Peptides and methods for treating disease

Abstract

To provide bioactive peptides and methods for their use for preventing, treating, and / or reducing the incidence and / or symptoms of an autoimmune disease or disorder that may result from chronic inflammation, damage to β-cells of the pancreas, and other conditions that are implicated by the CD40-CD154 dyad.SOLUTION: In particular, provided are small peptides that are capable of interacting with CD40, thereby interfering with the ability of CD40 to interact with CD154, which impacts inflammation, autoimmunity, and disease progression. Provided are the use of such peptides in reducing diabetes mellitus, and in particular, the autoimmune inflammatory response that may be a driving factor thereof, and methods and materials for preventing and modulating diabetes mellitus and autoimmune diseases especially in dogs, cats, and horses.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to peptides and methods and / or uses thereof for the prevention, regulation, and treatment of disease in a subject.

[0002] Additionally, the present disclosure relates to peptides that affect, interact with, and / or inhibit the interaction of CD154 with the CD40 complex, and the use of such compounds in modulating T cell activity and treating disease. [Background technology]

[0003] Inflammation occurs when inflammatory cells, such as neutrophils, eosinophils, basophils, mast cells, macrophages, platelets, and endothelial cells, respond to inflammatory events or harmful stimuli, such as invading microorganisms, damaged cells, or other irritants. For example, when a microorganism invades the body, the inflammatory response is beneficial because it serves as a critical step in localizing the infectious agent for elimination by the immune system. However, in autoimmune diseases, severe inflammation is present or persists even in the absence of infection. This inflammation, called sterile chronic inflammation (ACI), is harmful because it destroys normal tissue. The consequences of this sterile inflammation can be life-threatening and, in some cases, life-threatening. Furthermore, like acute inflammation, this process is mediated by immune cells, including T cells.

[0004] A major concern in modern medicine is how to control ACI, such as that occurring during autoimmune diseases, as well as acute inflammation caused by trauma. Chronic and acute inflammation can lead to tissue degeneration and ultimately to the loss of function in major organs. ACI is not limited to a single disease but can help with numerous autoimmune diseases, including, but not limited to: type 1 diabetes (T1D), multiple sclerosis (MS), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Crohn's disease, inflammatory bowel disease (IBS), chronic obstructive pulmonary disease (COPD), including autoimmune forms of asthma, arteriosclerosis, vasculitis, hypertension, thyroiditis, including Hashimoto's disease and Graves' disease, primary biliary cirrhosis, Paget's disease, Addison's disease, acute respiratory distress syndrome (ARDS), acute lung injury, and the sterile chronic inflammation associated with organ transplantation.

[0005] Autoimmune diseases are classified into two types: organ-specific (directed primarily at one organ) and non-organ-specific (widespread throughout the body). Examples of organ-specific autoimmune diseases include insulin-dependent type 1 diabetes (T1D), which affects the pancreas; Hashimoto's thyroiditis and Graves' disease, which affect the thyroid; pernicious anemia, which affects the blood; Addison's disease, which affects the adrenal glands; chronic active hepatitis, which affects the liver; myasthenia gravis, which affects receptors in the joints between muscles and nerves; and multiple sclerosis, which affects tissues of the nervous system. An example of a non-organ-specific autoimmune disease is rheumatoid arthritis. Autoimmune diseases are always chronic, debilitating, and life-threatening. The National Institutes of Health (NIH) estimates that up to 23.5 million Americans suffer from autoimmune diseases, and their prevalence is rising. Autoimmune diseases are estimated to be among the top 10 leading causes of death for women of all ages up to age 65.

[0006] Acute inflammation, such as that seen during trauma or sepsis, is also immune cell-mediated. While a comprehensive, complete, and exhaustive list of molecular mediators in this process has yet to be identified, prominent roles for T cells, lymphocytes, neutrophils, macrophages, monocytes, eosinophils, basophils, mast cells, and other inflammatory cells have been strongly implicated. Therefore, processes regulating these cell types may control the inflammatory response.

[0007] Canines, felines, and horses are known to suffer from a variety of autoimmune diseases. While some of these diseases may have similar molecular drivers to those in humans, others have entirely different causes, many of which are still unknown. Regardless, the market for treating companion animal diseases is a growing sector, with researchers, pet owners, and veterinarians alike seeking treatments for the diseases that plague our beloved animals.

[0008] Diabetes mellitus is one of the most common endocrine disorders that can develop in canines. Furthermore, the incidence of canine diabetes has increased significantly, with one study showing an incidence rate of 19 cases per 10,000 dogs in 1970 to 64 cases per 10,000 dogs in 1999. Standard treatment typically requires that affected dogs be initially initiated on insulin therapy, regardless of the underlying cause and / or classification of diabetes. Currently, canine diabetes cases are classified into two types: insulin-deficient diabetes and insulin-resistant diabetes. While canines are currently believed to be less susceptible to developing the human equivalent of type 2 diabetes, it is notable that obesity is sometimes associated with insulin-resistant diabetes (ibid.).

[0009] Many diabetic dogs have insulin-deficient diabetes, which is caused by the loss or destruction of pancreatic beta cells, resulting in insufficient or complete absence of insulin secretion or production (ibid.). The role of autoimmunity in the development of canine diabetes is currently less clear than that of human T1D and non-obese diabetic (NOD) mice, but inflammatory processes and autoimmunity remain suspected in at least some cases of the disease (ibid.). Canine diabetes is often diagnosed when hyperglycemia (usually >250 mg / dL) and glycosuria are confirmed, but no single diagnostic indicator, benchmark, or standard currently exists.

[0010] Diabetes is also a common endocrine disorder in cats. The current classification of feline diabetes is based on that of human diabetes, including the mechanisms and causes involved in pancreatic beta cell damage. Therefore, it is currently assumed that cats suffer from both type 1 and type 2 diabetes, although type 1 diabetes is thought to be less common in felines than in humans and dogs (ibid.).

[0011] Horses also suffer from diabetes, and models of this disease state are again based on what is understood about diabetes in humans. Horses are prone to developing equine metabolic syndrome (EMS), which can in turn lead to the development of debilitating conditions such as laminitis that affect the equine's hooves. (Id.) The development of laminitis may require euthanasia. (Id.) Therefore, therapies that can ameliorate disease and inflammation-related conditions may be useful for preventing complications in equine subjects suffering from EMS.

[0012] This subject matter is also related to the discovery that a unique subset of T cells expressing the CD40 protein (hence referred to as Th40 cells) may play a role in autoimmune inflammation. Furthermore, the involvement of Th40 cells in autoimmune processes may depend on the interaction between the CD40 protein expressed on the T cell surface and the CD154 protein. The interaction between CD40 and CD154 transmits an activation signal between the cells, subsequently activating Th40 cells. Such activation leads to the proliferation of Th40 cells and increased inflammation (e.g., an increase in the number of immune cells and immunoregulatory molecules present in the system). Therefore, inhibition of CD40 / CD154 interaction may modulate the activity of Th40 cells, thereby affecting inflammation.

[0013] Thus, there is a need in the art for safer and more effective methods for the treatment and prevention of autoimmune diseases, including diabetes, in canines, felines, and equines. The present disclosure provides novel biologically active peptides, variants, and derivatives thereof. Furthermore, the present invention may address this need by describing peptides and methods for treating autoimmune diseases by administering therapeutically effective amounts of CD40 peptides. Furthermore, the present invention may provide the added benefit of preventing the production of autoantibodies, thus allowing normal immune function to resume.

[0014] This background statement is for informational purposes only and is not intended to be a complete or exhaustive description of all potentially relevant background. [Prior art documents] [Non-patent literature]

[0015] [Non-Patent Document 1] Catchpole, B., et al., Diabetologia, 2005, 48:1948-56 [Non-patent document 2] Gruptill, L., et al., Vet. J., 2003, 165:240-47 [Non-patent document 3] O'Kell, A, et al., Diabetes, 2017, 66(6): 1443-52 [Non-patent document 4] Catchpole, B., et al., Diabetologia, 2005, 48:1948-56 [Non-patent document 5] Matthews, CE., Pediatr. Diabetes, 2005, 6:165-77; Amer. Diabetes Association Diagnosis and Classification of Diabetes Mell itus, Diabetes Care, 2014, 37, Suppl.1:S81-S90 [Non-patent document 6] O'Kell, A, et al., Diabetes, 2017, 66(6): 1443-52 [Non-Patent Document 7] Gottlieb, S, et al, Vet. Med. (Auckl), 9:33-42, 2018 [Non-patent document 8] Johnons, PJ, et al. J. Diabetes Sci. Tecnol. 6(3): 534-40, 2012 Summary of the Invention [Problem to be solved by the invention]

[0016] The present invention relates to bioactive peptides and their use to treat subjects before, during, and / or after the onset of diabetes and / or autoimmune disease. [Means for solving the problem]

[0017] The present invention demonstrates that the peptides of the present invention can affect the inflammatory state present in or occurring simultaneously with many diseases. The peptides of the present invention can be used to provide therapeutic results to help control, manage, or influence conditions and diseases caused by inflammation, inflammatory cascades, or autoimmune-autoinflammatory pathways. Thus, the present invention relates to the use of the peptides of the present invention to control one or more of blood glucose levels, serum fructocyme levels, c-peptide levels, and other measurable markers of diseases, disorders, or inflammation described and disclosed herein.

[0018] These inventions may relate to the understanding that the CD40-CD154 inflammatory dyad may play an important role in the development and progression of diabetes and autoimmune diseases. The invention may also relate to reducing Th40 cell levels and hyperglycemia. The invention may also relate to the therapeutic effects of lowering serum fructosamine levels and increasing c-peptide levels.

[0019] Thus, the present disclosure provides bioactive peptides and their uses for preventing, treating, and / or reducing the incidence and / or symptoms of autoimmune diseases or disorders that may result from chronic inflammation, damage to pancreatic beta cells, and other conditions involving the CD40-CD154 dyad. The peptides disclosed herein provide variants, homologs, orthologs, and / or other derivatives that may be useful in treating the disease. The methods disclosed herein include administering to a subject in need of treatment an effective amount of a peptide that affects, modulates, inhibits, regulates, and / or interacts with CD40. This interaction may be in a manner that disrupts the binding and interaction of the CD40 and CD154 proteins. In these methods, the autoimmune disease may include: type 1 diabetes (T1D), multiple sclerosis (MS), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Crohn's disease, inflammatory bowel disease (IBS), chronic obstructive pulmonary disease (COPD) including autoimmune forms of asthma, arteriosclerosis, vasculitis, hypertension, thyroiditis including Hashimoto's disease and Graves' disease, primary biliary cirrhosis, Paget's disease, Addison's disease, acute respiratory distress syndrome (ARDS), acute lung injury, and sterile chronic inflammation (ACI) associated with organ transplantation.

[0020] The peptides of the present invention may also be used in methods of treating or preventing hyperglycemia in a subject by administering to the subject a composition comprising a peptide of the present disclosure, and functional fragments and / or homologs and analogs thereof, in an amount and under conditions effective to reduce hyperglycemia.

[0021] Peptides useful in these methods can be peptides that bind to, interact with, and / or affect CD40. Additionally, peptides of the present invention can affect or disrupt the interaction between CD40 and CD154. Furthermore, in some embodiments, administration of peptides of the present invention can reduce the number of Th40 cells in a subject. These short interfering peptides (SIPs) can comprise an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15. The inhibitor peptides may contain modifications selected from phosphorylation and glycosylation, and / or may be linked to a polyethylene glycol (PEG) molecule, and / or may be linked to one or more domains of the Fc region of a mouse, human, canine, feline, or equine animal, an IgG immunoglobulin, or its equivalent in each species, and / or may be linked to an epitope tag polypeptide comprising 6 to 50 amino acid residues. Furthermore, the inhibitor peptides of the present invention may also contain terminal modifications such as acetylation and amidation. Furthermore, the peptides of the present invention may include several salt forms, including hydrochloride, acetate, TFA, and sodium chloride.

[0022] This Summary is not intended to, and should not be construed as, representative of the entire scope of the present disclosure. Moreover, references made herein to "the disclosure" or aspects thereof should be understood to refer to particular embodiments of the disclosure and should not necessarily be construed as limiting all embodiments to the particular description. The disclosure is described in various levels of detail in this Summary as well as in the Figures and Detailed Description, and no limitation on the scope of the disclosure is intended by either the inclusion or exclusion of elements. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting, and they should not be construed as limiting the embodiments.

