Proinsulin peptide for type 1 diabetes

Abstract

The present invention relates to a peptide. The peptide can be used to treat or prevent type 1 diabetes (T1D), especially patients with DR3-DQ2 haplotype. The present invention also relates to a method for diagnosing or determining therapeutic efficacy using the peptide, as well as a method for identifying TID-related antigen drivers, identifying subjects as being at high risk for T1D and identifying patients suitable for and / or responsive to T1D treatment.

Description

Technical Field

[0001] The present invention relates to one or more peptides that can be used to treat or prevent type 1 diabetes (T1D). Background Art

[0002] Type 1 diabetes (T1D) is an autoimmune disease characterized by metabolic dysfunction, most notably dysregulated glucose metabolism, with characteristic long-term vascular and neurological complications. T1D is one of the most common autoimmune diseases, affecting one in 250 individuals in the United States, with approximately 10,000 to 15,000 new cases reported annually in the United States, and incidence is increasing. The highest prevalence of T1D is in Northern Europe.

[0003] T1D is characterized by absolute insulin deficiency, rendering patients dependent on exogenous insulin for survival. The acute clinical onset of T1D with symptoms of hyperglycemia is preceded by a long asymptomatic clinical phase during which the insulin-producing β cells are progressively destroyed.

[0004] Once developed, lifelong treatment with synthetic insulin injections is required because beta cells do not regenerate. Once established, diabetes is a major burden on the patient, his or her family, and society. Although modern insulin doses, formulations, and delivery systems can maintain blood sugar within reasonable limits, complications of the disease inevitably develop within a few years. The most common serious complications of diabetes are kidney failure, blindness, and loss of neurological function. The life expectancy of patients with diabetes is shortened by an average of 10 years. Given this background, it is important to consider new means of treating or preventing T1D.

[0005] Like all autoimmune diseases, T1D is caused by inappropriate activation of the immune system in response to encounters with self-antigens, in particular short peptide sequences presented to it by specific HLA molecules. Although the condition can be increasingly better managed clinically, it still has a negative impact on quality of life and life expectancy.

[0006] The cause of loss of self-tolerance in type 1 diabetes remains controversial, but it is well established that the HLA system constitutes a major genetic risk factor. HLA-DR3-DQ2 (DRB1*03:01-DQA1*05:01-DQB1*02:01) is the most common haplotype in patients with type 1 diabetes, accounting for approximately 34% (Erlich H et al. (2008) Diabetes 57(4):1084-1092), thus delineating a major, identifiable disease group. Given the antigen presentation properties of HLA molecules, it is hypothesized that the diabetes risk associated with HLA-DR3-DQ2 is related to the selective presentation of peptide epitopes of potentially diabetogenic autoantigens. Therefore, identification of these autoantigens and specific disease-determining regions is an important step in understanding the etiology of the disease.

[0007] Attempts to discover disease-associated epitopes in clinical studies typically involve eluting or predicting peptides that bind to candidate HLA molecules on antigen-presenting cells and / or in vitro examination of T cell responses in subjects with type 1 diabetes. This approach has generated several potentially relevant HLA-DR3-DQ2-restricted epitopes from a number of pancreatic islet antigens, including glutamic acid decarboxylase 65 (GAD65) and islet antigen-2 (IA-2) (Di Lorenzo TP et al. (2007) Clin Exp Immunol 148(1):1-16). Interestingly, proinsulin, which is generally considered to be associated with the early stages of type 1 diabetes in humans and mice (Narendran P, et al. (2003) Autoimmun Rev 2(4):204-210), has not yet been identified as a robust source of diabetes-associated epitopes presented by HLA-DR3-DQ2. However, a recent study highlighted the region of the proinsulin C-peptide that generates HLA-DQ2-restricted CD4 + T cell response (So M et al. (2018) Proc Natl Acad Sci USA 115(42):10732-10737).

[0008] Preclinical data in mouse models often suggest the presence of "driver T cells," i.e., populations of T cells that focus on a single peptide region of a self-antigen. The importance of these is that they have a greater impact on the disease. As a therapeutic corollary, if the driver T cells can be rendered ineffective, this may be superior to using second-line, subdominant targets / T cells to control the disease. It may be difficult to find driver T cells and relevant peptides from the antigen in human disease. As the disease progresses over time, many subdominant T cell clones and targets may become involved, thereby obscuring the driver T cell / target.

[0009] This invention addresses how to identify molecular targets of driver T cells associated with human type 1 diabetes. By identifying these peptides, peptide immunotherapy can disable the most important disease drivers. This is particularly important and effective for individuals who have not yet initiated autoimmunity.

[0010] The inventors used HLA transgenic mice susceptible to diabetes and used in vivo methods to discover epitopes. The disease relevance of antigens and peptides was examined by the ability of antigens and peptides to promote the development of autoimmune diabetes in susceptible animals. HLA-DR3-DQ2 transgenic mice used in the art do not spontaneously develop diabetes (de Kauwe AL et al. (2009) J Immunol 182 (12): 7440-7450). In order to promote this disease phenotype, on the C57BL / 6 background, the inventors constructed a new HLA transgenic mouse model, which expresses a high-risk gene for type 1 diabetes (DRB1*03:01-DQA1*05:01-DQB1*02:01) and human CD80β cell-specific expression of the costimulatory molecule CD80 (B7.1) under the rat insulin promoter (RIP) and human CD4. The use of adjuvanted antigen priming as a mechanism to accelerate and prolong diabetes penetrance demonstrates the importance of specific islet antigens as diabetogenic drivers in an HLA-DR3-DQ2 background. The inventors highlight a new HLA-guided approach to identify disease-associated antigenic drivers in vivo and identify novel non-naturally occurring peptides that can be used for peptide immunotherapy to induce tolerance. The inventors have pinpointed the diabetogenic effects of proinsulin to a previously unreported region beginning at the C-terminus of the B chain.

[0011] Peptides from the B23-C20 region of the proinsulin of the present invention from mice or humans have not been previously identified as being HLA-DR3 or HLA-DQ2 restricted. In addition, previous studies have not directly demonstrated the diabetogenicity of this region associated with any haplotype. In fact, methods for exploring the potential diabetogenicity and relative preference of antigens and epitopes using primary immunization with adjuvant antigens against a selected HLA background have not yet been proposed.

[0012] Few studies have examined the reactivity of T cells to BC ligation, and there are still few studies on HLA-DR3 / DQ2-restricted responses. Semana et al. previously found HLA-DR-restricted CD4+T cell responses to proinsulin peptide C3-16 in diabetic patients, but did not analyze HLA (Semana G, et al. (1999) J Autoimmun 12 (4): 259-267). In autoantibody-positive individuals with the HLA-DRB1*0401 / DQB1*0302 genotype (HLA-DR4 / DQ8 haplotype), long B11-C24 peptides are often recognized (Durinovic-Bello I, et al. (2002) J Autoimmun 18 (1): 55-66). So et al. identified CD4+ T cell responses to C2-11 and C3-14 in 3 of 22 T cell clones, but with HLA-DQ8 restriction (So M, et al. (2018) Proc Natl Acad Sci USA 115 (42): 10732-10737.). The HLA-DQ2-restricted peptides capable of generating T cell responses were all located near the C-terminus of the C-peptide. Rudy et al. (Rudy G, et al. (1995) Mol Med 1: 625-633) examined T cell responses to a single proinsulin peptide, B24-C4, in subjects who did not have diabetes but were at high risk of developing the disease due to the presence of autoantibodies. T cell responses were present in 6 / 10 subjects. The subjects were HLA-DR3 or -DR4 or heterozygotes of these, and the response was not associated with any particular haplotype. Raju et al. (Raju R et al. (1997) Hum Immunol 58:21-29) examined T cell responses to each of 10 overlapping peptides of proinsulin in HLA-DQ8 transgenic mice following immunization with the same peptides. DQ8-restricted responses to B1-B24 and B20-C10 were observed in DQ8 mice. These mice were not diabetic and this study did not provide any disease insights.

