering to the subject a composition comprising an effective amount of beta cells, thereby reducing type 1 diabetes risk in a subject in need thereof.

[009] In another embodiment, the present invention further provides a method of transplanting a composition comprising pancreatic beta cells in a subject in need thereof, comprising the steps of: (a) inducing a state of immune tolerance to a pancreatic beta cell in the subject, comprising the step of administering to the subject a composition comprising an effective amount of beta cells; and (b) transplanting a composition comprising pancreatic beta cells; thereby, transplanting a composition comprising pancreatic beta cells in a subject in need thereof .

DETAILED DESCRIPTION OF THE INVENTION

[010] In one embodiment, the present invention provides a method of inducing a state of immune tolerance to a pancreatic beta cell in a subject in need thereof, comprising the step of administering to a subject a composition comprising an effective amount of beta cells, thereby inducing a state of immune tolerance to a pancreatic beta cell in a subject. In another embodiment, the present invention provides a method of inducing a state of immune tolerance to proteins produced by a pancreatic beta cell in a subject in need thereof, comprising the step of administering to a subject a composition comprising an effective amount of beta cells, thereby inducing a state of immune tolerance to a pancreatic beta cell in a subject. In another embodiment, the present invention provides a method of inducing a state of immune tolerance to antigens present within a pancreatic beta cell and on a membrane thereof in a subject in need thereof, comprising the step of administering to a subject a composition comprising an effective amount of beta cells, thereby inducing a state of immune tolerance to a pancreatic beta cell in a subject. In another embodiment, the term immune tolerance as used herein includes the ability to decrease an immune response against self beta cell antigens. In another embodiment, the term immune tolerance as used herein includes "induced tolerance", where tolerance to internal beta cell antigens is achieved by manipulating the immune system. In one embodiment, the present invention provides a preventive measure taken against type 1 diabetes. In another embodiment, beta cells are pancreatic beta cells. In another embodiment, beta cells are pancreatic beta cells located in the islets of Langerhans. In another embodiment, beta cells are pancreatic insulin-producing cells. In another embodiment, beta cells are pancreatic primary beta cells. In another embodiment, inducing a state of immune tolerance to a pancreatic beta cell is inducing a state of immune tolerance to a differentiated pancreatic beta cell. In another embodiment, inducing a state of immune tolerance to a pancreatic beta cell is inducing a state of immune tolerance to a · pancreatic beta cell antigen.

[Oi l] In another embodiment, the present invention provides a method of preventing future loss of beta cells in a diabetic subject or a subject at risk of being afflicted with type I diabetes, comprising the step of administering to a subject a composition comprising an effective amount of beta cells. In another embodiment, the present invention provides a method of reversing or inhibiting the loss of beta cells due to an autoimmune response directed against beta cells in a subject, comprising the step of administering to a subject a composition comprising an effective amount of beta cells. In another embodiment, the present invention provides a method of inhibiting the loss of beta cells due to an autoimmune response directed against beta cells in a type-1 diabetic subject, comprising the step of administering to the subject a composition comprising an effective amount of beta cells. In another embodiment, the present invention provides a method of reversing or inhibiting the loss of beta cells due to an autoimmune response directed against beta cells in a type-1 pre- diabetic subject, comprising the step of administering to the subject a composition comprising an effective amount of beta cells. In another embodiment, the present invention provides a method of preventing the loss of beta cells due to an autoimmune response directed against beta cells in a type-1 pre-diabetic subject, comprising the step of administering to the subject a composition comprising an effective amount of beta cells. In another embodiment, the present invention provides a method of preventing the loss of beta cells due to an autoimmune response directed against beta cells in a subject having a risk of being afflicted with type-1 diabetes, comprising the step of administering to the subject a composition comprising an effective amount of beta cells. In another embodiment, a diabetic subject has reduced amounts of beta cells. In another embodiment, a diabetic subject has no beta cells.

[012] In another embodiment, the present invention provides a method for prolonging the lifespan of a diabetic patient. In another embodiment, the present invention provides a method for improving the quality of life of a diabetic patient. In another embodiment, the present invention provides a method for reducing the frequency of diabetes within a population. In another embodiment, the present invention provides a method for reducing the tremendous costs associated with diabetes treatment and complications including secondary complications.

[013] In another embodiment, the invention provides a method of preventing, treating, or abrogating symptoms related to diabetes in a subject. In another embodiment, the invention provides a method of preventing, treating, or abrogating symptoms related to diabetes related to vascular deterioration in a subject. In another embodiment, the invention provides a method of preventing, treating, or abrogating neuropathy. In another embodiment, the invention provides a method of preventing, treating, or abrogating diabetes induced neuropathy.

[014] In another embodiment, the present invention provides a method of inducing a state of immune tolerance to beta cells. In another embodiment, the present invention provides a method of inducing a state of immune tolerance in a subject with antibodies to beta cell proteins. In another embodiment, the present invention provides a method of inhibiting antibody synthesis by B-cells (lymphocytes) which target beta cells. In another embodiment, immune tolerance is the reduction in the activity of T-cells which target beta cells. In another embodiment, immune tolerance is the reduction in the quantity of T-cells which target beta cells. In another embodiment, immune tolerance comprises the elimination of T-cells which target beta cells. [015] In another embodiment, immune tolerance is the reduction in the activity of antibodies directed against beta cell antigens. In another embodiment, immune tolerance is the reduction in the quantity of antibodies directed against beta cell antigens. In another embodiment, immune tolerance is the elimination of antibodies directed against beta cell antigens.

[016] In another embodiment, the subject is afflicted with diabetes mellitus. In another embodiment, the subject is afflicted with type 1 diabetes. In another embodiment, the subject is afflicted with immune-mediated diabetes. In another embodiment, the subject has antibodies directed to beta cell proteins. In another embodiment, the subject has antibodies that recognize beta cell proteins. In another embodiment, the subject is diagnosed with type 1 diabetes risk. In another embodiment, the subject suffers from a reduction or loss of the insulin-producing beta cells of the islets of Langerhans in the pancreas. In another embodiment, the subject suffers from insulin deficiency.

