WO2003092619A2

WO2003092619A2 - Prevention and treatment of type 2 diabetes

Info

Publication number: WO2003092619A2
Application number: PCT/US2003/013820
Authority: WO
Inventors: George Rainer Siber; Subramonia Padmanabha Pillai; Philip David Fernsten
Original assignee: Wyeth Holdings Corporation
Priority date: 2002-04-30
Filing date: 2003-04-30
Publication date: 2003-11-13
Also published as: AU2003231279A8; AU2003231279A1; WO2003092619A3

Abstract

Methods and compositions are provided to prevent or treat Type 2 diabetes. Such methods entail administering an agent that induces in the patient a beneficial immune response against islet amyloid fibrils, protofibrils and/or amyloid plaque deposited in the islets of the human pancreas. In such methods, a suitable agent is an antigen that is surface-exposed only in islet amyloid fibrils, protofibrils, protofibrils and/or amyloid plaque but not in soluble, native islet amyloid polypeptide (IAPP), or an antibody thereto.

Description

PREVENTION AND TREATMENT OF TYPE 2 DIABETES

FIELD OF THE INVENTION

The present invention is directed to methods and compositions for treating or preventing Type 2 diabetes using an antigen that is surface-exposed only in islet amyloid fibrils, protofibrils and/or amyloid plaque, but not in soluble, native islet amyloid polypeptide, or an antibody thereto.

BACKGROUND OF THE INVENTION Type 1 (insulin-dependent) diabetes usually results from an autoimmune disorder in which the immune system mistakenly attacks the body's insulin-producing pancreatic cells, reducing and ultimately stopping all insulin production. Sufferers need to test their blood sugar levels and inject insulin several times daily.

In contrast, Type 2 diabetes is a metabolic disorder resulting from the body's inability to make enough, or properly use, insulin. Type 2 diabetes used to be known as "adult-onset diabetes" or "non insulin dependent diabetes mellitus (NIDDM)", because it usually occurred in mature adults, but that term has been dropped as the disease increasingly strikes children in their teens or younger. Patients with the more severe cases of Type 2 diabetes must supplement their natural insulin production with insulin injections. Type 2 diabetes is characterized by impaired β-cell function, hyperglycemia and islet amyloid deposition.

Epidemiology and Pharmacoeconomics

The worldwide prevalence of diabetes has risen dramatically over the past two decades. It is projected that the number of individuals with diabetes will continue to increase in the near future, and the number having Type 2 diabetes is expected to rise more rapidly because of increasing obesity and reduced activity levels. It has been shown that between 1976 and 1994 the prevalence of diabetes among adults in the U.S. increased from 8.9% to 12.3% (Reference 1). In 1998, 15.7 million people or 5.9% of the population in the U.S. met the criteria for diabetes. While 10.3 million are currently diagnosed, another 5.4 million people are non-diagnosed or under- diagnosed. Each day approximately 2,200 new cases of diabetes are diagnosed, or about 798,000 new diagnoses per year (2). The vast majority of cases (90%) are Type 2. In 1994 diabetes was the seventh leading cause of death in the U.S. For certain high-risk groups, such as American Indians (fourth overall cause of death) and Hawaiians (fifth overall cause of death), the impact is even greater. Irrespective of ethnic group, diabetes ranked higher as a leading cause of death in females than in males (3). In addition, diabetes is the leading cause of acquired blindness in people ages 20-74. Each year, 12 to 24 thousand people lose their sight due to diabetes. It has also been estimated that approximately 30,000 people per year initiate treatment for end-stage renal disease resulting from diabetes. A high percentage of diabetes patients (60-70%) have some form of diabetic nerve damage, which in severe cases can lead to lower limb amputations. The risk of a leg amputation is 15-40 times greater for a person with diabetes. More than 56,000 amputations are performed yearly among diabetes patients. In addition, diabetes patients are 2-4 times more likely to have heart disease and 2-4 times more likely to suffer a stroke (2, 3).

In 1997, the annual economic cost of diabetes was estimated to be $98 billion, including $44 billion in direct medical costs and $54 billion for indirect costs attributed to disability and mortality. In 1997, the per capita cost of health care for people with diabetes amounted to $10,071 , as compared with $2,669 for non- diabetes people. Diabetes-related hospitalization totaled 13.9 million days in 1997, with an approximate cost of $27.5 billion. The cost of outpatient care for diabetes is also high, with 30.3 million office visits yearly (2). It is estimated that diabetes accounts for 88 million disability days and 14 million work-loss days from jobs outside of the home yearly. On average, diabetes patients lost 8.3 days from work per year as compared with 1.7 days for people without diabetes.

Pathogenesis of Type 2 diabetes

Type 2 diabetes is a multifactorial disease in terms of abnormalities associated with insulin resistance and reduced secretion of insulin resulting from β- cell failure (4). The etiology of Type 2 diabetes is still being debated. While peripheral insulin resistance appears to be a primary defect, impaired insulin secretion and loss of β-cell mass also appears to contribute to the pathogenesis of Type 2 diabetes. It is generally believed that insulin resistance in target organs, such as the liver and skeletal muscle, may initially be compensated for by the over-production of insulin, leading to hyperinsulinemia and eventual β-cell failure, resulting in insulin dependence (7). The deterioration of β-cell function and eventual loss of 40-50% β- cell mass that occurs in Type 2 diabetes patients appears to be correlated with islet amyloidosis, or the deposition of amyloid plaque material in pancreatic islets (4).

The relative contribution of these abnormalities varies among patients, as well as during the course of the disease, and may depend on the racial or ethnic background of the patient (5, 6). Genetic defects and environmental factors such as diet, sedentary life style, and obesity may also aggravate insulin resistance. Organ- specific amyloid deposition has been observed in certain other disease states in which the amyloid deposits are composed of unique products produced at the site, with other organs being spared (9,10). Examples of localized organ specific- amyloidosis include Alzheimer's disease (11), medullary thyroid carcinoma (12) and Type 2 diabetes (13). Unique products have been shown to be associated with organ specific amyloidosis: Aβ polypeptide in Alzheimer's disease (13); calcitonin in medullary thyroid carcinoma (14) and islet amyloid polypeptide (I APP), which is also known as amylin, in Type 2 diabetes (15, 16).

A major component of pancreatic islet amyloid is islet amyloid polypeptide (IAPP), or amylin, a 37-amino acid peptide (15, 16) with the following amino acid sequence:

Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-His-Ser- Ser-Asn-Asn-Phe-Gly-Ala-lle-Leu-Ser-Ser-Thr-Asn-Val-Gly-Ser-Asn-Thr-Tyr (SEQ ID No. 1).

This polypeptide is about 45% homologous to calcitonin gene related peptide (CGRP), but differs significantly from CGRP in the middle portion of the molecule. Abundant evidence suggests that the middle domain of IAPP (amino acids 20-29) is critical for amyloidosis and pathogenicity (17, 30, 49-52, 54, 55). IAPP is released in a pulsatile pattern similar to insulin and fulfills several physiological and pharmacological functions, such as inhibition of gastric emptying and suppression of arginine-induced glucagon secretion (27). The pancreatic islet β- cells cosecrete IAPP from the same β-cell granules as insulin. The gene for human IAPP is located on the short arm of chromosome 12, contains three exons and two introns, and transcribes an 89 amino acid precursor peptide (pro-IAPP). This precursor molecule contains a secretory type signal peptide and proregions which flank the IAPP and provide cleavage sites for proprotein convertases (19). The nucleotide sequence of the IAPP gene in normal subjects (17) is identical to that of diabetes patients (21), suggesting that a change in IAPP amino acid sequence is not the pathogenic mechanism that leads to the formation of amyloid fibrils. This finding is further supported by the absence of a genetic linkage of the IAPP locus on chromosome 12 with the development of the disease in families with Type 2 diabetes (22), as well as by the absence of disease-related mutations in the gene for IAPP in patients with Type 2 diabetes.

Similar promoters control both the insulin and IAPP genes and contain glucose-regulated elements. Hyperglycemia stimulates insulin and IAPP biosynthesis (23). After translation of mRNA, both proinsulin and pro-IAPP traverse the Golgi apparatus and get sorted into either secretory granules of the regulated secretory pathway or constitutive secretory pathway (24). Pro-IAPP is released from constitutive vesicles due to lack of endopeptidases in these vesicles. Pro-IAPP in the regulated vesicles, however, is cleaved by endogenous peptidases and released as mature IAPP within 120 minutes after the formation of the vesicles, as has been demonstrated for insulin (25, 26, 65). Because both proinsulin and pro-IAPP are likely to be cleaved by the same endopeptidases, increased release of proinsulin in Type 2 diabetes should be associated with similar increases in pro-IAPP (18). However, it is not clear whether the increased production of pro-IAPP contributes to the deposition of amyloid fibrils.

IAPP is C-terminally amidated and also contains a highly conserved disulfide linkage between cysteine residues 2 and 7. Several threonine residues at the N- terminus appear to provide the substrate for O-linked glycosylation (18).

IAPP is a major component of amyloid deposits of the pancreas. Recent experimentation using CD spectroscopy indicates that native IAPP molecules exist primarily in random coil conformation (59). During fibril assembly, IAPP undergoes conformational changes and assembles into helical β-pleated sheets with some additional -helical structure (29, 30, 49, 50, 55, 59). The β-pleated IAPP oligomerizes transversely to the axis of 5nm wide protofibrils, which intertwine to form polymorphic higher order fibrils up to 18 nm in diameter (30, 31 ). Similar protofibril formation has been demonstrated during the fibrillogenesis of Aβ peptide of Alzheimer's disease (32). These β-sheeted fibrils have characteristic features, including green birefringence under polarized light following Congo red staining, and also exhibit staining affinity for thioflavin S (28). Although IAPP fibrils form spontaneously in solution (29-31 , 59), the process is significantly accelerated in vitro by the presence of preformed fibrils or aggregates of IAPP, which serve as nucleation sites for the deposition of IAPP into the β-sheeted conformation (29, 52).

The regions of the IAPP molecule critical for fibrillogenesis have been extensively analyzed. There is abundant evidence that the central region of the molecule spanning residues 20-29, or even shorter peptides containing residues 23- 27, is sufficient to spontaneously form fibrils (17, 30, 49-52, 54, 55), although the morphologies of fibrils obtained with truncated IAPP are distinct from those composed of full length IAPP, with 3.6nm protofibrils predominating (30). More recent studies utilizing CD/FTIR spectroscopy and X-ray diffraction suggest that two additional domains of IAPP, residues 8-20 and 30-37, can also undergo a conformational transition to β-sheet secondary structure upon aggregation and may play a role in vivo in the deposition or stabilization of amyloid fibrils (57,58). Peptides derived from both the 8-20 and 30-37 peptides can also assemble in vitro into amyloid-like fibrils (57-58). A monoclonal antibody whose epitope maps to the 8-20 domain of native IAPP fails to react with amyloid plaques, suggesting that the conformation or accessibility of this domain is altered in amyloid deposits (53).

