WO2019104391A1

WO2019104391A1 - Type 1 diabetes treatment

Info

Publication number: WO2019104391A1
Application number: PCT/AU2018/051282
Authority: WO
Inventors: Stuart MANNERING; Michelle SO; Thomas Kay
Original assignee: St Vincents Institute of Medical Research
Current assignee: St Vincents Institute of Medical Research
Priority date: 2017-12-01
Filing date: 2018-11-30
Publication date: 2019-06-06
Anticipated expiration: 2020-06-01

Abstract

The present invention relates to peptides, compositions and methods for the treatment, prevention or monitoring the progression of type 1 diabetes.

Description

Type 1 diabetes treatment

Field of the invention

The present invention provides peptides, compositions and methods for treating, preventing or monitoring risk of type 1 diabetes. Related application

This application claims priority from Australian provisional application AU 2017904853, the contents of which are hereby incorporated by reference in their entirety.

Background of the invention

Type 1 diabetes (T1 D) is an autoimmune disorder that results from the T-cells of the body's immune system attacking the b-cells of the islets of Langerhans in the pancreas. While it represents 5%-10% of all diabetes cases, type 1 diabetes accounts for over 90% of diabetes cases in children.

The main aetiology of type 1 diabetes is a drop in insulin levels due to the destruction of the insulin-producing pancreatic b-cells, which leads to abnormally high blood glucose levels. This can lead to symptoms of the disease such as frequent urination; extreme hunger and thirst; weight loss; fatigue and irritability. However, over time, sustained hyperglycemia leads to a number of other more serious complications such as heart disease, high blood pressure, kidney disease and nervous system disease. It is these complications from diabetes that ultimately lead to death if left untreated.

As an autoimmune disease, self-reactive T cells infiltrate the pancreatic islets, causing inflammation (insulitis) and progressively destroying insulin-producing b-cells. Unfortunately, the pathogenic mechanisms behind the initiation of T1 D are poorly understood, and without a cure, it remains one of few diseases where incidence and prevalence continue to rise annually.

There remains a need for the development of new methods and compositions for treating, preventing and/or monitoring the onset of type 1 diabetes. Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and/or combined with other pieces of prior art by a skilled person in the art.

Summary of the invention

The present invention provides a method of treating type 1 diabetes in an individual in need thereof, the method comprising administering to the individual an effective amount of a polypeptide comprising, consisting essentially of, or consisting of the C-peptide of proinsulin or a fragment thereof, thereby treating type 1 diabetes in the individual.

The present invention provides a method of inhibiting progression of type 1 diabetes in an individual in need thereof, the method comprising administering to the individual an effective amount of a polypeptide comprising, consisting essentially of, or consisting of the C-peptide of proinsulin or a fragment thereof, thereby inhibiting progression of type 1 diabetes in the individual.

The present invention provides a method of inducing regulatory T cells in an individual having type 1 diabetes, the method comprising administering to the individual an effective amount of a polypeptide comprising, consisting essentially of, or consisting of the C-peptide of proinsulin or a fragment thereof, thereby inducing regulatory T cells in the individual.

The present invention provides a method of inducing deletion of pathogenic CD4+ T cells in an individual having type 1 diabetes, the method comprising administering to the individual an effective amount of a polypeptide comprising, consisting essentially of, or consisting of the C-peptide of proinsulin or a fragment thereof, thereby inducing deletion of pathogenic CD4+ T cells in an individual.

The present invention also provides a method of inducing anergy or exhaustion of CD4+ T cells in an individual having type 1 diabetes, the method comprising administering to the individual an effective amount of a polypeptide comprising, consisting essentially of, or consisting of the C-peptide of proinsulin or a fragment thereof, thereby inducing anergy or exhaustion of pathogenic CD4+ T cells in an individual.

The present invention also provides a method of inducing or promoting the generation of C-peptide regulatory CD4+ T cells in an individual having type 1 diabetes, or at risk of developing type 1 diabetes, the method comprising administering to the individual an effective amount of a polypeptide comprising, consisting essentially of, or consisting of the C-peptide of proinsulin or a fragment thereof, thereby inducing or promoting the generation of C-peptide regulatory CD4+ T cells in the individual. Preferably, the regulatory CD4+ T cells have a‘dominant’ effect and suppress the activity or generation of bystander pathogenic T cells in the individual.