[017] In another embodiment, a subject as described herein is treated with exogenous insulin. In another embodiment, according to the methods as described herein, exogenous insulin treatment continues during the application of the immune tolerance methods.

[018] In another embodiment, a pre-diabetic subject is a subject with Type 1 diabetes risk. In another embodiment, a pre-diabetic subject is a subject with Type 1 diabetes risk. In another embodiment, a pre-diabetic subject is a subject with a genetic predisposition based on HLA types (particularly types DR3 and DR4). In another embodiment, a pre-diabetic subject is a subject afflicted with uncontrolled autoimmune response that attacks the insulin producing beta cells. In another embodiment, a pre-diabetic subject is a subject with antibodies to beta cell proteins but no overt diabetes.

[019] In another embodiment, the methods as described herein reduce the risk of long term complications associated with type 1 diabetes. In another embodiment, one of skill in the art can readily identify long-term and short-term complications and risks associated with type 1 diabetes. [020] In another embodiment, a composition as described herein comprises 500-100,000 Beta cells. In another embodiment, a composition as described herein comprises 2,000- 20,000 Beta cells. In another embodiment, a composition as described herein comprises 5,000-10,000 Beta cells. In another embodiment, a composition as described herein comprises 2,000-5,000 Beta cells. In another embodiment, a composition as described herein comprises 5,000-10,000 Beta cells. In another embodiment, a composition as described herein comprises 6,000-8,000 Beta cells. In another embodiment, a composition as described herein comprises 10,000-15,000 Beta cells. In another embodiment, a composition as described herein comprises 10,000-20,000 Beta cells.

[021] In another embodiment, an effective single dosage of beta cells comprises 500- 100,000 Beta cells. In another embodiment, an effective single dosage of beta cells comprises 2,000-20,000 Beta cells. In another embodiment, an effective single dosage of beta cells comprises 5,000-10,000 Beta cells. In another embodiment, an effective single dosage of beta cells comprises 2,000-5,000 Beta cells. In another embodiment, an effective single dosage of beta cells comprises 5,000-10,000 Beta cells. In another embodiment, an effective single dosage of beta cells comprises 6,000-8,000 Beta cells. In another embodiment, an effective single dosage of beta cells comprises 10,000-15,000 Beta cells. In another embodiment, an effective single dosage of beta cells comprises 10,000-20,000 Beta cells.

[022] In another embodiment, a composition as described herein comprises a cell culture nutrient solution. In another embodiment, a composition as described herein comprises a pH modifying agent. In another embodiment, a pH modifying agent maintains the composition as described herein within a pH range of 5-10. In another embodiment, a pH modifying agent maintains the composition as described herein within a pH range of 6-8. In another embodiment, a pH modifying agent maintains the composition as described herein within a pH range of 6.8-7.8. In another embodiment, pH modifying agents as described herein are readily available to one of average skill in the art. [023] In another embodiment, a composition as described herein comprises cell culture media. In another embodiment, a composition as described herein comprises animal-free cell culture media. In another embodiment, a composition as described herein comprises glucose. In another embodiment, a composition as described herein comprises growth factors. In another embodiment, a composition as described herein comprises nutrients. In another embodiment, a composition as described herein comprises growth factors derived from animal blood. In another embodiment, a composition as described herein comprises serum. In another embodiment, a composition as described herein comprises passaged beta cells. In another embodiment, a composition as described herein is a sterile composition. In another embodiment, a composition as described herein comprises an antibiotic (e.g. penicillin or streptomycin). In another embodiment, a composition as described herein comprises an antifungal (e.g. Amphotericin B). In another embodiment, a composition as described herein comprises a pH indicator. In another embodiment, a pH indicator is added to the medium in order to measure nutrient depletion.

[024] In another embodiment, a composition as described herein comprises DL- Dithiothreitol. . In another embodiment, a composition as described herein comprises citric acid. . In another embodiment, a composition as described herein comprises ethanolamine. In another embodiment, a composition as described herein comprises glycerol 2-phosphate disodium salt. In another embodiment, a composition as described herein comprises HEPES solution. In another embodiment, a composition as described herein comprises potassium citrate tribasic. In another embodiment, a composition as described herein comprises potassium phosphate monobasic. In another embodiment, a composition as described herein comprises sodium acetate. In another embodiment, a composition as described herein comprises sodium bicarbonate. In another embodiment, a composition as described herein comprises sodium chloride. In another embodiment, a composition as described herein comprises sodium phosphate dibasic. In another embodiment, a composition as described herein comprises sodium phosphate monobasic.

[025] In another embodiment, a composition as described herein comprises 2'- Deoxyadenosine monohydrate. In another embodiment, a composition as described herein comprises 2'-Deoxycytidine hydrochloride. In another embodiment, a composition as described herein comprises 2'-Deoxyguanosine monohydrate. In another embodiment, a composition as described herein comprises 2-Deoxy-D-ribose. In another embodiment, a composition as described herein comprises 2-Mercaptoethanol. In another embodiment, a composition as described herein comprises 4-Nitrophenyl phosphate disodium salt hexahydrate. In another embodiment, a composition as described herein comprises D-(+)- Galactosamine hydrochloride. In another embodiment, a composition as described herein D- (+)-Gluconic acid δ-lactone. In another embodiment, a composition as described herein comprises D-(+)-Galactose anhydrous. In another embodiment, a composition as described herein comprises D-(+)-Raffinose pentahydrate. In another embodiment, a composition as described herein comprises D-(+)-Trehalose dihydrate. In another embodiment, a composition as described herein comprises D-Glucuronic acid sodium salt monohydrate. [026] In another embodiment, a composition as described herein comprises L-2- Aminobutyric acid. In another embodiment, a composition as described herein comprises L- Glutamic acid monosodium salt hydrate. In another embodiment, a composition as described herein comprises L-Glutamic acid potassium salt monohydrate. In another embodiment, a composition as described herein comprises L-Glutathione oxidized disodium salt. In another embodiment, a composition as described herein comprises L-a-Phosphatidylcholine Type III- S. In another embodiment, a composition as described herein comprises acetylcholine chloride. In another embodiment, a composition as described herein comprises adenine hemisulfate. In another embodiment, a composition as described herein comprises adenine hydrochloride. In another embodiment, a composition as described herein comprises adenosine 5 '-monophosphate sodium salt. In another embodiment, a composition as described herein comprises adenosine 5 '-triphosphate disodium salt. In another embodiment, a composition as described herein comprises a bioreactor pH adjustment solution. In another embodiment, a composition as described herein comprises Calcium L-lactate. In another embodiment, a composition as described herein comprises carboxyethyl-Y-aminobutyric acid. In another embodiment, a composition as described herein comprises casein. In another embodiment, a composition as described herein comprises Cobalt (II) chloride hexahydrate. In another embodiment, a composition as described herein comprises coenzyme A sodium , salt hydrate.