The IAPP of cats and monkeys can also undergo conformational changes to form β-pleated sheet structures, which in turn form amyloid fibrils (35, 69,70), but the IAPP of mice and rats does not undergo this change. This is most likely because the central amyloidogenic region of rat and mouse IAPP is substituted with proline at residues 25, 28 and 29 (54, 71). Studies of the effects of proline substitutions within the residue 20-29 amyloidogenic domain of IAPP suggest that a single proline replacement at residue 28 almost completely inhibits fibril formation (17, 54). Amyloid fibrils, including those formed by short peptides, but not soluble amylin, appear to be directly toxic to β-cells (37, 55, 80-83). The mechanism of β-cell destruction in vitro appears to be primarily apoptotic in nature and requires direct contact of the fibril with the cell surface (37, 80). At cytotoxic concentrations, amylin forms nonselective voltage-dependent ion channels across phospholipid bilayers, resulting in increased cytosolic free calcium (81 , 82). Other studies suggest that amyloid deposits may also promote the inflammatory activity of eosinophils (79).

Although a large body of evidence on the role of IAPP in islet amyliodosis is available, other components such as apolipoprotein E, serum amyloid P component, and the heparan sulfate proteoglycan, perlecan, have also been identified in amyloid deposits in Type 2 diabetes (18, 66). Interestingly, these components have been identified in of other forms of amyloid (34), and have been hypothesized to enhance the deposition or stabilize the fibril structures of amyloid (67).

While islet amyloid is a feature of the islet pathology in the vast majority of individuals with Type 2 diabetes, it has been questioned whether islet amyloid is a critical component of the pathogenesis of hyperglycemia or simply an epiphenomenon. Part of the difficulty in answering this question has been the inability to study islet amyloid formation in a large number of Type 2 diabetes patients longitudinally. However, longitudinal studies on diabetic monkeys have shown a significant correlation between deposition of islet amyloid and metabolic deterioration, with impaired animals progressing to overt diabetes when islet amyloid exceeded 50 to 60% (35, 69). Transgenic mouse strains that overexpress human IAPP generally do not show deposition of islet amyloid (72, 73), although some fibrillar material is detected in β-cell granules (74), which may lead to β-cell death and impaired insulin secretion (76). However, if the IAPP transgene is expressed against a background of obesity, or the transgenic animals are subjected to increased dietary fat or cortical steriods, the animals develop extensive islet amyloid deposits and β- cell degeneration progressing to hyperglycemia (36, 75, 77).

It has been demonstrated that macrophages are present in the endocrine pancreas in Type 2 diabetes and may be involved in the catabolism of islet amyloid derived from IAPP in vivo (39, 78). There is evidence that phagocytosis of amyloid fibrils does occur, but the process of removing islet amyloid deposits is inefficient, most likely due to inefficient degradation by tissue macrophages. It is also possible that activation of phagocytosis is reduced by diabetes-related conditions or amyloid- associated compounds such as amyloid P component (39).

Asn and asp residues can undergo deamidation (asn), isomerization (asn or asp), and racemization (asn or asp) rearrangements through a succinimidyl intermediate to a mixture or L- and D-asp and -isoasp residues, with L-isoasp typicially predominating (61 -63, 64). These rearrangements are favored by gly, ser, or thr residues on the C-terminal side. A rearrangement of asn or asp to isoasp has the effect of adding an additional carbon atom to the polypeptide backbone and has been demonstrated to alter conformation, epitopes, and biological activity (60, 63). In the case of the amyloid-β peptide of Alzheimer's disease (Aβ₄₂), isomerization of asp residues at positions 1 and 7 to isoasp residues results in alteration of epitopes and increased resistance to serum peptidases and may impact the deposition or stability of amyloid plaques (60, 61). IAPP has NT dipeptides at positions 3-4 and 35-36, which may be susceptible to rearrangements of this type. Such alterations of residues 3 or 35 or of other asn residues at positions 14, 21 , 22, or 31 of IAPP could affect the conformation of IAPP and promote fibril deposition, as well as confer resistance to serum or macrophage lysosomal peptidases and increase the stability of islet amyloid plaques. Such altered residues, if they exist, may also result in unique epitopes not found on native, soluble IAPP. Four immunological domains of IAPP have been defined using monoclonal antibodies elicited by IAPP and C-terminal peptide conjugates: residues 1 -10, another N-terminal epitope requiring an intact 2-7 disulfide bond, 18-29, and 30-37 (33). Of particular interest is another monoclonal antibody that recognizes an epitope within the 8-20 domain of native IAPP, but does not react with amyloid plaque material (53, 70).

Progressive deposition of islet amyloid plays a role in the depletion of islet β- cells via cytotoxicity (37). Any factor that impairs pro-IAPP processing, sorting, or storage may result in the initiation of islet amyloid formation and deposition. Once a focus of amyloid fibrils has formed, the process leading to the amyloid deposition is progressive. This progressive accumulation of amyloid is associated with a further β-cell mass reduction (38). It is hypothesized that a progressive reduction in islet mass by increased amyloid deposition is associated with a progressive impairment in insulin secretion, reduction in glucose tolerance and eventually the development of fasting hyperglycemia (18).

Results obtained with Aβ₄₂ of Alzheimer's disease suggest that active and passive immunization can reduce the level of amyloid plaques in animal models and attenuate the pathology of the disease. PDAPP transgenic mice overexpress mutant human amyloid precursor protein and progressively develop many of the neuropathological manifestations of Alzheimer's disease. Immunization of PDAPP mice with the Aβ₄₂ peptide induced a significant immune response against the amino terminal portion of the 42mer peptide and reduced the Alzheimer's disease-like pathology in these mice (40). Passively administered polyclonal and monoclonal antibodies generated against this amino terminal region of Aβ₄₂ have been shown to reduce amyloid burden and decorate neuritic plaques in vivo and induce clearing of preexisting plaques in an ex vivo phagocytosis assay (41). Additional studies collectively indicate that antibodies against the amino terminus of Aβ peptide are sufficient to reduce and prevent the amyloid deposition in PDAPP mice (42, 43).

Current modalities of therapy for Type 2 diabetes

At this time there are no prophylactic or therapeutic immunogenic compositions or monoclonal antibody products available for treating or preventing Type 2 diabetes. Rather, Type 2 diabetics are treated with a variety of modalities listed in Table 1 , including oral hypoglycemia drugs, including insulin secretion stimulators in the class of sulfonylureas [e.g., G UCOTROL® (glipizide)], insulin sensitivity enhancers [e.g., GLUCOPHAGE® (metformin hydrochloride)] and glycogen breakdown inhibitors [e.g., GLYSET™ (miglitol) and PRECOSE® (acarbose)]. A combination therapy of glyburide and metformin hydrochloride

(GLUCOVANCE™) is also available. Although most of these drugs are considered to be safe, a constant monitoring of liver functions is critically important for certain patient populations. In one exemplary instance, a drug was taken off the market due to liver toxicity. Patient compliance is another major issue with drug therapy. In addition to the unwanted side effects of currently available drugs, patients with the more severe cases of Type 2 diabetes must supplement their natural insulin production with insulin injections. Therefore, there is a need for new prophylactic and improved therapeutic approaches in the management of Type 2 diabetes. SUMMARY OF THE INVENTION

To this end, the present invention provides a method of preventing or treating Type 2 diabetes in a patient capable of forming islet amyloid, which entails immunizing the patient with a novel composition that induces a beneficial immune response against islet amyloid fibrils, protofibrils and/or amyloid plaque without inhibiting the biological activity of native islet amyloid polypeptide (IAPP).

More specifically, the present invention is directed to a pharmaceutical composition comprising an agent in an amount effective to induce an immune response against islet amyloid fibrils, protofibrils, and/or amyloid plaque, but not against soluble native islet amyloid polypeptide, together with a pharmaceutically acceptable carrier. In some compositions, such as an immunogenic composition, the agent is an antigen that is surface-exposed only in islet amyloid fibrils, protofibrils and/or amyloid plaque but not in soluble, native IAPP. In other compositions, the agent is an antibody, such as a monoclonal antibody, that binds specifically to epitopes exposed only in islet amyloid fibrils, protofibrils, and/or amyloid plaque, but not in soluble, native IAPP. In still other compositions, the agent is a peptide mimic of an epitope present in islet amyloid fibrils, protofibrils, and/or amyloid plaque but not in soluble, native IAPP. In yet other compositions, the agent is an antibody mimic that binds specifically to epitopes exposed only in islet amyloid fibrils, protofibrils, and/or amyloid plaque, but not in soluble, native IAPP.

The pharmaceutical composition is typically administered intranasally, intradermally, subcutaneously, intramuscularly or intravenously, although other conventional routes are also acceptable. Also contemplated is a method of preventing Type 2 diabetes in a patient capable of forming islet amyloid, which comprises inducing an immune response against islet amyloid fibrils, protofibrils, and/or amyloid plaque, but not against soluble, native IAPP, by administration of an antigen or passively by administration of an antibody, wherein the immune response is sufficient for inhibiting the formation of islet amyloid fibrils, protofibrils and/or amyloid plaque.

Also contemplated is a method of treating Type 2 diabetes in a patient capable of forming islet amyloid, which comprises inducing an immune response against islet amyloid fibrils, protofibrils, and/or amyloid plaque, but not against soluble, native IAPP, by administration of an antigen or passively by administration of an antibody, wherein the immune response comprises clearance or reduction of the islet amyloid fibrils, protofibrils and/or amyloid plaque.

In some methods, the patient is monitored following administration to assess the immune response. If the monitoring indicates a reduction of the immune response over time, the patient can be boosted with one or more additional doses of the antigen or antibody.

Also contemplated is a method of preserving β-cell function in a patient having or susceptible to Type 2 diabetes, comprising inhibiting or reversing islet amyloid formation by administering to the patient a composition comprising an effective amount of a purified antigen that is surface-exposed only in islet amyloid fibrils, protofibrils, and/or amyloid plaque, but not in soluble, native IAPP, or by administering a peptide mimic of an epitope present in islet amyloid fibrils, protofibrils, and/or amyloid plaque, but not in native IAPP, together with a pharmaceutically acceptable carrier.

Also contemplated is a method of preserving β-cell function in a patient having Type 2 diabetes, comprising clearing islet amyloid and amyloid plaque from said patient's islet tissues by administering to the patient a monoclonal antibody that binds specifically to epitopes exposed only in islet amyloid fibrils, protofibrils, and/or amyloid plaque but not in IAPP, thereby inducing clearance of the islet amyloid and amyloid plaque, such that the progressive loss of insulin-producing β cells associated with Type 2 diabetes is arrested.