[0013] The prior art does not disclose the peptides claimed in the present invention, which have significant therapeutic potential, especially in the DR3-DQ2 haplotype. Summary of the invention

[0014] In a first aspect of the invention, a peptide having at least 78% homology to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 is provided.

[0015] SEQ ID NO: 1 is a fragment of PI comprising residues B30-C13.

[0016] SEQ ID NO: 2 is a fragment of PI comprising residues B29-C11.

[0017] SEQ ID NO:3 is a fragment of PI comprising residue C -1 -C14.

[0018] SEQ ID NO:4 is a fragment of PI comprising residues B29-C14.

[0019] SEQ ID NO:5 is a fragment of PI comprising residues B23-C20.

[0020] The inventors have surprisingly found that these novel non-naturally occurring peptides can be used in peptide immunotherapy to induce tolerance.The inventors have pinpointed the diabetogenic effects of proinsulin to a previously unreported region starting at the C-terminus of the B chain.

[0021] The peptide is non-naturally occurring due to the presence of at least one R from an R:R motif that is naturally cleaved by PC1 / 3 (proprotein convertase 1, also known as prohormone convertase; and prohormone convertase 3, or neuroendocrine convertase 1, often abbreviated as PC1 / 3), which cleaves the R:R motif between the B chain and the C chain as part of the normal processing of the prohormone proinsulin into insulin and C peptide.

[0022] These peptides have not been previously identified as being HLA-DR3 or HLA-DQ2 restricted.

[0023] The term "peptide" as used herein refers to any peptide comprising amino acids linked to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. Peptides will generally contain naturally occurring amino acids, but may include amino acid sequences modified by natural processes (e.g., post-translational processing) or by chemical modification techniques well known in the art. Such modifications are well described in the basic text. Modifications may occur anywhere in the peptide, including the peptide backbone, amino acid side chains, and amino or carboxyl termini. It should be understood that the same type of modification may be present in several sites in a given peptide to the same or varying extent. In addition, a given peptide may contain many types of modifications.

[0024] The peptide has at least 78% homology to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5. In some embodiments, the peptide has at least 80% homology to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5. In some embodiments, the peptide has at least 85% homology to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5. In some embodiments, the peptide has at least 90% homology to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5. In some embodiments, the peptide has at least 95% homology to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5. In some embodiments, the peptide has at least 99% homology to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In a specific embodiment, the peptide has the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In another embodiment, the peptide has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0025] When the amino acid does not have the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, preferably the amino acid change is conservative, that is, the amino acid is changed to a different amino acid with similar biochemical properties (e.g., charge, hydrophobicity and size).

[0026] Preferably, the peptides that make up the peptide combination are isolated peptides. The term "isolated" refers to taking the peptide out of its original environment. For example, a peptide present in a living animal is not isolated, but the same peptide or a fragment of such a peptide separated from some or all coexisting materials in the natural system is isolated. Such a peptide can be part of a carrier and / or the peptide can be part of a composition and still be isolated because such a carrier or composition is not part of its natural environment.

[0027] In a preferred embodiment, a peptide combination comprising two or more of the above-mentioned peptides is provided.

[0028] A second aspect of the present invention relates to a pharmaceutically acceptable composition comprising a peptide or peptide combination of the present invention and one or more pharmaceutically acceptable excipients.

[0029] Preferably, the pharmaceutical composition comprises cysteine. The presence of free cysteine ​​in the composition stabilizes any tendency of the peptides of the invention to form interchain disulfide bonds and thus precipitate. The pharmaceutical composition may comprise 1 to 5 mg of L-cysteine ​​per 2 mg of peptide, preferably 2 to 4 mg of L-cysteine ​​per 2 mg of peptide, most preferably, 2.5 mg of L-cysteine ​​per 2 mg of peptide.

[0030] Pharmaceutical composition can be used for human or animal in human and veterinary medicine and will usually comprise one or more suitable excipients. Acceptable excipients for therapeutic use are well-known in the pharmaceutical field, and are described in, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co. (AR Gennaro, 1985). The selection of pharmaceutical excipients can be selected according to the expected route of administration and standard pharmaceutical practice. Pharmaceutical composition can comprise any suitable adhesive, lubricant, suspending agent, coating agent or solubilizing agent as excipient or also comprise any suitable adhesive, lubricant, suspending agent, coating agent or solubilizing agent except excipient.

[0031] Preservatives, stabilizers and dyes may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may also be used.

[0032] Depending on different delivery systems, there may be different composition / formulation requirements. For example, the pharmaceutical composition of the present invention can be formulated into parenteral delivery, wherein the composition is formulated into an injectable form for delivery by, for example, intravenous, intradermal, intramuscular, subcutaneous or intraperitoneal routes. For parenteral administration, the composition is preferably used in the form of a sterile aqueous solution, which may contain other substances (e.g., enough salts or monosaccharides) to make the solution isotonic with the blood. The composition can also be formulated into administration by oral or topical routes, including nasal, oral or epidermal administration. Preferably, the composition is formulated into delivery by an intradermal route.

[0033] Intradermal routes of administration include any means of dermal access, for example, needle-free or needle-free ballistic injection of fluids or powders into the intradermal space using microneedle-based injection and infusion systems (or other means of precisely targeting the intradermal space), Mantoux-type intradermal injections, iontophoresis enhanced by microdevices, and direct deposition of fluids, solids, or other dosage forms into the skin, including the use of patches to deposit the composition onto the skin.

[0034] Typically, the doctor will determine the actual dosage that is most suitable for the individual subject, and will vary with the disease, age, weight and response of the particular patient. Those skilled in the art can, for example, use body surface area (BSA) normalization to determine the appropriate dosage for a person. For example, the pharmaceutical composition can include about 0.1 μg to 15 mg of total peptide per single dose, preferably 1 μg to 12 mg of total peptide per single dose. In a preferred embodiment, 240 μg (1 μg dose BSA standardized for 60 kg adults) of total peptides are administered per single dose. In another preferred embodiment, 12 mg of total peptides are administered per single dose. If more than one peptide is administered, it is preferred that the peptides are present in an equimolar ratio.

[0035] In a preferred embodiment, the pharmaceutical composition of the present invention is administered at least once a month, preferably at least once and up to twelve times a month.

[0036] The pharmaceutical composition may also include an adjuvant that promotes tolerance and / or a cell that promotes tolerance. Adjuvants that promote tolerance include IL-10, recombinant cholera toxin B subunit (rCTB), ligands of Toll-like receptor 2, and biological preparations and monoclonal antibodies that regulate immune responses, such as anti-CD3, co-stimulation blockers, and immune checkpoint regulators, which can be co-administered with peptide combinations. Cells that promote tolerance include regulatory T cells, immature dendritic cells, and dendritic cells treated with vitamin D3, (1α, 25-dihydroxyvitamin D3) or its analogs. Preferably, one or more peptides of the present invention are conjugated to the surface of dendritic cells treated with vitamin D3 or its analogs.

[0037] The peptide or peptide combination of the present invention can be coated on nanoparticles in a pharmaceutically acceptable composition. The nanoparticles can be carbon-based nanoparticles, ceramic nanoparticles, metal nanoparticles, semiconductor nanoparticles, polymer nanoparticles or lipid-based nanoparticles or mixtures thereof.

[0038] In one embodiment, the peptide or combination of peptides is coated on the nanoparticle by binding to an MHC complex attached to said nanoparticle.