[027] In another embodiment, a composition as described herein comprises concanavalin A. In another embodiment, a composition as described herein comprises Copper(II) chloride dihydrate. In another embodiment, a composition as described herein comprises copper(II) sulfate pentahydrate. In another embodiment, a composition as described herein comprises demecolcine solution. In another embodiment, a composition as described herein comprises diisopropylamine. In another embodiment, a composition as described herein comprises ferric citrate. In another embodiment, a composition as described herein comprises fetuin. In another embodiment, a composition as described herein comprises flavin adenine dinucleotide disodium salt hydrate. In another embodiment, a composition as described herein comprises Gly-Gln solution. In another embodiment, a composition as described herein comprises guanosine. In another embodiment, a composition as described herein comprises heparin sodium salt. In another embodiment, a composition as described herein comprises hydrochloric acid. In another embodiment, a composition as described herein comprises Iron(II) sulfate heptahydrate. In another embodiment, a composition as described herein comprises Iron(III) nitrate nonahydrate. In another embodiment, a composition as described herein comprises Iron(III) phosphate. In another embodiment, a composition as described herein comprises Lactalbumin enzymatic hydrolysate. In another embodiment, a composition as described herein comprises lactoferrin. In another embodiment, a composition as described herein comprises lectin. In another embodiment, a composition as described herein comprises Manganese(II) chloride tetrahydrate. In another embodiment, a composition as described herein comprises Manganese(II) sulfate monohydrate. [028] In another embodiment, a composition as described herein comprises sodium hydroxide. In another embodiment, a composition as described herein comprises sodium propionate. In another embodiment, a composition as described herein comprises sodium pyruvate. In another embodiment, a composition as described herein comprises sodium succinate dibasic hexahydrate. In another embodiment, a composition as described herein comprises succinic acid. In another embodiment, a composition as described herein comprises thrombin. In another embodiment, a composition as described herein comprises Thymidine. In another embodiment, a composition as described herein comprises Uracil. In another embodiment, a composition as described herein Uridine. In another embodiment, a composition as described herein comprises water. In another embodiment, a composition as described herein comprises zinc chloride. In another embodiment, a composition as described herein comprises zinc sulfate heptahydrate. In another embodiment, a composition as described herein comprises a-Lactose monohydrate.

[029] In another embodiment, beta cells of the invention are grown and maintained at 37°C. In another embodiment, beta cells of the invention are grown in an incubator and supplemented with 5% C0₂.

[030] In another embodiment, a composition as described herein comprises beta cells and a buffer. In another embodiment, a composition as described herein comprises beta cells and a phosphate buffered saline. In another embodiment, a composition as described herein is prepared 1 minute to 14 days prior to administration. In another embodiment, a composition as described herein is prepared 1-48 hours prior to administration. In another embodiment, a composition as described herein is prepared instantly before administration. In another embodiment, a composition as described herein is prepared few minutes (1-30 minutes) before administration. In another embodiment, a composition as described herein is prepared 30 minutes to an hour before administration. In another embodiment, a composition as described herein is prepared 1-5 hours prior to administration. In another embodiment, a composition as described herein is prepared 3-10 hours prior to administration. In another embodiment, a composition as described herein is prepared 5-15 hours prior to administration. In another embodiment, a composition as described herein is prepared 10-20 hours prior to administration. In another embodiment, a composition as described herein is prepared 20-48 hours prior to administration.

[031] In another embodiment, a composition as described herein comprises syngeneic beta cells. In another embodiment, a composition as described herein comprises normal syngeneic beta cells. In another embodiment, a composition as described herein comprises normal syngeneic precursor beta cells. In another embodiment, a composition as described herein comprises syngeneic differentiated beta cells.

[032] In another embodiment, a composition as described herein comprises allogeneic beta cells. In another embodiment, a composition as described herein comprises normal allogeneic beta cells. In another embodiment, a composition as described herein comprises normal allogeneic precursor beta cells. In another embodiment, a composition as described herein comprises allogeneic differentiated beta cells. In another embodiment, a composition as described herein comprises viable beta cells. In another embodiment, a composition as described herein comprises lysed beta cells.

[033] In another embodiment, a composition as described herein comprises xenogeneic beta cells. In another embodiment, a composition as described herein comprises normal xenogeneic beta cells. In another embodiment, a composition as described herein comprises normal xenogeneic precursor beta cells. In another embodiment, a composition as described herein comprises xenogeneic differentiated beta cells.