[0023] Additional features and aspects of the present invention will become apparent from the following detailed description and claims. [Brief explanation of the drawings]

[0024] [Figure 1] 1 is a graph showing the effect of various peptides of CD154 on the development of diabetes in NOD mice, including 6-mer (SEQ ID NO:46), 8-mer (SEQ ID NO:22), 10-mer (SEQ ID NO:41), 13-mer (SEQ ID NO:42), 15-mer (SEQ ID NO:24), and 24-mer (SEQ ID NO:43). [Figure 2A] 1 is a chart showing the effect of a 15-mer peptide from CD154 on the CD4 / CD8 ratio in NOD mice. [Figure 2B] 1 is a graph of the effect of a 15-mer peptide on treated versus control pancreata that were excised, examined, and scored. [Figure 3] 1 is a graph of the reversal of diabetes in NOD mice using a 15-mer peptide from CD154. [Figure 4] FIG. 1 is a dot plot of detection of Th40 cells using a 15-mer peptide from CD154. [Figure 5] 1 is a dot plot of screening of B cells with a 15-mer peptide from CD154. [Figure 6] 1 is a graph showing a comparison of Th40 cell levels in diabetic and non-diabetic mice. [Figure 7] 1 is a graph showing the effect of treatment with 15-mer peptides on pancreatic insulin granulation. [Figure 8] 1 is a graph showing the effect of mutations in a 15-mer peptide on the ability of the 15-mer peptide to suppress the development of diabetes in NOD mice. [Figure 9] 1 is a graph showing the number of CD3+CD4+CD40+ cells (×106) in different mouse models. [Figure 10]1 is a graph showing the percentage of TH40 cells in the peripheral blood of human subjects in control and diabetic groups. [Figure 11] 1 is a dot plot of the detection of Th40 cells in canine blood (dogs known to have diabetes) using a 6-mer peptide from CD154 (SEQ ID NO: 46). [Figure 12] 1 is a graph of pre- and post-treatment blood glucose levels in diabetic canine subjects. [Figure 13] 1 is a graph of pre- and post-treatment fructosamine serum levels in diabetic canine subjects. [Figure 14a] 1 is a graph of c-peptide levels in diabetic canine subjects before, during, and at the end of treatment. [Figure 14b] 1 is a graph of c-peptide levels in diabetic canine subjects before, during, and at the end of treatment. [Figure 14c] 1 is a graph of c-peptide levels in diabetic canine subjects before, during, and at the end of treatment. [Figure 14d] 1 is a graph of c-peptide levels in diabetic canine subjects before, during, and at the end of treatment. [Figure 14e] 1 is a graph of c-peptide levels in diabetic canine subjects before, during, and at the end of treatment. [Figure 15] 1 provides a graph of Th40 percentage in canine peripheral blood. [Figure 16] 1 provides a graph showing the average daily blood glucose levels of male diabetic canine subjects before, during, and after treatment. [Figure 17] 1 provides a graph showing the daily blood glucose averages of female diabetic canine subjects before, during, and after treatment. [Figure 18] 1 provides a graph comparing peripheral Th40 cell percentages in mouse, human, and canine subjects. [Figure 19] 1 provides a table of pre-treatment and on-treatment blood counts for diabetic canine subjects. [Figure 20] 1 provides a table of pre-treatment and on-treatment blood counts for diabetic canine subjects. [Figure 21] 1 is a graph showing the effect of peptide treatment on hyperglycemia in diabetic NOD mice. [Figure 22] 1 is a graph of peptide binding to live cells in culture over time. [Figure 23] 1 provides a graph showing that SEQ ID NO:11 regulates the inflammatory cytokine IFNγ without affecting the production of the non-inflammatory cytokine IL-4. [Figure 24] 1 provides a graph showing that different concentrations of the peptide of SEQ ID NO:46 affect or influence levels of IL-2, IFNγ, and IL-17A. [Figure 25] 1 provides a graph showing the effect of treatment with SEQ ID NO: 13 on ketones measured in the urine of canine subjects. DETAILED DESCRIPTION OF THE INVENTION

[0025] The present disclosure provides bioactive peptides that are useful for preventing, modulating, and / or treating a variety of indications and diseases, as discussed in detail below. In some embodiments, the peptides of the present invention affect the interaction and / or binding of the CD40 complex with its ligand, CD154.

[0026] The present invention provides novel compositions and methods for treating, modulating, and / or reducing the severity of diabetes in animal subjects. In some instances, diabetes can result from the infiltration and destruction of beta cells, resulting in a loss or diminished ability to produce insulin. The present invention also relates to the treatment of these conditions, as evidenced by the effects on blood glucose levels, serum fructosamine levels, c-peptide levels, and peripheral Th40 levels. Accordingly, the present invention also relates to methods for treating subjects before, during, and / or after the onset of diabetes with specific peptides or pharmaceutical compositions that can alleviate the signs and symptoms of diabetes, reduce blood Th40 levels, and reduce, modulate, and / or regulate hyperglycemia.

[0027] Accordingly, aspects and embodiments disclosed herein can include compositions of matter and methods for treating and / or reducing diabetes. These methods involve administering to a subject in need thereof an effective amount of a peptide to inhibit, disrupt, interact, and / or affect binding or signaling between proteins associated with the CD40 complex, including, but not limited to, CD154. It is understood that the relevant disease or disorder can also include autoimmune conditions such as type 1 diabetes (T1D), multiple sclerosis (MS), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Crohn's disease, inflammatory bowel disease (IBS), chronic obstructive pulmonary disease (COPD), including autoimmune forms of asthma, arteriosclerosis, vasculitis, hypertension, thyroiditis, including Hashimoto's disease and Graves' disease, primary biliary cirrhosis, Paget's disease, Addison's disease, acute respiratory distress syndrome (ARDS), acute lung injury, and sterile chronic inflammation (ACI) associated with organ transplantation.

[0028] The present invention is based on the discovery that a unique subset of T cells expressing the CD40 protein (hence referred to as Th40 cells) may contribute to autoimmune inflammation. Furthermore, the involvement of Th40 cells in autoimmune processes may depend on the interaction between the CD40 protein expressed on the T cell surface and the CD154 protein. The interaction between the CD40 complex and CD154 transmits an activation signal between cells, subsequently activating Th40 cells. Such activation leads to the proliferation of Th40 cells and increased inflammation (e.g., an increase in the number of immune cells and immunoregulatory molecules present in the system). Therefore, inhibition of the CD40 / CD154 interaction may regulate Th40 cell activity and thereby affect inflammation. Therefore, the present invention relates to peptides and their administration that can affect the interaction between the CD40 protein and the CD154 protein, thereby modulating inflammation. Furthermore, the present invention relates to peptides that affect the interaction between the CD40 protein and the CD154 protein expressed on the surface of T cells, thereby affecting T cell activity, controlling inflammation, and ultimately preventing, modulating, and reducing diabetes and / or other autoimmune diseases. The subject matter of the present invention also encompasses the use of such peptides for detecting Th40 cells.

[0029] Unique T cell subsets have been shown to play a role in the development of autoimmune diseases. These cells are phenotypically characterized as CD4 CD40 (Waid, DM, et al., Eur. J. of Immunol., 34:1488, 2004; Vaitaitis, GM, et al., Cutting Edge, J. Immunol., 170:3455, 2003; Wagner, DH, Jr., et al., Proc. Nat'l. Acad. Sci. USA, 99:3782, 2002; Wagner, DH, Jr., et al., Int'l J. of Mol. Med. 4:231, 1999) and have been called Th40 cells (Waid, DM, et al. (2004) Eur. J. of Immunol. 34:1488; Vaitaitis, GM, et al., Cutting Edge, J. Immunol. 170:3455, 2003; Wagner, DH, Jr., et al., Proc. Nat'l Acad. Sci. USA 99:3782, 2002; Wagner, DH, Jr., et al., Int'l J. of Mol. Med. 4:231, 1999). Th40 cells can be any CD4+ T cells that co-express CD40 and CD4hi or CD4lo.CD40 expression is generally associated with antigen-presenting cells, and the majority of prior art describes CD40 as being expressed on B cells, macrophages, monocytes, and other cells; however, CD40 protein is also expressed on T cells (Waid, DM, et al., 2004. Eur. J. of Immunol., 34:1488, 2004; Vaitaitis, GM, et al., Cutting Edge, J. Immunol., 170:3455, 2003; Wagner, DH, Jr., et al., Proc. Nat'l Acad. Sci. USA, 99:3782, 2002; Wagner, DH, et al., Int'l. J. of Mol. Med., 4:231, 1999; Bourgeois, C., et al., Science, 297:2060, 2002; Fanslow, WC, et al., J. of Immun., 152:4262, 1994; Ramsdell, F., et al., J. of Immunol. 152:2190, 1994; Grabstein, KH, et al., J. of Immunol., 150:3141, 1993; Armitage, RJ, et al., Sem. in Immun., 5:401, 1993; Cooper, CJ, et al., J of Immunol., 173:6532, 2004).Th40 cells comprise a portion of the peripheral CD4+ compartment in naive, non-autoimmune mice (Waid, DM, et al., Eur. J. of Immunol., 34:1488, 2004; Wagner, DH, Jr., et al., Int'l J. of Mol. Med., 4:231, 1999) and humans (Waid, DM, et al., Clin. Immunol., 124:138, 2007), but are not abundant in autoimmune-prone mice (Waid, DM, et al., Eur. J. of Immunol., 34:1488, 2004; Wagner, DH, Jr., et al., Proc. Nat'l Acad. Sci. USA 99:3782, 2002; Wagner, DH, et al., Int'l J. of In mice (Waid, DM, et al., Eur. J. of Immunol. 34:1488, 2004; Waid, DM, et al., Clin. Immunol. 124:138, 2007), this proportion dramatically expands to 50% of the CD4+ compartment. These T cells do not express early activation markers and present as a naive phenotype in unchallenged mice.

[0030] In NOD (non-obese diabetic) mice, Th40 cells develop at exaggerated levels in the spleen, lymph nodes, and pancreas, even before the onset of diabetes (Waid, DM, et al., Eur. J. of Immunol. 34:1488, 2004; Wagner, DH, Jr., et al., Proc. Nat'l Acad. Sci. USA 99:3782, 2002). Increased numbers and percentages of these T cells are found in the peripheral blood of type 1 diabetic (T1D) patients compared with non-autoimmune controls and type 2 diabetic patients (Waid, DM, et al., Clin. Immunol., 124:138, 2007).

[0031] The observed increase in Th40 cells may indicate that these T cells are antigen-responsive or that CD40 expression is activation-induced. Furthermore, some diabetogenic T cell clones are CD40+ (Wagner, DH, Jr., et al., Proc. Nat'l Acad. Sci. USA 99:3782, 2002). Purified primary Th40 cells from NOD mice and prediabetic NOD (12-week-old) mice successfully transfer type 1 diabetes to NOD / Scid (non-obese diabetic / severe combined immunodeficiency) recipient mice, directly demonstrating the pathogenic role of the Th40 T cell subset (Waid, DM, et al., Eur. J. of Immunol. 34:1488, 2004; Wagner, DH, Jr., et al., 2002. Proc. Nat'l Acad. Sci. USA, 99:3782, 2002). It has been shown that Th40 cells infiltrate pancreatic islet β cells and disrupt insulin production, thereby suggesting islet antigen specificity (Waid, DM, et al., Eur. J. of Immunol. 34:1488, 2004; Wagner, DH, Jr., et al., Proc. Nat'l Acad. Sci. USA 99:3782, 2002). It has also been shown that Th40 cells are required for diabetes progression. Peripheral (spleen and draining lymph node) T cells depleted of CD40, followed by CD25 and Tregs, failed to induce diabetes in Scid (severe combined immunodeficiency) recipients. Depletion of Treg cells, but not autoaggressive CD40+ T cell subsets, prevented disease progression.