[0039] A third aspect of the invention relates to a pharmaceutically acceptable composition of the invention for use in therapy.

[0040] The fourth aspect of the present invention relates to a pharmaceutically acceptable composition of the present invention for use in the treatment or prevention of type 1 diabetes (T1D).

[0041] When T1D is "treated," it means that one or more clinical manifestations of T1D are improved. This does not mean that the symptoms of T1D are completely cured so that the patient no longer has those symptoms, although this may be the case in some approaches. "Treatment" results in one or more symptoms of T1D being less severe than before treatment.

[0042] Preferably, the pharmaceutical composition of the invention is used to treat or prevent (T1D), wherein the patient has a DR3 haplotype. Preferably, the patient has a DR3-DQ2 haplotype. The inventors unexpectedly and specifically demonstrated the importance of the peptides claimed in the present invention as diabetogenic drivers on the HLA-DR3-DQ2 background.

[0043] The fifth aspect of the present invention relates to a pharmaceutically acceptable composition of the present invention, which is used in the manufacture of a medicament for treating or preventing type 1 diabetes (T1D).

[0044] The sixth aspect of the present invention relates to a method for treating or preventing type 1 diabetes (T1D), wherein the pharmaceutically acceptable composition of the present invention is administered to a patient suffering from T1D or a non-diabetic individual identified as being at high risk of T1D.

[0045] Preferably, the pharmaceutically acceptable compositions of this invention are administered to patients having beta cell mass remaining.

[0046] The seventh aspect of the present invention relates to a kit for treating or preventing type 1 diabetes (T1D), comprising the peptide or peptide combination of the present invention.

[0047] The eighth aspect of the present invention relates to a nucleotide sequence encoding the peptide of the present invention.

[0048] The ninth aspect of the present invention relates to a vector for expressing any peptide of the present invention.

[0049] The vector can be any suitable vector for expressing the peptide of the present invention, including viral and non-viral vectors. Viral vectors include parvovirus, adenovirus, retrovirus, lentivirus or herpes simplex virus. Parvovirus can be adenovirus-associated virus (AAV). The vector is preferably a recombinant adeno-associated virus (rAAV) vector or a lentiviral vector. More preferably, the vector is a rAAV vector.

[0050] The vector according to the present invention can be a gene delivery vector. Such a gene delivery vector can be a viral gene delivery vector or a non-viral gene delivery vector.

[0051] Therefore, the present invention provides gene delivery vectors based on animal parvoviruses, in particular dependent viruses (such as infectious human or simian AAV) and their components (e.g., animal parvovirus genomes), which are used to introduce and / or express the peptides of the present invention in mammalian cells. Therefore, the term "parvovirus" as used herein encompasses dependent viruses, such as any type of AAV.

[0052] The tenth aspect of the present invention relates to a transgenic animal that spontaneously develops T1D, wherein the animal has a DR3 haplotype, preferably a DR3-DQ2 haplotype. Preferably, the animal is a non-human mammal, in particular a primate. Alternatively, the animal may be a rodent, in particular a mouse; or the animal may be a canine, feline, ovine or porcine. Preferably, the animal comprises cells expressing human CD80 in the presence of a rat insulin promoter (RIP).

[0053] The eleventh aspect of the present invention relates to a method for identifying a TID-associated antigenic driver, comprising priming the above-mentioned animal with a peptide from the B23-C20 region of proinsulin, and if disease progression is accelerated, identifying the peptide as an antigenic driver.

[0054] In one embodiment, disease progression is considered accelerated if disease progression is 50% faster than when no primary immunization is performed. Preferably, the peptide is selected from a peptide having at least 78% homology to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5. In one embodiment, the peptide is administered in combination with an adjuvant disclosed above. The peptide can be administered at a dose of up to 1 mg per peptide.

[0055] A twelfth aspect of the present invention relates to a method for diagnosing or determining the efficacy of a treatment, comprising: (a) providing CD4 lymphocytes from an individual suspected of having or being susceptible to T1D; (b) providing a population of antigen presenting cells (APCs) carrying on their surface a class II MHC molecule of an allele identical to an allele expressed by the individual, the population of APCs having been contacted with a peptide or a combination of peptides according to any one of claims 1 to 5, and the class II MHC molecules being bound to one or more of the peptides according to claims 1 to 4; or (c) providing a soluble peptide-HLA multimer reagent comprising a class II MHC molecule of an allele identical to an allele expressed by the individual, the MHC molecule having bound thereto one or more peptides of the peptide combination according to claims 1 to 5; (d) contacting the population of APCs of step (b) or the peptide-HLA multimer of step (c) with the CD4 lymphocytes of step (a); and (e) determining whether the CD4 lymphocytes recognize the class II MHC-bound peptide as an indication that the individual suffers from or is susceptible to T1D.

[0056] Such APCs can be B lymphocytes, monocytes, macrophages or dendritic cells, or whole peripheral blood mononuclear cells (PBMCs). APCs can also be immortalized cell lines derived from B lymphocytes, monocytes, macrophages or dendritic cells. When the subject is a human, APCs can also be T cells, because human T cells can express class II MHC molecules. The method also includes administering a peptide or peptide combination of the present invention to an individual if the CD4 lymphocytes recognize class II MHC binding peptides.

[0057] The thirteenth and fourteenth aspects of the present invention relate to methods for identifying a subject at high risk for T1D and identifying a patient as suitable for T1D treatment, respectively, the method comprising: when an immune response to any one of the peptides or peptide combinations of the present invention is detected, determining whether the subject or patient has a DR3 haplotype, preferably a DR3-DQ2 haplotype.

[0058] Those skilled in the art will be familiar with methods for measuring CD4 T cell immune responses to the peptides of the invention. Examples include enzyme-linked immunospot assays (ELISPOT), peptide-HLA tetramer assays, or in vitro stimulation assays followed by detection of proliferating or cytokine-producing CD4 T cells.

[0059] A fifteenth aspect of the invention relates to a method of identifying a subject as a responder to immunotherapy or in need of re-treatment with a peptide or peptide combination of the invention, the method comprising: determining whether an immune response to any of the peptides or peptide combinations of the invention is detected.

[0060] Likewise, the immune response can be detected, for example, by ELISPOT, peptide-HLA tetramer assays or in vitro stimulation assays followed by detection of proliferating or cytokine-producing CD4 T cells. If there is no immune response or in fact the subject's immune response is reduced, the subject is responding to immunotherapy. If the subject's immune response is increased, the subject may require further treatment with the peptide or peptide combination of the invention.

[0061] It will be appreciated by the skilled person that all aspects of the present invention, whether or not they relate to, for example, peptides, combinations of peptides, uses thereof, pharmaceutically acceptable compositions or methods of treatment, are equally applicable to all other aspects of the present invention. In particular, for example, aspects of peptides may have been described in more detail than other aspects of the present invention (e.g., uses of peptides). However, it will be appreciated by those skilled in the art that, in the case of having provided more detailed information for a particular aspect of the present invention, this information is generally equally applicable to other aspects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] The present invention will now be described in detail, by way of example only, with reference to the accompanying drawings, in which:

[0063] Figure 1 It is shown that DR3DQ2xRIP-B7.1 mice spontaneously develop immune-mediated diabetes.

[0064] (a) Thirty-two males and 24 females were monitored weekly for diabetes from 6-7 weeks of age to 35 weeks of age. Statistical analysis was performed using the Mantel-Cox Log-rank test. ns = not significant.

[0065] (b) Representative images of double immunohistochemical staining of frozen pancreatic sections from diabetic mice. Insulin is stained red and immune cells are stained brown. 20x objective. Arrows indicate Ly6G + cell.

