Proinsulin Peptide for Type 1 Diabetes

Abstract

The present invention relates to peptides that can be used for the treatment or prevention of type 1 diabetes (T1D), particularly in patients with the DR3-DQ2 haplotype. The present invention also relates to methods for determining diagnostic or therapeutic efficacy, methods for utilizing the peptides, and methods for identifying T1D-associated antigenic drivers, identifying subjects as at high risk for T1D, and identifying patients as 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 for the treatment or prevention of type 1 diabetes (T1D).

Background Art

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

[0003] T1D is characterized by absolute insulin deficiency, making the patient's survival dependent on exogenous insulin. Before the acute clinical onset of T1D with symptoms of hyperglycemia, there is a long asymptomatic pre-clinical period during which insulin-producing β-cells are gradually destroyed.

[0004] Since β-cells do not regenerate, once the disease develops, treatment by injection of synthetic insulin is required throughout life. Once established, diabetes becomes a significant burden for the patient, the patient's family, and society. Although the latest dosages, formulations, and delivery systems of insulin can maintain blood glucose within a reasonable range, over the years, the complications of the disease will inevitably occur. The most common and severe complications of diabetes are kidney failure, blindness, and loss of neurological function. The life expectancy of diabetic patients is shortened by an average of 10 years. Against this background, it is important to explore new means for treating or preventing T1D.

[0005] Like all autoimmune diseases, T1D results from inappropriate activation of the immune system in response to self - antigen encounter, particularly by short peptide sequences presented to the immune system by specific HLA molecules. Although the condition is increasingly clinically manageable, it still negatively impacts quality of life and average life expectancy.

[0006] The cause of the loss of self - tolerance in type 1 diabetes continues to be debated, 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 found in approximately 34% of patients with type 1 diabetes (Erlich H et al. (2008) Diabetes 57(4):1084 - 1092) and thus represents a major, identifiable disease cohort. Considering the antigen - presenting properties of HLA molecules, the risk of diabetes associated with HLA - DR3 - DQ2 is hypothesized to be related to the selective presentation of peptide epitopes of potential diabetes - inducing self - antigens. Therefore, the identification of these self - antigens and specific disease - determining regions is an important step in understanding the disease etiology.

[0007] Attempts to identify disease-related epitopes in clinical studies generally involve eluting or predicting peptides bound to candidate HLA molecules on antigen-presenting cells and / or in vitro testing of T cell responses in subjects with type 1 diabetes. This approach has yielded multiple potentially relevant HLA-DR3-DQ2-restricted epitopes from several pancreatic 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 thought to be involved in the early stages of type 1 diabetes in both humans and mice (Narendran P, et al. (2003) Autoimmun Rev 2(4):204-210), has not been identified as a strong source of diabetes-related epitopes presented by HLA-DR3-DQ2. However, a recent study has highlighted that regions of the proinsulin C-peptide generate HLA-DQ2-restricted CD4 + T cell responses in subjects with type 1 diabetes (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., a cohort of T cells focused on a single peptide region of a self-antigen. Their importance lies in their having a greater impact on the disease. As a therapeutic consequence, if driver T cells can be inactivated, the effect on disease control will be better than using subdominant targets / T cells, which are the second option. Driver T cells and the related peptides derived from the antigen can be very difficult to find in human diseases. As the disease progresses over time, many subdominant T cell clones and targets can become involved, potentially masking the driver T cell / target.

[0009] The present invention addresses methods for identifying molecular targets of driver T cells associated with human type 1 diabetes. By identifying these peptides, peptide immunotherapy may be able to neutralize the most important disease drivers. This is particularly important and effective in individuals in whom autoimmunity has not yet been initiated.

[0010] The inventors have utilized an in vivo approach to epitope discovery using diabetes-susceptible HLA transgenic mice. The disease relevance of antigens and peptides was investigated by their ability to promote the development of autoimmune diabetes in susceptible animals. HLA-DR3-DQ2 transgenic mice used in the art do not develop diabetes spontaneously (de Kauwe AL et al. (2009) J Immunol 182(12):7440 - 7450). To promote this disease phenotype, the inventors generated a novel HLA transgenic mouse expressing the high-risk genes for type 1 diabetes (DRB1*03:01 - DQA1*05:01 - DQB1*02:01) on a C57BL / 6 background and having human CD80β cell-specific expression of the costimulatory molecule CD80 (B7.1) under the rat insulin promoter (RIP) and human CD4. The application of adjuvant antigen priming as a mechanism to accelerate and expand the diabetes penetrance indicates the importance of specific islet antigens as diabetes-inducing drivers in the HLA-DR3-DQ2 background. The inventors highlight a novel HLA-based approach for identifying disease-associated antigenic drivers in vivo and have identified novel unnatural peptides that can be used to induce tolerance in peptide immunotherapy. The inventors have pointed out the diabetogenic action of proinsulin on the region starting at the C-terminus of the B-chain, which has not been reported previously.

[0011] Peptides derived from the B23-C20 region of murine or human proinsulin of the present invention have not been previously identified as restricted by either HLA-DR3 or HLA-DQ2. Furthermore, no previous study has directly demonstrated the diabetogenicity of this region in association with any haplotype, and in fact, an approach to investigate the potential diabetogenicity and relative dominance of antigens and epitopes using antigen adjuvant priming against a selected HLA background has not been proposed.