Also contemplated is a method of inhibiting the formation of islet amyloid, comprising blocking apolipoprotein E, serum amyloid P component or perlecan from binding to developing islet amyloid by administering to a patient capable of developing islet amyloid a composition comprising an effective amount of an agent to induce an immune response against islet amyloid fibrils, protofibrils, and/or amyloid plaque, but not against soluble, native IAPP, together with a pharmaceutically acceptable carrier. Also contemplated is a composition comprising an antigen conjugated to a carrier molecule wherein the resulting conjugate promotes an immune response against the antigen in a patient, wherein the antigen is surface-exposed only in islet amyloid fibrils, protofibrils, and/or amyloid plaque, but not in soluble, native IAPP. Also contemplated is a method of identifying peptide mimics of epitopes present in islet amyloid fibrils, protofibrils, and/or amyloid plaque, but not in soluble, native IAPP, which comprises screening phage display libraries expressing 7-10mer random peptide sequences, 7-12mer E. coli constrained random peptide sequences, or synthetic combinatorial libraries.

Also contemplated is a method of identifying antibody mimics that bind specifically to epitopes exposed only in islet amyloid fibrils, protofibrils, and/or amyloid plaque, but not in soluble, native IAPP, which comprises screening a candidate antigen comprising a fibronectin type III domain having at least one randomized loop, the antigen being characterized by its ability to bind to an antigen that is not bound by the corresponding naturally-occurring fibronectin.

Also contemplated is an agent for use as a medicament wherein said agent induces an immune response against islet amyloid fibrils, protofibrils and/or amyloid plaque, but not against soluble, native IAPP. Preferably said agent is selected from the group comprising:

(i) a surface-exposed antigen of islet amyloid fibrils, protofibrils and/or amyloid plaque wherein said antigen is absent in soluble native IAPP; (ii) an antibody that binds to a surface-exposed epitope of islet amyloid fibrils, protofibrils and/or amyloid plaque wherein said epitope is absent in soluble native IAPP;

(iii) a mimic of an antibody according to (ii); and (iv) a peptide mimic of an epitope according to (ii).

An antigen in accordance with this use suitably comprises an amino acid sequence according to SEQ ID NO:1 or an amyloidogenic part thereof, wherein said amino acid sequence is conjugated to a β-sheeted protein carrier. Amyloidogenic parts of this amino acid sequence preferably comprise peptides spanning residues 8 to 37 of SEQ ID NO:1. More preferably said amyloidogenic parts are selected from the group of sequences comprising amino acid residues 8-20, 8-29, 8-37, 20-29, 20- 37 and 30-37. The central region spanning amino acids 20-29 as been identified as a particularly important region in the formation of islet amyloid, hence an antigen in accordance with this use preferably comprises amino acids 20-29 of SEQ ID NO:1.

Also contemplated is the use of an agent in the manufacture of a medicament for the treatment and/or prevention of type 2 diabetes, wherein said agent induces an immune response against islet amyloid fibrils, protofibrils and/or amyloid plaque, but not against soluble, native IAPP.

These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description and appended claims.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

In order for the full scope of the invention to be clearly understood, the following definitions are provided.

The term "soluble" in relation to native IAPP is intended to refer to molecules that exist primarily in random coil conformation when viewed with CD spectroscopy. Unlike the β-pleated and α-helical IAPP structures associated with amyloid deposits, the random coil conformaton allows the native IAPP to remain in solution within the physiological conditions of the pancreas.

The term "adjuvant" means a substance that enhances, nonspecifically, the immune response to an antigen, or which causes an individual to respond to an antigen who would otherwise without the adjuvant not respond to the antigen.

The term "antibody" encompasses any immunoglobulin, including intact antibodies and binding fragments thereof, that binds a specific epitope.

An "antibody combining site" is that structural portion of an antibody molecule comprised of heavy and light chain variable and hypervariable regions that specifically binds antigen.

The term "antigen" includes any substance that may be specifically bound by an antibody molecule. Thus, the term "antigen" encompasses biologic molecules including, but not limited to, simple intermediary metabolites, sugars, lipids, autoacids, and hormones, as well as macromolecules such as complex carbohydrates, phospholipids, nucleic acids and proteins.

The term "CRM₁₉₇" refers to a non-toxic mutant of diphtheria toxin with one amino acid change in its primary sequence. The glycine present at the amino acid position 52 of the molecule is replaced with a glutamic acid due to a single nucleic acid codon change. Due to this change, the protein lacks ADP-ribosyl transferase activity and becomes non-toxic. CRM₁₉₇ has a molecular weight of 58,390 daltons. Conjugations of saccharides as well as peptides to CRMι₉₇ are carried out by linking through the epsilon amino groups of lysine residues.

The term "epitope" refers to a site on an antigen recognized by an antibody or antigen receptor; epitopes are also called "antigenic determinants." The term "Fv" designates the antigen binding fragment of an antibody, including the V_H and V_L chains. The term "scFv" is the single-chain Fv, intended to include the minimal light chain variable region linked to the minimal heavy chain variable region necessary to form a binding polypeptide capable of interacting with an epitope. The term "immune response" refers to the development of a beneficial humoral (antibody-mediated) and/or a cellular (mediated by antigen-specific T cells or their secretion products) response directed against islet amyloid and amyloid plaque, but not against islet amyloid polypeptide (IAPP) in a recipient patient. Such a response can be an active response induced by administration of an antigen or a passive response induced by administration of an antibody.

The term "immunogen" means a macromolecular antigen that is capable of initiating lymphocyte activation resulting in an antigen-specific immune response. An immunogen therefore includes any molecule that contains one or more epitopes that will stimulate a host's immune system to initiate a secretory, humoral and/or cellular antigen-specific response.

The phrase "monoclonal antibody" in its various grammatical forms refers to an antibody having only one species of antibody combining site capable of immunoreacting with a particular antigen. A monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts. For example, the monoclonal antibody as used herein can be selected from recombinant human, humanized murine, chimerized murine, and transgenic murine antibodies. The phrase "patient capable of forming islet amyloid" includes mammalian subjects, such as humans, cats and monkeys, whose IAPP contains an amyloidogenic sequence in positions 20-29. Three proline (P) residues substituted at positions 25, 28 and 29 in rat and mouse IAPP prevent rats and mice from forming islet amyloid.

The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologially tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.

The term "purified" is defined herein as free from at least some of the components with which it naturally occurs. "Purified" as used herein also means altered "by the hand of man" from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polypeptide naturally present in a living organism is not "purified," but the same polypeptide separated from the coexisting materials of its natural state is "purified," as the term is employed herein.

II. Therapeutic Agents

The present invention is based on the discovery that an immune response comprising antibodies which bind specifically to epitopes exposed only in islet amyloid fibrils, protofibrils, and/or amyloid plaque but not in soluble native islet amyloid polypeptide (IAPP), will induce clearance of the islet amyloid fibrils and amyloid plaque, such that the progressive loss of insulin-producing β cells associated with Type 2 diabetes is inhibited, without inhibiting the biological activity of IAPP.

Therapeutic agents for use in the present invention induce an immune response against islet amyloid fibrils and amyloid plaque but not against soluble, native IAPP. These agents include purified antigen that is surface exposed only in islet amyloid fibrils and amyloid plaque, but not in soluble, native IAPP, variants thereof, analogs and mimics of this antigen that induce and/or cross-react with antibodies to this antigen, and antibodies reactive with this antigen, hereinafter denoted as "the antigen." Induction of an immune response can be active, as when the antigen is administered to elicit antibodies reactive with the antigen in a patient, or passive, as when an antibody is administered that itself binds to the antigen in a patient.

Islet amyloid polypeptide (IAPP), also known as amylin, is a major component of islet amyloid. Human IAPP is a 37-amino acid peptide and has the following amino acid sequence: Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-His-Ser-

Ser-Asn-Asn-Phe-Gly-Ala-lle-Leu-Ser-Ser-Thr-Asn-Val-Gly-Ser-Asn-Thr-Tyr (SEQ ID

No:1).

The central region spanning amino acids 20 to 29 has been identified as a major amyloidogenic region within the human IAPP molecule that is critical for the formation of islet amyloid. The regions spanning amino acids 8-20 and 30-37 also appear to participate in the deposition of amyloid fibrils.

Therapeutic agents of this invention therefore include IAPP (with and without intrachain disulfide bonds linking cysteine residues at positions 2 and 7) and peptides spanning one or more of the amyloidogenic regions of IAPP peptides spanning residues 8-20, 8-29, 8-37, 20-29, 20-37, or 30-37, as well as smaller peptides derived from these regions selected from the group comprising FGAIL, NFGAIL, GAILS, FGAILS, and NFGAILS (SEQ ID NOS:3-7). These agents are either conjugated to a protein carrier or administered as fibrils, protofibrils, or other β-sheeted aggregates. Additional therapeutic agents also include IAPP and peptides derived from residues 1-8 (with and without intrachain disulfide bonds linking cysteine residues at positions 2 and 7) and peptides derived from residues 8-37, 20-37, and 30-37 of IAPP, with L- or D-isoaspartic acid or aspartic acid replacing the asparagine residues at position 3, position 35, or both positions. These substituted peptides would also be administered conjugated to a protein carrier as described below, or as fibrils, protofibrils, or other β-sheeted aggregates.

These peptides are synthesized by solid phase peptide synthesis. Automatic peptide synthesizers are commercially available from numerous suppliers, such as Applied Biosystems, Foster City, California. Recombinant expression can be in bacteria, such as E. coli, yeast, insect cells or mammalian cells. Procedures for recombinant expression are described by Sambrook et al., Molecular Cloning: A Laboratory Manual (C.S.H.P. Press, NY 2d ed., 1989). Human IAPP or IAPP fragments are also available commercially (e.g., Sigma Chemical Co., St. Louis, MO).

Therapeutic agents also include peptides and other compounds that do not necessarily have a significant linear amino acid sequence homology with the antigen but nevertheless serve as mimics of the antigen and induce a similar immune response. Such mimics are identified by screening phage display libraries expressing 7-10mer random peptide sequences (See, e.g., Devlin, WO 91/18980) or 7-12mer E. coli constrained random peptide sequences (83) with antibodies specific for epitopes present in islet amyloid fibrils, protofibrils, and/or amyloid plaque, but not in soluble, native IAPP.

Random combinatorial libraries of peptides or other compounds are also screened for suitability. Combinatorial libraries are synthesized in a step-by-step fashion from many types of compounds including peptides, peptoids, beta-turn mimics, saccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, heterocyclic compounds, benzodiazepines, oligomeric N-substituted glycines, and oligocarbamates. Large combinatorial libraries of compounds are constructed by the encoded synthetic libraries (ESL) method described in Affymax, WO 95/12608, Affymax, WO 93/06121 , Columbia University, WO 94/08051 , Pharmacopeia, WO 95/35503 and Scripps, WO 95/30642 (each of which is incorporated by reference for all purposes).