[0066] (cd) IgG autoantibody ELISA against human GAD65 protein (c) and against overlapping 30-mer peptides of mouse proinsulin-2 (d). c+d: Analysis of sera obtained from cross sections of mice aged 8 to 23 weeks. Horizontal lines represent medians. Dunnett's multiple comparison test. 10 to 22 samples per age group. ELISAs were performed in parallel on the same samples. *p<0.05, ***p<0.001.

[0067] Figure 2 It is shown that primary immunization with murine proinsulin-2 peptide adjuvant promotes diabetes.

[0068] (a) DR3DQ2xRIP-B7.1 mice aged 7 to 12 weeks were subcutaneously administered 100 μg of murine proinsulin-2 peptide dissolved in TiterMax Gold adjuvant on days 0 and 14. Human GAD65 protein or the 377-aa C-terminal region of IA-2 and PBS only control 200 ng of pertussis toxin was administered intraperitoneally on day 0 and day 1 or day 2. Mice were monitored weekly for diabetes until 20 weeks after the initial immunization. The gray dashed line indicates the expected level of spontaneous diabetes in 12-week-old mice on day 0 of the experiment (based on Figure 1 Data in and normalized for sex). Statistical analysis was performed using the Mantel-Cox log-rank test. **p<0.01, ***p<0.001.

[0069] (be) Blood glucose level of each mouse in the groups immunized with PBS (b), mouse proinsulin-2 peptide (c), human GAD 65 (d), or human IA-2 fragment (e).

[0070] Figure 3 Murine proinsulin-2 peptide B23-C20 is shown to mediate diabetogenicity.

[0071] (a) DR3DQ2xRIP-B7.1 mice aged 7-8 weeks (in each group ) were administered 100 μg of murine 30-mer proinsulin-2 peptide B1-30, B23-C20, C10-A6, or C25-A21 in TiterMax Gold adjuvant subcutaneously on days 0 and 14, with 200 ng of pertussis toxin administered intraperitoneally on days 0 and 1 or 2. Mice were monitored weekly for diabetes until 20 weeks after the initial immunization. The gray dashed line indicates the expected level of spontaneous diabetes in 8-week-old mice on day 0 of the experiment, based on Figure 1 Data were normalized for sex. Statistical analysis was performed using the Mantel-Cox log-rank test. ***p<0.001 for B23-C20 versus B1-30 or C10-A6. p<0.01. The rest were not significant.

[0072] (be) Blood glucose level of each mouse in the groups immunized with B1-30 (b), B23-C20 (c), C10-A6 (d), or C25-A21 (e).

[0073] Figure 4 Analysis and comparison of proinsulin-2B23-C20 are shown.

[0074] (a) Top: Alignment of equivalent regions of mouse proinsulin-2 and human proinsulin. Boxed amino acids are identical, shaded are similar. Bottom: Six overlapping 14-15-mer mouse peptides were generated for more detailed analysis.

[0075] (b) Binding predictions for all six murine peptides to HLA-DR3 and HLA-DQ2. For the VD-14 peptide, a leading glutamine (Q) was added to the sequence for prediction to achieve the required minimum length of 15 amino acids. Predicted HLA binding cores and affinities were generated using the NetMHCIIpan prediction method with the online IEDB analysis resource (http: / / tools.immuneepitope.org / mhcii / ).

[0076] (c) Binding predictions of the human equivalents of the murine peptides to HLA-DR3 and HLA-DQ2. The predicted HLA binding cores and affinities were generated using the NetMHCIIpan prediction method with the online IEDB analysis resource (http: / / tools.immuneepitope.org / mhcii / ).

[0077] Figure 5 The ME-15 peptide (B29-C11) is shown to cover the core region of B23-C20 for diabetogenicity.

[0078] (a) DR3DQ2xRIP-B7.1 mice aged 10 to 14 weeks were subcutaneously administered 100 μg of GP-15 in TiterMaxGold adjuvant on days 0 and 14. YA-15 ME-15 RG-15 DA-15 and VD-14 Peptide, wherein 200 ng of pertussis toxin was administered intraperitoneally on day 0 and day 1 or day 2. Mice were monitored weekly for diabetes until 20 weeks after the initial immunization. The gray dashed line represents the expected level of spontaneous diabetes in 14-week-old mice on day 0 of the experiment (based on Figure 1 Data from the 24-hour study were normalized for sex). Statistical analysis was performed using the Mantel-Cox log-rank test. *p<0.05 for RG-15 versus YA-15, **p<0.01 for ME-15 versus GP-15, YA-15, or VD-14. p<0.05. The rest were not significant.

[0079] (bf) Blood glucose level of each mouse in the groups immunized with GP-15 (b), YA-15 (c), ME-15 (d), RG-15 (e), DA-15 (f), and VD-14 (g).

[0080] Figure 6 : R Tolerance induction against accelerated diabetes in RIP.CD80xDR3DQ2 mice.

[0081] (a) Dosing schedule. Mice aged 7 to 8 weeks (n=7 per group) were treated with subcutaneous (sc) administration of soluble 14 / 15-mer peptide in PBS before and after accelerated diabetes development by antigen primary immunization with 30-mer murine proinsulin-2 peptide B23-C20 in TiterMax Gold adjuvant on days 0 and 14. Pertussis toxin was administered intraperitoneally (ip) on days 0 and 2. (b) Mice were treated with 14 / 15-mer peptide ME-15 YA-15 or VD-14 Diabetes incidence in treated mice. Statistical analysis was performed using the Mantel-Cox log-rank test. ME-15 vs. YA-15, p<0.01; ME vs. VD-14, p<0.05.

[0082] Figure 7 Antigen-specific responses in human donors detected by enzyme-linked immunospot (ELISPOT) assay are shown.

[0083] Response to proinsulin B30-C13 in human donors. PBMCs from individuals with HLA-DR3-DQ2 (a) or any other haplotype (b) were stimulated in vitro with human proinsulin-derived peptides B30-C13 or C19-A3 prior to detection of IFNγ by ELISPOT analysis. HLA-DR3-DQ2 individuals, n=3; non-HLA-DR3-DQ2, n=6. *p<0.05, paired t-test for ratios.

[0084] FIG. 8 shows antigen-specific responses in human donors recently diagnosed with type 1 diabetes and having the HLA-DRB1*03:01-DQA1*05:01-DQB1*02:01 genotype (HLA-DR3 / DQ2) as detected by ELISPOT assay.

[0085] The results for proinsulin B23-C20, B29-C11, B29-C14, B30-C13 and C -1The human proinsulin-derived peptides B23-C20, B29-C11, B29-C14, B30-C13 and C14 were used before the detection of IFN-γ and IL-17 (proinflammatory response) and IL-10 (regulatory response) by ELISPOT assay. -1 -C14 stimulated PBMCs from 5 patients with type 1 diabetes (identified as #1-#5) with HLA-DR3-DQ2 in vitro. (a) Data are shown as raw counts of responder CD4 T cells per million PBMCs for each peptide assay. Grey shading shows the upper limit of the negative control for each cytokine assay (average 0.4, 0.2, and 0.2 CD4 T cells responding to diluent control for IFN-γ, IL-17, and IL-10 assays, respectively). (b) A summary table of positive responses (shaded boxes) for each patient / peptide is shown. DETAILED DESCRIPTION

[0086] Example 1

[0087] Materials and methods

[0088] animal

[0089] HLA-DR3-DQ2-transgenic animals (B6.hCD4.DR3-DQ2.DQ2.MHCII - / - Mice (described previously (de Kauwe AL et al. (2009) J Immunol 182(12):7440-7450) and obtained from J. McCluskey) were crossed with RIP-B7.1 transgenic animals (B6.Cg-Tg(Ins2-CD80)3B7Flv / Orl, EMMA, Orleans, France; ID00216) to obtain HLA-DR3-DQ2 + huCD4 + IA / IE - / - RIP.B7.1 + Mice, hereinafter referred to as DR3DQ2xRIP-B7.1. tm1GruDR4xRIP-B7.1 mice generated by crossing Tg(HLA-DRA / H2-Ea, HLA-DRB1*0401 / H2-Eb)1Kito mice with B6.Cg-Tg(Ins2-CD80)3B7Flv / Orl have been described previously (Verhagen J, et al. (2018) Sci Rep 8(1):14106). All animals were housed in individually ventilated cages under specific pathogen-free conditions at the KCL Biological Services Unit with a 12-hour light / dark cycle, with food and water provided ad libitum. Experiments were performed under a project license held by M. Peakman in accordance with UK Home Office regulations. All work was assessed and approved locally by the Guy Animal Welfare and Ethical Review Board (AWERB).