[0012] There have been few studies examining T cell reactivity against the B-C junction, and even fewer for HLA-DR3 / DQ2 restricted responses. Semana et al. have previously found HLA-DR restricted CD4 + T cell responses to proinsulin peptide C3-16 in diabetic patients, but these have not been analyzed for 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), the long B11-C24 peptide is frequently recognized (Durinovic-Bello I, et al. (2002) J Autoimmun 18(1):55-66). So et al. found CD4 +T cell responses were identified and were HLA-DQ8 restricted (So M, et al. (2018) Proc Natl Acad Sci USA 115(42):10732-10737.). All HLA-DQ2 restricted peptides capable of generating T cell responses were 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 out of 10 subjects. The subjects were HLA-DR3 or HLA-DR4, or heterozygous for these, and the responses were not associated with any specific haplotype. Raju et al. (Raju R et al. (1997) Hum Immunol 58:21-29) tested T cell responses to each of 10 overlapping peptides of proinsulin in transgenic mice for HLA-DQ8 after immunization with the same peptide. DQ8 restricted responses were seen to B1-B24 and B20-C10 in these mice. These mice did not have diabetes and this study provided no insight into any disease.

[0013] None of the available prior art discloses the claimed peptides having significant therapeutic potential, particularly in the DR3-DQ2 haplotype.

Summary of the Invention

Problems to be Solved by the Invention

[0014] The present invention provides peptides that can be used for the treatment or prevention of type 1 diabetes (T1D), particularly in patients having the DR3-DQ2 haplotype.

Means for Solving the Problems

[0015] In a first aspect of the present 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.

[0016] SEQ ID NO: 1 is a fragment of PI that includes residues B30 - C13.

[0017] SEQ ID NO: 2 is a fragment of PI that includes residues B29 - C11.

[0018] SEQ ID NO: 3 is a fragment of PI that includes residues C -1 - C14.

[0019] SEQ ID NO: 4 is a fragment of PI that includes residues B29 - C14.

[0020] SEQ ID NO: 5 is a fragment of PI that includes residues B23 - C20.

[0021] The inventors have surprisingly discovered that these novel non - natural peptides can be used to induce tolerance in peptide immunotherapy. The inventors have pointed out the diabetes - inducing effect of proinsulin on this region starting at the C - terminus of the B - chain, which has not been reported previously.

[0022] The peptide is not naturally present due to the presence of at least one R derived from the R:R motif that is naturally cleaved by PC1 / 3 (proprotein convertase 1, which is also known as prohormone convertase and prohormone convertase 3, or often abbreviated as PC1 / 3, neuroendocrine convertase 1). PC1 / 3 cleaves the R:R motif between the B - chain and the C - chain as part of the normal processing of prohormone proinsulin to insulin and C - peptide.

[0023] These peptides have not been previously identified as being restricted by either HLA - DR3 or HLA - DQ2.

[0024] As used herein, the term "peptide" refers to any peptide containing peptide bonds or modified peptide bonds, i.e., amino acids linked to each other by peptide isosteres. Peptides generally contain naturally occurring amino acids, but may also contain amino acid sequences modified either by natural processes such as post-translational processing or by chemical modification techniques well known in the art. Such modifications are fully described in basic texts. Modifications can occur at any location on the peptide, including the peptide backbone, amino acid side chains, and amino or carboxyl termini. It will be understood that the same type of modification may be present to the same or varying degrees at several sites on a given peptide. Also, a given peptide may contain multiple types of modifications.

[0025] 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 certain embodiments, 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 further embodiments, the peptide has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0026] 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, the amino acid change is conservative, that is, the amino acid is preferably changed to a different amino acid having similar biochemical properties (e.g., charge, hydrophobicity, and size).

[0027] Preferably, the peptides constituting the peptide combination are isolated peptides. The term "isolated" means that the peptide has been removed from 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 of the materials that coexist in a natural system is isolated. Such a peptide may be part of a vector and / or the peptide may be part of a composition, and still is isolated in that such a vector or composition is not part of its natural environment.

[0028] In a preferred embodiment, a combination of peptides comprising two or more of the above peptides is provided.

[0029] A second aspect of the present invention relates to a pharmaceutically acceptable composition comprising the peptide or peptide combination of the present invention and one or more pharmaceutically acceptable excipients.

[0030] Preferably, a pharmaceutical composition containing cysteine. The presence of free cysteine in the composition stabilizes any tendency for the peptides of the present invention to form intermolecular disulfide bonds and thus precipitate. The pharmaceutical composition may contain 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, and most preferably 2.5 mg of L-cysteine per 2 mg of peptide.

[0031] The pharmaceutical composition can be for use in humans or animals in human and veterinary medicine and will typically contain one or more suitable excipients. Excipients acceptable for therapeutic use are well known in the pharmaceutical field and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985). The choice of pharmaceutical excipient can be selected in relation to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition can contain, as excipients or in addition thereto, any suitable binder, lubricant, suspending agent, coating agent, or solubilizing agent.

[0032] 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 can also be used.

[0033] Depending on the different delivery systems, different composition / formulation requirements may exist. As an example, the pharmaceutical composition of the present invention may be formulated to be delivered parenterally, in which case the composition is formulated in an injectable form for delivery by, for example, intravenous, intradermal, intramuscular, subcutaneous, or intraperitoneal routes. In the case of parenteral administration, the composition is most preferably used in the form of a sterile aqueous solution that may contain other substances, such as salts or monosaccharides sufficient to render the solution isotonic with blood. The composition may also be formulated to be administered by oral or topical routes, including nasal, oral, or transdermal. Preferably, the composition is formulated to be delivered by the intradermal route.