More specifically, such mimics are identified by the steps of: (a) providing phage display libraries expressing 7-1 Omer random peptide sequences, libraries expressing 7-12mer E. coli constrained random peptide sequences, or synthetic combinatorial libraries, said libraries containing epitopes present on islet amyloid fibrils, protofibrils, and/or amyloid plaque, but not in soluble, native IAPP; (b) contacting the libraries with antibodies that specifically bind to epitopes exposed only in islet amyloid fibrils, protofibrils, and/or amyloid plaque, but not in soluble, native IAPP; and (c) determining the peptide sequences that bind to the specific antibodies. Reactive peptides with sequences that are non-homologous to IAPP are then conjugated to protein carriers and tested for the ability to elicit antibodies specific for epitopes exposed only in islet amyloid fibrils, protofibrils, and/or amyloid plaque, but not in soluble, native IAPP. Multiple dilutions of sera are tested on microtiter plates that have been precoated with islet amyloid fibrils, protofibrils and/or plaque, and a standard ELISA is carried out to detect reactive antibodies to those antigens, thereby identifying mimics of epitopes present on islet amyloid fibrils, protofibrils and/or plaque, but not on native, soluble IAPP. Mimics are then tested for prophylactic and therapeutic efficacy in transgenic animals predisposed to Type 2 diabetes, such as transgenic mice expressing human IAPP, as described in the Examples.

Anti-idiotypic antibodies are also used as therapeutic agents. Anti- idiotypicantibodies are elicited by immunizing with monoclonal antibodies specific for epitopes present in islet amyloid fibrils, protofibrils, and/or amyloid plaque but not in soluble, native IAPP. The second generation antibodies are then tested for their ability to block the binding of the monoclonal antibody used as the immunogen and for their ability to elicit a response against epitopes present in islet amyloid fibrils, protofibrils, and/or amyloid plaque but not in soluble, native IAPP. Such anti-idiotypic antibodies, which elicit a response to the original antigen, are believed to display an image of an epitope of that antigen.

Therapeutic agents of the present invention also include monoclonal antibodies that specifically bind to the antigen. The production of monoclonal antibodies, e.g., murine or rat, is accomplished by immunizing mice or rats with the antigen of interest, confirming a serological response to the antigen, harvesting and isolating splenocytes 3-5 days after a final interperitoneal boost, fusing the splenocytes with a suitable fusion partner (HGPRT-deficient myeloma line), selecting for growth of hybrid cells in HAT medium, and testing spent culture media for the presence of antibodies to the antigen of interest. . See Harlow & Lane, Antibodies, A Laboratory Manual (CSHP NY, 1988) (incorporated by reference for all purposes). Immunogens are obtained from natural sources, by peptide synthesis or by recombinant expression. Humanized forms of mouse antibodies are generated by splicing the complementarity determining regions (CDR) of non-human antibodies to human constant regions by recombinant DNA techniques. See Queen et al., Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989) and WO 90/07861 (incorporated herein by reference). Human antibodies are obtained using phage-display methods. See, e.g.,

Dower et al., WO 91/17271 ; McCafferty et al., WO 92/01047. In these methods, libraries of phage are produced in which members display recombinant scFv or Fab antibodies on their minor coat proteins. Phage displaying antibodies with a desired specificity are selected by multiple rounds of affinity enrichment to the antigen, or fragments thereof. Human antibodies against the antigen are also produced by immunizing mice having transgenes encoding at least a portion of the human immunoglobulin repertoire and an inactivated endogenous immunoglobulin locus. See, e.g., Lonberg et al., W093/12227 (1993); Kucheriapati, WO 91/10741 (1991) (each of which is incorporated herein by reference). Following immunization, human antibodies are produced using standard hybridoma methodology and are selected by competitive binding experiments, or otherwise, to have the same epitope specificity as a particular mouse antibody. Such antibodies are likely to share the useful functional properties of the mouse antibodies.

Recombinant antibodies are expressed in E. coli as Fab fragments or as single chain antibodies, scFv, in which heavy and light chain variable domains are linked through a spacer. Human or humanized IgG antibodies are designed to have lgG1, lgG2, lgG3, or lgG4 constant regions and must be expressed in eukaryotic cells. Stably transfected mammalian cells (e.g., CHO or Sp2/0) or transgenic animals or plants can be used to produce recombinant human or humanized IgG or IgA .

Therapeutic agents of the present invention also include antibody mimics that specifically bind to the antigen. The structure of these antibody mimics provides optimal folding, stability and solubility, even under conditions that normally lead to the loss of structure and function in antibodies. Such mimics include a fibronectin type III domain having at least one randomized loop, the mimic being characterized by its ability to bind to an antigen that is not bound by the corresponding naturally-occurring fibronectin. (See WO 00/34784 and WO 01/64942, each incorporated herein by reference). The antibody mimics are identified, for example, by the steps of: (a) providing an antigen that is surface-exposed only in islet amyloid fibrils, protofibrils and/or amyloid plaque, but not in soluble, native IAPP; (b) contacting the antigen with a candidate protein that comprises a fibronectin type III domain having at least one randomized loop, the contacting being carried out under conditions that allow antigen-protein complex formation; and (c) determining from the complex, the protein which binds to the antigen.

Carrier Proteins Some agents for inducing an immune response may contain the appropriate epitope for inducing an immune response against islet amyloid and amyloid plaques but are too small to be immunogenic or lack T-cell epitopes. In this situation, the agent is linked to a suitable carrier to help elicit an immune response. Suitable carriers include serum albumins such as bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), thyroglobulin, ovalbumin, tetanus toxoid, or a toxoid from other pathogenic bacteria, such as diphtheria (including an attenuated toxin derivative such as CRM*|₉₇), E. coli, cholera, or H. pylori. Other carriers for stimulating or enhancing an immune response include cytokines such as IL-1 , IL-1 α and β peptides, IL-2, γINF, IL-10, GM-CSF, and chemokines, such as MlPlα and β and RANTES. Immunogenic agents are also linked to peptides that enhance transport across tissues, as described in WO 97/17613 and WO 97/17614.

Immunogenic agents are coupled to carriers by chemical cross-linking. A list of cross-linking agents, the groups they react with, and their advantages and disadvantages is presented in Table II below. Techniques for linking an immunogen to a carrier include using heterobifunctional reagents to crosslink through cysteine sulfhydryl groups on one peptide/protein and through the lysine ε-amino groups or N- terminal amino groups of the other peptide/protein. A variety of such heterobifunctional sulfhydryl/amine-linking reagents are described by Immun. Rev. 62, 185 (1982). Commonly employed heterobifunctional reagents may form stable thioether linkages with cysteine sulfhydryl groups through reactive esters of maleimide, 2-bromoacetic acid, or 2-iodoacetic acid; or may form disulfide linkages with cysteine sulfhydryl groups through pyridyldithio groups. These heterobifunctional reagents commonly employ N-hydroxysuccinimide esters to form amide linkages with lysine or N-terminal amino groups, or amidine linkages may be formed through imidoester groups. Many of these reagents, such as N- succinimidyl- 3-(2-pyridyl-thio) propionate (SPDP), succinimidyl 4-(N- maleimidomethyl)cyclohexane-l-carboxylate (SMCC), m-maleimidobenzoyl-N- hydroxysuccinimide (MBS), succinimidyl 3-[bromoacetimido]proprionate (SBAP), and N-succinimidyl iodoacetate (SIA) are commercially available.

If the peptide lacks a sulfhydryl group, this can be provided by addition of a cysteine residue to the synthetic peptide or by coupling SPDP or N-succinimidyl S- acetylthioacetate (SAT A) with a peptide amino group and reducing the disulfide bond of SPDP or deprotecting the sulfhydryl group of SATA. For example, the lysine residues present in CRM₁₉₇ or other carrier polypeptide can be derivatized with N- hydroxy succinimidyl bromoacetate to form a bromoacetylated polypeptide. The bromoacetylated polypeptide is purified from other reactants either by gel filtration or diafiltration and stored frozen in the presence of a cryoprotectant. In the case of CRM₁₉₇, only about 18-24 out of 39 lysine residues are modified. Quantitation of bromoacetylation is accomplished by MALDI-MS molecular weight determination of the activated polypeptide. Peptide/carrier ratios are optimized and controlled by titration of the cross-linker and by adjusting the pH of the reaction. Following conjugation, the remaining reactive bromoacetyl groups are capped with N-acetyl cysteamine. The efficiency of the coupling is evaluated by hydrolyzing the peptide conjugates in strong acid and determining the carboxy methyl cysteine (CMC)/carboxy methyl cysteamine (CMCA) ratios by amino acid analysis.

Immunogenic peptides are be expressed as fusion proteins with carriers. The immunogenic peptide can be linked at the amino terminus, the carboxyl terminus, or internally to the carrier. Multiple repeats of the immunogenic peptide can also be present in the fusion protein.

III. Patients Amenable to Treatment Patients amenable to treatment include individuals at risk of developing Type

2 diabetes but not showing symptoms and having impaired glucose tolerance, as well as patients presently showing symptoms. In light of the fact that Type 2 diabetes has reached epidemic proportions in the United States, due to an increased number of older Americans, and a greater prevalence of obesity and sedentary lifestyles, virtually anyone is at risk of suffering from Type 2 diabetes. Therefore, the present methods can be administered prophylactically to the general population without any assessment of the genetic risk of Type 2 diabetes in the subject patient. The present methods are especially useful for individuals who do have a known genetic risk. Such individuals include those having relatives who have experienced this disease, and those whose risk is determined by analysis of genetic or biochemical markers. Representative markers of risk include family history of Type 2 diabetes, obesity, hyperglycemia, high blood pressure and high cholesterol. In addition, a number of diagnostic tests are available for identifying individuals who have Type 2 diabetes, including the measurement of fasting blood sugar and insulin levels. The amount of islet amyloid is generally correlated with the level of β-cell depletion, and humans without evidence of Type 2 diabetes have little or no islet amyloid.

In asymptomatic patients, treatment can begin at any age (e.g., 10, 20, 30). Treatment typically entails multiple dosages over a period of time. Treatment is monitored by assaying antibody, or activated T-cell or B-cell responses to the therapeutic agent over time. If the response falls, a booster dosage is indicated.

IV. Treatment Regimens In prophylactic applications, pharmaceutical compositions or medicaments are administered to a patient susceptible to, or otherwise at risk of, a particular disease in an amount sufficient to eliminate or reduce the risk or delay the onset of the disease. In therapeutic applications, compositions or medicaments are administered to a patient suspected of, or already suffering from such a disease in an amount sufficient to cure, or at least partially arrest, the symptoms of the disease and its complications. An amount adequate to accomplish this is defined as a therapeutically- or pharmaceutically-effective dose. In both prophylactic and therapeutic regimens, agents are usually administered in several dosages until a sufficient immune response has been achieved. Typically, the immune response is monitored and repeated dosages are given if the immune response starts to fade. Effective doses of the compositions of the present invention may vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the patient is a human, but in some cases, the patient can be a nonhuman mammal, such as a cat or monkey. Treatment dosages need to be titrated to optimize safety and efficacy. The amount of immunogen depends on whether adjuvant is also administered, with higher dosages possibly being required in the absence of adjuvant. The amount of an immunogen for administration sometimes varies from about 0.1 μg to about 500 μg per patient and more usually from about 1 μg to about 250 μg per injection for human administration. Typically about 10, 20, 50 or 100 μg is used for each human injection. Occasionally, a higher dose of 1-2 mg per injection is used. The timing of injections can vary significantly from once a day, to once a year, to once a decade. On any given day that a dosage of immunogen is given, the dosage is greater than or equal to 1 μg/patient and usually greater than 10 μgl patient if adjuvant is also administered, and greater than or equal to 10 μg/patient and usually greater than 100 μg/patient in the absence of adjuvant. A typical regimen consists of an immunization followed by booster injections at 3 to 12 months later. Alternatively, booster injections can be on an irregular basis as indicated by monitoring of immune response.