[0090] Primary immunization antigen

[0091] All mouse proinsulin-2 peptides were custom-made by Almac (Edinburgh, UK) or GLS Biochem (Shanghai, China) with purity >95%. The 377 amino acid C-terminal fragment of human IA-2 was produced by ProteoGenix (Schiltigheim, France). Recombinant human GAD65 (T-cell GAD) was purchased from Diamyd Medical (Stockholm, Sweden). The predicted HLA binding core and avidity were generated using the NetMHCIIpan prediction method with the online IEDB analysis resource (http: / / tools.immuneepitope.org / mhcii / ).

[0092] Induction and monitoring of diabetes

[0093] All mice were regularly monitored for unstimulated glucosuria using Diastix strips (Bayer, Basel, Switzerland). From 6-7 weeks of age, the hyperglycemia of some mice was monitored weekly by minimally puncturing the tail vein on the alternating sides of the tail and analyzing using OneTouch Verio meter and strips (Lifescan, High Wycombe, UK) to closely monitor the progress towards spontaneous diabetes. In addition to confirming glucosuria, mice were considered to be diabetic after blood glucose readings>16.7mmol / l (300mg / dl). For disease acceleration experiments, animals were distributed in groups of equal or similar size to achieve a comparable distribution of age and sex in each group. Mice (6-14 weeks of age) were immunized with 100 μg of peptide or protein in TiterMax Gold adjuvant (TiterMax, Norcross, GA, USA) subcutaneously (sc) at the base of the tail, and received a second dose subcutaneously in the inguinal region on the 14th day. On day 0 and day 1 or day 2, mice received 200 ng pertussis toxin (Sigma, Poole, UK) dissolved in PBS intraperitoneally (ip). Mice were then monitored weekly for hyperglycemia and glycosuria. In experiments examining tolerance induction, mice received subcutaneous injections of 10 μg of an aqueous peptide solution at intervals of 3 to 4 days for a total of 6 injections. The tolerance regimen was initiated one week prior to the primary immunization with adjuvanted antigen. Upon detection of diabetes, mice were immediately humanely sacrificed according to ethical approval.

[0094] Histology

[0095] Pancreases embedded in OCT compound (Cellpath, Newtown, UK) were frozen in isopentane (Sigma) cooled with liquid nitrogen. 10 μm sections were fixed in acetone, detected with Vector Impress anti-rabbit AP (Vector Labs, Peterborough, UK), and developed with Vector Impress Red before first staining with rabbit anti-mouse insulin (Abcam). Immune cells in the tissue were stained with biotinylated antibodies against mouse CD4 (clone GK1.5, which also recognizes human CD4), CD8, CD11b, CD11c, Ly6G, and B220 (all from eBioscience / ThermoFisher, Altrincham, UK), detected using ABC reagent and DAB solution from Vector Labs. Nuclei were stained with hematoxylin (Sigma) before mounting slides with VectaMount (Vector Labs). Images were acquired on a Zeiss Axiovert A1 microscope using Zen software. No cropping or alteration of images was used.

[0096] Autoantibody ELISA

[0097] Proinsulin-2 peptide or recombinant human GAD65 was coated onto Maxisorp plates (Nunc, Roskilde, Denmark) in ELISA coating buffer (eBioscience). Diluted serum (in PBS 5% BSA, Sigma) or controls (mouse IgG2a anti-insulin clone ICBTACLS and isotypes from ThermoFisher, mouse IgG1 anti-GAD65 clone N-GAD65 and isotypes from Biolegend) were incubated at room temperature for 2 hours, then detected with biotin-anti-mouse IgG, streptavidin-HRP and high-sensitivity TMB solution (all from eBioscience) and read at 450nm. The titration concentrations of the relevant control antibodies on each plate (1-1000ng / ml) were used to normalize the data and assign arbitrary units to serum antibody levels.

[0098] Analysis of C-peptide fragments released from human β cells

[0099] To examine the full potential of C-peptide fragments available in vivo, we analyzed (i) islet cell secretion into the supernatant and (ii) secretion into intracellular granules.

[0100] (i) Secretion

[0101] Human islets were washed in 1X DPBS containing 0.5 mM EDTA and pretreated in Krebs-Ringer buffer containing 2.5 mM glucose at 37°C, 5% CO2 for 30 minutes. The buffer was then changed to Krebs-Ringer buffer containing 2.5 mM or 25 mM glucose, and the islets were incubated at 37°C, 5% CO2 for 30 minutes. The secretion in the supernatant of each condition was collected and centrifuged at 400 × g for 5 minutes at 4°C to pellet any residual cells. The secretion supernatant was collected and the protein / fragment was isolated and purified by C18 reverse phase resin purification.

[0102] (ii) Intracellular granules

[0103] Islets are washed 3 times at room temperature with 300 × g in 1X DPBS containing 0.5mM EDTA for 3 minutes. Islet cells are dissolved in Accutase (preheated to 37 ° C) at a concentration of 0.5ml / 1000IEQ (islet equivalent), and incubated in 37 ° C water baths for 10 minutes. The tube is inverted every minute, and in addition, after 5 minutes and 10 minutes, a 5ml serological pipette is used to pipette up and down five times to produce a single cell suspension. The suspension is filtered through a 35 μm nylon mesh and washed with 300xg for 3 minutes in 1X DPBS 0.5mM EDTA. Cells are resuspended in 2ml of 1X DBPS 0.5mM EDTA. Use a Balch homogenizer (Isobiotech, Germany) with a 14 μm pore size to destroy the cell membrane while keeping the intracellular compartment intact. Single cell lysate is passed through the homogenizer 10 times. The homogenized cells were centrifuged at 1000 × g for 15 minutes at 4°C to release the collected intracellular contents. The pellet was resuspended in 1X DPBS 0.5mM EDTA and centrifuged again. The intracellular contents in the supernatant were collected again and combined, and then centrifuged at 5000 × g for 15 minutes at 4°C to produce a precipitate containing a high-density intracellular compartment rich in crinosomes. The supernatant from this rotation was centrifuged again at 20000 × g for 45 minutes at 4°C to produce a low-density intracellular compartment precipitate rich in insulin secretory granules. The crinosome-rich and granule-rich precipitates were placed in 1X DPBS, quickly frozen and stored at -80°C. The supernatant was thawed on ice and the protease inhibitor (cOmplete TM , Roche, Switzerland), the contents of these intracellular compartments were released, and this freeze-thaw cycle was repeated a total of five times.