[0034] As an intradermal administration route, those using direct attachment of a fluid, solid, or other dosage form to the skin are included, such as any skin access means, for example, a micro-needle-based injection and infusion system (or other means that accurately target the intradermal space), needleless or needle-free ballistic injection of a liquid or powder into the intradermal space, Mantoux-type intradermal injection, enhanced iontophoresis via a microdevice, and the use of a patch for attachment of a composition to the skin.

[0035] Typically, the actual dosage amount most suitable for an individual subject is determined by a physician, and the dosage amount will vary depending on the specific patient's disease, age, weight, and response. A suitable dosage amount for humans can be determined by those skilled in the art, for example, using normalization of body surface area (BSA). For example, the pharmaceutical composition may contain from about 0.1 μg to 15 mg of total peptide per single dose, preferably from 1 μg to 12 mg of total peptide per single dose. In a preferred embodiment, 240 μg of total peptide (a dosage of 1 μg normalized for a 60 kg adult human in terms of BSA) is administered per single dose. In another preferred embodiment, 12 mg of total peptide is administered per single dose. When two or more peptides are administered, preferably, the peptides are present in an equimolar ratio.

[0036] In a preferred embodiment, the pharmaceutical composition of the present invention is administered at least once a month, preferably at a maximum of 12 administrations, at least once a month.

[0037] The pharmaceutical composition may also contain a tolerance-promoting adjuvant and / or tolerance-promoting cells. Examples of tolerance-promoting adjuvants include IL-10, recombinant cholera toxin B subunit (rCTB), ligands of Toll-like receptor 2, and biological agents and monoclonal antibodies that regulate the immune response, such as anti-CD3, costimulatory blockers, and immune checkpoint regulators, which can be co-administered with the peptide combination. Examples of tolerance-promoting cells 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 of the peptides of the present invention are conjugated to the surface of dendritic cells treated with vitamin D3 or its analogs.

[0038] The peptide or peptide combination of the present invention can be coated on nanoparticles within a pharmaceutically acceptable composition. The nanoparticles can be carbon-based nanoparticles, ceramic nanoparticles, metal nanoparticles, semiconductor nanoparticles, polymer nanoparticles, lipid-based nanoparticles, or mixtures thereof.

[0039] In one embodiment, the peptide or peptide combination is coated on the nanoparticles by binding to the MHC complex bound to the nanoparticles.

[0040] The third aspect of the present invention relates to a pharmaceutically acceptable composition of the present invention for use in therapy.

[0041] 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).

[0042] When T1D is "treated", this means that one or more clinical symptoms of T1D are alleviated. It does not mean that the symptoms of T1D are completely improved and as a result the symptoms no longer exist in the patient, although in some ways this may be the case. As a result of "treatment", one or more of the T1D symptoms become less severe than before treatment.

[0043] Preferably, the pharmaceutical composition of the present invention is for use in the treatment or prevention of (T1D) in a patient having the DR3 haplotype. Preferably, the patient has the DR3-DQ2 haplotype. The inventors have surprisingly and specifically demonstrated the importance of the claimed peptides as diabetes-inducing drivers against the HLA-DR3-DQ2 background.

[0044] A fifth aspect of the present invention relates to a pharmaceutically acceptable composition of the present invention for use in the manufacture of a medicament for the treatment or prevention of type 1 diabetes (T1D).

[0045] A sixth aspect of the present invention is a method for the treatment or prevention of type 1 diabetes (T1D), wherein the pharmaceutically acceptable composition of the present invention is administered to a patient having T1D or a non-diabetic individual identified as being at high risk of T1D.

[0046] Preferably, the pharmaceutically acceptable composition of the present invention is administered to a patient in whom β-cell mass remains.

[0047] A seventh aspect of the present invention relates to a kit for the treatment or prevention of type 1 diabetes (T1D) comprising the peptide or combination of peptides of the present invention.

[0048] An eighth aspect of the present invention relates to a nucleotide sequence encoding the peptide of the present invention.

[0049] A ninth aspect of the present invention relates to a vector expressing any one of the peptides of the present invention.

[0050] The vector can be any suitable vector for expressing the peptide of the present invention, including viral vectors and non-viral vectors. Viral vectors include parvovirus, adenovirus, retrovirus, lentivirus, or herpes simplex virus. The parvovirus can be adeno-associated virus (AAV). The vector is preferably a recombinant adeno-associated virus (rAAV) vector or a lentivirus vector. More preferably, the vector is an rAAV vector.

[0051] 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.

[0052] Accordingly, the present invention provides a parvovirus of an animal, particularly a dependovirus such as infectious human AAV or simian AAV, and a gene delivery vector based on the same (e.g., the animal parvovirus genome) for use as a vector for introducing and / or expressing the peptide of the present invention in mammalian cells. Accordingly, the term "parvovirus" as used herein encompasses dependoviruses such as any type of AAV.

[0053] The tenth aspect of the present invention relates to a genetically modified animal that spontaneously develops T1D, and the animal has a DR3 haplotype, preferably a DR3-DQ2 haplotype. Preferably, the animal is a non-human mammal, particularly a primate. Alternatively, the animal may be a rodent, particularly a mouse, or a dog, a cat, a sheep, or a pig. Preferably, the animal contains cells that express human CD80 under the rat insulin promoter (RIP).