The present invention provides a method for reducing, preventing, slowing or halting the decrease in insulin production that occurs in an individual having type 1 diabetes, the method comprising, consisting essentially of, or consisting of administering to the individual an effective amount of a polypeptide comprising, consisting essentially of, or consisting of the C-peptide of proinsulin or a fragment thereof, thereby reducing, preventing, slowing or halting the decrease in insulin production in the individual.

The present invention provides a method for maintaining insulin production in an individual having type 1 diabetes, the method comprising, consisting essentially of, or consisting of administering to the individual an effective amount of a polypeptide comprising, consisting essentially of, or consisting of the C-peptide of proinsulin or a fragment thereof, thereby maintaining insulin production in the individual.

The present invention also provides a method for restoring insulin production in an individual, the method comprising, consisting essentially of, or consisting of administering to the individual an effective amount of a polypeptide comprising, consisting essentially of, or consisting of the C-peptide of proinsulin or a fragment thereof, thereby restoring insulin production in an individual.

In any aspect of the invention, the individual may have recent-onset type 1 diabetes or long-standing type 1 diabetes. In any aspect of the invention, the individual may have stage 1 , 2 or 3 type 1 diabetes. In any aspect of the invention, the individual has T cells expressing the T cell receptor genes as shown in Table 2. For example, in one embodiment, the individual has CD4+ T cells which have a T-cell receptor with a sequence as shown in Table 2. For example, in any embodiment, the individual has CD4+ T cells which have a T-cell receptor with a CDR3 sequence as shown in Table 2. Further, in any embodiment, the individual has CD4+ T cells which have a TCR alpha variable (TRAV) sequence as shown in Table 2. Alternatively or in addition, the individual has CD4+ T cells which have a TCR beta variable (TRBV) sequence as shown in Table 2.

In any aspect of the invention, the polypeptide comprising or consisting essentially of the C-peptide of proinsulin or a fragment thereof does not include any more than 3, 2 or 1 amino acid from the B or A chain of insulin. More preferably, the polypeptide does not include any amino acids from the B or A chain. As used herein, the polypeptide comprising the C-peptide of proinsulin or a fragment thereof does not include full length proinsulin.

In any aspect of the invention, the C-peptide is the contiguous sequence of amino acids that separates the B and A chain in proinsulin prior to cleavage. Preferably, the C-peptide is the contiguous sequence of amino acids that is cleaved from proinsulin to form mature insulin. Preferably, the C-peptide is derived from human proinsulin. In one embodiment, the C-peptide has the amino acid sequence:

EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ (SEQ ID NO: 1 ).

In any aspect of the invention, C -peptide or a fragment of a C-peptide comprises a peptide that can stimulate T cell proliferation. In any embodiment, the C-peptide or fragment thereof may be presented by an FILA molecule FILA-DQ8, HLA-DQ2, HLA- DR3 or HLA-DR4. In any embodiment, the DQ8 molecule may be DQA1 ^*03:01/DQB1 ^*03:02. The DQ2 molecule may be DQA1 ^*05:01 /DQB1 ^*02:01. In alternative embodiments, the peptide, or fragment thereof is presented by an HLA molecule FILA-DR4. The DR molecule may be DRB^*04:01 or DRB^*03:01.

In certain embodiments, the individual to whom the C-peptide, or fragments or derivatives thereof, are administered, has the alleles: HLA-DQ8 (HLA-DQA 1 ^*03:01, HLA-DQB1 ^*03:02), HLA-DQ2 (HLA-DQA 1 ^*05:01 , HLA-DQB1 ^*02:01), HLA-DQ8 frans {HLA-DQA 1 ^*05:01, HLA-DQB1 ^*03:02), HLA-DQ2 trans {HLA-DQA 1 ^*03:01, HLA- DQB1 ^*02:01), or HLA-DR4 (HLA-DRA^*01 :01, HLA-DRB1 ^*04:0X, wherein ΌC’ indicates any allele of this class, and thus includes DRB1 ^*04:01 , -02, -03, -04, -05 etc).