[0032] While Th40 cells are important in the development of autoimmunity, another important factor is the expression of the CD40 ligand, CD154. CD154 is transiently induced on activated T cells in response to CD3 / TCR stimulation (Lederman, S. et al., J. of Exp. Med., 175:1091, 1992). CD154 expression has also been confirmed on platelets, monocytes, basophils, eosinophils, dendritic cells, fibroblasts, smooth muscle cells, and endothelial cells (Russo, S. et al., J. Immunol. 171:5489, 2003; Stumpf, C., et al., Eur. J. Heart Fail., 5:629, 2003; Schonbeck, U., et al., Cell Mol. Life Sci. 58:4, 2001). CD154 is a member of the tumor necrosis factor (TNF) superfamily, and a soluble form of CD154 (sCD154) has been described (Russo, S., et al., J. Immunol. 171:5489 2003; Stumpf, C., et al., Eur. J. Heart Fail 5:629, 2003; Toubi, E., et al., Autoimmunity 37:457, 2004). Thus, sCD154 may act like a cytokine (Stumpf, C., et al., Eur. J. Heart Fail 5:629, 2003). Although CD154 has not been genetically linked in studies of T1D, sCD154 is significantly elevated in T1D and may play a role in the disease process (Varo, N. et al., Circulation 107:2664, 2003; Cipollone, F., et al., Diabetologia 48:1216, 2005; Devaraj, S., et al., Diabetes 55:774, 2006).The importance of CD40-CD154 interactions in autoimmunity has been established (Wagner, DH, Jr., et al., Proc. Nat'l Acad. Sci. USA 99:3782, 2002; Kobata, T., et al., Rev. Immunogenet. 2:74, 2000; Homann, D., et al., Immunity 16:403, 2002; Goodnow, CC, et al., Lancet 357:2115, 2001; Balasa, B., et al., J. of Immunol. 159:4620, 1997). Inhibition of CD40-CD154 interaction can prevent collagen-induced arthritis in the NOD mouse model (Durie, FH, et al., Science 281:1328, 1993), experimental autoimmune encephalitis (Howard, LM, et al., Autoimmunity 37:411, 2004), prostatitis (Grossman, ME, et al., J. Immunother. 24:237, 2001), and type 1 diabetes (Durie, FH et al., Science 281:1328, 1993; Balasa, B. et al., Journal of Immunology 159:4620, 1997; Howard, LM, et al., Autoimmunity 37:411, 2004; Grossman, ME et al., J. Immunother. 24:237, 2001). In a diabetic model, administration of CD154-blocking antibodies to 3-week-old NOD mice was necessary because blocking antibodies did not have a diabetes-preventing effect at 9 weeks of age (Balasa, B. et al., J. of Immunol. 159:4620, 1997).

[0033] Previous studies have shown that the Th40 cell subset induces transcription, translation, and nuclear translocation of RAG1 and RAG2 (recombination activating genes) upon CD40 engagement (Vaitaitis, GM et al., Cutting Edge, J. Immunol. 170:3455, 2003), whereas CD3 engagement does not induce RAG1 and RAG2 in T cells (Vaitaitis, GM, et al., Cutting Edge, J. Immunol. 170:3455, 2003). Following RAG1 / RAG2 induction, CD40-mediated T-cell receptor (TCR) revision occurs in peripheral T cells (Vaitaitis, GM et al., Cutting Edge, J. Immunol. 170:3455, 2003). CD40 induction of TCR revision is RAG-dependent. T cells isolated from TCR-Tg mice undergo TCR revision upon CD40 engagement, whereas T cells from TCR-Tg.RAG- / - mice do not undergo TCR revision upon CD40 engagement (Wagner, DH, Jr. et al., Int'l J. of Mol. Med. 4:231, 1999).

[0034] CD40 is a 50-kDa membrane protein of the tumor necrosis factor receptor (TNF-R) family. It is constitutively expressed as a homotrimer (Foy TM, et al., Ann. Rev. of Immunol., 14:591, 1996). In general, stimulation of all CD40-expressing cell types induces events that contribute to inflammation, such as the enhancement of costimulatory and adhesion molecules and the upregulation of proteolytic enzymes (Mach, F. et al., Atherosclerosis. 137 Suppl:S89-95, 1998).

[0035] The ligand for CD40, CD154, is a 39 kDa protein belonging to the tumor necrosis factor (TNF) family. CD40 forms a trimer that binds to CD154 at the interface of three monomers. CD154 is generally expressed on cells beyond surface-expressed CD154, as it also exists in a soluble, biologically active form (sCD154) that is shed from the cell surface after activation. The primary source of sCD154 is platelets (Foy TM, et al., Ann. Rev. of Immunol., 14:591, 1996).

[0036] Because NOD mice spontaneously develop T1D, genetically engineered mouse models such as NOD mice are utilized in research and development of autoimmune diseases, including diabetes. Previous studies in humans and mice have attempted to block the CD40 / CD154 interaction using monoclonal antibodies. While this approach proved effective in several mouse model studies and some human studies, the use of monoclonal antibodies targeting the CD40 / CD154 dyad was discontinued due to thromboembolic events that may be related to the function of CD154 in thrombus stabilization. CD154 stabilizes thrombi by interacting with integrin αIIbβ3, and inhibiting CD154 is speculated to reduce thrombus stability, resulting in the shedding of emboli that lead to thrombosis.

[0037] Multiple treatment options have been proposed to address and control chronic and acute inflammation. Many approaches use nonsteroidal anti-inflammatory drugs (NSAIDs), which attack the production of leukotrienes and prostaglandins, intracellular substances that cause local inflammation. Other treatments use more powerful immunosuppressants, such as cyclophosphamide, methotrexate, and azathioprine, to suppress the immune response and halt disease progression. Still other treatments involve the use of monoclonal antibodies (mAbs), which are designed to alter the immune response against self-tissues, as occurs during autoimmune diseases. However, all of these treatments invariably have potentially severe long-term side effects.

[0038] Current immunomodulatory therapies may rely on monoclonal antibody therapy, which can lead to complications. For example, antibodies administered to a subject may cross-react with unintended targets and cause serious renal complications, and antibodies specifically directed against CD154 may cause embolic complications. Furthermore, CD40-CD154 interaction may play an important role in antibody production, suggesting that administration of monoclonal antibodies may induce autoantibody production and further complications, inhibiting the recovery of normal immune function (see generally Banchereau, J. et al., Annu. Rev. of Immunol. 12:881, 1994).

[0039] Before the present invention is further described, it is to be understood that this invention is not limited to the precise embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting, since the scope of the present invention will be limited only by the claims.

[0040] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. As used herein, it should be further understood that the term "a" or "an" refers to one or more of that thing. For example, a nucleic acid molecule refers to one or more nucleic acid molecules. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably. Similarly, the terms "comprising," "including," and "having" can be used interchangeably.

[0041] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described herein. All publications mentioned herein are incorporated by reference to disclose and describe the methods and materials with which the publications are related. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which must be independently confirmed.

[0042] It will be appreciated that certain features of the present invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the present invention that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable combination. All combinations of embodiments are specifically embraced by the present invention and are disclosed herein as if each combination were individually and expressly disclosed. Furthermore, all subcombinations are also specifically embraced by the present invention and are disclosed herein as if each subcombination were individually and expressly disclosed.

[0043] It should further be noted that the claims are written to the exclusion of any optional elements. As such, this statement is intended to serve as a prelude to using exclusive terminology such as "solely," "only," and the like in connection with reciting claim elements, or to using a "negative" limitation.

[0044] Furthermore, as used herein, the term "animal" refers to a vertebrate, preferably a mammal. Mammals suitable for use with the methods of the present invention include, but are not limited to, livestock, sport animals, pets, primates, mice, rats, horses, dogs, cats, and humans. The term "animal" can be used interchangeably with the term "subject" or "patient." One embodiment of the present subject matter is a peptide that interacts with the CD40 protein in a manner that prevents diabetes and / or autoimmune disease. As used herein, the terms "interact" and "interaction" refer to two molecules being in sufficient physical proximity to cause modulation of inflammation. One such type of interaction is a binding interaction. In such an interaction, the peptide binds to CD40 to form a complex. An example of complex formation is the binding of an antigen with an antibody. According to the present subject matter, the binding of the peptide of the present invention to the CD40 protein can be reversible (e.g., a non-covalent interaction) or irreversible (e.g., a covalent interaction). Furthermore, reversible interactions can be strong or weak, with the strength of the interaction being measured by the force (e.g., ionic charge, hydrogen bonds, van der Wall interactions, etc.) that each protein exerts on the other proteins in the complex. Factors that influence the strength of the interaction between two molecules are known to those of skill in the art. One useful measure of the strength of binding between two molecules, such as a peptide and a protein, is the dissociation constant (Kd). Preferred peptides of the present invention have a dissociation constant of about 1×10 -6 M, about 1 x 10 -7 M, or approximately 1 x 10 -8 Particularly preferred peptides are those that bind to the CD40 protein with a Kd of no more than about 1 x 10 -9 In one embodiment, the peptides of the invention bind to CD40 protein with a Kd of less than 100 nM, less than 50 nM, less than 25 nM, less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, or less than 1 nM. Methods for measuring and analyzing binding interactions between peptides and CD40 protein are known to those skilled in the art.

[0045] As used herein, modulating inflammation means altering or affecting the interaction of CD40 and / or the CD40 complex, and / or modifying or affecting inflammation more generally. As used herein, the terms level, number, count, and concentration can be used interchangeably. Modulation of inflammation can refer to an increase or decrease in the number of Th40 cells present in an inflammatory environment. Consequently, modulation can be referred to as positive or negative. Positive modulation (also referred to as upregulation) of inflammation can result in an increase in the number of Th40 cells in an inflammatory environment. Negative modulation (also referred to as downregulation) of inflammation can result in a decrease in the number of Th40 cells present in an inflammatory environment. Preferred peptides can downregulate inflammation, thereby decreasing the number of Th40 cells present in an inflammatory environment. Positive and negative modulation of inflammation may or may not result in a change in the type and amount of immunoregulatory molecules present in the inflammatory environment.

[0046] It will be understood by those skilled in the art that both cell culture systems and animal immune systems contain basal levels of immune cells and immunoregulatory molecules. The terms basal level and normal level can be used interchangeably. As used herein, with respect to an animal's immune system, the basal level of a type of immune cell (e.g., Th40 cell) or immunoregulatory molecule refers to the average number of that cell type or immunoregulatory molecule present in a population considered healthy (i.e., a population free of metabolic, autoimmune, or infectious diseases). As used herein, with respect to a cell culture system, the basal level of a type of immune cell or immunoregulatory molecule refers to the average level of that cell type or immunoregulatory molecule present in a population of non-activated cells. One skilled in the art can determine whether T cells or a population of such cells are activated. For example, in some embodiments, expression of CD69, CD25, and / or CD154 proteins by cells indicates that the cells are activated.

[0047] A basal level of a cell or molecule can be a specific amount (e.g., a specific concentration) or can encompass a range of amounts. Basal levels or ranges of immune cells and immunoregulatory molecules are known to those skilled in the art. For example, in healthy individuals, the normal level of CD4+ T cells present in human blood is 500-1500 cells / ml. Variation in this measurement can be due to differences in the method used to measure cell count. Furthermore, normal levels of cells can be reported as a percentage of the total cell population. For example, in healthy individuals, Th40 cells may account for less than 25% of the total T cell population. Therefore, as used herein, the term "inflammation" can refer to an inflammatory environment in which Th40 cells account for about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, or about 80% or more of the total T cell population. Furthermore, preferred peptides herein are those that can reduce the level of Th40 cells to a level equal to less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 27%, or less than about 25% of the total T cell population. Methods for measuring different types of T cells in a T cell population are known to those skilled in the art. Furthermore, a novel method for detecting Th40 cells using the peptides of the present invention is disclosed herein.

[0048] As used herein, the term "inflammatory environment" refers to the overall population of immune cells and associated immunoregulatory molecules present in a cell culture or in an animal. Thus, the term "inflammatory environment" encompasses the types and / or relative amounts of immune cells and immunoregulatory molecules (e.g., cytokines) present in a cell culture or in an animal that are involved in influencing the inflammatory response. Examples of cells encompassed by the term "inflammatory environment" include, but are not limited to, T cells, neutrophils, macrophages, granulocytes, and the like. The inflammatory environment is associated with cells and molecules that mediate both acute and chronic inflammation. It will be understood by those skilled in the art that the inflammatory environment refers to the system to which the peptides of the present invention are administered. In one embodiment, the system is a cell culture system. In one embodiment, the system is an entire animal.