[0054] The eleventh aspect of the present invention is a method for identifying a T1D-related antigen driver, comprising priming the above-mentioned animal with a peptide derived from the B23-C20 region of proinsulin, and identifying the peptide as an antigen driver when the progression of the disease is accelerated.

[0055] In one embodiment, the progression of the disease is considered to be accelerated when it progresses 50% faster than when priming is not performed. Preferably, the peptide is selected from peptides 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 as described above. The peptide can be administered at a dosage of up to 1 mg per peptide.

[0056] The twelfth aspect of the present invention is a method for determining diagnostic or therapeutic efficacy, comprising: (a) providing CD4 lymphocytes derived from an individual having T1D or suspected of being T1D-sensitive; (b) providing a population of antigen-presenting cells (APCs) having on their surface class II MHC molecules of the same allele as those expressed by the individual, wherein the population of APCs is in contact with the peptide or combination of peptides of the present invention and class II MHC molecules bound to one or more of the peptides of the present invention; or (c) providing a soluble peptide-HLA multimer reagent comprising class II MHC molecules of the same allele as those expressed by the individual, wherein the MHC molecules are bound to one or more peptides of the combination of peptides of the present invention; (d) contacting the population of APCs of (b) or the peptide-HLA multimer of (c) with the CD4 lymphocytes of (a); and (e) determining whether the CD4 lymphocytes recognize class II MHC-binding peptides as an indicator of whether the individual has T1D or is sensitive to T1D.

[0057] Such APCs can be B lymphocytes, monocytes, macrophages, or dendritic cells, or whole peripheral blood mononuclear cells (PBMCs). The APCs can also be immortalized cell lines derived from B lymphocytes, monocytes, macrophages, or dendritic cells. When the subject is human, since human T cells can express class II MHC molecules, the APCs can also be T cells. The method further comprises administering to the individual the peptide or combination of peptides of the present invention when CD4 lymphocytes recognize class II MHC-binding peptides.

[0058] The 13th and 14th aspects of the present invention relate to a method for identifying a subject as being at high risk of T1D and a method for identifying a patient as being suitable for T1D treatment, respectively. The method comprises determining whether the subject or patient has the DR3 haplotype, preferably the DR3-DQ2 haplotype, when an immune response to any one of the peptides or combinations of peptides of the present invention is detected.

[0059] One skilled in the art would be familiar with methods for measuring the immune response by CD4 T cells to the peptides of the present invention. Examples include enzyme-linked immunosorbent spot assay (ELISPOT), peptide-HLA tetramer assay, or detection of proliferating or cytokine-producing CD4 T cells following in vitro stimulation assay.

[0060] The 15th aspect of the present invention relates to a method for identifying a subject as either a responder to immunotherapy or in need of retreatment with the peptides or combinations of peptides of the present invention. The method comprises determining whether an immune response to any one of the peptides or combinations of peptides of the present invention is detected.

[0061] Here too, the immune response can be detected, for example, by ELISPOT, peptide-HLA tetramer assay, or detection of proliferating or cytokine-producing CD4 T cells following in vitro stimulation assay. If there is no immune response, or if the immune response of the subject is actually decreased, the subject is responsive to immunotherapy. If the immune response of the subject has increased, they may require further treatment with the peptides or combinations of peptides of the invention.

[0062] One of ordinary skill in the art will understand that all aspects of the invention are equally applicable to all other aspects of the invention, regardless of whether they relate, for example, to peptides, combinations of peptides, their use, pharmaceutically acceptable compositions, or methods of treatment. In particular, for example, aspects of peptides may have been described in more detail than other aspects of the invention, such as the use of peptides. However, one of ordinary skill in the art will understand that when more detailed information is provided for a particular aspect of the invention, this information is generally equally applicable to other aspects of the invention.

[0063] The invention will now be described in detail by way of example only with reference to the following figures. BRIEF DESCRIPTION OF THE DRAWINGS

[0064]

Figure 1

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Figure 8A

Figure 8B

[0065] Example 1 Materials and methods

[0066] Animals HLA-DR3-DQ2 transgenic (B6.hCD4.DR3-DQ2.MHCII - / - mice (previously described (de Kauwe AL et al. (2009) J Immunol 182(12):7440 - 7450), obtained from J. McCluskey) were used. HLA-DR3-DQ2 + huCD4 + IA / IE - / - RIP.B7.1 + To obtain RIP-B7.1 transgenic animals (B6.Cg-Tg(Ins2-CD80)3B7Flv / Orl, EMMA, Orleans, France; ID 00216), they were mated with RIP-B7.1-transgenic animals. B6.129S2-H2-Abl tmlGru Tg(HLA-DRA / H2-Ea, HLA-DRB1*0401 / H2-Eb)1Kito mice were mated with B6.Cg-Tg(Ins2-CD80)3B7Flv / Orl to obtain DR4xRIP-B7.1 mice, which have been previously described (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, on a 12-hour light / dark cycle, with free access to food and water. Experiments were conducted in accordance with UK Home Office regulations under a project license held by M. Peakman. All work was subject to review and was locally approved by the Guy's Animal Welfare and Ethical Review Board (AWERB).

[0067] Priming antigen All mouse proinsulin-2 peptides were custom-made with a purity exceeding 95% by either Almac (Edinburgh, UK) or GLS Biochem (Shanghai, China). 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). Predicted HLA binding cores and affinities were generated using the NetMHCIIpan prediction method on the online IEDB analysis resource (http: / / tools.immuneepitope.org / mhcii / ).