[034] In another embodiment, the methods as described herein comprise drinking isotonic liquid prior to the administration of a composition comprising beta cells. In another embodiment, the methods as described herein comprise drinking water prior to the administration of a composition comprising beta cells. In another embodiment, the methods as described herein comprise drinking 100 ml or more of water or isotonic liquid prior to the administration of a composition comprising beta cells. In another embodiment, the methods as described herein comprise drinking 0.2-2.0 liters of water or isotonic liquid prior to the administration of a composition comprising beta cells. In another embodiment, the methods as described herein comprise drinking 0.2-0.5 liters of water or isotonic liquid prior to the administration of a composition comprising beta cells. In another embodiment, the methods as described herein comprise drinking 0.5-1.0 liters of water or isotonic liquid prior to the administration of a composition comprising beta cells. In another embodiment, the methods as described herein comprise drinking 1.0-1.5 liters of water or isotonic liquid prior to the administration of a composition comprising beta cells. In another embodiment, the methods as described herein comprise drinking 1.0-2.0 liters of water or isotonic liquid prior to the administration of a composition comprising beta cells.

[035] In another embodiment, the methods as described herein comprise the administration of a composition comprising beta cells 1-3 times a day. In another embodiment, the methods as described herein comprise the administration of a composition comprising beta cells once a day. In another embodiment, the methods as described herein comprise the administration of a composition comprising beta cells once every 2 days. In another embodiment, the methods as described herein comprise the administration of a composition comprising beta cells once every 3 days. In another embodiment, the methods as described herein comprise the administration of a composition comprising beta cells once every 4 days. In another embodiment, the methods as described herein comprise the administration of a composition comprising beta cells once every 5 days. In another embodiment, the methods as described herein comprise the administration of a composition comprising beta cells once every 6 days. In another embodiment, the methods as described herein comprise the administration of a composition comprising beta cells once every 7 days. In another embodiment, the methods as described herein comprise the administration of a composition comprising beta cells once every 8 days. In another embodiment, the methods as described herein comprise the administration of a composition comprising beta cells once every 9 days. In another embodiment, the methods as described herein comprise the administration of a composition comprising beta cells once every 10 days. In another embodiment, the methods as described herein comprise the administration of a composition comprising beta cells once every 1 1 days. In another embodiment, the methods as described herein comprise the administration of a composition comprising beta cells once every 12 days. In another embodiment, the methods as described herein comprise the administration of a composition comprising beta cells once every 13 days. In another embodiment, the methods as described herein comprise the administration of a composition comprising beta cells once every 14 days. [036] In another embodiment, the methods as described herein comprise the administration of a composition comprising beta cells once every two to three weeks. In another embodiment, the methods as described herein comprise the administration of a composition comprising beta cells once every two weeks to one month. In another embodiment, the methods as described herein comprise the administration of a composition comprising beta cells once every one month to three months.

[037] In another embodiment, the methods as described herein comprise orally administrating a composition comprising beta cells as described herein. In another embodiment, the methods as described herein comprise parenterally administrating a composition comprising beta cells as described herein. In another embodiment, the methods as described herein comprise intra-intestinal administration of a composition comprising beta cells as described herein. In another embodiment, the methods as described herein comprise intra-duodenal administration of a composition comprising beta cells as described herein. In another embodiment, the methods as described herein comprise intra-jejunum administration of a composition comprising beta cells as described herein. In another embodiment, the methods as described herein comprise intra-ileum administration of a composition comprising beta cells as described herein. [038] In another embodiment, provided herein a method of reducing type 1 diabetes risk in a subject in need thereof, comprising the step of administering to a subject a composition comprising an effective amount of beta cells, thereby reducing type 1 diabetes risk in a subject in need thereof. In another embodiment, provided herein a method of preventing type 1 diabetes in a subject, comprising the step of administering to a subject a composition comprising an effective amount of beta cells. In another embodiment, reducing type 1 diabetes risk in a subject comprises preventing diabetes. In another embodiment, type 1 diabetes risk comprises several factors. In another embodiment, type 1 diabetes risk in humans comprises early exposure to cow's milk in infancy and not being breastfed. In another embodiment, type 1 diabetes risk comprises having a parent with type 1 diabetes. In another embodiment, type 1 diabetes risk comprises having an older mother. In another embodiment, type 1 diabetes risk comprises having a mother who had preeclampsia during pregnancy. In another embodiment, type 1 diabetes risk comprises having another immune abnormality. In another embodiment, type 1 diabetes risk comprises having an autoimmune disease. In another embodiment, type 1 diabetes risk comprises having Grave's disease. In another embodiment, type 1 diabetes risk comprises having Hashimoto's thyroiditis. In another embodiment, type 1 diabetes risk comprises having Addison's disease. In another embodiment, type 1 diabetes risk comprises having multiple sclerosis (MS). In another embodiment, type 1 diabetes risk comprises having pernicious anemia. In another embodiment, type 1 diabetes risk comprises exposure to mumps virus. In another embodiment, type 1 diabetes risk comprises exposure to a cytomegalovirus. In another embodiment, type 1 diabetes risk includes low vitamin D levels. In another embodiment, type 1 diabetes risk includes being born with jaundice or experiencing a respiratory infection just after birth.

[039] In another embodiment, a subject diagnosed as having type 1 diabetes risk comprises antibodies to beta cell proteins. In another embodiment, a subject diagnosed as having type 1 diabetes risk comprises the allele DR3, DR4, DQ2.5, DR7, DR8, DR15, or any combination thereof.

[040] In another embodiment, provided herein a method of transplanting a composition comprising pancreatic beta cells in a subject in need thereof, comprising the steps of: inducing a state of immune tolerance to a pancreatic beta cell in the subject, comprising the step of administering to the subject a composition comprising an effective amount of beta cells; and transplanting a composition comprising pancreatic beta cells; thereby, transplanting a composition comprising pancreatic beta cells in a subject in need thereof. In another embodiment, provided herein a method of reducing the risk of graft rejection in a subject, wherein the graft comprises pancreatic beta cells, comprising the step of inducing immune- tolerance to pancreatic beta cells or antigens of pancreatic beta cells in the subject who's destined to pass pancreatic beta cells transplantation. In another embodiment, the subject is destined to pass pancreatic beta cells transplantation. In another embodiment, the subject is afflicted with diabetes. In another embodiment, the subject is afflicted with diabetes type-1.