[0049] Preferred peptides of the present invention selectively interact with the CD40 protein in solution or on the surface of T cells, as measured using assays such as immunosorbent assays. As used herein, the terms selective, specific, and the like indicate that the peptide has a higher affinity for the CD40 protein than for proteins unrelated to the CD40 protein. More specifically, the terms selective, specific, and the like indicate that the affinity of the peptide for CD40 is statistically significantly higher than the affinity for a negative control (e.g., an unrelated protein such as albumin) measured using a standard assay (e.g., ELISA). Suitable techniques for assaying the ability of a peptide to selectively interact with the CD40 protein are known to those of skill in the art. Such assays can be in vitro or in vivo assays. Examples of useful assays include, but are not limited to, enzyme-linked immunoassays, competitive enzyme-linked immunoassays, radioimmunoassays, fluorescent immunoassays, chemiluminescent assays, lateral flow assays, flow-through assays, agglutination assays, microparticle-based assays (e.g., using microparticles such as, but not limited to, magnetic particles or plastic polymers (such as latex or polystyrene beads)), immunoprecipitation assays, immunoblot assays (e.g., Western blots), phosphorescence assays, flow-through assays, chromatography assays, polyacrylamide gel electrophoresis (PAGE)-based assays, surface plasmon resonance assays, spectrophotometry assays, microparticle-based assays, electronic sensor assays, and flow cytometry assays. Methods for performing such assays are well known to those skilled in the art. In one embodiment, the assays can be performed using cells in culture or in whole animals. The assays can be designed to provide qualitative, quantitative, or semi-quantitative results, depending on how they are used and the type of results desired.

[0050] One embodiment of the present invention is a peptide that interacts with the CD40 protein in a manner that affects the interaction between the CD40 protein and the CD154 protein. The effect of the peptide on the CD40 / CD154 interaction can be positive or negative. For example, the peptide can interact with the CD40 protein in a manner that increases the strength of the interaction between the CD40 protein and the CD154 protein. Alternatively, the peptide can interact with the CD40 protein in a manner that decreases the strength of the interaction between the CD40 protein and the CD154 protein. Methods for measuring the strength of binding between a peptide and the CD40 protein are known to those of skill in the art. Preferred peptides of the present invention are those that decrease the strength of the interaction between the CD40 protein and the CD154 protein. Preferred peptides of the present invention can decrease the strength of binding between the CD40 protein and the CD154 protein by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. Particularly preferred peptides are those that completely inhibit the binding of CD40 to CD154. Complete inhibition of the binding of CD40 to CD154 means that when a peptide of the present invention is brought into close proximity with CD40 and CD154 proteins under conditions that normally allow the interaction of CD40 and CD154, such interaction does not occur and activation signals are not stimulated in CD40-expressing cells. As a result, CD40 / CD154-mediated regulation of inflammation does not occur. In one embodiment, the peptide interacts with the CD40 protein in a manner that reduces the level of inflammation in the system. In one embodiment, the peptide interacts with the CD40 protein in a manner that suppresses the onset of inflammation in the system.

[0051] Although the peptides of the present invention can interact with any site on the CD40 protein, preferred peptides interact with the CD40 protein at a position overlapping the CD154-binding site. In one embodiment, the peptides of the present invention interact with the CD40 protein at the CD154-binding site. An example of such a peptide is a CD40 ligand competitive antagonist. As used herein, peptides that interfere with or inhibit the binding of the CD154 protein to the CD40 protein are referred to as small interfering peptides (SIPs). As used herein, small interfering peptides are peptides that, through physiochemical properties, interfere with the interaction of the CD40 protein with the CD154 protein, thereby preventing activation signals from being delivered to CD40-bearing cells, thereby limiting the activation and inflammation of CD40-bearing cells. As demonstrated herein, the result of such interference is the prevention of T cell activation and dissemination, and the prevention or reduction of inflammation. As demonstrated herein, in some examples, the results of such inhibition or prevention of the interaction between CD40 and CD154 can include observable data demonstrating the prevention, modulation, and / or reduction of diabetes and related disease characteristics.

[0052] As used herein, small interfering peptides, through their physiochemical properties, interfere with the interaction between CD40 and CD154 proteins, thereby preventing activation signals from being delivered to CD40-bearing cells, thereby limiting the activation of CD40-bearing cells and thereby regulating, suppressing, and preventing diabetes and / or autoimmune disease. As demonstrated herein, the result of such interference is the prevention of T cell activation and propagation, and the prevention, reduction, or regulation of diabetes-related progression.

[0053] The peptides of the present invention may also be modified, conjugated, and / or administered to include phosphorus, sulfur, manganese, magnesium, calcium, halogens, metals, and the like.

[0054] Amino acid mimetics may be used to produce polypeptides, and thus, polypeptides of the disclosure may include amino acid mimetics with enhanced properties, such as resistance to degradation.

[0055] In the methods disclosed herein, the bioactive peptide may be administered intramuscularly (IM), intravenously (IV), subcutaneously (SC), orally, via gavage, ointment / skin, transdermal patch, and / or intranasally.

[0056] The peptides and methods of the present invention may also involve administering prodrugs that are metabolized to the active forms of these peptides. As used herein, "prodrug" refers to a compound that a living system metabolizes into an active compound as a result of natural chemical reactions, enzyme-catalyzed reactions, and / or metabolic chemical reactions, or a combination of each. Standard prodrugs are formed using groups that are attached to functional groups, such as HO-, HS-, HOOC-, and RN-, that are cleaved in vivo on the drug-linked group. Standard prodrugs include, but are not limited to, carboxylic acid esters where the group is alkyl, aryl, aralkyl, acyloxyalkyl, or alkoxycarbonyloxyalkyl, as well as hydroxides, thiol, and amine esters where the attached group is acyl, alkoxycarbonyl, aminocarbonyl, phosphate, or sulfate. The exemplified groups are exemplary and not exhaustive; one of ordinary skill in the art can generate other known types of prodrugs. Prodrugs must undergo some form of chemical transformation to produce a compound that is biologically active or a precursor to a biologically active compound. In some cases, prodrugs are biologically active, usually less so than the drug itself, and serve to enhance the efficacy or safety of the drug through improvements in bioavailability, pharmacodynamic half-life, etc.

[0057] Methods herein can include treating a subject who has, is suspected of developing, or is at high risk of developing diabetes to reduce or prevent complications and problems associated with the development of diabetes and / or autoimmune disease. In these methods, the subject's blood glucose level can be reduced to less than 160 mg / dl, less than 150 mg / dl, less than 140 mg / dl, less than 130 mg / dl, less than 120 mg / dl, or less than 110 mg / dl.

[0058] These methods are not limited to use solely with type 1 diabetes, as they may be used to treat hyperglycemia resulting from any condition.

[0059] When administered or co-administered, the compounds of the present invention are provided to a subject in a therapeutically effective amount. As used herein, the term "therapeutically effective amount" refers to an amount that has any beneficial effect in treating a condition, disease, or symptom, or in preventing or alleviating the onset of a disease, condition, syndrome, or disorder. Determining the therapeutically effective amount of a bioactive peptide of the present invention to be administered is a matter of optimization and titration within the skill of the art and may vary depending on characteristics such as, but not limited to, the degree or extent of progression of the disease, condition, syndrome, or disorder to be treated, the age and condition of the subject, the subject's weight and body surface area, and other such identifiable traits that may be appropriate and relevant to the treatment of the subject. The present invention provides novel bioactive peptides, their variants, homologs, orthologs, and / or derivatives related to the peptides disclosed herein. The sequences of some of these peptides are shown in Table 1 below.

[0060] [Table 1]

[0061] In one aspect, the invention features a peptide six amino acids in length, wherein the peptide comprises the amino acid sequence of Formula I: X 1 -X 2 -X3 -X 4 -X 5 -X 6 where: X 1 is K, E, P, or Q, or a natural or non-naturally occurring small amino acid; X 2 is K or a natural or non-naturally occurring small amino acid; X 3 is G or a natural or non-naturally occurring small amino acid; X 4 is Y; X 5 is Y; X 6 is T.

[0062] In some embodiments, the amino acid sequence of the peptide differs by at least one, or at least two, or at least three amino acids compared to the amino acid sequence set forth in SEQ ID NOs:7-10.

[0063] In another aspect, the invention features a peptide 15 amino acids in length, wherein the peptide comprises the amino acid sequence of Formula II: X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -X 12 -X 13 -X14-X15 where: X 1 is V; X 2 is L; X 3 is Q or R; X 4 is W; X 5 is A; X6 is K, E, P, or Q; X 7 is K; X 8 is G; X 9 is Y; X 10 is Y; X 11 is T. X 12 is M or I; X 13 is K or S; X 14 is S or N; X 15 is N.

[0064] In some embodiments, the amino acid sequence of the peptide differs by at least one, or at least two, or at least three amino acids compared to the amino acid sequence set forth in SEQ ID NOs:11-15.

[0065] The present invention provides novel biologically active peptides, their variants, homologs, orthologs, and / or derivatives related to the peptides as disclosed herein. The sequences of such peptides are shown in Table 2 below.

[0066] [Table 2]

[0067] In some embodiments, the peptides disclosed herein may be peptides that bind to CD40 and / or the CD40 complex. Additionally, the peptides of the present invention may be short interfering peptides that can disrupt, affect, or alter the interaction between CD40 and CD154.

[0068] In some embodiments, the peptide is isolated from a protein that is recombinantly produced in a cell. The cells used for recombinant production can be prokaryotic or eukaryotic. In some embodiments, the peptide is chemically synthesized. A preferred embodiment is a chemically synthesized peptide.

[0069] In some embodiments, the peptide is phosphorylated at a serine, threonine, or tyrosine residue.

[0070] In some embodiments, the peptide is modified at its amino terminus. Examples of amino terminus modifications include N-glycosylation, N-alkylation, N-acetylation, or N-acylation of an amino acid. In some embodiments, the peptide comprises a C-terminal amidated amino acid. In other embodiments, the peptide does not comprise an amidated amino acid at its carboxy terminus. In some embodiments of the peptide, the C-terminus may comprise a protecting group.

[0071] In some embodiments, the peptide is pegylated.

[0072] In another embodiment, the invention includes a pharmaceutical composition comprising any one of the peptides of SEQ ID Nos. 7-15 and a pharmaceutically acceptable carrier.

[0073] In another embodiment, the invention includes a pharmaceutical composition comprising any one of the peptides of SEQ ID Nos. 21-25; SEQ ID Nos: 41-48; and SEQ ID Nos: 50-52 in a pharmaceutically acceptable carrier.

[0074] In some embodiments, the peptide is less than 225, 200, 175, 150, 125, 100, 75, 50, 30, 25, 20, 15, 10, 9, 8, 7, 6, or 5 amino acids in length.

[0075] The peptides of the present invention may be modified to include one or more modifications. Thus, in some embodiments, the C-terminus is amidated. In other embodiments, the N-terminus is acetylated. In other embodiments, both modifications are present. In still other embodiments, neither modification is present.

[0076] The present invention may also relate to the treatment of human, canine, feline and equine subjects for a variety of autoimmune diseases, including, but not limited to, diabetes, both insulin-deficient and insulin-resistant, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, chronic obstructive pulmonary disease, and / or acute respiratory distress syndrome.

[0077] The present invention is based on the finding that the interaction between CD40 ligand (CD154 protein) and CD40 protein expressed on T cells (Th40 cells) is important in the development of autoimmune diseases and type 1 diabetes.

[0078] The present invention may be based on the elucidation of key residues in CD40 and CD154 that are important for this interaction. The present invention relates to influencing, modulating, and / or modifying the interaction between CD40 and CD154 proteins through the use of small peptides that interact with the CD40 protein at the site where the CD154 protein would normally bind. In some aspects, the present invention may also relate to the use of such peptides to reduce the levels of Th40 cells, thereby reducing the severity of disease.

[0079] One embodiment of the present invention is a method for preventing diabetes in a subject, comprising contacting a CD40 protein with a peptide that interacts with the CD40 protein. Preferred peptides are those that are less than 25 amino acids in length and that bind to the CD40 protein, thereby inhibiting its interaction with the CD154 protein.

[0080] One embodiment of the present invention is a method for preventing autoimmune disease in a subject, comprising contacting a CD40 protein with a peptide that interacts with the CD40 protein. Preferred peptides are those that are less than 25 amino acids in length and that bind to the CD40 protein, thereby inhibiting its interaction with the CD154 protein.

[0081] One embodiment of the present invention is a method of preventing, reducing, ameliorating, and / or modulating diabetes in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of a peptide that reduces, modulates, and / or affects the subject's blood glucose levels.

[0082] One embodiment of the present invention is a method for preventing, reducing, ameliorating, and / or modulating diabetes in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of a peptide that reduces, modulates, and / or affects the subject's serum fructosamine levels.