For passive immunization with an antibody, the dosage ranges from about 0.001 to 100 mg/kg, and more usually 0.1 to 15 mg/kg of the host body weight. Agents for inducing an immune response can be administered by conventional routes including, but not limited to, intravenous, subcutaneous, intranasal, intramuscular or intradermal means for prophylactic and/or therapeutic treatment. The most typical route of administration is subcutaneous although others can be equally effective. The next most common is intramuscular injection. This type of injection is most typically performed in the arm or leg muscles. In some methods, agents are injected directly into a particular tissue where deposits have accumulated. Passive immunization with antibody is most often carried out using highly diluted antibody administered intravenously over a prolonged period, although intramuscular injection is sometimes used. Agents of the invention can optionally be administered in combination with other agents, including another antibody, that are at least partly effective in treating Type 2 diabetes.

Immunogenic agents of the invention, such as peptides or peptide conjugates, are sometimes administered in combination with an adjuvant to elicit an immune response. These adjuvants augment the intrinsic response to an immunogen without causing conformational changes in the immunogen. Adjuvants can be administered as a component of a therapeutic composition with an active agent or can be administered separately, before, concurrently with, or after administration of the therapeutic agent. Examples of adjuvants contemplated in the present invention include, but are not limited to, 3-O-deacylated monophosphoryl lipid A (MPL™), which is produced by Corixa (Hamilton, MT). MPL™ is described in U.S. Patent No. 4,912,094, which is incorporated herein by reference. Also suitable for use as adjuvants are aminoalkyl glucosamine phosphate compounds (AGP), or derivatives or analogs thereof, which are produced by Corixa (Hamilton, MT), and which are described in U.S. Patent No. 6,113,918, which is incorporated herein by reference. One such AGP is 2-[(R)-3- Tetradecanoyloxytetradecanoylaminojethyl 2-Deoxy-4-O-phosphono-3-O-[(R)-3- tetradecanoyoxytetradecanoyl]-2-[(R)-3-tetradecanoyoxytetradecanoylamino]-b-D- glucopyranoside, which is also known as 529 (formerly known as RC529).

Various cytokines and lymphokines are suitable for use as adjuvants. One such adjuvant is granulocyte-macrophage colony stimulating factor (GM-CSF), which has a nucleotide sequence as described in U.S. Patent No. 5,078,996, which is incorporated herein by reference. A plasmid containing GM-CSF cDNA has been transformed into E. coli and has been deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110-2209, under Accession Number 39900. The cytokine lnterleukin-12 (IL-12) is another adjuvant which is described in

U.S. Patent No. 5,723,127, which is incorporated herein by reference.

Other cytokines or lymphokines have been shown to have immune modulating activity, including, but not limited to, the interleukins 1 -alpha, 1 -beta, 2, 4, 5, 6, 7, 8, 10, 13, 14, 15, 16, 17 and 18, the interferons-alpha, beta and gamma, granulocyte colony stimulating factor, and the tumor necrosis factors alpha and beta, and are suitable for use as adjuvants.

Still other adjuvants include alum (aluminum hydroxide or aluminum phosphate), Stimulon™ QS-21 (Antigenics Inc., Framingham, MA), described in U.S. Patent No. 5,057,540, which is incorporated herein by reference, Mycobacterium tuberculosis, Bordetella pertussis, bacterial lipopolysaccharides, synthetic polynucleotides such as oligonucleotides containing a CpG motif (U.S. Patent No. 6,207,646, which is hereby incorporated by reference), the heat-labile toxin of E. coli, and cholera toxin (either in a wild-type or mutant form, for example, wherein the glutamic acid at amino acid position 29 is replaced by another amino acid, preferably a histidine, in accordance with published International Patent Application Number WO 00/18434, which is incorporated herein by reference).

An adjuvant can be combined with an immunogen and administered as a single therapeutic composition, or the adjuvant can be administered separately before, concurrent with or after administration of the immunogen. Immunogen and adjuvant can be packaged and supplied in the same vial or can be packaged in separate vials and mixed before use. Immunogen and adjuvant are typically packaged with a label indicating the intended therapeutic application. If immunogen and adjuvant are packaged separately, the packaging typically includes instructions for mixing before use. The choice of an adjuvant and/or carrier depends on the stability of the vaccine containing the adjuvant, the route of administration, the dosing schedule, the efficacy of the adjuvant for the species being vaccinated, and, in humans, a pharmaceutically acceptable adjuvant is one that has been approved or is approvable for human administration by pertinent regulatory bodies. For example, Complete Freund's adjuvant is not suitable for human administration. Optionally, two or more different adjuvants can be used simultaneously. Some combinations are alum with MPL™, alum with Stimulon™ QS21 , MPL™ with Stimulon™ QS21, and alum, Stimulon™QS21 and MPL™ together. Agents of the invention are often administered as pharmaceutical compositions comprising an active therapeutic agent together with a variety of other pharmaceutically acceptable components. See Remington's Pharmaceutical Science (15^th ed., Mack Publishing Company, Easton, Pennsylvania, 1980). The form of the composition depends on the intended mode of administration and therapeutic application. The compositions can also include, depending on the formulation desired, pharmaceutically acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, or non-toxic, nontherapeutic, nonimmunogenic stabilizers and the like. Pharmaceutical compositions can also include large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids and copolymers (such as latex f unctionalized sepharose, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes). Additionally, these carriers can function as immunostimulating agents (i.e., adjuvants). For parenteral administration, agents of the invention can be administered as injectable dosages of a solution or suspension of the substance in a physiologically acceptable diluent with a pharmaceutical carrier which can be a sterile liquid such as water, oils, saline, glycerol or ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, surfactants, pH buffering substances and the like can be present in compositions. Other components of pharmaceutical compositions are those of petroleum, animal, vegetable, or synthetic origin, for example, soybean oil, and mineral oil. In general, glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.

Typically, compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above (see Langer, Science 249, 10 1527 (1990) and Hanes, Advanced Drug Delivery Reviews 28, 97- 119 (1997). The agents of this invention can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient. Intradermal delivery is facilitated by co-administration of the agent with cholera toxin, detoxified derivatives or subunits thereof, or other similar bacterial toxins (See Glenn et al., Nature 391, 851 (1998)). Co-administration is achieved by using the components as a mixture or as linked molecules obtained by chemical cross-linking or expression as a fusion protein. Also contemplated is intranasal delivery, which can be achieved, for example, by spraying a mist of the composition into the nose.

III. Methods of Diagnosis

The invention provides methods of detecting an immune response against islet amyloid and amyloid plaques in a patient suffering from or susceptible to Type 2 diabetes. The methods are particularly useful for monitoring a course of treatment being administered to a patient. The methods are used to monitor both therapeutic treatment of symptomatic patients and prophylactic treatment of asymptomatic patients.

Some methods entail determining a baseline value of an immune response in a patient before administering a dosage of agent, and comparing this with a value for the immune response after treatment. A significant increase (i.e., greater than is the typical margin of experimental error in repeat measurements of the same sample, expressed as one standard deviation from the mean of such measurements) in value of the immune response signals a positive treatment outcome (i.e., that administration of the agent has achieved or augmented an immune response). If the value for immune response does not change significantly, or decreases, a negative treatment outcome is indicated. In general, patients undergoing an initial course of treatment with an agent are expected to show an increase in immune response with successive dosages, which eventually reaches a plateau. Administration of agent is generally continued while the immune response is increasing. Attainment of a plateau is an indicator that the administration of treatment can be discontinued or reduced in dosage or frequency.

In other methods, a control value (i.e., a mean and standard deviation) of immune response is determined for a control population. Typically the individuals in the control population have not received prior treatment. Measured values of immune response in a patient after administering a therapeutic agent are then compared with the control value. A significant increase relative to the control value (e.g., greater than one standard deviation from the mean) signals a positive treatment outcome. A lack of significant increase or a decrease signals a negative treatment outcome. Administration of agent is generally continued while the immune response is increasing relative to the control value. As before, attainment of a plateau relative to control values in an indicator that the administration of treatment can be discontinued or reduced in dosage or frequency.

In other methods, a control value of immune response ( i.e., a mean and standard deviation) is determined from a control population of individuals who have undergone treatment with a therapeutic agent and whose immune responses have plateaued in response to treatment. Measured values of immune response in a patient are compared with the control value. If the measured level in a patient is significantly greater (e.g., more than one standard deviation) than the control value, treatment can be discontinued or reduced in dosage. If the level in a patient is significantly below the control value, continued administration of agent is warranted. If the level in the patient persists below the control value, then a change in treatment regime, for example, use of a different adjuvant may be indicated. In other methods, a patient who is not presently receiving treatment but has undergone a previous course of treatment is monitored for immune response to determine whether a resumption of treatment is required. The measured value of immune response in the patient can be compared with a value of immune response previously achieved in the patient after a previous course of treatment. A significant decrease relative to the previous measurement (i.e., greater than a typical margin of error in repeat measurements of the same sample) is an indication that treatment can be resumed. Alternatively, the value measured in the patient can be compared with a control value (mean plus standard deviation) determined in a population of patients after undergoing a course of treatment. Alternatively, the measured value in a patient can be compared with a control value in populations of prophylactically treated patients who remain free of symptoms of disease, or populations of therapeutically treated patients who show amelioration of disease characteristics. In all of these cases, a significant decrease relative to the control level (i.e., more than a standard deviation) is an indicator that treatment should be resumed in a patient. The tissue sample for analysis is typically blood, plasma, serum, mucus or cerebral spinal fluid from the patient. The sample is analyzed for evidence of an immune response to islet amyloid fibrils and amyloid plaques but not to native, soluble IAPP. The immune response is determined from the presence of, e.g., antibodies that specifically bind to islet amyloid fibrils and amyloid plaques, but not to native, soluble IAPP. ELISA methods of detecting antibodies specific to islet amyloid and amyloid plaques, but not to native IAPP, are described in the Examples section.

IV. Materials and Methods

A. Monoclonal Antibodies Three different approaches are used to develop therapeutic human monoclonal antibodies specific for human islet amyloid peptides, plaques, and fibrils. The first approach involves generating murine monoclonal antibodies specific for islet amyloid fibrils, using conventional hybridoma technology and subsequent "humanization." The second approach is to use transgenic mice expressing the human immunoglobulin repertoire to isolate human antibodies directed against islet amyloid sequences. The third approach is to screen phage libraries expressing human immunoglobulin V_Hand V domains as scFv for phages expressing single chain antibodies that bind to islet amyloid sequences. Each of these approaches offers specific advantages in the selection and development of a panel of human antibodies for efficacious passive immunization and potential therapeutic applications.