[041] In another embodiment, this invention relies upon routine techniques in the field of cell culture, and suitable methods can be determined by those of skill in the art using known methodology (see, e.g., Freshney et al., Culture of Animal Cells (3rd ed. 1994)). In general, the cell culture environment includes consideration of such factors as the substrate for cell growth, cell density and cell contact, the gas phase, the medium, and temperature.

[042] In another embodiment, beta cells of the invention are grown under conditions that provide for maximal cell to cell contact. In another embodiment, the cell-to-cell contact occurs to a greater degree than found in monolayer cell cultures. In another embodiment, the cells are grown in suspension as three dimensional aggregates. In another embodiment, the cells are grown in Costar dishes that have been coated with a hydrogel to prevent them from adhering to the bottom of the dish. In another embodiment, the cells are cultured under adherent conditions, plastic dishes, flasks, roller bottles, or microcarriers in suspension. In another embodiment, artificial substrates are used such as glass and metals. In another embodiment, the substrate is treated by etching, or by coating with substances such as collagen, chondronectin, fibronectin, and laminin. In another embodiment, the type of culture vessel depends on the culture conditions, e.g., multi-well plates, petri dishes, tissue culture tubes, flasks, roller bottles, and the like. In another embodiment, cultured cells are normally grown in an incubator that provides a suitable temperature, e.g., the body temperature of the animal from which is the cells were obtained, accounting for regional variations in temperature. In another embodiment, an incubator is a humidified incubator. In another embodiment, atmospheric oxygen tensions are used for cell cultures. In another embodiment, carbon dioxide plays a role in pH stabilization, along with buffer in the cell media and is typically present at a concentration of 1-10% in the incubator. [043] In another embodiment, known cell media are used. In another embodiment, defined cell media comprises premixed powders or presterilized solutions. In another embodiment, cell media comprises DME, RPMI 1640, DMEM, Iscove's complete media, or McCoy's Medium. In another embodiment, cell media comprises low glucose DME or RPMI 1640. In another embodiment, cell media is supplemented with 5-20% serum, typically heat inactivated, e.g., human horse, calf, and fetal bovine serum. In another embodiment, cell media is supplemented with 10% fetal bovine serum. In another embodiment, cell media is buffered to maintain the cells at a pH preferably from 7.0-7.8. In another embodiment, cell media is supplemented with antibiotics, amino acids, sugars, growth factors, or any combination thereof. [044] In another embodiment, a subject is a human subject. In another embodiment, a subject is a pet. In another embodiment, a subject is a laboratory animal. In another embodiment, a subject is a rodent. In another embodiment, a subject is a farm animal. In another embodiment, a subject is a diabetic subject. In another embodiment, a subject is a subject afflicted with any type of diabetes, including primary and secondary diabetes, type 1 IDDM, type 2 IDDM-transient, and type 2 MODY, as described in Harrison's Internal Medicine, 14th ed. 1998. [045] In some embodiments, the beta cells are transformed beta cells. In another embodiment, the beta cells are cultured under effective conditions, which allow for the expression of high amounts a certain protein of interest. In another embodiment, effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production. In some embodiments, a medium typically includes an aqueous solution having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. In some embodiments, cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes and petri plates. In some embodiments, culturing is carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. In some embodiments, culturing conditions are within the expertise of one of ordinary skill in the art.

[046] In one embodiment, the present invention provides combined preparations. In one embodiment, "a combined preparation" defines especially a "kit of parts" in the sense that the combination partners as defined above can be dosed independently or by use of different fixed combinations with distinguished amounts of the combination partners i.e., simultaneously, concurrently, separately or sequentially. In some embodiments, the parts of the kit of parts can then, e.g., be administered simultaneously or chronologically staggered, that is at different time points and with equal or different time intervals for any part of the kit of parts. The ratio of the total amounts of the combination partners, in some embodiments, can be administered in the combined preparation. In one embodiment, the combined preparation can be varied, e.g., in order to cope with the needs of a patient subpopulation to be treated or the needs of the single patient which different needs can be due to a particular disease, severity of a disease, age, sex, or body weight as can be readily made by a person skilled in the art. [047] In another embodiment, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which can be interchangeably used, refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered beta cells. An adjuvant is included under these phrases. [048] In another embodiment, "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of the beta cells. In one 3 embodiment, excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

[049] Techniques for formulation and administration of cells are readily available to one of average skill in the art. [050] In another embodiment, suitable routes of administration, for example, include oral, rectal, transmucosal, intestinal or parenteral delivery, including intravenous or intraperitoneal injections.

[051] In another embodiment, treatment according to the methods as described herein last for at least 10 weeks. In another embodiment, treatment according to the methods as described herein last for at least 20 weeks. In another embodiment, treatment according to the methods as described herein last for at least 30 weeks. In another embodiment, treatment according to the methods as described herein last for 10-40 weeks. In another embodiment, treatment according to the methods as described herein last for 20-40 weeks. In another embodiment, treatment according to the methods as described herein last for 30-40 weeks. In another embodiment, treatment according to the methods as described herein comprises administering 500-50000 beta cells to a subject in need. In another embodiment, treatment according to the methods as described herein comprises weekly administration of 500-50000 beta cells to a subject in need. In another embodiment, treatment according to the methods as described herein comprises biweekly administration of 500-50000 beta cells to a subject in need. In another embodiment, treatment according to the methods as described herein comprises weekly administration of 500- 10000 beta cells to a subject in need.

[052] In another embodiment, the preparation is administered in a local rather than systemic manner, for example, via injection of the preparation directly into a specific region of a patient's body. In another embodiment, the preparation is administered in a local rather than systemic manner, for example, via injection of the preparation directly into a specific region of a patient's intestine.