[0083] One embodiment of the present invention is a method for preventing, reducing, ameliorating, and / or modulating diabetes in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of a peptide that reduces, modulates, and / or affects c-peptide levels in the subject.

[0084] Yet another embodiment of the present invention is a method for preventing, modulating, and / or reducing diabetes, the method comprising inhibiting the interaction of CD40 protein with CD154 protein with a peptide that interacts with the CD40 protein. Preferred peptides interact with the CD40 protein at the CD154 binding site. Preferably, such peptides are less than 25 amino acids in length. Further preferred peptides are amino acid sequences selected from SEQ ID NOs: 7-15, 21-25, 41-48, and 50-52.

[0085] Yet another embodiment of the present invention is a method for preventing, modulating, and / or reducing autoimmune disease, the method comprising inhibiting the interaction of CD40 protein with CD154 protein with a peptide that interacts with the CD40 protein. Preferred peptides interact with the CD40 protein at the CD154 binding site. Preferably, such peptides are less than 25 amino acids in length. Further preferred peptides are amino acid sequences selected from SEQ ID NOs: 7-15, 21-25, and 41-48.

[0086] One embodiment of the present invention is a method for preventing, modulating, and / or reducing diabetes, comprising administering to a subject in need thereof a therapeutically effective amount of a peptide that affects the interaction of CD40 with CD154 / CD40-ligand. One aspect of the invention may be that the peptide binds to CD40. In this embodiment, the peptide may bind to the CD40 protein with a Kd of less than 10. Furthermore, in this embodiment, the peptide may affect the interaction between CD40 and CD154. Furthermore, a preferred embodiment may inhibit the binding of CD40 to CD154. Furthermore, in this embodiment, the peptide binds to CD40 at the site where CD40 interacts with CD154. In this embodiment, the peptide may affect the interaction between CD40 and CD154 in a manner that prevents the expansion of Th40 cells. In this embodiment, the peptide may affect the interaction between CD40 and CD154 in a manner that reduces the number of Th40 cells. In this embodiment, the peptide may affect the interaction of CD40 with CD154 in such a way as to alter the cytokine expression profile of a cell population treated with said peptide.

[0087] One embodiment of the present invention is a method for preventing, modulating, and / or reducing autoimmune disease, comprising administering to a subject in need thereof a therapeutically effective amount of a peptide that affects the interaction of CD40 with CD154 / CD40-ligand. One aspect of the invention may be that the peptide binds to CD40. In this embodiment, the peptide may bind to the CD40 protein with a Kd of less than 10. Furthermore, in this embodiment, the peptide may affect the interaction between CD40 and CD154. Furthermore, a preferred embodiment may inhibit the binding of CD40 to CD154. Furthermore, in this embodiment, the peptide binds to CD40 at the site where CD40 interacts with CD154. In this embodiment, the peptide may affect the interaction between CD40 and CD154 in a manner that prevents the expansion of Th40 cells. In this embodiment, the peptide affects the interaction between CD40 and CD154 in a manner that reduces the number of Th40 cells. In this embodiment, the peptide may affect the interaction of CD40 with CD154 in such a way as to alter the cytokine expression profile of a cell population treated with said peptide.

[0088] One embodiment of the present invention is a method for modulating and / or alleviating diabetes in an animal, the method comprising administering to the animal a peptide that interacts with the CD40 protein in a manner that modulates IFNγ (interferon gamma). Preferred peptides interact with the CD40 protein at the CD154 binding site, thereby modulating IFNγ. Preferred peptides can modulate inflammation by reducing the level of Th40 cells to 25% or less of the total T cell population. Such methods can be used more generally to prevent and / or alleviate diabetes and symptoms that may accompany diabetes. In this embodiment, the peptide can be selected from SEQ ID NOs: 7-15, 21-25, 41-48, and 50-52.

[0089] One embodiment of the present invention is a method for identifying a subject at risk of developing diabetes and / or an autoimmune disease, the method comprising obtaining a sample containing T-cells from the subject to be tested, contacting the sample with a peptide that binds to CD40 protein, detecting the CD40-binding peptide, and determining the level of Th40 cells from the amount of CD40 binding, wherein a level of Th40 cells greater than 25% of the total T-cell population indicates that the subject is at risk of developing diabetes and / or an autoimmune disease.

[0090] Another aspect of the present invention is a method of administering a bioactive peptide of the present invention to prevent, regulate, and / or alleviate diabetes and / or autoimmune diseases, comprising selecting one or more of the peptides of SEQ ID NOs. 7-15, 21-25, 41-48, and 50-52; selecting a delivery method selected from the group including intramuscular (IM) delivery, intravenous (IV) delivery, subcutaneous (SC) delivery, oral delivery, gavage delivery, emollient / dermal delivery, or transdermal patch; and delivering a therapeutically effective amount of the peptide using one of the above delivery methods.

[0091] Another aspect of the present invention is a method of administering a bioactive peptide of the present invention to prevent, regulate, and / or alleviate diabetes and / or autoimmune diseases, comprising selecting one or more of the peptides of SEQ ID NOs.: 7-15; selecting from the group including intramuscular (IM) delivery, intravenous (IV) delivery, subcutaneous (SC) delivery, oral delivery, gavage delivery, emollient / dermal delivery, or transdermal patch; and delivering a therapeutically effective amount of the peptide using one of the above delivery methods.

[0092] Another embodiment of the present invention is a method of administering a CD40 peptide to prevent, modulate, and / or alleviate diabetes and / or autoimmune disease in an animal, comprising selecting a peptide that interacts with the CD40 protein and the CD154 binding site, and using a wide range of delivery methods selected from the group including implantable devices, hydrophilic polymer formulations, permeable polymer membranes, injectable gel implants, solvent extraction systems, phase inversion systems, temperature-sensitive gels, pH-dependent in situ gels, microparticles, microspheres, nanoparticles, nanospheres, biodegradable implants, or photoactivated depots.

[0093] Peptides useful in practicing the methods of the present invention should be of a size sufficient to interact with the CD40 protein / CD40 complex in a manner that regulates diabetes. Those skilled in the art will understand that preferred peptides are relatively short because they may be easier and less expensive to produce. Preferred peptides may be less than 20 amino acids in length. Preferred peptides may be 4, 6, 8, 10, 13, and 15 amino acids in length.

[0094] Interaction between the CD40 and CD154 proteins has been shown to occur at specific regions within each protein. The inventors have now surprisingly discovered that peptides comprising only a short portion of the CD154 region that interacts with CD40 can bind to, affect, and interact with the CD40 protein, thereby modulating diabetes. Accordingly, one embodiment of the present invention is a peptide comprising at least a portion of the amino acid sequence of the CD154 protein, such that it interacts with the CD40 protein in a manner that modulates diabetes. In one embodiment, interaction of the peptide with the CD40 protein results in negative regulation of diabetes. In one aspect, the peptide comprises at least a portion or fragment of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5.

[0095] In one aspect, the peptide is as short as possible and contains enough CD154 protein to allow interaction with CD40 protein in a diabetes-modulating manner. In one embodiment, a peptide of the invention comprises 6, 13, or 15 contiguous amino acids from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5 and interacts with CD40 in a diabetes-modulating manner. In one embodiment, a peptide of the invention comprises 6, 13, or 15 contiguous amino acids from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5 and interacts with CD40 in a diabetes-modulating manner. Preferred peptides contain a core sequence of lysine-glycine-tyrosine-tyrosine (KGYY; SEQ ID NOs:6 and 20), corresponding to amino acids 142-145 of SEQ ID NO:1, amino acids 143-146 of SEQ ID NO:2, amino acids 142-145 of SEQ ID NO:3, amino acids 142-145 of SEQ ID NO:4, and amino acids 143-146 of SEQ ID NO:5. Useful peptides can contain additional regions of sequence from SEQ ID NOs:1, 2, 3, 4, and 5 flanking the core sequence, so long as the peptide is capable of modulating diabetes and / or autoimmune disease. In one embodiment of the present invention, the peptide comprises at least one sequence selected from SEQ ID NOs: 7-15, 21-25, 41-48 and 50-52, so long as the peptide interacts with the CD40 protein in a manner that modulates diabetes.

[0096] The peptides of the present invention may be entirely selected from sequences responsible for interaction with the CD40 protein, or may be selected from sequences responsible for interaction with the CD40 protein, but may also contain additional amino acid sequences that do not interact with the CD40 protein but have other useful functions. Any useful additional amino acid sequence can be added to the CD40-interacting sequence, as long as the additional sequence does not undesirably affect the ability of the CD40-interacting sequence to interact with the CD40 protein. For example, in addition to the amino acid sequence responsible for interaction with the CD40 protein, the peptides of the present invention may contain an amino acid sequence useful for visualizing or purifying the peptide. Such a sequence acts as a label (e.g., an enzyme) or a tag (antibody binding site). Examples of such labels and tags include, but are not limited to, B-galactosidase, luciferase, glutathione-transferase, thioredoxin, HIS-tag, biotin tag, and fluorescent tag. Other useful sequences for labeling and tagging proteins are known to those skilled in the art.

[0097] Similarly, the peptides of the present invention may be conjugated or fused to another peptide, polypeptide, or nanoparticle, so long as such modifications do not affect the ability of the peptide to modulate diabetes or autoimmune disease.

[0098] Similarly, the peptides of the present invention can be modified as long as such modifications do not significantly affect the peptide's ability to regulate diabetes, autoimmune diseases, or the peptide's function of regulating and influencing blood glucose levels, serum fructosamine levels, and / or c-peptide levels.Such modifications can be made, for example, to improve protein stability, solubility, or absorption.Examples of such modifications include, but are not limited to, pegylation, glycosylation, acetylation, amidation, N-terminal protection, C-terminal protection, and chemical modification of peptides.

[0099] N-terminal modifications can include those provided herein, so long as such modifications do not significantly affect the functionality of the peptide in performing its desired role and providing its intended therapeutic effect. Such N-terminal modifications can include, but are not limited to, 5-FAM, 5-FAM-ahx, Abz, acetylation, acryl, Alloc, benzoyl, biotin, biotin-Ahx, BOC, Br-Ac-, BSA, CBZ, dansyl, dansyl-Ahx, decanoic acid, DPTA, fatty acid, FITC, FITC-Ahx, Fmoc, formylation, hexanoic acid, HYNIC, KLH (-NH at the N-terminus), lauric acid, lipoic acid, maleimide, MCA, myristoyl, octanoic acid, OVA (-NH at the N-terminus), palmitoyl, PEN, stearic acid, succinylation, and / or TMR.

[0100] C-terminal modifications may include those provided herein, so long as such modifications do not significantly affect the functionality of the peptide in performing its desired role and providing its intended therapeutic effect. Such C-terminal modifications may include, but are not limited to, the following: AFC, AMC, amidation, BSA (-COOH at the C-terminus), Bzl, cysteamide, ester (Oet), ester (OMe), ester (OtBu), ester (OTBzl), KLH (-COOH at the C-terminus), MAPS asymmetric 2-, 4-, or 8-branched, Me, NHEt, NHisopen, NHMe, OSU, OVA (-COOH at the C-terminus), p-nitroanilide, and / or tBu.

[0101] The peptides of the present invention may be derivatives or fragments of those occurring in nature (e.g., obtained from plants, animals, or microorganisms), or may be produced in a laboratory (e.g., recombinantly or synthetically). Preferred peptides are synthetic. Also encompassed are peptides that are combinations of natural and synthetic molecules. General methods for producing and isolating recombinant or synthetic peptides are known to those skilled in the art. It should be noted that, as used herein, an isolated or biologically pure molecule is one that has been removed from its natural environment. Therefore, terms such as isolated and biologically pure do not necessarily reflect the extent to which the protein has been purified.

[0102] As described herein, the interaction between the CD40 protein and the CD154 protein may be necessary for the involvement of Th40 cells in diabetes and autoimmune diseases. Therefore, inhibiting the interaction between the CD40 protein and the CD154 protein and / or the CD40 complex using the peptides of the present invention is a useful method for affecting diabetes and autoimmune diseases. Accordingly, one embodiment is a method for reducing the interaction between the CD40 protein and the CD154 protein, comprising introducing a peptide that interacts with the CD40 protein into an environment containing the CD40 protein and the CD154 protein, such that the interaction between the CD40 protein and the CD154 protein is reduced. In one aspect of the present invention, the peptide reduces the interaction between the CD40 protein and the CD154 protein by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In one embodiment, the peptide reduces the interaction between CD40 and CD154 proteins by a factor of at least 10, at least 100, at least 1,000, or at least 10,000. Methods for measuring the strength of the interaction between CD40 and CD154 proteins have been previously discussed and are known to those skilled in the art.