1. Development of therapeutic human monoclonal antibodies using hybridoma technology Monoclonal antibodies (mAbs) can be produced using classical hybridoma technology. The two approaches are to humanize conventional mouse mAbs or to start with a humanized mouse (Abgenix or Medarex) and isolate human mAbs. Either approach allows for using standard immunization and fusion methods and fusion partners. The advantage to either of these approaches is that full affinity maturation of the antibodies takes place in vivo, and antibodies of much higher affinity are likely to be obtained. Primary screening is for antibodies that bind to amyloid fibrils and in vivo plaques, but not to soluble native IAPP. Murine antibodies are also immediately screened for in vivo functional acitivity in transgenic mouse model systems, as well as for in vitro functional activity without additional modification. The primary advantage to using humanized mice is that there is minimal loss of affinity following humanization, no residual murine sequences, and no delay while the antibody is being humanized and expressed in a production cell.

a. Humanization of mouse monoclonal antibodies Monoclonal antibodies can be produced using standard hybridoma technology. Briefly, Balb/c or other mice (outbred ND4 mice reportedly give stronger responses to IAPP) (38) are immunized at 4-week intervals with appropriate antigen/adjuvant combinations, and serum samples are screened two weeks after immunizations for antibody titers using EIA or functional assays. When satisfactory titers are achieved, a final interperitoneal boost is administered, and three days later splenocytes are harvested and fused with HAT-sensitive P3X63Ag8 myeloma cells using standard methodology. Seven to fourteen days later, individual hybridoma cultures are screened for desired antibody specificities in EIA or functional assays. Cultures with desired specificities are propagated, cryopreserved, and subcloned at limiting dilution. Murine monoclonal antibodies are suitable for preclinical in vivo and ex vivo studies, but are too immunogenic for therapeutic use. Monoclonal antibodies with desired functional activity will therefore be humanized. Typically, V_H and V_κ genes are cloned from the hybridoma using PCR methodology and sequenced. Human V_H and V_κ genes with high homology are selected from a database and computer modeled with mouse CDR replacing existing CDR. Human amino acid residues that make close contact with mouse CDR are usually also identified in the model. Human V_H and V_κ genes are then synthesized with CDR from the mouse V_H and V_κ genes and interacting amino acid residues replacing the human homologs and are cloned into vectors encoding constant domains of selected isotype. Additional framework residues may also be replaced to optimize affinity and reduce immungenicity, and a series of constructs are transiently expressed and evaluated (typically in COS cells) prior to transfection of a manufacturing line (typically SP2/0 or CHO), and high yielding transfectants are selected (43, 44). Humanized antibodies are purified from spent culture medium using conventional affinity (protein A or protein G) or ion- exchange methods.

An alternative approach is to de-immunize the murine antiibodies. Biovation (UK) sequences the murine V_H and V_κ region and models their interaction with human MHC class II antigens. Sequences predicted to interact with human MHC class II antigens are eliminated, which reduces the immunogenicity of the murine V_H and V_κ regions. The engineered V_H and V_κ genes are synthesized and cloned into constructs encoding constant domains of human γ and K chains, respectively.

b. Generation of monoclonal antibodies in humanized mice As an alternative approach, fully human monoclonal antibodies can be produced in transgenic strains of mice in which murine Ig synthesis is knocked out and replaced by the human V_H, D, J_H, C_γ, V_κ, J_κ, and C_κ repertoire. Immunization, fusion, screening, selection, subcloning, production, and purification methods are all as described above. Advantages to using humanized mice include minimal loss of affinity following humanization, minimal residual murine sequences, and no delay while the antibody is being humanized and expressed in a production line. Humanized mice can be obtained from the suppliers. The Abgenix Xenomouse has the mouse JH (4) and either the C_κ (1 ) or J_κ (4) regions knocked out. In 1 Mb YAC constructs are 1 human C_γ (γ1 , γ2, or γ4), all D and J regions and 34 functional V_H genes; C_κ, all J regions, and 18 functional V_κ genes (45). Based on utilization, -80% of human V repertoire is represented on a B6/129 background.

The Medarex Humanized Mouse has the mouse C_κ, and J_κ and either C_μ (neo insertion in CH1 domain) or J_H knocked out. Model 1 is transgenic for 8 human V_H genes (the most frequently utilized), all J_H, half (-15) of the D_H regions (the most frequently utilized), and either C_γ1 or C_γ3 (46). This represents about 30% of human VH repertoire. Model 2 carries an episomal chromosome fragment encoding the entire human heavy chain repertoire. Both models are transgenic for 17 functional human V_κ regions (about 50%), all J_κ, and C_κ.

2. Development of Therapeutic Recombinant Human Antibodies by Screening of Phage-Displayed Libraries Phage display library screening provides antibody specificity independent of an immunogenicity/antigen-induced repertoire of antibodies, and numerous laboratories, such as Cambridge Antibody Technologies, have established the utility of very large antibody libraries derived from human donors that can be used to select antibodies capable of binding to a variety of antigens.

Phages displaying recombinant antibodies can be selected on solid phase antigen (IAPP fragments or peptides) using -4 rounds of panning, recovery, and amplification; and can then be cloned. Gene III is excised from the cloned phagemid vectors, and the recombinant antibodies are then expressed as soluble scFv and evaluated for affinity and specificity. For ease of analysis and application development, the V_H and V_L sequences can be excised from selected phagemids, reinserted into expression vectors with appropriate C_γι and O_ domains, and retransfected into competent cells for expression of Fab antibodies. Since both in vitro functional assays and any in vivo use requires intact IgG, the V_H and V_L sequences are excised from selected phagemid vectors, reinserted into full length heavy and light chain cassettes, and transfected into mammalian cells for expression of intact IgG and subsequent functional analyses and production.

B. Immunoqens and Antigens The following materials can be used as immunogens for the generation in mice or in humanized mice of monoclonal antibodies specific for human islet amyloid (fibrils) or amyloid plaques. Appropriate adjuvants are used as describedabove.

• Commercially available human IAPP or IAPP fragments (Sigma Chemical Co., St. Louis, MO). This material is aggregated into fibrils (spontaneous in low ionic strength buffer at neutral pH) prior to administration with adjuvants.

The IAPP may also be assembled into fibrils with carriers found associated with islet amyloid plaques, such as apolipoprotein E, perlecan, or serum amyloid P component.

• Peptides conjugated to protein carriers, such as CRM-|₉₇, tetanus toxoid, KLH, BSA and thyroglobulin. These conjugates are administered with appropriate adjuvants. The focus is on peptides derived from the amyloidogenic midregion of IAPP, residues 20-29 (SNNFGAILSS - SEQ ID NO:2) and/or other amyloidogenic domains of IAPP (residues 8-20 and 30-37) with and without flanking residues and spacers (GAGA). • Peptides derived from the amyloidogenic midregion of IAPP, with and without flanking residues, smaller peptides derived from the region that have been shown to be critical for fibril formation (FGAIL - SEQ ID NO:3, NFGAIL - SEQ ID NO:4, GAILS - SEQ ID NO:5, FGAILS - SEQ ID NO:6, NFGAILS - SEQ ID NO:7), and peptides derived from other amyloidogenic domains of IAPP (residues 8-20 and 30-37) with and without flanking residues are assembled into fibrils, chemically cross-linked, if necessary, and administered with appropriate adjuvants. If necessary, these aggregates can be coupled to carrier polypeptides.

• Human IAPP and peptides spanning the N and C terminal domains of IAPP, with the asparagine residues at positions 3 and/or 35 replaced by L- or D- isoaspartic acid or aspartic acid, are coupled to carrier polypeptides and will also be assembled into fibrils or protofibrils, chemically cross-linked, if necessary, and administered with appropriate adjuvants. The following materials can be used as screening antigens for the murine fusions and for screening phage-displayed libraries of recombinant antibodies.

• Commercially available human IAPP or IAPP fragments. Sigma Chemical Co., St. Louis, MO) This material is tested as fibrils/aggregates as well as in the soluble, native form..

• Peptides derived from the amyloidogenic midregion of IAPP (SNNFGAILSS - SEQ ID NO:2) and other amyloidogenic domains, with and without flanking residues, and smaller peptides derived from the region that have been shown to be critical for fibril formation (FGAIL, NFGAIL, GAILS, FGAILS, NFGAILS - SEQ ID NOS:3-7), and peptides derived from other amyloidogenic domains of

IAPP (residues 8-20 and 30-37). These peptides are assembled into fibrils and chemically cross-linked, if necessary. The smaller peptides are also useful for defining/mapping the epitopes of the monoclonal antibodies and for determining relative affinities. • Human IAPP and peptides spanning the N and C terminal domains of IAPP, with the asparagine residues at positions 3 and/or 35 replaced by L- or D- isoaspartic acid or aspartic acid.

• Frozen pancreatic tissue from hyperglycemic mice transgenic for human IAPP (positive for islet amyloid plaques), diabetic macaques (Macaca nigra), or diabetic cats are used in immunohistochemical testing. Deparaffinized pancreatic tissue sections from human Type 2 diabetes patients may also be useful.

The following materials can be used as negative control screening antigens for the murine fusions and the recombinant human antibodies. • Commercially available human IAPP or IAPP fragments in soluble, nonaggregated form, pramlintide, rat amylin. Sigma Chemical Co., St. Louis,

MO; Amylin Pharmaceuticals, La Jolla, CA)

• Peptides derived from non-amyloidogenic regions of IAPP; scrambled analogs of peptides derived from the amyloidogenic midregion of IAPP, with and without flanking residues; scrambled analogs of smaller peptides critical for fibril formation (FGAIL, NFGAIL, GAILS, FGAILS, NFGAILS - SEQ ID NOS:3-7). • Frozen pancreatic tissue from normal mice, normal cats, and normal macaques for immunohistochemical staining. Deparaffinized sections of normal human pancreas from young individuals may also be useful.

C. Selection Criteria

Regardless of the source, antibodies with potential utility for therapeutic applications should possess the following characteristics.

1. The antibodies should react with aggregated/fibrillar human IAPP, aggregated IAPP fragments, certain aggregated peptides from the midregion or other amyloidogenic domains of IAPP or peptides spanning the N and C terminal domains of IAPP, with the asparagine residues at positions 3 and/or 35 replaced by L- or D-isoaspartic acid or aspartic acid. The antibodies should not react with soluble/nonaggregated/native human IAPP or soluble IAPP fragments or peptides. They should not react with soluble peptides derived from the C-terminal or N-terminal domain of human IAPP. The ability to block the aggregation of soluble IAPP, IAPP fragments, and peptides is a highly desirable trait.

2. The antibodies should stain amyloid regions surrounding the β cells/islets in hyperglycemic mice transgenic for human IAPP and in diabetic humans. They should not, however, stain β cells or β cell secretory granules in these tissues. The antibodies should not stain the islet regions of normal nonhuman primates or humans.