More preferably, the fragment comprises or consists of any one of the following amino acid sequences, or functional derivatives or homologues thereof:

PGAGSLQPLALE (SEQ ID NO: 2);

SLQPLALEGSL (SEQ ID NO: 3);

QPLALEGSL (SEQ ID NO: 4);

QPLALEGSLQ (SEQ ID NO: 5); PGAGSLQPLAL (SEQ ID NO: 6);

GQVELGGGPGAG (SEQ ID NO: 7);

AEDLQVGQVEL (SEQ ID NO: 8);

GSLQPLALEGSLQ (SEQ ID NO: 9);

SLQPLALEGS (SEQ ID NO: 10); AGSLQPLAL (SEQ ID NO: 1 1 );

VELGGGPGAG (SEQ ID NO: 12);

EDLQVGQVELGG (SEQ ID NO: 13);

LQVGQVELGGGPGAGSLQ (SEQ ID NO: 14);

RREAEDLQVGQVELGGGP (SEQ ID NO: 15); GAGSLQPLALEGSLQKRG (SEQ ID NO: 16); or any peptide as shown in the figures and tables included herein (such as any of the peptides having sequences as shown in SEQ ID NOs: 59 to 145). In any method of the invention, the method further comprises the step of diagnosing the individual as having type 1 diabetes.

In any method of the invention, the method further comprises determining whether the individual has T cells expressing the T cell receptor genes as shown in Table 2. For example, in one embodiment, the method includes determining whether the CD4+ T cells of the individual have a T-cell receptor with a CDR3 sequence as shown in Table 2 (more specifically, a sequence as shown in any of SEQ ID NOs: 17 to 58).

The present invention also provides a method of monitoring beta-cell specific T cell response in an individual the method comprising

- providing a population of T cells derived from the individual in whom the betacell specific T cell response is to be determined; wherein the individual does not display any signs or symptoms of type 1 diabetes,

- contacting the population of T cells with a polypeptide comprising, consisting essentially of, or consisting of the C-peptide of proinsulin or a fragment thereof.

In certain embodiments, an increase in the activation or proliferation of T cells upon contact with the polypeptide indicates the individual is at risk of developing type 1 diabetes. It will be understood that an increase in activation of T cells may include an increase in cytokine secretion therefrom.

Preferably, the C-peptide or fragment thereof is presented by an HLA molecule HLA-DQ8, HLA-DQ2, HLA-DR3 or HLA-DR4. For example, the FILA-DQ8 molecule may be DQA1 ^*03:01 /DQB1 ^*03:02. The DQ2 molecule may be DQA1 ^*05:01 /DQB1 ^*02:01. In alternative embodiments, the peptide, or fragment thereof is presented by an FILA molecule HLA-DR4. The DR molecule may be DRB^*04:01 or DRB^*03:01.

In certain embodiments, the individual, whose T cells are contacted with C- peptide, or fragments thereof, has the following HLA alleles: HLA-DQ8 (A1 ^*0301 , B1 ^*0302), HLA-DQ2 (A1 ^*0501 , B1 ^*0201 ), HLA-DQ8 frans (A1 ^*0501 , B1 ^*0302), HLA- DQ2 trans (A1 ^*0301 ), B1 ^*0201 ), or HLA-DR4 (HLA-DRA^*01 :01, HLA-DRB1 ^*04:0X, wherein ΌC’ indicates any allele of this class, and thus includes DRB1 ^*04:01 , -02, -03, - 04, -05 etc).

In any embodiment of the invention, the population of T cells is obtained from a sample of the individual’s blood, including a preparation of peripheral blood mononuclear cells (PBMC) comprising the T cells.