[0103] One embodiment of the present invention is a method for modulating diabetes, comprising contacting a CD40 protein with a peptide that interacts with the CD40 protein in a manner that modulates inflammation. In one aspect, interaction of the CD40 protein with the peptide can increase or decrease the number of Th40 cells by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In one embodiment, interaction of the CD40 protein with the peptide can increase or decrease the number of Th40 cells by a factor of at least 10, at least 100, at least 1,000, or at least 10,000.

[0104] One aspect is a method of preventing, alleviating, or modulating diabetes in a subject, the method comprising administering to the patient a peptide of the invention. In one embodiment, the peptide comprises an amino acid sequence selected from SEQ ID NOs: 7-15, 21-25, 41-48, and 50-52. In one embodiment, the peptide is an amino acid sequence selected from SEQ ID NOs: 7-15. In preferred embodiments, interaction of the CD40 protein with the peptide can reduce the number of Th40 cells by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In another embodiment, interaction of the CD40 protein with the peptide can reduce the number of Th40 cells by a factor of at least 10, at least 100, at least 1,000, or at least 10,000. In preferred embodiments, the level of Th40 cells may be reduced such that Th40 cells do not exceed about 20%, about 25%, about 30%, about 35%, or about 40% of the total T-cell population.

[0105] The peptides and methods of the present invention are suitable for use not only in treating patients but also in cell culture. As used herein, the term "patient" refers to any animal in need of such treatment. The animal may be a human or a non-human animal. Preferred animals for treatment are mammals. The peptides can be administered or applied per se or as pharmaceutical compositions. The peptides of the present invention or pharmaceutical compositions thereof can be administered to patients by various routes, including, but not limited to, injection (e.g., intravenous, intramuscular, subcutaneous, intrathecal, intraperitoneal), inhalation, oral administration (e.g., in tablets, capsules, powders, syrups, solutions, suspensions, films, dispersions, or emulsions), transdermal, transmucosal, pulmonary, buccal, intranasal, sublingual, intracerebral, rectal, or topical administration, or any other convenient method known to those skilled in the art.

[0106] The effective amount of the peptides of the present invention and / or pharmaceutical compositions thereof can be determined by standard clinical techniques known in the art. Such amounts will depend, among other factors, on the patient being treated (including, but not limited to, the patient's weight, age, and condition, the intended effect of the compound, the method of administration, and the judgment of the prescribing physician or veterinarian). Also, in this context, it should be noted that when treating a patient exhibiting a disorder of interest, a therapeutically effective amount of a drug or drugs such as these is administered. A therapeutically effective amount refers to that amount of a compound that results in an improvement in one or more symptoms or an extension of survival in a patient.

[0107] The peptides of the present invention or pharmaceutical compositions thereof can be administered alone or in combination with one or more other pharmaceutical compositions containing other compounds of the present disclosure. The peptides utilized in the methods may be administered to a subject in a pharmaceutically acceptable carrier, adjuvant, or excipient. Selected examples of pharmaceutically acceptable carriers, adjuvants, or excipients known in the art are disclosed in Remington: The Science and Practice of Pharmacy, 21st Ed., Hendrickson, R., et al., Eds., Lippincott Williams & Wilkins, Baltimore, Md. (2006). The choice of pharmaceutically acceptable carrier, adjuvant, or excipient will depend on various factors, including, but not limited to, the route of administration, dosage level, and the age, weight, and / or condition of the subject. The particular pharmaceutical composition will depend on the desired mode of administration, as is well known to those skilled in the art.

[0108] Because the present invention discloses that Th40 cells are closely involved in the development of autoimmune diseases and diabetes, the peptides and methods disclosed herein can be used to affect inflammation and other symptoms resulting from such diseases. Accordingly, one embodiment of the present invention is a method for treating diabetes in a patient in need of such treatment, comprising administering to the patient a peptide that interacts with the CD40 protein, thereby reducing diabetes. In one embodiment, the peptide interacts with the CD40 protein in a manner that affects the interaction between CD40 and CD154, thereby reducing diabetes. In a preferred embodiment, the interaction of the peptide with the CD40 protein reduces the number of Th40 cells in the patient to levels equivalent to those observed in subjects without diabetes. The present invention is suitable for treating any patient with autoimmune diseases and / or diabetes, the development of which is dependent on Th40 cells. More specifically, the peptides of the present invention are suitable for reducing the level of Th40 cells in such patients. In a preferred embodiment, the peptides of the present invention reduce the level of Th40 cells in patients with diabetes to approximately 25% or less of the total T cell population.

[0109] The inventors have also surprisingly found that the peptides of the present invention can reverse the disease process in individuals who already exhibit symptoms of diabetes. Accordingly, one aspect of the present subject matter is a method for reversing diabetes, comprising administering a peptide of the present invention to a patient diagnosed with diabetes. In one embodiment, the peptide comprises an amino acid sequence selected from SEQ ID NOs: 7-15, 21-25, 41-48, and 50-52, so long as it is capable of downregulating inflammation. As used herein, reversing diabetes means reducing diabetic symptoms to levels comparable to observably low levels of symptoms and measurements that may be more common in non-diabetic subjects. A subject may be required to continue therapy indefinitely to continue achieving the desired results from the therapy. In other instances, a subject may taper off the therapy, allowing the positive effects or changes induced by the therapy to continue. This may depend on many factors, including, but not limited to, the condition at the time of initial presentation, the progression of the condition, and other factors, as appropriate, as determined by a physician or veterinarian.

[0110] As described, the peptides of the present invention selectively interact with and / or affect CD40-expressing cells. Thus, the peptides of the present invention can be used to identify Th40 cells. Accordingly, one embodiment of the present invention is a method for detecting Th40-dependent diabetes, comprising contacting a T-cell population with a peptide of the present invention. In a preferred embodiment, the peptide is labeled with a detectable marker, such as luciferase or alkaline phosphatase. Such detection can be performed using assay techniques known to those skilled in the art. Generally, an assay for detecting Th40 cells using the peptides of the present invention comprises: (a) obtaining a cell sample; (b) contacting the cells with a peptide of the present invention under conditions suitable for allowing binding of the peptide to the Th40 cells, if present; (c) washing the cells under conditions that disrupt nonspecific interactions and remove unbound peptide; and (d) detecting the peptide bound to the cells. Detection of the bound peptide can be achieved directly or indirectly. For example, direct detection can be achieved using a peptide labeled with a detectable marker, as disclosed herein. Following the washing step, the cells are simply screened for the presence of the detectable marker. The presence of the detectable marker in the cell sample indicates the presence of Th40 cells, and therefore Th40-dependent diabetes. Alternatively, indirect detection involves the use of a second molecule, such as an antibody, that binds to the peptide. In an indirect detection assay, following the washing step, a detection molecule that binds to the peptide is added to the cell sample. This detection molecule is labeled with a detectable marker. After washing away unbound detection molecule, the cells are screened for the presence of the detectable marker. The presence of the detectable marker in the cell sample indicates the presence of Th40 cells. It should be understood that the assays described herein are intended as examples of useful assays, and other assay techniques can be employed.Suitable assay techniques are known to those skilled in the art and are also disclosed, for example, in Molecular Cloning: A Laboratory Manual, Sambrook, J., Fritsch, EF, and Maniatis, T, Cold Spring Harbor Laboratory Press; 2nd Edition (December 1989). All references cited herein are incorporated herein in their entirety.

[0111] The peptides utilized in the above detection methods and described can also include modifications to the peptide, such as stable isotope-labeled peptides or fluorescent peptide modifications / FRET pairs, allowing the peptides to be utilized in several detection methods and applications. For example, stable isotope-labeled peptides such as Arg (C6, N4), Ile (C6, N), Leu (C6, N), Lys (C6, N2), and / or Val (C5, N) can be utilized. Additionally, fluorescent peptide modification / FRET pairs such as 1-pyrenemethylamine HCl, 5-FAM (N-terminus), 5-FAM-Ahx (N-terminus), Abz (N-terminus), Abz / DNP, Abz / Tyr(3-NO), DABCYL, DABCYL / Glu(EDANS)-NH, Dansyl (N-terminus), Dansyl-Ahx (N-terminus), EDANS / DABCYL, FITC (N-terminus), FITC-Ahx (N-terminus), Glu(EDANS)-NH, MCA (N-terminus), MCA / DNP, quenched fluorescent peptides, Tyr(3-NO), TMR, and / or AMC may be utilized. Thus, the peptides of the present invention may be so modified, whether with stable isotope peptides or fluorescent peptide modifications / FRET pairs, so that the peptides of SEQ ID NOs:7-15, 21-25, 41-48, and 50-52, or others disclosed herein, can be used in assays or other useful procedures.

[0112] The above assay techniques can also be used to identify other molecules that affect the interaction between CD40 and CD154 proteins. Examples of such molecules include, but are not limited to, proteins, peptides, and small molecules. For example, an assay can be designed to test the ability of a molecule to compete with the peptides of the present invention for binding to Th40 cells. For example, a peptide labeled with a detectable marker can be mixed with the test molecule and a population of cells known to contain Th40 cells under conditions that allow binding of the peptide to Th40 cells. After a suitable incubation period, the cells are washed to remove unbound peptide, and the cells are screened for the presence of the detectable marker. Alternatively, the labeled peptide can be first bound to Th40 cells, and after a washing step to remove unbound peptide, the test molecule can be added to the cells containing the bound peptide. Following an incubation period and a washing step to remove unbound molecules or released peptide, the cells are screened for the presence of the detectable marker. In either case, the absence of a detectable marker in the cell sample indicates that the test molecule can compete with the peptide for binding to Th40 cells, and the presence of a detectable marker indicates that the test molecule does not inhibit the binding of the peptide to Th40 cells. Inhibition of binding need not be 100%, as such assays will also be useful for identifying molecules that partially inhibit the binding of peptides to Th40 cells. It will be understood by those skilled in the art that such assays involve the use of positive controls (e.g., unlabeled peptides) and negative controls (e.g., proteins / molecules known not to bind to Th40 cells).

[0113] Because increased levels of Th40 cells are associated with the development of autoimmune diseases, the present invention can be used to identify patients at risk of developing autoimmune diseases and autoimmune-related diabetes. Accordingly, one embodiment of the present invention is a method for identifying patients at risk of developing autoimmune-related diabetes. In one embodiment, patients at risk of developing diabetes are identified by obtaining a sample from the patient to be tested, contacting the T-cell portion of the sample with a peptide of the present invention, and measuring the level of Th40 cells present in the sample, wherein a level of Th40 cells greater than about 25% of the total population of T cells indicates that the patient is at risk of developing autoimmune diseases or diabetes. In one embodiment, the peptide comprises an amino acid sequence selected from SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, so long as it binds to CD40 protein. In one embodiment, the peptide is an amino acid sequence selected from SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15. In another embodiment, the amino acid sequence is selected from one of SEQ ID NOs:7-15, 21-25, 41-48, and 50-52. In one embodiment, the peptide is labeled with a suitable detectable marker, such as, for example, luciferase or alkaline phosphatase.

[0114] The present invention also includes kits useful for practicing the methods disclosed herein, the kits comprising a peptide that interacts with the CD40 protein in a manner that modulates diabetes. In one embodiment, the peptide comprises an amino acid sequence selected from SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, so long as the peptide is capable of downregulating diabetes. In one embodiment, the peptide is an amino acid sequence selected from SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15. In one embodiment, the peptide is an amino acid sequence selected from SEQ ID NOs:7-15, 21-25, 41-48, and 50-52. Another embodiment is a kit for measuring levels of Th40 cells, including peptides that interact with CD40 protein and methods for detecting CD40-binding peptides. The kit may also include associated reagents and components, such as, but not limited to, buffers, labels, containers, inserts, tubes, vials, syringes, etc. The kit may also include stable isotope-labeled amino acids or peptides with fluorescent peptide modifications / FRET pairs, as described elsewhere herein.

[0115] Another embodiment of the invention includes a method of modulating and / or reducing interleukin 17 (IL-17) in a cell or a subject, comprising administering a peptide selected from SEQ ID NOs: 7-15, 21-26, 41-48, and 50-52 in an amount sufficient to reduce or inhibit interleukin 17 (IL-17) signaling, where more generally, IL-17 signaling is associated with a condition selected from the group including type 1 diabetes, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, Crohn's disease, inflammatory bowel disease, chronic obstructive pulmonary disease, asthma, arteriosclerosis, vasculitis, hypertension, thyroiditis, primary biliary cirrhosis, Paget's disease, Addison's disease, acute respiratory distress syndrome, acute lung injury, and / or sterile chronic inflammation.