[0068] Induction and monitoring of diabetes All mice were regularly monitored for non - induced glycosuria using Diastix strips (Bayer, Basel, Switzerland). Some mice were monitored weekly for hyperglycemia from 6 - 7 weeks of age by minimal tail vein puncture in the alternating caudal part and analysis using a OneTouch Verio meter and strips (Lifescan, High Wycombe, UK) to closely monitor the progression to spontaneous diabetes. Mice were considered diabetic after a blood glucose reading exceeded 16.7 mmol / L (300 mg / dL) in addition to confirmed glycosuria. For disease acceleration experiments, animals were assigned to groups of equal or similar size as shown to achieve an equal spread of age and sex for each group. Mice (6 - 14 weeks old) were primed subcutaneously (s.c.) at the base of the tail with 100 μg of peptide or protein in TiterMax Gold adjuvant (TiterMax, Norcross, GA, USA) and received a second dose by subcutaneous injection into the inguinal area on day 14. Mice received an intraperitoneal (i.p.) injection of 200 ng of pertussis toxin (Sigma, Poole, UK) in PBS on days 0 and 1 or 2. Mice were then monitored weekly for hyperglycemia and glycosuria. In experiments to examine tolerance induction, mice received subcutaneous injections of 10 μg of peptide in aqueous solution at 3 - 4 day intervals for a total of 6 injections. The tolerance regimen was started 1 week before adjuvant antigen priming. As soon as diabetes was detected, mice were humanely euthanized in accordance with ethical approval.

[0069] Histology The pancreas embedded in OCT compound (Cellpath, Newtown, UK) was frozen in liquid nitrogen-cooled isopentane (Sigma). 10-μm sections were fixed in acetone and then first stained with rabbit anti-mouse insulin (Abeam), detected with Vector Impress anti-rabbit AP (Vector Labs, Peterborough, UK), and developed with Vector ImmPress Red. 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 obtained from eBioscience / ThermoFisher (Altrincham, UK)) and detected using ABC reagent and DAB solution obtained from Vector Labs. After staining the nuclei with hematoxylin (Sigma), the slides were mounted with VectaMount (Vector Labs). Images were acquired with a Zeiss Axiovert A1 microscope using Zen software. No cropping or alteration of the images was used.

[0070] Autoantibody ELISA Proinsulin-2 peptide or recombinant human GAD65 was coated onto Maxisorp plates (Nunc, Roskilde, Denmark) in ELISA coating buffer (eBioscience). Diluted serum (in 5% BSA in PBS, Sigma) or controls (mouse IgG2a anti-insulin clone ICBTACLS and isotype obtained from ThermoFisher, mouse IgG1 anti-GAD65 clone N-GAD65 and isotype obtained from Biolegend) were incubated at room temperature for 2 hours and then detected with biotin-anti-mouse IgG, streptavidin-HRP, and high-sensitivity TMB solution (all obtained from eBioscience) and read at 450 nm. The data were normalized using the titration concentrations (1 - 1000 ng / mL) of the relevant control antibodies in each plate, and arbitrary units were assigned to the serum antibody levels.

[0071] Analysis of C-Peptide Fragments Released from Human β-Cells To investigate the full capacity of C-peptide fragments available in vivo, the inventors analyzed (i) secretion into the supernatant by pancreatic islet cells and (ii) secretion into intracellular granules. (i) Secretion Human islets were washed with 1× DPBS containing 0.5 mM EDTA and pretreated for 30 minutes at 37 °C and 5% CO2 in Krebs-Ringer buffer containing 2.5 mM glucose. The buffer was then exchanged for Krebs-Ringer buffer containing either 2.5 mM or 25 mM glucose, and the islets were incubated for 30 minutes at 37 °C and 5% CO2. Secretions in the supernatant were collected under each condition and centrifuged at 400 xg for 5 minutes at 4 °C to pellet any remaining cells. The secreted supernatant was collected, and proteins / fragments were isolated and purified by C18 reverse-phase resin purification. (ii) Intracellular Granules Islets were washed three times at room temperature for 3 minutes at 300 x g in 1x DPBS containing 0.5 mM EDTA. Islet cells were taken up in Accutase (preheated to 37°C) at a concentration of 0.5 mL / 1,000 IEQ (islet equivalent) and incubated in a 37°C water bath for 10 minutes. The tube was inverted every minute and further pipetted up and down 5 times with a 5 mL serological pipette at 5 and 10 minutes to obtain a single cell suspension. The suspension was filtered through a 35 μm nylon mesh and washed at 300 x g for 3 minutes in 1x DPBS 0.5 mM EDTA. The cells were resuspended in 2 mL of 1x DBPS 0.5 mM EDTA. A Balch homogenizer (Isobiotech, Germany) with a pore size of 14 μm was used to disrupt the cell membrane while keeping the intracellular components intact. The single cell lysate was passed through the homogenizer 10 times. The homogenized cells were centrifuged at 1,000 x g for 15 minutes at 4°C to release and collect the intracellular contents. The pellet was resuspended in 1x DPBS 0.5 mM EDTA and centrifuged again. The intracellular contents in the supernatant were collected again, pooled, and then centrifuged at 5,000 x g for 15 minutes at 4°C to obtain a pellet containing a high-density intracellular compartment enriched in crinosomes. The supernatant obtained from this spin was centrifuged again at 20,000 x g for 45 minutes at 4°C to obtain a pellet containing a low-density intracellular compartment enriched in insulin secretory granules. Both the pellet enriched in crinosomes and the pellet enriched in granules were taken up in 1x DPBS, snap frozen, and stored at -80°C. They were thawed on ice and the contents of these intracellular compartments were released by the addition of a protease inhibitor (cOmplete™, Roche, Switzerland), and this freeze-thaw cycle was repeated a total of 5 times. (i) The secreted supernatant and (ii) the proteins and fragments obtained in the intracellular granules were moistened with 50% acetonitrile (ACN) / 50% ddH2O, equilibrated with 0.1% trifluoroacetic acid (TFA) / 99.9% ddH2O, rinsed with 2% ACN / 0.1% TFA / 97.9% ddH2O, purified by C18 reverse-phase resin (Waters, US), and then the sample (adjusted to a final concentration of 0.2% TFA) was loaded onto a C18 column. The sample was washed with 2% ACN / 0.1% TFA / 97.9% ddH2O, and the proteins and protein fragments were eluted with 80% ACN / 0.1% TFA / 19.9% ddH2O and dried using a vacuum centrifugal concentrator. The dried sample was resuspended in 2% ACN / 0.1% formic acid / 97.9% ddH2O. Chromatographic separation was performed using an Ultimate 3000 NanoLC system (ThermoFisher Scientific, UK). Peptides were separated by reverse-phase chromatography on a 75 μm × 50 cm C18 column using a linear liquid chromatography gradient of water in 0.1% formic acid (A) and 80% acetonitrile in 0.1% formic acid (B). The gradient was delivered at a flow rate of 300 nL / m from 2% B to 50% B over 65 minutes to elute the peptides. The eluate was ionized by electrospray ionization using an Orbitrap-Fusion-Lumos (ThermoFisher Scientific, UK) operating with Xcalibur v4.1. The instrument was programmed to acquire data using a "universal" method by defining a 3-second cycle time between full MS scans and MS / MS fragmentation. 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 / ).