[053] Oral administration, in one embodiment, comprises a unit dosage form comprising, capsules, suspensions, emulsions and the like. Such unit dosage forms comprise a safe and effective amount of a composition comprising beta cells of the invention as described herein. The pharmaceutically-acceptable carriers suitable for the preparation of unit dosage forms for peroral administration are well-known in the art. [054] Peroral compositions, in some embodiments, comprise liquid solutions, emulsions, suspensions, and the like. In some embodiments, pharmaceutically-acceptable carriers suitable for preparation of such compositions are well known in the art.

[055] In another embodiment, the pharmaceutical compositions are administered by intravenous, intra-arterial, or intramuscular injection of a liquid preparation. In some embodiments, liquid formulations include solutions, suspensions, dispersions, emulsions, oils and the like. In one embodiment, the pharmaceutical compositions are administered intravenously, and are thus formulated in a form suitable for intravenous administration. In another embodiment, the pharmaceutical compositions are administered intra-arterially, and are thus formulated in a form suitable for intra-arterial administration. In another embodiment, the pharmaceutical compositions are administered intramuscularly, and are thus formulated in a form suitable for intramuscular administration.

[056] In another embodiment, pharmaceutical compositions of the present invention are manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[057] In In one embodiment, injectables, of the invention are formulated in aqueous solutions. In one embodiment, injectables, of the invention are formulated in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. In some embodiments, for transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[058] In another embodiment, the preparations described herein are formulated for parenteral administration, e.g., by bolus injection or continuous infusion. In some embodiments, formulations for injection are presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. In some embodiments, compositions are suspensions, solutions or emulsions in oily or aqueous vehicles, and contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

[059] The compositions also comprise, in some embodiments, preservatives, such as benzalkonium chloride and thimerosal and the like; chelating agents, such as edetate sodium and others; buffers such as phosphate, citrate and acetate; tonicity agents such as sodium chloride, potassium chloride, glycerin, mannitol and others; antioxidants such as ascorbic acid, acetylcystine, sodium metabisulfite and others; aromatic agents; viscosity adjustors, such as polymers, including cellulose and derivatives thereof; and polyvinyl alcohol and acid and bases to adjust the pH of these aqueous compositions as needed. The compositions also comprise, in some embodiments, local anesthetics or other actives. The compositions can be used as sprays, mists, drops, and the like.

[060] In one embodiment, determination of a therapeutically effective amount of beta cells is well within the capability of those skilled in the art.

[061] Some examples of substances which can serve as pharmaceutically-acceptable carriers or components thereof are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and methyl cellulose; powdered tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil of theobroma; polyols such as propylene glycol, glycerine, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as the Tween™ brand emulsifiers; wetting agents, such sodium lauryl sulfate; coloring agents; flavoring agents; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline; and phosphate buffer solutions. The choice of a pharmaceutically-acceptable carrier to be used in conjunction with the compound is basically determined by the way the compound is to be administered. If the subject compound is to be injected, in one embodiment, the pharmaceutically-acceptable carrier is sterile, physiological saline, with a blood-compatible suspending agent, the pH of which has been adjusted to about 7.4.

[062] In addition, the compositions further comprise binders (e.g. acacia, cornstarch, gelatin, carbomer. ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone), disintegrating agents (e.g. cornstarch, potato starch, alginic acid, silicon dioxide, croscarmelose sodium, crospovidone, guar gum, sodium starch glycolate), buffers (e.g., Tris- HCL, acetate, phosphate) of various pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), protease inhibitors, surfactants (e.g. sodium lauryl sulfate), permeation enhancers, solubilizing agents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g. hydroxypropyl cellulose, hyroxypropylmethyl cellulose), viscosity increasing agents(e.g. carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum), sweeteners (e.g. aspartame, citric acid), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), lubricants (e.g. stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow-aids (e.g. colloidal silicon dioxide), plasticizers (e.g. diethyl phthalate, triethyl citrate), emulsifiers (e.g. carbomer, hydroxypropyl cellulose, sodium lauryl sulfate), polymer coatings (e.g., poloxamers or poloxamines), coating and film forming agents (e.g. ethyl cellulose, acrylates, polymethacrylates) and/or adjuvants.

[063] Typical components of carriers for syrups, elixirs, emulsions and suspensions include ethanol, glycerol, propylene glycol, polyethylene glycol, liquid sucrose, sorbitol and water. For a suspension, typical suspending agents include methyl cellulose, sodium carboxymethyl cellulose, cellulose (e.g. Avicel™, RC-591), tragacanth and sodium alginate; typical wetting agents include lecithin and polyethylene oxide sorbitan (e.g. polysorbate 80). Typical preservatives include methyl paraben and sodium benzoate. In another embodiment, peroral liquid compositions also contain one or more components such as sweeteners, flavoring agents and colorants disclosed above.

[064] In some embodiments, preparation of effective amount or dose can be estimated initially from in vitro assays. In one embodiment, a dose can be formulated in animal models and such information can be used to more accurately determine useful doses in humans.

[065] In one embodiment, toxicity and therapeutic efficacy of beta cells described herein can be determined by standard pharmaceutical procedures such as in experimental animals. In one embodiment, the dosages vary depending upon the dosage form employed and the route of administration utilized. In one embodiment, the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition or response. [See e.g., Fingl, et al., (1975) "The Pharmacological Basis of Therapeutics", Ch. 1 p.l].

[066] In one embodiment, depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks, months, or until cure is effected or diminution of the disease state is achieved.

[067] In another embodiment, the amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc. [068] In one embodiment, compositions including the preparation of the present invention are formulated in a compatible pharmaceutical carrier, placed in an appropriate container, and labeled for treatment of an indicated condition as described herein.

[069] In another embodiment, the pharmaceutical composition as described herein is stabilized when placed in buffered solutions having a pH between about 4 and 7.2. In another embodiment, the pharmaceutical composition as described herein is stabilized with an amino acid as a stabilizing agent and in some cases a salt (if the amino acid does not contain a charged side chain).

[070] In another embodiment, the pharmaceutical composition as described herein is a liquid composition comprising a stabilizing agent at between about 0.3% and 5% by weight which is an amino acid.