[0116] Another embodiment of the invention includes a method of modulating and / or reducing interleukin-17 (IL-17) in a cell or a subject, comprising administering a peptide selected from SEQ ID NOs: 7-15, 21-26, 41-48, and 50-52 in an amount sufficient to reduce or inhibit interleukin-17 (IL-17) signaling, wherein IL-17 is associated with diabetes. One aspect of the invention includes an embodiment in which the peptides of SEQ ID NOs: 7-15, 21-26, 41-48, and 50-52 can be provided as a sterile solution in phosphate buffered saline (PBS). In this embodiment, the pH is about 5.5.

[0117] The following examples are offered for illustrative purposes and are not intended to limit the scope of the present invention. [Example]

[0118] This example demonstrates the effect of various peptide fragments of CD154 on the CD4 / CD8 ratio and the development of diabetes in NOD mice.

[0119] Peptides were designed based on the amino acid sequence of mouse CD154 protein (SEQ ID NO:1) from the SwissPro database. Peptides (8-mer (SEQ ID NO:22; SEQ ID NO:23), 10-mer (SEQ ID NO:41), 13-mer (SEQ ID NO:42), 15-mer (SEQ ID NO:24), 24-mer (SEQ ID NO:26), scrambled (SEQ ID NO:40), and RGD (arginylglycerol-aspartic acid) were then purified by the New England The RGD peptide was ordered from Peptide. The RGD peptide is a 15-amino acid sequence derived from the CD154 sequence and does not contain a CD40-binding motif. The lyophilized peptide was suspended in sterile PBS at 1 mg / ml. Next, 25 μg (1 mg / kg) of the specific peptide was injected into the tail vein of 6-week-old NOD mice. Control mice received 100 μl of sterile PBS. This was before the onset of diabetes (and atherosclerosis), but after pancreatic islet damage had begun. After the initial injection, another 25 μg of peptide (100 μl of PBS for control mice) was injected into the tail vein once a week. At 10 weeks of age, mice were monitored for diabetes, indicated by blood glucose levels exceeding 250 mg / dL for three consecutive days. The results of this study are shown in Figure 1. During this time, blood was collected from the tail vein or by submandibular vein puncture, and CD4+ and CD8+ cell levels were measured by flow cytometry using antibodies against CD4 and CD8 proteins. The results of this analysis are shown in Figure 2A.

[0120] Pancreases were excised and examined histologically for cellular infiltration, and a score was assigned based on observable, measurable, and quantifiable data: 0 = no infiltration; 1 = unipolar infiltration; 2 = peri-insular inflammation, bipolar infiltration; 3 = 75% infiltration, and 4 = complete infiltration. The results of this analysis are shown in Figure 2B.

[0121] These results demonstrate that treatment with peptides unrelated to the CD154 protein did not reduce the onset of diabetes in NOD mice. In contrast, treatment of mice with a 15-mer peptide derived from the CD154 protein prevented the onset of diabetes. Furthermore, a 13-mer peptide derived from the CD154 protein had a significant effect on the onset of diabetes. Furthermore, these data demonstrate that the 15-mer peptide did not result in impaired immune system function, as measured by the CD4 / CD8 ratio. [Example]

[0122] This example demonstrates the effect of a 15-mer peptide on hyperglycemia in newly diabetic NOD mice.

[0123] Six mice that received the 6-mer peptide in Example 1 and subsequently developed diabetes were injected intravenously with 100 μg of the 15-mer peptide. These mice received weekly tail vein injections of the 15-mer peptide, and blood glucose levels were monitored twice weekly. After a total of 10 weeks of 15-mer peptide administration, treatment was discontinued. The results of this study are shown in Figure 3. This study demonstrates that injection of the 15-mer peptide into mice that already had diabetes can reverse hyperglycemia. It also demonstrates that hyperglycemia reverts within 7 weeks of discontinuing treatment. [Example]

[0124] This example demonstrates the ability of a 15-mer peptide to bind to Th40 cells and B cells.

[0125] Total lymphocytes were isolated from 9-week-old NOD mice. Lymphocytes were incubated with anti-CD4, anti-CD8, and FITC-labeled 15-mer peptides and analyzed by flow cytometry. Cells were gated for CD4 (including both CD4hi and CD4lo populations) and CD40 versus the 15-mer peptide. The results of this analysis are shown in Figure 4.

[0126] B cells were isolated from the spleens of NOD mice. Sorted MHC-II+ cells were purified from total lymphocytes. Cells were stained with FITC-labeled 15-mer peptide, anti-CD40, and B cell markers CD19 and CD21. MHC-II+ cells were gated for CD19+ and CD21+ and then quantified using 15-mer peptide versus CD40 antibody. The results of this study are shown in Figure 5.

[0127] This study shows that a substantial majority of CD40+ T cells, as much as 90%, bound the 15-mer peptide, thereby demonstrating that the 15-mer peptide is highly specific for CD40+ cells. It also shows that while 90% of B cells were CD40 positive, only 8% of B cells bound the 15-mer peptide. [Example]

[0128] This example demonstrates the levels of CD40-positive cells in the blood of type 1 diabetic and non-diabetic (control) subjects.

[0129] One milliliter of whole blood was obtained from each individual and incubated with the biotin-conjugated 15-mer peptide. The cells were then exposed to horseradish peroxidase (HRP) avidin, washed, and the absorbance at 405 nm was measured using a spectrophotometer. The results of the study are shown in Figure 6. This study demonstrates that blood cells from type 1 diabetic patients had higher 15-mer peptide binding activity than blood cells from non-diabetic controls. [Example]

[0130] This example demonstrates the level of insulin granulation observed in the pancreas of NOD mice treated with either a 15-mer peptide or a peptide from ovalbumin.

[0131] At the onset of diabetes, six NOD mice were injected with 100 μg / ml of a 15-mer peptide (SEQ ID NO:24), resulting in reversal of hyperglycemia in 80% of recipients. Six weeks after reversal of hyperglycemia, the mice were sacrificed and the pancreases were removed for analysis. The pancreases were fixed, sectioned, and stained using an aldehyde / fuchsin stain, which allows for the detection of insulin granules. Tissue granulation was scored as follows: 4 = completely granulated; 3 = 75% islet granulation; 2 = 50% islet granulation and peri-insular inflammation; 1 = 25% islet granulation; and 0 = no insulin granules detected. The results of this analysis are shown in Figure 7.

[0132] This analysis demonstrates that the 15-mer peptide preserved insulin granules in the majority of mice and was significantly improved in diabetic mice with the peptide reversal compared to diabetic mice receiving an irrelevant peptide. [Example]

[0133] This example demonstrates that mutations in the 15-mer peptide affect its ability to prevent the onset of diabetes. Figure 8 provides results related to this Example 6.

[0134] Peptides were designed and produced as described in Example 1. Variant peptides were produced such that, in each variant, the amino acids corresponding to amino acids at positions 1 to 9 of SEQ ID NO:24 were substituted with glycine as follows: Gly-1 GLQWAKKGYYTMKSN (SEQ ID NO:28) Gly-2 VGQWAKKGYYTMKSN (SEQ ID NO:29) Gly-3 VLGWAKKGYYTMKSN (SEQ ID NO:30) Gly-4 VLQGAKKGYYTMKSN (SEQ ID NO:31) Gly-5 VLQWGKKGYYTMKSN (SEQ ID NO:32) Gly-6 VLQWAGKGYYTMKSN (SEQ ID NO:33) Gly-7 VLQWAKGGYYTMKSN (SEQ ID NO:34) Gly-9 VLQWAKKGGYTMKSN (SEQ ID NO:35) Gly-10 VLQWAKKGYGTMKSN (SEQ ID NO:36) Gly-11 VLQWAKKGYYGMKSN (SEQ ID NO:37) Gly-12 VLQWAKKGYYTGKSN (SEQ ID NO:38)

[0135] NOD mice were placed in groups of 10 and each group received a weekly intravenous injection of 25 μg of wild-type (WT; Legend) peptide or the above-mentioned variant peptides (in PBS, pH 7.2). The onset of diabetes was monitored by weekly blood glucose monitoring. Mice were considered "diabetic" when their blood glucose levels were 250 mg / dL or higher on two consecutive measurements. Injections began at 6 weeks of age, indicating prediabetes.

[0136] This example demonstrates that substitutions with glycine at either positions 1-7 or 9-12 reduce the ability of a 15-mer peptide to inhibit the onset of diabetes, and also shows that such mutations do not completely abolish the ability of the mutated 15-mer peptide to inhibit the onset of diabetes. [Example]

[0137] This example demonstrates that the same increase in Th40 cell levels in the ApoE-deficient mouse model of atherosclerosis is also markedly elevated in human type 1 diabetes (T1D).

[0138] Peripheral blood was assayed for total CD3+CD4+CD40+ cell counts in NOD, NOR (non-obese diabetes resistant), and BALB / c (control) mice, as shown in Figure 9. This was compared to the percentage of Th40 cells in the peripheral blood of human subjects from control, diabetic / new onset, and long-term diabetic patient populations, as shown in Figure 10. [Example]

[0139] Canine blood was obtained from a known diabetic dog. Peripheral blood mononuclear cells were isolated. Cells were stained with CD3, CD4, CD40, and the peptide of SEQ ID NO: 46. The results are shown in Figure 11. [Example]

[0140] Five diabetic dogs, including four with new-onset diabetes and one with long-term diabetes (diagnosed for 3 years or more), were recruited for the study. These dogs were enrolled and studied at Colorado State University Veterinary Hospital (CSU-VH) in Fort Collins, Colorado. Each dog was evaluated by a board-certified veterinarian. Blood samples were collected from each dog and analyzed with a chemistry panel to determine general health, and a complete physical examination was performed. Each dog was confirmed to have diabetes, with blood glucose levels ranging from 300 to 600 mg / dL, but otherwise appeared to be in good general health. One dog was diagnosed with high blood pressure, two dogs had eye problems and were partially blind, and the long-term diabetic dog was completely blind due to diabetes.

[0141] The dogs were of various breeds and ranged in size from 8 kg to 27 kg. Four dogs were diagnosed within 3 months, and one dog was diagnosed 3.5 years ago. All dogs were treated with daily insulin. The administration route was SEQ ID NO: 13, administered intravenously over 30 minutes at 2 mg / kg on days 0, 4, and 8, followed by weekly infusions for a total of 8 weeks. Dogs were treated with canine-sequence ((SEQ ID NO: 13 - VLRWAPKGYYTISSN); acetate salt form; administered in sterile PBS for injection), and the injections were administered as a slow infusion over 30 minutes. All five dogs achieved favorable outcomes during treatment: reduction in blood and urinary glucose, elimination of ketonuria in all dogs, and elimination of urinary glucose in two dogs. A dog diagnosed 3.5 years ago showed an improvement in time-in-range (the percentage of 24-hour periods when blood glucose levels were between 80 and 160) from 10% to 80% for five consecutive days in the fifth week, achieving this goal over the final three study weeks. All five dogs showed a typical response, with blood glucose levels improving within three to five days of treatment. For example, blood glucose levels moved from 400 to 120, remained stable for two days, and then returned to elevated levels. In all treated dogs, glucose spikes were lower, with highs of 300 mg / dL compared with pretreatment levels of >500 mg / dL. In fact, no dog recorded blood glucose levels below 350 before treatment. During treatment, most dogs maintained blood glucose levels below 200 mg / dL, often below 150 mg / dL. There were no adverse effects on the dog's WBC and RBC profiles, and all chemistry panel values remained normal as before.