[0072] ELISPOT assay The ELISPOT assay was performed using human peripheral blood mononuclear cells (PBMCs) isolated from fresh heparinized blood. Informed consent was obtained from all participants. 1×10 6 PBMCs evenly divided across three wells of a 96-well plate were stimulated for 48 hours with human proinsulin B30-C13 (SEQ ID NO: 1), C19-A3 (SEQ ID NO: 49), B23-C20 (SEQ ID NO: 5), B29-C11 (SEQ ID NO: 2), B29-C14 (SEQ ID NO: 4), or C -1 -C14 (SEQ ID NO: 3) peptides (all used at 10 μg / mL, custom manufactured by ThermoFisher Scientific), or diluent alone. The cytokine secretion assay was performed using the ELISPOT kits for IFN-γ, IL-17, and IL-10 (U-CyTech) according to the manufacturer's instructions, and the plates were analyzed using an automated ELISPOT Bioreader 6000 (Bio-Sys) and the associated software. The data was represented as the total number of spots in triplicate wells after peptide stimulation divided by the total number of spots in the control wells with diluent alone (stimulation index [SI]), or responder CD4 T cells / 10 6 PBMCs.

[0073] Statistical analysis All analyses were performed using the appropriate tests indicated in GraphPad Prism8 software.

[0074] Example 2 Spontaneous diabetes in DR3DQ2xRIP-B7.1 mice

[0075] Unlike the DR4xRIP-B7.1 model, which does not show spontaneous pancreatitis or diabetes as previously described by the inventors (Verhagen J, et al. (2018) Sci Rep 8(1):14106), DR3DQ2xRIP-B7.1 mice spontaneously develop diabetes (Figure 1a). Both models show relatively high baseline blood glucose levels at all ages, but only DR3DQ2xRIP-B7.1 mice develop levels above 16.7 mmol / L in addition to glycosuria. By 35 weeks of age, 46% (26 out of 56) of all monitored animals developed autoimmune diabetes. In male and female animals, the disease incidence (16 / 32 vs 10 / 24, respectively) and mean age of onset (24.2 weeks ± 7.3 (standard deviation) vs 24.8 ± 8.5, respectively) were similar. No obvious signs of other immune-mediated conditions evidenced by splenomegaly, cachexia, lethargy, or skin / eye abnormalities were detected. All mice with diabetes showed severe immune infiltration in the islets. This infiltration was highly diverse, with both lymphoid and myeloid cells expressing CD4, CD8, B220, CD11b, and CD11c and being abundantly found in all islets (Figure 1b). Ly6G +Granulocytes were found in smaller numbers in some islets. No significant immune cell infiltration was observed in non-diabetic mice examined histologically by the inventors at 6-8 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), except for one 6-week-old male in which moderate infiltration of some islets was detected. This suggests that the insulitis phenotype is disease-associated, leading to rapid progression to diabetes and being strongly associated with the disease. Since the onset of type 1 diabetes in human HLA-DR3-DQ2 patients is characterized in the majority by the presence of autoantibodies against GAD65 and insulin, mouse sera were tested for antibodies against a mouse 30-mer peptide spanning the length of mouse proinsulin-2, the isoform most similar to human proinsulin, and for antibodies against human GAD65 with >95% sequence homology to the mouse counterpart. As shown in FIGS. 1c-1d, when comparing groups of mice at increasing weeks of age, there was a significant increase in the level of anti-proinsulin-2 antibodies, but no increase in the level of anti-GAD65 antibodies.