[0104] The proteins and fragments obtained in (i) secreted supernatants and (ii) intracellular particles were purified by C18 reverse phase resin (Waters, US), which was wetted with 50% acetonitrile (ACN) / 50% dd H2O, equilibrated with 0.1% trifluoroacetic acid (TFA) / 99.9% dd H2O, and washed with 2% ACN / 0.1% TFA / 97.9% dd H2O, and then the sample (adjusted to a final concentration of 0.2% TFA) was loaded onto the C18 column. The sample was washed with 2% ACN / 0.1% TFA / 97.9% dd H2O, and the proteins and protein fragments were eluted with 80% ACN / 0.1% TFA / 19.9% ​​dd H2O and dried using a vacuum centrifugal concentrator. The dried sample was resuspended in 2% ACN / 0.1% formic acid / 97.9% dd H2O. Chromatographic separation was performed using an Ultimate 3000 NanoLC system (Thermo Fisher Scientific, UK). Peptides were analyzed by reverse phase chromatography on a 75 pm * 50 cm C18 column using a linear LC gradient of 80% acetonitrile (B) in 0.1% formic acid and water (A) 0.1% formic acid. The gradient was delivered to elute the peptides at a flow rate of 300 nl / min in 2% B to 50% B in 65 minutes. The eluent was ionized by electrospray ionization using an Orbitrap-Fusion-Lumos (Thermo Fisher Scientific, UK) operated under Xcalibur v4.1. The instrument was programmed to acquire using a "universal" method by defining a 3 s cycle time between full MS scans and MS / MS fragmentation. The MS / MS data were analyzed using PeaksStudio (version 7.5; Bioinformatics Solutions, Canada) against the current version of the reviewed Swissprot Homo sapiens database downloaded from Uniprot (http: / / www.uniprot.org / uniprot / ).

[0105] ELISPOT assay

[0106] ELISPOT analysis was performed using human peripheral blood mononuclear cells (PBMCs) isolated from fresh heparinized blood. Informed consent was obtained from all participants. 1×10 50 μL of 1 ... 6 PBMCs were subjected to 48 hours. Cytokine secretion analysis was performed using IFN-γ, IL-17 and IL-10 ELISPOT kits (U-Cy tech) according to the manufacturer's instructions, and the plates were analyzed using the automated ELISPOT Bioreader 6000 (Bio-Sys) and associated software. Data are expressed as the total number of spots in triplicate wells after peptide stimulation divided by the total number of spots in the diluent-only control wells (stimulation index [SI]) or as responder CD4 T cells / 10 6 PBMCs.

[0107] Statistical analysis

[0108] All analyses were performed using GraphPad Prism 8 software using the appropriate assays as indicated.

[0109] Example 2

[0110] Spontaneous onset of diabetes in DR3DQ2xRIP-B7.1 mice

[0111] Unlike our previously described DR4xRIP-B7.1 model (Verhagen J, et al. (2018) Sci Rep 8(1):14106), which does not display spontaneous insulitis or diabetes, DR3DQ2xRIP-B7.1 mice spontaneously develop diabetes ( Figure 1a). Although both models exhibited relatively high baseline blood glucose levels at any age, only DR3DQ2xRIP-B7.1 mice developed levels >16.7 mmol / l in addition to glycosuria. By 35 weeks of age, 46% of all animals monitored (26 of 56) developed autoimmune diabetes. Disease incidence (16 / 32 vs. 10 / 24, respectively) and mean age of onset (24.2 weeks ± 7.3 (SD) vs. 24.8 ± 8.5, respectively) were similar in male and female animals. No overt signs of other immune-mediated disorders were detected, as evidenced by splenomegaly, cachexia, lethargy, or skin / ocular abnormalities. All diabetic mice exhibited severe immune infiltration in the pancreatic islets. This infiltrate was highly diverse, with both lymphoid and myeloid cells expressing CD4, CD8, B220, CD11b, and CD11c, found in large numbers in all islets ( Figure 1 b) Lower amounts of Ly6G were found in some islets + Granulocytes. No significant immune cell infiltration was observed in non-diabetic mice examined histologically at 6 to 8 weeks of age (n=5), 10 weeks of age (n=10), 12 weeks of age (n=7), 16-20 weeks of age (n=5), or even 35 weeks of age (n=13), with moderate infiltration of some islets detected in one 6-week-old male. This suggests that the insulin secretory phenotype is pathological, leads to a rapid progression toward diabetes, and is strongly associated with the disease. Because the onset of type 1 diabetes in human HFADR3-DQ2 patients is characterized by the presence of autoantibodies to GAD65 and insulin in the majority of cases, antibodies in mouse sera were tested against a murine 30-mer peptide spanning the length of murine proinsulin-2 (the isoform most similar to human proinsulin) and against human GAD65, which has more than 95% sequence homology to the murine equivalent. As Figure 1 As shown in cd, when the groups of mice with increasing age were compared, the levels of anti-proinsulin 2 antibodies increased significantly, while the levels of anti-GAD65 antibodies did not increase significantly.

[0112] Example 3

[0113] Proinsulin is a diabetogenic antigen in DR3DQ2xRIP-B7.1 mice

[0114] Spontaneous onset of diabetes in mice carrying high-risk transgenic HLA suggests that autoimmune processes are central to disease development and prompted us to address the question of whether specific autoantigens are disease drivers. If the antigen has "driver" properties, the hypothesis that priming animals against the candidate molecule with an adjuvant will accelerate disease progression has been tested. Thus, mice were primed with individual 30-mer peptides overlapping and spanning the length of murine proinsulin 2 in TiterMax Gold adjuvant; recombinant human GAD65; the 377 amino acid intracellular region of human islet antigen-2 (IA-2); or PBS alone ( Figure 2 af)( Figure 2 Importantly, in these conditions, primary immunization with proinsulin peptide alone significantly accelerated the onset of diabetes and increased the incidence beyond that observed with control stimulation or by spontaneous onset. This was an unexpected finding. Previous studies have primarily used HLA-DR3 and HLA-DQ2 restriction elements to identify GAD65 and IA-2 as being expressed by CD4 T cells from subjects with type 1 diabetes. + Target pancreatic islet antigens recognized by T cells or T cell clones (Di Lorenzo TP, et al. (2007) Clin Exp Immunol 148 (1): 1-16.). In addition, the environmental determinants of diabetes in the Young study showed that anti-GAD65 autoantibodies usually first appeared in disease-susceptible individuals with the HLA-DR3-DQ2 haplotype. In contrast, anti-insulin autoantibodies usually first appeared in risk subjects with the HLA-DR4-DQ8 haplotype (Krischer JP et al. (2017) Diabetes Care 40 (9): 1194-1202 and Krischer JP, et al. (2015) Diabetologia 58 (5): 980-987). These represent potentially important disease endotypes, characterized by different pathological processes involving loss of immune tolerance to GAD65 and insulin, respectively. Given this, one might have predicted that in the new DR3DQ2xRIP-B7.1 model, GAD65 would be the most potent anti-genetic driver of disease, but this turned out not to be the case.

[0115] At the end of the 20-week experiment, every mouse primed with proinsulin peptide (10 / 10) in adjuvant had developed diabetes (mean onset time 77 ± 38.6 (SD) days after primary immunization). The intensity or diversity of pancreatic immune infiltration in these antigen-challenged animals did not differ from that observed in mice with spontaneous diabetes (not shown). These findings suggest the importance of immune recognition of proinsulin in the context of HFA-DR3-DQ2 as a diabetes-driving event in this model.

[0116] Example 4

[0117] Specific regions of proinsulin are responsible for diabetes induction in this model

[0118] The inventors next examined whether there are major regions of proinsulin that interact to promote diabetes in DR3DQ2xRIP-B7.1 mice by repeating these adjuvant primary immunization experiments with each of the four proinsulin-2 peptides ( Figure 3 af). Primary immunization with the B23-C20 peptide provided the most pronounced disease exacerbation, with diabetes developing very rapidly (100% incidence at day 84 post-primary immunization, mean onset time post-primary immunization 49 ± 16.2 (SD) days post-primary immunization). In contrast, the remaining three peptides showed disease incidence that was not significantly different from the expected spontaneous disease incidence.