[071] In another embodiment, the pharmaceutical composition as described herein provides dosing accuracy and product safety. In another embodiment, the pharmaceutical composition as described herein provides a biologically active, stable liquid formulation. [072] In another embodiment, the pharmaceutical composition as described herein provides a liquid formulation permitting storage for an extended period of time in a liquid state facilitating storage and shipping prior to administration.

[073] In one embodiment, compositions of the present invention are presented in a pack or dispenser device, such as an FDA approved kit. In one embodiment, the pack, for example, comprise metal or plastic foil, such as a blister pack. In one embodiment, the pack or dispenser device is accompanied by instructions for administration. In one embodiment, the pack or dispenser is accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, in one embodiment, is labeling approved by the U.S. Food and Drug Administration for prescription drugs or an approved product insert.

[074] In one embodiment, it will be appreciated that the compositions of the present invention can be provided to the individual with additional active agents to achieve an improved therapeutic effect as compared to treatment with each agent by itself. In another embodiment, measures (e.g., dosing and selection of the complementary agent) are taken to adverse side effects which are associated with combination therapies.

[075] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

EXAMPLES

[076] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical and microbiological techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes i-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide, to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801 ,531 ; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791 ,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; 1 000023

"PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document. EXAMPLE 1

THE INDUCTION OF IMMUNE SYSTEM TOLERANCE TO A UTOANTIGEN (self- antigen) AND TO ALLERGENS

[077] The ability to induce beta cell specific immune-tolerance was shown in a model of type la diabetes (immune mediated). [078] Human patients afflicted with type la diabetes were treated as following: 5,000- 10,000 Beta cells derived from islets of Langerhans were suspended in 5ml of a nutrients solution comprising DMEM or solution N°199. The composition comprising 5,000-10,000 Beta cells and nutrients solution was administered orally or intra- intestinally.

Frequency of introduction [079] The composition comprising 5,000-10,000 Beta cells in 5 ml nutrients solution was administered once a week.

Duration of introduction

[080] The composition comprising 5,000-10,000 Beta cells in 5ml nutrients solution was administered until the titer of antibodies directed against Beta cells, declined below 10. [081] The titer of antibodies directed against Beta cells declined below 10 within 11 to 13 months.

Gastroduodenoscopic administration

[082] A composition comprising 5,000-10,000 beta cells in 5 ml nutrients solution was administered in the morning hours on an empty stomach (after 12 hours of starvation) and then additional 200-300 ml of a physiological solution (0.9% solution NaCl) was administered through a gastroduodenoscope so that the cells washed down the gastroduodenoscope could reach the distal part of the small intestine. Oral administration

[083] A composition comprising 5,000-10,000 Beta cells in 5 ml nutrients solution was administered in the morning hours on an empty stomach (after 12 hours of starvation). Prior to administration of the composition comprising 5,000-10,000 Beta cells and nutrients solution, 200 ml of water were orally administered. Within 15-20 minutes after water administration a composition comprising 5,000-10,000 Beta cells in 5 ml nutrients solution was orally administered and then additional 1.0-1.5 liters of water were administered to ensure "washing down" the composition comprising 5,000-10,000 Beta cells in 5 ml nutrients solution. EXAMPLE 2

USE OF -CELL CULTURES OF RABBITS FOR THE INDUCTION OF THE IMMUNE SYSTEM TOLERANCE TO THE ANTIGENS OF B-CELLS IN PATIENTS

WITH DIABETES MELLITUS TYPE 1A

[084] A method for the induction of immune system tolerance to autoantigens has been developed. The method includes the introduction of antigens to which patients develop an autoimmune reaction into the distal small intestine. The procedure is performed on an empty stomach, after a minimum of 12 hours of fasting with obligatory exception of the impact of hydrochloric acid of the stomach and pancreatic enzymes for proper antigens introduction.

[085] The current experimental models of autoimmune and allergic diseases have been developed in rodents (rats, rabbits, guinea pigs). However, the current method of inducing immune system tolerance to auto-antigens can not be applied to rodents due to the anatomical structure of the intestine in rodents, which includes sites such as pouches which are always filled with food debris.

[086] Considering the lack of valid animal model to perform this study, the method was successfully tested on human volunteers afflicted with diabetes mellitus type la.

The Patients

[087] ' Specifically, the study involved 10 patients diagnosed with diabetes mellitus type la (autoimmune). [088] All patients had elevated islet-cell antibody (ICA) titers. None of the patients suffered from a gastrointestinal disease. This is a long study and 80 weeks passed since the beginning of this study.

Induction of tolerance [089] For the induction of immune tolerance to beta cell antigens, β-cells were obtained from newborn rabbits by using the previously described collagenase/dispase method of cell isolation. The number of β-cells in the culture medium (RPMI-1640) was 1000 β-cells per 1 ml medium.

[090] Beta-cell cultures of newborn rabbits were used in the present study for the induction of immune tolerance to beta cells antigens. These cells are also widely used in diabetology for as a transplantation material to patients with diabetes mellitus (xenotransplantation) into abdominal subcutaneous fat. The β-cell cultures were tested for the absence of pathogenic bacteria and viruses.

[091] Each volunteer patient received 5000 β-cells in a volume of 5 ml RPMI-1640 once a week for at least 7 months. This composition of cells was administered orally, on an empty stomach, after a 12-hour fasting. The suspension of cells was washed down by the patients with two liters of drinking water.

[092] Every two weeks (for 80 weeks) the patients were examined for islet-cell antibody (ICA) titers and the level of C-peptide was measured. [093] The C-peptide level has been examined to confirm the presence of at least small amounts of the patient's own β-cells, because the antibodies to the β-cell membranes in patients with autoimmune diabetes disappear after the elimination of all β-cells.

[094] Table 1 presents data generated from four patients treated by the method of the invention. The data includes: indication of treatment with β-cells, the dynamics of islet-cell antibody titers and the C-peptide level.