3. The antibodies should induce the clearance and degradation of amyloid plaques from islet tissue derived from hyperglycemic obese mice transgenic for human IAPP, possibly through Fc receptor-mediated phagocytosis, in ex vivo assays.

4. Upon in vivo administration to hyperglycemic obese mice transgenic for human IAPP, the antibodies should decorate islet amyloid plaques and induce clearance or reduction of the plaques, relative to a control group not receiving passively administered antibodies. Nonhyperglycemic obese mice transgenic for human IAPP receiving passively administered antibodies should also maintain improved oral glucose tolerance and glucose clamp results, decreased fasting sugar levels, increased production of insulin, improved insulin/glucose ratios in homeostasis model assessments, decreased islet amyloid deposition, and decreased progression to insulin dependence over time, relative to a control group not receiving passively administered antibodies. 5. Human subjects with Type II diabetes receiving a therapeutic antibody should maintain improved oral glucose tolerance and glucose clamp results, decreased fasting sugar levels, increased production of insulin, improved insulin/glucose ratios in homeostasis model assessments, decreased glycation of hemoglobin A1 c, decreased islet amyloid deposition (evaluated by noninvasive imaging), and decreased progression to insulin dependence over time, relative to control subjects not receiving the passively administered antibody.

D. Epitope Characterization Epitopes recognized by antibodies specific for IAPP fibrils or plaques (i.e., islet amyloid) reside within one of the amyloidogenic domains in β-conformation or sequences containing de-amidated asparagine residues (i.e., L- or D-isoaspartic acid or aspartic acid). The fine specificity of these antibodies is analyzed by testing against a series of truncated aggregated peptides derived from IAPP plus flanking residues. Preferably, these assays are carried out in competition format against solid-phase IAPP fibrillar material or aggregated fragments. The effect of single amino acid replacements within the competing peptides are similarly evaluated in a competition format to gauge the contribution of the each residue within the epitope. If it is necessary to test the antibodies against aggregated peptides to ensure β-conformation (fragments down to FGAIL will aggregate into fibrils), antibody binding directly to immobilized/aggregated/cross-linked peptides can be evaluated.

In order that this invention may be better understood, the following examples are set forth. The examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention.

EXAMPLES

Preparation of a Therapeutic Vaccine A safe and efficacious therapeutic vaccine for Type 2 diabetes will likely elicit a humoral response directed against epitopes from within one of the amyloidogenic domains in β conformation. It will not elicit an immune response to IAPP in random coil conformation, to the flanking C-terminal and N-terminal domains of IAPP, to soluble/nonaggregated IAPP, or to normal β cells.

A. Peptide Conjugate Vaccines

CRM₁₉₇or other polypeptide carriers (e.g., tetanus toxoid, KLH, BSA, thyroglobulin) are bromoacetylated through lysine residues using N-hydroxy succinimidyl bromoacetate. Peptides derived from the amyloidogenic midregion of IAPP (SNNFGAILSS), with and without flanking residues, smaller peptides derived from the region that has been shown to be critical for fibril formation (FGAIL, NFGAIL, GAILS, FGAILS, NFGAILS - SEQ ID NOS: 3-7), and peptides spanning the N and C terminal domains of IAPP, with the asparagine residues at positions 3 and/or 35 replaced by L- or D-isoaspartic acid or aspartic acid are synthesized with and without linker sequences (GAGA) and with N-terminal or C-terminal cysteine residues and coupled through a thioether bond to the activated protein carriers.

The effect of peptide linkers, peptide orientation, peptide sequence and size, peptide density (peptide/carrier ratios), and choice of adjuvants on immunogenicity and on specificity for aggregated/fibrillar IAPP is evaluated in animal models.

Peptides conjugated to a polypeptide carrier may maintain a random coil conformation, rather than a β-conformation, and may therefore elicit an immune response to IAPP in its native, nonaggregated state. The various peptides described above are therefore allowed to assemble into small aggregates/fibrils in low ionic strength buffer and are chemically cross-linked as described below, if necessary. The aggregated peptides are then evaluated for immunogenicity and specificity for aggregated/fibrillar IAPP. If the peptide aggregates are found to be poorly immunogenic, small aggregates are coupled to a carrier polypeptide as described above.

B. Peptide Mimics/Single Chain Anti-idiotypic Antibodies Monoclonal antibodies directed against the amyloidogenic domain of IAPP for therapeutic purposes and appropriate control antibodies (matched for isotype and repertoire V genes) are used to probe phage or E. coli constrained random peptide libraries (7 to 12mer) or even synthetic random peptide libraries (on beads). Potential mimics of IAPP that are nonhomologs are conjugated to a protein carrier in different orientations with and without spacer peptides and tested in vivo for the generation of a specific immune response to IAPP fibrils or plaques.

A related approach is to probe a phage displayed combinatorial antibody library for scFv with potential anti-idiotypic activity. Again, nonhomologs are tested in mice for generation of a specific immune response to IAPP fibrils or plaques. The anti-idiotypic scFv is a component of an immunogenic composition.

C. Evaluation of Immunogenic Compositions

1. The immunogenic compositions described above are evaluated for immunogenicity, specificity and safety, initially in outbred Swiss Webster mice. Selection criteria for successful candidate compositions are similar to the criteria for potential therapeutic antibodies. Immunogenic compositions should elicit high serum EIA titers against aggregated/fibrillar (β conformation) IAPP, IAPP fragments and peptides from the amyloidogenic domains of IAPP, or peptides spanning the N and C terminal domains of IAPP, with the asparagine residues at positions 3 and/or 35 replaced by L- or D-isoaspartic acid or aspartic acid, but should not induce titers against native/soluble

(random coil) IAPP, IAPP fragments, or peptides derived from nonamyloidogenic domains of IAPP.

2. The serum antibodies elicited by a composition should stain amyloid regions surrounding the β cells/islets in hyperglycemic obese mice transgenic for human IAPP and in diabetic macaques, and humans. However, they should not stain β cells or β cell secretory granules in these tissues. The antibodies should not stain the islet regions of normal nonhuman primates, or humans.

3. The serum antibodies elicited by a composition should induce the ex vivo clearance or reduction of amyloid plaques from islet tissue derived from hyperglycemic mice transgenic for human IAPP.

4. Upon in vivo administration to hyperglycemic obese mice transgenic for human IAPP, passively administered serum antibodies elicited by an immunogenic composition should decorate islet amyloid plaques and induce clearance or reduction of the plaques, relative to a control group not receiving passively administered antibodies. Nonhyperglycemic obese mice transgenic for human IAPP receiving passively administered serum antibodies elicited by a composition should also maintain improved oral glucose tolerance and glucose clamp results, decreased fasting sugar levels, increased production of insulin, improved insulin/glucose ratios in homeostasis model assessments, decreased islet amyloid deposition, and decreased progression to insulin dependence over time, relative to a control group not receiving passively administered antibodies. Upon in vivo administration to hyperglycemic obese mice transgenic for human IAPP, an immunogenic composition should elicit serum antibodies that decorate islet amyloid plaques ex vivo and induce clearance or reduction of the plaques, relative to a control group not receiving the composition. Nonhyperglycemic obese mice transgenic for human IAPP receiving an immunogenic composition should also maintain improved oral glucose tolerance and glucose clamp results, decreased fasting sugar levels, increased production of insulin, improved insulin/glucose ratios in homeostasis model assessments, decreased islet amyloid deposition, and decreased progression to insulin dependence over time, relative to a control group not receiving the composition. Human subjects with Type II diabetes receiving an immunogenic composition should maintain improved oral glucose tolerance and glucose patch results, decreased fasting sugar levels, increased production of insulin, improved insulin/glucose ratios in homeostasis model assessments, decreased glycation of hemoglobin A1 c, decreased islet amyloid deposition (evaluated by noninvasive imaging), and decreased progression to insulin dependence over time, relative to control subjects not receiving the composition or a passively administered antibody.

Table I: Current modalities of treatment for Type 2 diabetes: BRAND NAME COMPANY MECHANISM OF ACTION

Glucophage Bristol-Myers Not related to sulfonylureas Bristol-Myers Reduce basal & Post-prandial glucose Decrease hepatic glucose production, decrease intestinal absorption & improve insulin sensitivity. Does not induce hyperinsulinemia.

Glucovance Bristol-Myers Glyburide + Metform HCI

Glyset Pharmacia (miglitol) - alpha glucosidase inhibitor Precose Bayer Delays digestion of ChO

Reduce glycosylated hemoglobin

Does not enhance insulin production glucosidase inhibitor reduce basal membrane bound glucosidase activity

Prandin Novo Nordisk Insulin stimulators - doses ATP dependent potassium channel in beta cells - opens calcium channel - Increases insulin

Actos Takeda Improve insulin sensitivity. Peroxisome proliferator Avandia SKB activated receptor - gamma (PPARalpha)) - agonist of PPARalpha)- Decreased insulin resistence pioglitazone. Decrease in triglycerides associated with diabetes

Amaryl Aventis Increase weight stimulates insulin production by

DiaBeta Aventis beta cells

Diabinese Pfizer

Glucotrol Pfizer

Glucotrol XL Pfizer

Glucovance Bristol Myers

Table II: Cross-linking agents for protein stabilization and conjugation:

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Claims

What is claimed is:

1. A pharmaceutical composition, comprising: an effective amount of an agent to induce in a patient an immune response against islet amyloid fibrils, protofibrils and/or amyloid plaque, but not against soluble, native islet amyloid polypeptide (IAPP), together with a pharmaceutically acceptable carrier.

2. The composition of claim 1 , wherein the composition is administered intranasally, intradermally, subcutaneously, intramuscularly or intravenously.

3. The pharmaceutical composition of claim 1 , wherein the agent is a purified antigen that is surface-exposed only in islet amyloid fibrils, protofibrils and/or amyloid plaque, but not in soluble, native IAPP.

4. The pharmaceutical composition of claim 1 , wherein the agent is an antibody that binds specifically to epitopes exposed only in islet amyloid fibrils, protofibrils and/or amyloid plaque, but not in soluble, native IAPP.

5. The pharmaceutical composition of claim 4, wherein the antibody is a monoclonal antibody.

6. The pharmaceutical composition of claim 5, wherein the monoclonal antibody is selected from recombinant human, humanized murine, chimerized murine, and transgenic murine antibodies.

7. The pharmaceutical composition of claim 1 , wherein the agent is an anti- idiotypic antibody.

8. The pharmaceutical composition of claim 1 , wherein the agent is a peptide mimic of an epitope present in islet amyloid fibrils, protofibrils and/or amyloid plaque, but not in soluble, native IAPP.

9. The pharmaceutical composition of claim 1 , wherein the agent is an antibody mimic that binds to an epitope present in islet amyloid fibrils, protofibrils and/or amyloid plaque, but not in soluble, native IAPP.