[0119] The inventors next wanted to find the core sequence in B23-C20 that mediates the disease acceleration (APDA) effect of antigen primary immunization. One way to solve this problem is to prepare multiple peptides on the region of interest, offset by 1 amino acid. However, hundreds of potential options of any suitable length need to be tested in this method, which requires tens of thousands of mice to be tested in studies with the correct power. Therefore, the inventors used innovative steps to identify the driving region of B23-C20.

[0120] The beta cells are examined to specifically identify whether naturally occurring C-peptide fragments are present (or indeed whether C-peptide is always produced intact). From this, it can be determined what peptide species occur naturally from the B23-C20 region and may be drivers of disease in vivo.

[0121] The inventors purified different types of particles from human β cells (n=6 total samples) and examined the contents using mass spectrometry.

[0122] Table 1 below shows peptides in the region of interest (from B23 to C peptide) in human β-cell granules:

[0123]

[0124]

[0125] There are no peptides that contain residues from both the B chain and the C-peptide. The reason for this is that PC1 / 3 (proprotein convertase 1, also called prohormone convertase; and prohormone convertase 3, or neuroendocrine convertase 1, often abbreviated as PC1 / 3) is very efficient, cleaving the R:R in the sequence B23-C20 as part of the normal processing of the prohormone proinsulin into insulin and C-peptide. The first species in the sequence actually produced in vivo begins at proinsulin residue 32 (REA). Therefore, in some cases, the N-terminal R is retained. In addition, there are many subsequences of the C-peptide produced by beta cells. These can bind directly to HLA-DR3 / DQ2 without the need for conventional antigen processing, bind to T cells and drive disease.

[0126] Then, to further examine the translational extent of the mouse findings and to understand potential interactions between driver antigens / epitopes and HLA, the inventors aligned the relevant regions of mouse proinsulin-2 and human proinsulin ( Figure 4 a). The B23-C20 region of human proinsulin and mouse proinsulin-2 showed high homology, with 23 / 30 amino acids (77%) identical and an additional 3 / 30 amino acids (10%) similar between species. Next, to further pinpoint the region with major disease-inducing effects, six overlapping 14 / 15-mer peptides spanning the length of B23-C20 were generated. Sequences with N-terminal glutamine (Q) or glutamic acid (E) residues were avoided to avoid the formation of pyroglutamate, which would produce peptides with unpredictable properties. One of the sequences, RG-15, was highly insoluble in aqueous solution (although soluble in dimethyl sulfoxide). For HLA-DR3 and HLA-DQ2, it had the same predicted binding core as Me-15 ( Figure 4 b) Based on predicted binding affinities, none of the 14 / 15-mer peptides were classified as strong binders to HLA-DR3 or HLA-DQ2 by the prediction tools used. Importantly, the predicted binding cores and affinities of the human equivalents of the peptides were generally comparable to the mouse sequences ( Figure 4 c).

[0127] Primary immunization with the ME-15 (B29-C11) peptide adjuvant resulted in significantly higher disease incidence during follow-up than would be expected spontaneously. Disease was also significantly accelerated ( 1.0 mmol / l) compared to any other 14 / 15-mer peptide tested in this experiment. Figure 5af), thus strongly suggesting that the ME-15 peptide contains a minimal core amino acid sequence that binds HLA and interacts with immune cells to drive diabetes. RG-15 can also drive disease, but the peptide cannot be further excursed to the C-terminus (DA-15 and VD-14). Thus, the most terminal region of the C-peptide that includes a single R defines a region that contains a genetic driver resistant to type 1 diabetes generated on an HLA-DR3 / DQ2 background.

[0128] Another important data from preclinical models shows that this approach can indeed identify peptides with tolerogenic properties that have potential value in human disease on a DR3 / DQ2 background. The inventors used B23-C20 to induce disease and observed the tolerogenicity of ME-15 ( Figure 6 ). The tolerogenic effect of the ME-15 peptide was demonstrated. Thus, the peptide with the strongest disease-driving properties in this B:C junction region was also tolerogenic. This suggests that peptides with HFA-DR3 / DQ2 binding properties (such as ME-15) may be important in breaking immune tolerance and accelerating disease in the natural course of diabetes development; and may also have therapeutic potential if given early enough and via a tolerogenic (highly soluble, adjuvant-free) route.

[0129] The similarities between the human and mouse equivalents of the proinsulin region of interest suggest that these findings are highly relevant to human therapy. In support of this, human CD4 + T cell responses to the human proinsulin peptide B30-C13. This showed that HFA-DR3-DQ2 donors showed a greater response to B30-C13 than those who did not have this haplotype ( Figure 7 In addition, the response to B30-C13 was greater than the response to the control peptide C19-A3, which was previously shown to be HFA-DR4 restricted ((Arif S, et al. (2004) J Clin Invest 113(3):451-463).

[0130] Example 5

[0131] Five peptides were tested in HLA-DR3 / DQ2 positive patients with new onset type 1 diabetes (Figure 8). Inflammatory cytokine responses were observed for all peptides, indicating that these peptides are highly immunogenic in T1D. In addition, IFN-γ and IF-17 responses were observed, which are associated with cellular damage in the disease. There were also immunomodulatory (IF-10) responses in these subjects, indicating that these peptides are tolerable in the T1D disease setting.

[0132] All patents and literature references cited in this specification are incorporated herein by reference in their entirety.

[0133] Sequence Listing

[0134]