Table 1

+ +

+ 40 0.45 + 160 0.22

+ +

+ 20 0.23 + 160 0.07

+ +

+ 10 0.1 + 80 0.25

+ +

+ 5 0.15 + 80 0.1

+ +

+ 2.5 0.16 + 160 0.1

+ +

+ 5 0.18 + 160 0.08

+ +

+ 2.5 0.17 + 80 0.05

+ +

+ 5 0.17 + 80 0.06

+ +

+ 0 0.07 + 80 0.05

+ +

+ 5 0.07 + 40 0.09

+ +

+ 0 0.28 + 40 0.08

+ +

+ 0 0.15 + 20 0.08

+ +

+ 0 0.23 + 40 0.04

+ +

0 0.15 + 40 0.09

+

0 0.12 + 40 0.06

+

0 0.18 + 40 0.14

+

0 0.26 + 40 0.14

+

0 0.09 + 20 0.04

+

0 0.07 + 20 0.07

+

0 0.1 1 + 10 0.1 1

+

0 0.15 + 40 0.14

+

0 0.19 + 20 0.15

+

0 0.18 + 10 0.16

+

0 0.16 + 20 0.15 50 +

51 0 0.16 + 20 0.15

52 +

53 0 0.16 + 0 0.15

54 +

55 0 0.14 + 10 0.13

56 +

57 0 0.16 + 5 0.15

58 +

59 0 0.16 + 0 0.15

60 +

61 0 0.15 + 0 0.15

62

63 0 0.12 0 0.16

64

65 0 0.18 0 0.14

66

67 0 0.17 0 0.17

68

69 0 0.17 0 0.17

70

71 0 0.16

72

73 0 0.17

74

75 0 0.15

76

77 0 0.15

Weeks of Patient K.S.V. Patient N.L.E.

ICA C- ICA C- study

β-cells treatment titer peptide β-cells treatment titer peptide

1 + 160 0.31 + 80 0.29

2 + +

3 + 160 0.51 + 80 0.67

4 + +

5 + 80 0.43 + 40 0.22

6 + +

7 + 80 0.41 + 80 0.2

8 + +

9 + 40 0.38 + 40 0.26

10 + +

1 1 + 80 0.5 + 80 0.27

12 + +

13 + 80 0.63 + 40 0.2

14 + +

15 + 80 0.42 + 40 0.42

16 + +

17 + 80 0.33 + 80 0.43 2011/000023

66

67 0 0.35

68

69 0 0.63

[095] As clearly shown in table 1, stable immune system tolerance to auto-antigens (β-cell membrane antigens that mimic human-self antigens) was induced in all patients within 23, 59, 59 and 27 weeks from the start of β-cells treatment-exposure to the antigens.

[096] This study demonstrates that stable immune tolerance to beta-cell antigens can be induced in humans. Moreover, this induced tolerance enables the de-novo introduction of beta cells (beta cells transplantation) to an immune tolerant recipient. Last, induction of immune tolerance by the administration of a composition comprising beta-cells can be used as a preventive measure to type-1 diabetes and can be preformed during the initial phases of type- 1 diabetes, thus inhibiting autoimmunity and beta-cells destruction. This process results in reversing this devastating disease during its development.

Claims

What is claimed is.

1. A method of inducing a state of immune tolerance to a pancreatic beta cell in a subject in need thereof, comprising the step of administering to said subject a composition comprising an effective amount of beta cells, thereby inducing a state of immune tolerance to a pancreatic beta cell in a subject.

2. The method of claim 1 , wherein said subject is afflicted with type 1 diabetes.

3. The method of claim 1 , wherein said subject has antibodies to beta cell proteins.

4. The method of claim 1, wherein said subject is diagnosed with type 1 diabetes risk.

5. The method of claim 1, wherein said composition comprises 1 ,000-100,000 Beta cells.

6. The method of claim 1, wherein said composition comprises a cell culture nutrient solution.

7. The method of claim 1, wherein the frequency of administration is between once a day to once every 14 days.

8. The method of claim 7, wherein said frequency of administration is once a week.

9. The method of claim 1, wherein said administering is orally administering or intra- intestinal administering.

10. A method of reducing type 1 diabetes risk in a subject in need thereof, comprising the step of administering to said subject a composition comprising an effective amount of beta cells, thereby reducing type 1 diabetes risk in a subject in need thereof.

1 1. The method of claim 10, wherein said subject has antibodies to beta cell proteins.

12. The method of claim 10, wherein said subject has the allele DR3, DR4, DQ2.5, DR7, DR8, DR15 or any combination thereof.

13. The method of claim 10, wherein said composition comprises 1,000-100,000 Beta cells.

14. The method of claim 10, wherein said composition comprises a cell culture nutrient solution.

15. The method of claim 10, wherein the frequency of administration is between once a day to once every 14 days.

16. The method of claim 15, wherein said frequency of administration is once a week.

17. The method of claim 10, wherein said administering comprises orally administering, or intra-intestinal administering.

18. A method of transplanting a composition comprising pancreatic beta cells in a subject in need thereof, comprising the steps of:

(a) inducing a state of immune tolerance to a pancreatic beta cell in said subject, comprising administering to said subject a composition comprising an effective amount of beta cells; and

(b) transplanting a composition comprising pancreatic beta cells; thereby, transplanting a composition comprising pancreatic beta cells in a subject in need thereof .

19. The method of claim 18, wherein said subject is afflicted with type 1 diabetes.

20. The method of claim 18, wherein said subject has antibodies to beta cell proteins.

21. The method of claim 18, wherein said composition comprises 1,000-100,000 Beta cells.

22. The method of claim 18, wherein said composition comprises a cell culture nutrient solution.

23. The method of claim 18, wherein said the frequency of administration is between once a day to once every 14 days.

24. The method of claim 23, wherein said frequency of administration is once a week.

25. The method of claim 18, wherein said administering comprises orally administering, intra-intestinal administering, or parenteral administering.