10. The pharmaceutical composition of claim 3, further comprising an adjuvant.

11. A method of treating Type 2 diabetes in a patient capable of forming islet amyloid, comprising: inducing an immune response by administering to the patient a composition comprising an effective amount of purified antigen that is surface-exposed only in islet amyloid fibrils, protofibrils and/or amyloid plaque, but not in solublenative IAPP, together with a pharmaceutically acceptable carrier, wherein the immune response comprises clearance of the islet amyloid fibrils, protofibrils and/or amyloid plaque.

12. The method of claim 11 , wherein the patient is a human.

13. The method of claim 11 , wherein the composition is administered intranasally, intradermally, subcutaneously, intramuscularly or intravenously.

14. The method of claim 11 , further comprising monitoring the patient for the immune response.

15. The method of claim 11 , further comprising administering an adjuvant.

16. The method of claim 15, wherein the adjuvant is administered with the antigen as a single composition.

17. The method of claim 15, wherein the adjuvant is administered before the composition is administered.

18. The method of claim 15, wherein the adjuvant is administered after the composition is administered.

19. The method of claim 15, wherein the adjuvant is administered concurrently with the composition.

20. The method of claim 11 , wherein the administering comprises administering multiple doses of the antigen until a sufficient immune response is achieved, monitoring the immune response, and administering an additional dose of the antigen when the immune response starts to fade.

21. The method of claim 20, wherein the monitoring comprises detecting antibodies that specifically bind to the antigen.

22. The method of claim 14, further comprising administering booster doses of the antigen as indicated by the monitoring.

23. A method of preventing Type 2 diabetes in a subject capable of forming islet amyloid, comprising: inducing an immune response by administering to the subject a composition comprising an effective amount of purified antigen that is surface-exposed only in islet amyloid fibrils, protofibrils and/or amyloid plaque but not in solublenative IAPP, together with a pharmaceutically acceptable carrier, wherein the immune response is sufficient for inhibiting the formation of islet amyloid fibrils, protofibrils and/or amyloid plaque, thereby preventing Type 2 diabetes in the subject.

24. The method of claim 23, wherein the subject is a human.

25. The method of claim 23, wherein the composition is administered intranasally, intradermally, subcutaneously, intramuscularly or intravenously.

26. The method of claim 23, further comprising monitoring the patient for the immune response.

27. The method of claim 23, further comprising administering an adjuvant.

28. The method of claim 27, wherein the adjuvant is administered with the antigen as a single composition.

29. The method of claim 27, wherein the adjuvant is administered before the composition is administered.

30. The method of claim 27, wherein the adjuvant is administered after the composition is administered.

31. The method of claim 27, wherein the adjuvant is administered concurrently with the composition.

32. The method of claim 23, wherein the administering comprises administering multiple doses of the antigen until a sufficient immune response is achieved, monitoring the immune response, and administering an additional dose of the antigen when the immune response starts to fade.

33. The method of claim 32, wherein the monitoring comprises detecting antibodies that specifically bind to the antigen.

34. The method of claim 26, further comprising administering booster doses of the antigen as indicated by the monitoring.

35. A method of treating Type 2 diabetes in a patient capable of forming islet amyloid, comprising: inducing an immune response by administering to the patient a composition comprising an effective amount of an antibody that binds specifically to epitopes exposed only in islet amyloid fibrils, protofibrils and/or amyloid plaque but not in soluble, native IAPP, together with a pharmaceutically acceptable carrier, wherein the immune response comprises clearance of the islet amyloid fibrils, protofibrils and/or amyloid plaque.

36. The method of claim 35, wherein the patient is a human.

37. The method of claim 35, wherein the antibody is a monoclonal antibody.

38. The method of claim 37, wherein the monoclonal antibody is selected from recombinant human, humanized murine, chimeriized murine, and transgenic murine antibodies.

39. A method of preventing Type 2 diabetes in a subject capable of forming islet amyloid, comprising: inducing an immune response by administering to the subject a composition comprising an effective amount of an antibody that binds specifically to epitopes exposed only in islet amyloid fibrils, protofibrils and/or amyloid plaque but not in soluble, native IAPP, together with a pharmaceutically acceptable carrier, wherein the immune response is sufficient for inhibiting the formation of islet amyloid fibrils, protofibrils and/or amyloid plaque, thereby preventing Type 2 diabetes in the subject.

40. The method of claim 39, wherein the subject is a human.

41. A method of treating Type 2 diabetes in a patient capable of forming islet amyloid, comprising: inducing an immune response by administering to the patient a composition comprising an effective amount of a peptide mimic of an epitope present in islet amyloid fibrils, protofibrils and/or amyloid plaque, but not in soluble, nativelAPP, together with a pharmaceutically acceptable carrier, wherein the immune response comprises clearance of the islet amyloid fibrils, protofibrils and amyloid plaque.

42. The method of claim 41 , wherein the patient is a human.

43. A method of preventing Type 2 diabetes in a subject capable of forming islet amyloid, comprising: inducing an immune response by administering to the subject a composition comprising an effective amount of a peptide mimic of an epitope present in islet amyloid fibrils, protofibrils and/or amyloid plaque, but not in soluble, nativelAPP, together with a pharmaceutically acceptable carrier, wherein the immune response is sufficient for inhibiting the formation of islet amyloid fibrils, protofibrils and/or amyloid plaque, thereby preventing Type 2 diabetes in the subject.

44. The method of claim 43, wherein the subject is a human.

45. A method of treating Type 2 diabetes in a patient capable of forming islet amyloid, comprising: inducing an immune response by administering to the patient a composition comprising an effective amount of an antibody mimic that binds specifically to epitopes exposed only in islet amyloid fibrils, protofibrils and/or amyloid plaque but not in soluble, nativelAPP, together with a pharmaceutically acceptable carrier, wherein the immune response comprises clearance of the islet amyloid fibrils, protofibrils and/or amyloid plaque.

50. The method of claim 45, wherein the patient is a human.

47. A method of preventing Type 2 diabetes in a subject capable of forming islet amyloid, comprising: inducing an immune response by administering to the subject a composition comprising an effective amount of an antibody mimic that binds specifically to epitopes exposed only in islet amyloid fibrils, protofibrils and/or amyloid plaque but not in soluble, nativelAPP, together with a pharmaceutically acceptable carrier, wherein the immune response is sufficient for inhibiting the formation of islet amyloid fibrils, protofibrils and/or amyloid plaque, thereby preventing Type 2 diabetes in the subject.

48. The method of claim 47, wherein the subject is a human.

49. A composition, comprising: an antigen conjugated to a carrier molecule wherein the resulting conjugate promotes an immune response against the antigen in a patient, wherein the antigen is surface-exposed only in islet amyloid fibrils, protofibrils and/or amyloid plaque but not in soluble, nativelAPP.

50. The composition of claim 49, wherein the carrier molecule is selected from attenuated diptheria toxin CRM₁₉₇, ovalbumin, thyroglobulin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), tetanus toxoid, diptheria toxoid, E. coli toxoid, cholera toxoid and H. pylori toxoid.

51. A method of identifying peptide mimics of epitopes present on islet amyloid fibrils, protofibrils and/or amyloid plaque but not on native, solublelAPP, the method comprising: providing phage display libraries expressing 7-1 Omer random peptide sequences or libraries expressing 7-12mer E. coli constrained random peptide sequences, said sequences comprising epitopes present on islet amyloid fibrils, protofibrils, and/or amyloid plaque, but not on soluble, native

IAPP; contacting the libraries with a test monoclonal antibody that specifically binds to epitopes exposed only in islet amyloid fibrils, protofibrils, and/or amyloid plaque, but not in soluble, native IAPP; and determining the peptide sequences that bind to the monoclonal antibody, thereby identifying peptide mimics of epitopes present on islet amyloid fibrils, protofibrils and/or plaque, but not on native, soluble IAPP.

52. The method of claim 51 , wherein the identified peptide mimics are conjugated to carrier molecules and tested for the ability to elicit antibodies specific for epitopes present in islet amyloid fibrils, protofibrils, and/or amyloid plaque, but not in soluble, native IAPP.

53. An immunogenic composition for preventing or treating Type 2 diabetes in a patient capable of forming islet amyloid, comprising: an effective amount of purified antigen that is surface-exposed only in islet amyloid fibrils, protofibrils and/or amyloid plaque but not in soluble, native IAPP, together with a pharmaceutically acceptable carrier.

54. A method of treating Type 2 diabetes in a patient capable of forming islet amyloid, comprising: administering to the patient the composition of claim 53.

55. A method of preventing Type 2 diabetes in a subject capable of forming islet amyloid, comprising: administering to the subject the composition of claim 53.

56. A monoclonal antibody that binds specifically to epitopes exposed only in islet amyloid fibrils, protofibrils and/or amyloid plaque, but not in soluble, native

IAPP.

57. An antibody mimic that binds specifically to epitopes exposed only in islet amyloid fibrils, protofibrils and/or amyloid plaque, but not in soluble, native IAPP.

58. A method of identifying antibody mimics that bind specifically to epitopes exposed only in islet amyloid fibrils, protofibrils and/or amyloid plaque, but not in soluble, native IAPP, the method comprising: providing an antigen that is surface-exposed only in islet amyloid fibrils, protofibrils and/or amyloid plaque, but not in soluble, native IAPP; contacting the antigen with a candidate protein that comprises a fibronectin type III domain having at least one randomized loop, the contacting being carried out under conditions that allow antigen-protein complex formation; and determining from the complex, the protein which binds to the antigen, thereby identifying antibody mimics that bind specifically to epitopes exposed only in islet amyloid fibrils, protofibrils and/or amyloid plaque, but not in soluble, native IAPP.

59. A method of preserving β-cell function in a patient having or susceptible to Type 2 diabetes, comprising the step of inhibiting or reversing islet amyloid formation by administering to the patient a composition comprising an effective amount of a purified antigen that is surface-exposed only in islet amyloid fibrils, protofibrils and/or amyloid plaque but not in soluble, native IAPP, together with a pharmaceutically acceptable carrier.

60. A method of preserving β-cell function in a patient having Type 2 diabetes, comprising: clearing islet amyloid fibrils, protofibrils and/or amyloid plaque from said patient's islet tissues by administering to the patient a monoclonal antibody that binds specifically to epitopes exposed only in islet amyloid fibrils, protofibrils and/or amyloid plaque, but not in soluble, native IAPP, thereby inducing clearance of the islet amyloid fibrils, protofibrils and amyloid plaque, such that the progressive loss of insulin-producing β-cells associated with Type 2 diabetes is arrested.

61. A method of inhibiting the formation of islet amyloid fibrils, protofibrils and/or amyloid plaque, comprising: blocking apolipoprotein E, serum amyloid P component or perlecan from binding to developing islet amyloid fibrils, protofibrils and/or amyloid plaque by administering to a patient capable of developing islet amyloid a composition comprising an effective amount of an agent to induce an immune response against islet amyloid fibrils, protofibrils and/or amyloid plaque, but not against soluble, native IAPP, together with a pharmaceutically acceptable carrier.