[0076] Example 3 Proinsulin is a diabetes-inducing antigen in DR3DQ2xRIP-B7.1 mice

[0077] The spontaneous development of diabetes in mice bearing high-risk transgenic HLA suggests that an autoimmune process is central to disease development, thereby prompting the inventors to address the question of whether specific autoantigens are disease drivers. If an antigen has "driver" properties, the hypothesis that priming animals with an adjuvant against a candidate molecule would accelerate disease progression was tested. Thus, mice were primed with either individual 30-mer peptides overlapping and spanning the length of murine proinsulin-2, recombinant human GAD65, the intracellular region of 377 amino acids of human islet antigen-2 (IA-2), or PBS alone in TiterMax Gold adjuvant (Figures 2a-2f). Importantly, of these conditions, only priming with the proinsulin peptide clearly promoted the development of diabetes, increasing the incidence above that observed with control stimulation or spontaneous development. This was an unexpected finding. Previous studies have primarily focused on GAD65 and IA-2 as CD4 from subjects with type 1 diabetes using HLA-DR3 and HLA-DQ2 restriction elements +It was identified as a target pancreatic antigen recognized by T cells or T cell clones (Di Lorenzo TP, et al. (2007) Clin Exp Immunol 148(1):1-16.). Furthermore, in the Environmental Determinants of Diabetes in the Young study, it has been suggested that anti-GAD65 autoantibodies typically first appear in disease-susceptible individuals with the HLA-DR3-DQ2 haplotype. In contrast, anti-insulin autoantibodies usually first appear in at-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, each characterized by a different pathological process involving loss of immune tolerance to GAD65 and insulin, respectively. Given this, it was predicted that GAD65 might be the most potent antigenic driver of disease in the new DR3DQ2xRIP-B7.1 model, but this was found not to be the case.

[0078] By the end of the 20-week experiment, all mice (10 / 10) primed with proinsulin peptide in adjuvant had developed diabetes (mean onset time was 77 ± 38.6 (standard deviation) days after priming). Pancreatic immune infiltration did not differ with respect to intensity or diversity in these antigen-challenged animals compared to that observed in mice with spontaneous diabetes (not shown). These findings indicate the importance of immune recognition of proinsulin in the HLA-DR3-DQ2 context as an event causing diabetes in this model.

[0079] Example 4 Specific regions of proinsulin are involved in the induction of diabetes in this model

[0080] Next, the inventors repeated these adjuvant priming experiments using each of the four proinsulin-2 peptides individually to examine whether there is a dominant region of proinsulin that interacts to promote diabetes in DR3DQ2xRIP-B7.1 mice (Figs. 3a - 3f). Priming with the B23 - C20 peptide was associated with a very rapid onset of diabetes and resulted in the most obvious disease exacerbation (100% incidence by 84 days after priming, mean onset was 49 ± 16.2 (standard deviation) days after priming). In contrast, the remaining three peptides showed disease incidence that was not significantly different from that expected to occur spontaneously.

[0081] Next, the inventors sought to identify the core sequence in B23 - C20 that mediates the disease - accelerating (APDA) effect by antigen priming. One systematic approach to this is to generate multiple peptides with one amino acid offset across the region of interest. However, this approach would require testing hundreds of possible options of any suitable length, and thousands of mice would be needed for a study with the correct statistical power. Therefore, the inventors used an innovative step to identify the driver region of B23 - C20.

[0082] β - cells were examined to specifically identify whether there are fragments of the naturally - produced C - peptide (or whether C - peptide is actually always produced intact). This can establish which peptide species are naturally present in the B23 - C20 region and have the potential to be drivers of disease in vivo.

[0083] The inventors purified different types of granules from human β - cells (total sample of n = 6) and examined the contents using mass spectrometry.

[0084] Table 1 below shows the peptides in the region of interest (from B23 to C - peptide) of human β - cell granules.

[0085]

Table 1

[0086] There are no peptides containing residues derived from both the B-chain and the C-peptide. The reason for this is the very high efficiency of PC1 / 3 (proprotein convertase 1, also known as prohormone convertase 1, and prohormone convertase 3, or neuroendocrine convertase 1, often abbreviated as PC1 / 3), which cleaves the R:R within the sequence B23-C20 to insulin and C-peptide as part of the normal processing of prohormone proinsulin. The first species in the sequence actually produced in vivo starts with proinsulin residue 32 (REA). Thus, in some situations, the N-terminal R remains. Furthermore, there are numerous partial sequences of C-peptide produced by β-cells. These can bind directly to HLA-DR3 / DQ2 without the need for conventional antigen processing and bind to T-cells to cause disease.

[0087] Next, to further examine the extent to which mouse findings can be extrapolated and to understand the potential interaction between the driver antigen / epitope and HLA, the inventors aligned the relevant regions of mouse proinsulin-2 and human proinsulin (Figure 4a). The B23-C20 region of human proinsulin and mouse proinsulin-2 has 23 / 30 (77%) amino acids identical, and a further 3 / 30 (10%) are interspecies-similar, showing high homology. Next, to further identify regions with a dominant disease-inducing effect, six overlapping 14 / 15-mer peptides spanning the length of B23-C20 were generated. To prevent the formation of pyroglutamic acid, sequences with an N-terminal glutamine (Q) residue or glutamic acid (E) residue were avoided, and peptides with unpredictable properties were obtained. One of the sequences, RG-15, was highly insoluble in aqueous solution (although soluble in dimethyl sulfoxide). This has the same predicted binding core as that of ME-15 for both HLA-DR3 and HLA-DQ2 (Figure 4b). Based on the predicted binding affinity, none of the 14 / 15-mer peptides were classified as strong binders to HLA-DR3 or HLA-DQ2 by the prediction tool used. Importantly, the predicted binding cores and affinities for the human equivalents of the peptides were generally equivalent to the mouse sequences (Figure 4c).