[0135] SEQUENCE LISTING <110> King's College London <120> Proinsulin peptide for type 1 diabetes <130> P536272GB <160> 49 <170> PatentIn version 3.5 <210> 1 <211> 16 <212> PRT <213> Homo sapiens <400> 1 Thr Arg Arg Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly 1 5 10 15 <210> 2 <211> 15 <212> PRT <213> Homo sapiens <400> 2 Lys Thr Arg Arg Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu 1 5 10 15 <210> 3 <211> 15 <212> PRT <213> Homo sapiens <400> 3 Arg Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly 1 5 10 15 <210> 4 <211> 18 <212> PRT <213> Homo sapiens <400> 4 Lys Thr Arg Arg Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu 1 5 10 15 Gly Gly <210> 5 <211> 30 <212> PRT <213> Homo sapiens <400> 5 Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp Leu Gln 1 5 10 15 Val Gly Gln Val Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser 20 25 30 <210> 6 <211> 30 <212> PRT <213> Mus musculus <400> 6 Gly Phe Phe Tyr Thr Pro Met Ser Arg Arg Glu Val Glu Asp Pro Gln 1 5 10 15 Val Ala Gln Leu Glu Leu Gly Gly Gly Pro Gly Ala Gly Asp 20 25 30 <210> 7 <211> 15 <212> PRT <213> Mus musculus <400> 7 Gly Phe Phe Tyr Thr Pro Met Ser Arg Arg Glu Val Glu Asp Pro 1 5 10 15 <210> 8 <211> 15 <212> PRT <213> House mouse (Mus musculus) <400> 8 Tyr Thr Pro Met Ser Arg Arg Glu Val Glu Asp Pro Gln Val Ala 1 5 10 15 <210> 9 <211> 15 <212> PRT <213> House mouse (Mus musculus) <400> 9 Met Ser Arg Arg Glu Val Glu Asp Pro Gln Val Ala Gln Leu Glu 1 5 10 15 <210> 10 <211> 15 <212> PRT <213> House mouse (Mus musculus) <400> 10 Arg Glu Val Glu Asp Pro Gln Val Ala Gln Leu Glu Leu Gly Gly 1 5 10 15 <210> 11 <211> 15 <212> PRT <213> House mouse (Mus musculus) <400> 11 Asp Pro Gln Val Ala Gln Leu Glu Leu Gly Gly Gly Pro Gly Ala 1 5 10 15 <210> 12 <211> 14 <212> PRT <213> House mouse (Mus musculus) <400> 12 Val Ala Gln Leu Glu Leu Gly Gly Gly Pro Gly Ala Gly Asp 1 5 10 <210> 13 <211> 15 <212> PRT <213> Homo sapiens <400> 13 Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp Leu 1 5 10 15 <210> 14 <211> 15 <212> PRT <213> Homo sapiens <400> 14 Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp Leu Gln Val Gly 1 5 10 15 <210> 15 <211> 15 <212> PRT <213> Homo sapiens <400> 15 Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro Gly Ala 1 5 10 15 <210> 16 <211> 14 <212> PRT <213> Homo sapiens <400> 16 Val Gly Gln Val Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser 1 5 10 <210> 17 <211> 9 <212> PRT <213> House mouse (Mus musculus) <400> 17 Phe Phe Tyr Thr Pro Met Ser Arg Arg 1 5 <210> 18 <211> 9 <212> PRT <213> House mouse (Mus musculus) <400> 18 Arg Glu Val Glu Asp Pro Gln Val Ala 1 5 <210> 19 <211> 9 <212> PRT <213> House mouse (Mus musculus) <400> 19 Glu Val Glu Asp Pro Gln Val Ala Gln 1 5 <210> 20 <211> 9 <212> PRT <213> House mouse (Mus musculus) <400> 20 Pro Gln Val Ala Gln Leu Glu Leu Gly 1 5 <210> twenty one <211> 9 <212> PRT <213> House mouse (Mus musculus) <400> twenty one Leu Glu Leu Gly Gly Gly Pro Gly Ala 1 5 <210> twenty two <211> 9 <212> PRT <213> House mouse (Mus musculus) <400> twenty two Met Ser Arg Arg Glu Val Glu Asp Pro 1 5 <210> 23 <211> 9 <212> PRT <213> Mus musculus <400> 23 Glu Asp Pro Gln Val Ala Gln Leu Glu 1 5 <210> 24 <211> 9 <212> PRT <213> Homo sapiens <400> 24 Phe Phe Tyr Thr Pro Lys Thr Arg Arg 1 5 <210> 25 <211> 9 <212> PRT <213> Homo sapiens <400> 25 Thr Arg Arg Glu Ala Glu Asp Leu Gln 1 5 <210> 26 <211> 9 <212> PRT <213> Homo sapiens <400> 26 Glu Ala Glu Asp Leu Gln Val Gly Gln 1 5 <210> 27 <211> 9 <212> PRT <213> Homo sapiens <400> 27 Leu Gln Val Gly Gln Val Glu Leu Gly 1 5 <210> 28 <211> 9 <212> PRT <213> Homo sapiens <400> 28 Val Glu Leu Gly Gly Gly Pro Gly Ala 1 5 <210> 29 <211> 9 <212> PRT <213> Homo sapiens <400> 29 Lys Thr Arg Arg Glu Ala Glu Asp Leu 1 5 <210> 30 <211> 9 <212> PRT <213> Homo sapiens <400> 30 Glu Asp Leu Gln Val Gly Gln Val Glu 1 5 <210> 31 <211> 25 <212> PRT <213> Homo sapiens <400> 31 Arg Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly 1 5 10 15 Pro Gly Ala Gly Ser Leu Gln Pro Leu 20 25 <210> 32 <211> 32 <212> PRT <213> Homo sapiens <400> 32 Arg Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly 1 5 10 15 Pro Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln 20 25 30 <210> 33 <211> 10 <212> PRT <213> Homo sapiens <400> 33 Glu Ala Glu Asp Leu Gln Val Gly Gln Val 1 5 10 <210> 34 <211> 11 <212> PRT <213> Homo sapiens <400> 34 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu 1 5 10 <210> 35 <211> 12 <212> PRT <213> Homo sapiens <400> 35 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu 1 5 10 <210> 36 <211> 13 <212> PRT <213> Homo sapiens <400> 36 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly 1 5 10 <210> 37 <211> 14 <212> PRT <213> Homo sapiens <400> 37 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly 1 5 10 <210> 38 <211> 20 <212> PRT <213> Homo sapiens <400> 38 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro 1 5 10 15 Gly Ala Gly Ser 20 <210> 39 <211> 22 <212> PRT <213> Homo sapiens <400> 39 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro 1 5 10 15 Gly Ala Gly Ser Leu Gln 20 <210> 40 <211> 23 <212> PRT <213> Homo sapiens <400> 40 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro 1 5 10 15 Gly Ala Gly Ser Leu Gln Pro 20 <210> 41 <211> 24 <212> PRT <213> Homo sapiens <400> 41 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro 1 5 10 15 Gly Ala Gly Ser Leu Gln Pro Leu 20 <210> 42 <211> 25 <212> PRT <213> Homo sapiens <400> 42 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro 1 5 10 15 Gly Ala Gly Ser Leu Gln Pro Leu Ala 20 25 <210> 43 <211> 26 <212> PRT <213> Homo sapiens <400> 43 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro 1 5 10 15 Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu 20 25 <210> 44 <211> 27 <212> PRT <213> Homo sapiens <400> 44 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro 1 5 10 15 Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu 20 25 <210> 45 <211> 28 <212> PRT <213> Homo sapiens <400> 45 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro 1 5 10 15 Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly 20 25 <210> 46 <211> 29 <212> PRT <213> Homo sapiens <400> 46 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro 1 5 10 15 Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser 20 25 <210> 47 <211> 30 <212> PRT <213> Homo sapiens <400> 47 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro 1 5 10 15 Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu 20 25 30 <210> 48 <211> 31 <212> PRT <213> Homo sapiens <400> 48 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro 1 5 10 15 Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln 20 25 30 <210> 49 <211> 18 <212> PRT <213> Homo sapiens <400> 49 Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg Gly 1 5 10 15 Ile Val

Claims

1. A peptide, the amino acid sequence of which is shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO:

5.

2. The peptide according to claim 1, wherein the amino acid sequence of the peptide is shown in SEQ ID NO:

1.

3. A peptide combination comprising two or more peptides according to claim 1.

4. A peptide combination comprising: A peptide having an amino acid sequence as shown in SEQ ID NO: 1; A peptide having an amino acid sequence as shown in SEQ ID NO: 2; A peptide having an amino acid sequence as shown in SEQ ID NO: 3; A peptide having an amino acid sequence as shown in SEQ ID NO:4; and A peptide having the amino acid sequence shown in SEQ ID NO:

5.

5. A pharmaceutically acceptable composition comprising the peptide according to claim 1 or the peptide combination according to claim 3 or 4 and one or more pharmaceutically acceptable excipients.

6. The pharmaceutically acceptable composition according to claim 5, further comprising cysteine.

7. The pharmaceutically acceptable composition according to claim 5, in, The peptide or the combination of peptides is coated on the nanoparticles.

8. The pharmaceutically acceptable composition according to claim 7, in, The peptide or the combination of peptides is coated on the nanoparticle by binding to the MHC complex attached to the nanoparticle.

9. The pharmaceutically acceptable composition according to any one of claims 5 to 8, in, The compositions are formulated for delivery by parenteral, oral or topical routes, including intravenous, intradermal, intramuscular, subcutaneous, intraperitoneal, nasal, oral or epidermal routes.

10. The pharmaceutically acceptable composition according to claim 9, in, The composition is formulated for delivery via the intradermal route.

11. A kit comprising the peptide according to claim 1 or the peptide combination according to claim 3 or 4.

12. A nucleic acid molecule, the nucleotide sequence of which is a nucleotide sequence encoding the peptide according to claim 1.

13. A vector expressing any one of the peptides described in claim 1 and a mixture thereof.

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