[0142] Pre-treatment blood samples were analyzed for blood glucose levels and peripheral blood Th40 cell percentages. These pre-treatment Th40 cell and blood glucose levels are shown in Figures 15, 16, and 17. In Figure 15, the control is a blood sample from the test beagle used by CSU-VH, all of which were confirmed to be non-diabetic (i.e., not diabetic). The control samples demonstrate that "normal" dogs have moderately low levels of Th40 cells (<25%), as reported for mice and humans. On day 0, before the first treatment, blood was collected from a leg vein and analyzed for the percentage of Th40 cells. Before treatment, all diabetic dogs demonstrated peripheral blood Th40 cells of 50% or greater, as shown in Figure 15. Samples were analyzed for Th40 cells by flow cytometry. Separated blood was further processed to obtain lymphocytes. Lymphocytes were stained with antibodies recognizing CD4 and CD40, which are by definition Th40 cells. The percentage of CD4+CD40+ cells as a subset of total CD4+ cells was measured and graphed. By day 6, after the third treatment, all dogs showed a decrease in Th40 cell levels, and by day 16 (the fifth treatment), all dogs had Th40 cell percentages within the normal range. Dogs were treated for 28 days. On day 35, one week after the last treatment, Th40 cell levels remained within the normal range. Treatment did not result in ablation; it merely reduced the number of Th40 cells. Three of the five dogs were retested two months later, and although Th40 cell levels were still reduced, they had begun to return to higher levels. None of the dogs experienced any side effects. Blood chemistry panels showed normal levels for most parameters, each an improvement from the pretreatment blood samples. Two example blood panels and results are provided in Figures 19 and 20.

[0143] Blood glucose measurements were performed on the diabetic dogs before, during, and after peptide treatment. Data were collected via blood analysis and continuous glucose monitoring. Continuous glucose monitoring measurements were obtained periodically, and the displayed measurements are daily averages obtained over a two-week period. Both male dog, subject 5, and female dog, subject 3, exhibited consistently high glucose readings prior to treatment. See Figure 16 for the results of male dog, subject 5. See Figure 17 for the results of female dog, subject 3. Blood samples were taken and glucose monitoring data was recorded during the 28-day treatment period. The results are plotted and shown in Figure 15. This graph shows daily averages from day 6 through day 28, the final day of treatment. Blood glucose levels were monitored for up to two months after treatment.

[0144] Figure 12 is a graph of blood glucose levels before and after treatment for four of the five dogs. Longer treatment may result in further glucose control. A multi-column t-test showed that each dog experienced a significant reduction in blood glucose, p=0.0042. Overall, all treatments significantly reduced blood glucose, p=0.0040.

[0145] Figure 13 provides a graph of glycosylated fructosamine serum level values before and after treatment for canine subjects. Glycosylated fructosamine is an indicator of systemic inflammation. In humans with T1D, A1C is measured. The data for the canine subjects in Figure 13 were treated as described above. A peripheral blood chemistry panel was performed and collected before peptide administration. Fructosamine levels were included. Systemic inflammation increases fructosamine levels. After 8 weeks of treatment, fructosamine levels were measured again and compared to the original values. Open squares represent pre-treatment values, and closed circles represent corresponding post-treatment values. Fructosamine levels decreased in each dog.

[0146] Figures 14a, 14b, 14c, 14d, and 14e provide graphs of c-peptide measurements during treatment for canine subjects 1 through 5. C-peptide is an indicator of potential beta cell recovery. As beta cells in pancreatic islets produce insulin, c-peptide levels increase. Plasma samples were collected before and after treatment. The plasma was analyzed for c-peptide using a commercially available test kit. The ELISA (enzyme-linked immunosorbent assay) method showed that c-peptide levels increased after treatment in both cases. This indicates that treatment affected and restored normal beta cell activity and the appropriate production of insulin. Figures 14a, 14b, 14c, 14d, and 14e provide the results of these c-peptide measurement studies. [Example]

[0147] The proportion of Th40 cells in peripheral blood was studied in mice, humans, and canine subjects. The results are shown in Figure 18. Thus, Figure 18 provides a graph of control and diabetic mouse, human, and canine subjects. In this study, peripheral blood from nondiabetic dogs (n=12) was compared with diabetic dogs (n=9). All dogs were from the Colorado State University College of Veterinary Medicine Teaching Hospital, where they were examined and examined in the clinic. Th40 measurements were the same for dogs, mice, and humans: CD4+CD3+CD40+ with confirmed TCR expression. [Example]

[0148] This example, performed in mice, demonstrates that KGYY-15 (SEQ ID NO: 11) can be utilized to significantly reduce and / or reverse hyperglycemia in diabetic NOD mice. In this study, NOD mice with blood glucose levels of 250-400 mg / dL were treated with KGYY-15. Control mice were treated with MR1 (an anti-CD154 antibody) or a scrambled 15-mer peptide (SEQ ID NO: 17). KGYY-15 (SEQ ID NO: 11) reduced blood glucose in 57% of treated mice, while the control MR1 and scrambled 15-mer peptide showed no effect. These results are presented in graphical format in Figure 21. [Example]

[0149] Figure 22 provides a graph showing the binding stability of KGYY-15 (SEQ ID NO:11). KGYY-15 bound to human or mouse CD4+CD40+ cells maintained in culture for 10 days. KGYY15 was highly stable through 5 days, shed by 7 days, and shed again by 10 days. Cell death was the primary cause of loss of binding. [Example]

[0150] 23 provides a graph showing that SEQ ID NO:11 regulates the inflammatory cytokine IFNγ without affecting the production of the non-inflammatory cytokine IL-4. In this study, human T cells isolated from the peripheral blood of T1D subjects were stimulated with autologous antigen-presenting cells loaded with human pancreatic islets in the absence / presence of KGYY15 (SEQ ID NO:11), after which intracellular IFNγ and IL-4 were measured by flow cytometry. P<0.001 for IFNγ. [Example]

[0151] Figure 24 provides a graph showing that different concentrations of the peptide of SEQ ID NO:46 affect or influence levels of IL-2, IFNγ, and IL-17A. In this study, CD3+CD4+CD40+ (Th40) spleen cells were purified from ApoE- / - mice with cardiovascular disease. The cells were cultured overnight in the absence or presence of various concentrations of the KGYY6 peptide. Treated cells demonstrated varying degrees of reduction in the inflammatory cytokines IL-2, IFNγ, and IL-17A compared to untreated cells. [Example]

[0152] 25 is a graph showing the effect of treatment with SEQ ID NO: 13 on ketones measured in urine from canine study subjects described in Example 9. Ketones were measured in urine at the start of the study and approximately every two weeks thereafter. Canine subjects treated with SEQ ID NO: 13 demonstrated that treatment with SEQ ID NO: 13 resulted in zero ketone measurements in all canine subjects by the end of the study.

[0153] As previously stated, it will be readily apparent that there have been described and illustrated herein novel and useful embodiments of methods that meet many needs in a surprisingly and unexpected manner. Of course, it will be understood that such modifications, variations, and adaptations which may readily occur to those skilled in the art upon facing this disclosure are within the spirit of this disclosure and are limited only by the scope of the appended claims.

Claims

1. A peptide that is 6 amino acids in length, said peptide comprising the amino acid sequence of SEQ ID NO:

50.

2. 10. The peptide of claim 1, conjugated or fused to a second peptide or polypeptide.

3. 3. The peptide according to claim 1 or 2, (a) disrupting or inhibiting the interaction between CD40 and CD154 (b) containing an amidated amino acid at the C-terminus (c) containing an acetylated amino acid at the N-terminus (d) 1 x 10 ６ Binds to CD40 protein with a Kd less than M (e) Approximately 45 kDa, including splenic CD4hi cells, CD8 cells, and antigen-presenting cells binds to multiple myeloid-derived cell types that express CD40; and / or (f) binding to antigen-presenting cells It is characterized by:

4. A peptide according to any one of claims 1 to 3, characterized in that the peptide comprises a modification at the N-terminus and / or a modification at the C-terminus.

5. 5. The peptide of claim 4, wherein the modifications at the N-terminus include 5-FAM, 5-FAM-ahx, Abz, acetylation, acryl, Alloc, benzoyl, biotin, biotin-Ahx, BOC, Br-Ac-, BSA, CBZ, dansyl, dansyl-Ahx, decanoic acid, DPTA, fatty acid, FITC, FITC-Ahx, Fmoc, formylation, hexanoic acid, HYNIC, KLH, lauric acid, lipoic acid, maleimide, MCA, myristoyl, octanoic acid, OVA, palmitoyl, PEN, stearic acid, succinylation, and / or TMR.

6. 5. The peptide of claim 4, wherein the modifications at the C-terminus include AFC, AMC, amidation, BSA, Bzl, cysteamide, Oet, OMe, OtBu, OTBzl, KLH, MAPS asymmetric 2-, 4-, or 8-branched, Me, NHEt, NHisopen, NHMe, OSU, OVA, p-nitroanilide, and / or tBu.

7. A pharmaceutical composition comprising the peptide of any one of claims 1 to 6 and a pharmaceutically acceptable carrier.

8. 8. The pharmaceutical composition of claim 7, wherein the pharmaceutical composition is formulated for intramuscular (IM) delivery, intravenous (IV) delivery, subcutaneous (SC) delivery, oral delivery, gavage delivery, skin delivery, transdermal patch delivery, and / or nasal delivery.

9. A peptide for use in preventing, treating, modulating, and / or alleviating diabetes in a subject.

10. 10. The peptide according to claim 9, wherein the subject is a dog, a cat, a horse, a mouse, or a human.

11. 11. The peptide according to claim 9 or 10, characterized in that the subject does not produce autoantibodies that react with said peptide and / or CD154.

12. 12. The peptide according to any one of claims 9 to 11, characterized in that the peptide is formulated for intramuscular (IM), intravenous (IV), subcutaneous (SC), oral, gavage, cutaneous, transdermal patch, and / or nasal delivery.

13. A peptide according to any one of claims 9 to 12, characterized in that the peptide affects the interaction between CD40 and CD154 in such a way as to alter the cytokine expression profile of a cell population treated with the peptide.

14. A method for identifying a subject at risk of developing an autoimmune disease and / or diabetes, said method comprising contacting a sample containing T-cells from the subject to be tested with a peptide according to any one of claims 1 to 6, detecting CD40 binding peptides and determining the level of Th40 cells from the amount of CD40 binding, wherein a level of Th40 cells greater than 25% of the total number of T cells indicates that the patient is at risk of developing an autoimmune disease and / or diabetes.

15. 7. A peptide according to any one of claims 1 to 6 for use in modulating and / or reducing one or more of IFN-γ, interleukin-2 and interleukin-17 (IL-17) signalling in a cell or a subject.

16. A peptide according to any one of claims 1 to 6 for use in modulating and / or reducing blood glucose levels, serum fructosamine levels and / or c-peptide levels in a subject.

17. 7. The peptide according to any one of claims 1 to 6, characterized in that the peptide binds to living cells for less than one day, or more than one day, or more than two days.

18. 9. The pharmaceutical composition according to claim 7 or 8, wherein the peptide binds to live cells for less than one day, or more than one day, or more than two days.

19. The peptide according to any one of claims 9 to 13, 15 and 16, characterized in that the peptide binds to living cells for less than one day, more than one day, or more than two days.

20. 15. The method of claim 14, wherein the peptide binds to live cells for less than one day, or more than one day, or more than two days.

21. The peptide according to any one of claims 1 to 6, characterized in that the peptide reduces IFNγ, reduces IL-2, and / or reduces IL-17a.

22. 9. The pharmaceutical composition according to claim 7 or 8, wherein the peptide reduces IFNγ, reduces IL-2, and / or reduces IL-17a.

23. The peptide according to any one of claims 9 to 13, 15 and 16, characterized in that the peptide reduces IFNγ, reduces IL-2 and / or reduces IL-17a.

24. The method of claim 14, wherein the peptide reduces IFNγ, reduces IL-2, and / or reduces IL-17a.

25. A pharmaceutical composition comprising at least one peptide according to any one of claims 1 to 6, a sterile solubilizing agent, and a buffering agent.

26. 26. The pharmaceutical composition of claim 25, wherein the solubilizing agent comprises water.

27. 27. The pharmaceutical composition according to claim 25 or 26, further comprising a sugar.

28. The pharmaceutical composition according to any one of claims 25 to 27, characterized in that it is for use in the treatment of a disease selected from the group consisting of type 1 diabetes, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, chronic obstructive pulmonary disease, atherosclerosis, and / or acute respiratory distress syndrome.

29. A pharmaceutical composition for the treatment of hyperglycemia, comprising at least one peptide according to any one of claims 1 to 6, characterized in that the peptide is dissolved in a sterile solution in phosphate buffered saline (PBS).

30. 4. The peptide according to claim 1, wherein the peptide comprises glycation, alkylation, acetylation, or acylation of the N-terminal residue, and the peptide comprises amidation of the C-terminal residue.

31. A pharmaceutical composition according to any one of claims 25 to 27, characterized in that the peptide is pegylated, glycosylated, contains a chemical agent or is N-terminally acetylated.

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