[0088] Adjuvant priming with the ME-15 (B29-C11) peptide led to a significantly higher disease incidence during follow-up than would be expected spontaneously. The disease was also significantly accelerated compared to any of the other 14 / 15-mer peptides tested in this experiment (Figures 5a - 5f), thus strongly suggesting that the ME-15 peptide contains the minimal core amino acid sequence that binds to HLA and interacts with immune cells to cause diabetes. RG-15 can also cause disease, but peptides offset further towards the C-terminal side (DA-15 and VD-14) do not. Thus, the most N-terminal region of the C-peptide containing a single R defines the region containing the antigenic driver of type 1 diabetes occurring in the HLA-DR3 / DQ2 background.

[0089] A further important part in the preclinical model was to show that this approach can actually identify peptides with potentially valuable tolerogenic properties in human disease in the DR3 / DQ2 background. The inventors induced disease using B23-C20 and examined the tolerogenic properties of ME-15 (Figure 6). The tolerogenic effect of the ME-15 peptide was demonstrated. Thus, the peptide with the most potent disease driver properties in this B:C junction region is also tolerogenic. This suggests that peptides such as ME-15 with HLA-DR3 / DQ2 binding properties can be important in the disruption of immune tolerance and the acceleration of disease in the natural course of diabetes development, and also have therapeutic capacity when administered early enough and via a tolerogenic (high solubility, non-adjuvant) route.

[0090] The similarity between the human and mouse equivalents of the targeted proinsulin region indicates that these findings are highly relevant to human therapy. To confirm this, human CD4 derived from donors with the HLA-DR3-DQ2 haplotype +The response of T cells to human proinsulin peptide B30-C13 was analyzed. This showed that HLA-DR3-DQ2 donors exhibited a higher response to B30-C13 than donors without this haplotype (Figure 7). Furthermore, the response to B30-C13 was higher than that to the control peptide C19-A3 of the present inventors, which has been shown to be HLA-DR4 restricted (Arif S, et al. (2004) J Clin Invest 113(3):451-463).

[0091] Example 5

[0092] Five peptides were tested in newly diagnosed patients with type 1 diabetes who are HLA-DR3 / DQ2 positive (Figure 8). An inflammatory cytokine response was seen in response to all of the peptides, indicating that they are strongly immunogenic in T1D. Furthermore, both IFN-γ and IL-17 responses were observed, which are associated with cell damage in the disease. In these subjects, an immunoregulatory (IL-10) response was also present, suggesting that these peptides are tolerogenic in the context of the T1D disease.

[0093] All patent and literature references cited herein are hereby incorporated by reference in their entirety.

[0094] Sequence Listing

Table 2-1

Table 2-2

Table 2-3

Claims

**Claim 1** A pharmaceutically acceptable composition for use in the treatment or prevention of type 1 diabetes (T1D) in a patient having the DR3-DQ2 haplotype, comprising a peptide consisting of an amino acid sequence having at least 95% identity 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. **Claim 2** The pharmaceutically acceptable composition according to claim 1, wherein the peptide consists of an amino acid sequence having at least 99% identity 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. **Claim 3** The pharmaceutically acceptable composition according to claim 1 or 2, wherein the peptide consists of 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. **Claim 4** The pharmaceutically acceptable composition according to claim 3, wherein the peptide consists of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

2. **Claim 5** The pharmaceutically acceptable composition according to any one of claims 1 to 4, comprising two or more of said peptides. **Claim 6** The pharmaceutically acceptable composition according to any one of claims 1 to 5, comprising one or more pharmaceutically acceptable excipients. **Claim 7** The pharmaceutically acceptable composition according to claim 6, further comprising cysteine. **Claim 8** The pharmaceutically acceptable composition according to any one of claims 1 to 7, wherein the peptide or combination of peptides is coated on nanoparticles. **Claim 9** The pharmaceutically acceptable composition according to claim 8, wherein the peptide or combination of peptides is coated on the nanoparticles by binding to an MHC complex bound to the nanoparticles. **Claim 10** The pharmaceutically acceptable composition according to any one of claims 1 to 9, formulated to be delivered by a parenteral, oral, or topical route, including intravenous, intradermal, intramuscular, subcutaneous, intraperitoneal, nasal, oral, or transdermal routes. **Claim 11** The pharmaceutically acceptable composition according to claim 10, formulated to be delivered by the intradermal route. **Claim 12** An in vitro method for identifying a subject as being at high risk of T1D, comprising determining whether the subject has the DR3-DQ2 haplotype and comparing, when an immune response to any one of a peptide or a combination of peptides is detected, with the criterion that the subject is at high risk of T1D, wherein the peptide consists of an amino acid sequence having at least 95% identity 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. **Claim 13** An in vitro method for identifying a patient as being suitable for T1D treatment, comprising determining whether the patient has the DR3-DQ2 haplotype and comparing, when an immune response to any one of a peptide or a combination of peptides is detected, with the criterion that the patient is suitable for T1D treatment, wherein the peptide consists of an amino acid sequence having at least 95% identity 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.

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