In any embodiment of the invention, the increase in the activation or proliferation of T cells can be determined by directly measuring T cell proliferation. In alternative embodiments, T cell activation can be determined by measuring for cytokine secretion from the T cells, or changes in the expression of genes in the T cells which are indicative of activation. In certain embodiments, changes in the expression of genes encoding cytokines, activation markers, or other immune related genes are measured in the T cells. Further, activation may be measured by analysing the proteins expressed by the antigen specific T cells, including proteins expressed on the surface of these cells.

Further, the present invention provides a method of determining whether an individual at risk of developing type 1 diabetes is displaying early signs or symptoms of the disease, the method comprising:

- providing a population of T cells derived from an individual who is at risk of developing type 1 diabetes;

- contacting the population of T cells with a polypeptide comprising, consisting essentially of, or consisting of the C-peptide of proinsulin or a fragment thereof;

- determining if there is a response of the T cells in the population to contact with the polypeptide; wherein an increase in the activation or proliferation of T cells upon contact with the polypeptide indicates the individual is displaying early signs or symptoms of type 1 diabetes.

Still further, the present invention provides a method of monitoring beta-cell specific T cell response in an individual who has early markers of type 1 diabetes, the method comprising: - providing a population of T cells derived from the individual in whom the betacell specific T cell response is to be determined;

- contacting the population of T cells with a polypeptide comprising, consisting essentially of, or consisting of the C-peptide of proinsulin or a fragment thereof; and determining if there is a response of the T cells in the population to contact with the polypeptide.

An increase in the activation or proliferation of T cells upon contact with the polypeptide indicates the progression of type 1 diabetes in the individual if the individual has early markers of type 1 diabetes, or who is the early stages of the disease.

The present invention also provides a method of determining the success of a treatment for type 1 diabetes in an individual, the method comprising:

- providing a population of T cells derived from the individual in whom the betacell specific T cell response is to be determined;

- contacting the population of T cells with a polypeptide comprising, consisting essentially of, or consisting of the C-peptide of proinsulin or a fragment thereof; and determining if there is a response of the T cells in the population to contact with the polypeptide, wherein an increase in the activation or proliferation of T cells upon contact with the polypeptide indicates the progression of type 1 diabetes in the individual, if the individual has stage 1 or early stage type 1 diabetes; wherein a decrease in the activation or proliferation of T cells upon contact with the polypeptide and an absence of clinical symptoms of type 1 diabetes indicates that the treatment for type 1 diabetes has been successful; and wherein a decrease in the activation or proliferation of T cells upon contact with the polypeptide and the presence of clinical symptoms of type 1 diabetes indicates that the treatment for type 1 diabetes has not been successful. The present invention also provides a method for determining the strength of an individual’s autoimmune response to proinsulin, the method comprising:

- providing a population of T cells derived from an individual for whom the autoimmune response is to be determined;

- determining the response of the T cells in the population to contact with the polypeptide;

- comparing the level of response of the T cells in the population to contact with the polypeptide, with the level of response in a control sample obtained from the individual wherein an increase in the proliferation or activation of the T cells compared with the control sample indicates an increase in the individual’s autoimmune response to proinsulin; and wherein a decrease in the proliferation or activation of the T cells compared with the control sample indicates a decrease in the individuals’ autoimmune response to proinsulin.

In any embodiment of the invention, the T cells may be derived from the periphery of the individual, for example, from a peripheral blood sample.

The present invention also provides use of a polypeptide comprising, consisting essentially of, or consisting of the C-peptide of proinsulin or a fragment thereof in the manufacture of a medicament for (a) treating type 1 diabetes, (b) inhibiting progression of type 1 diabetes, (c) inducing regulatory T cells, (d) inducing deletion, anergy or exhaustion of pathogenic CD4+ T cells, or (e) restoring insulin production, in an individual having type 1 diabetes.

The present invention also provides a polypeptide comprising, consisting essentially of, or consisting of the C-peptide of proinsulin or a fragment thereof for use in (a) treating type 1 diabetes, (b) inhibiting progression of type 1 diabetes, (c) inducing regulatory T cells, (d) inducing deletion, anergy or exhaustion of pathogenic CD4+ T cells, or (e) restoring insulin production, in an individual having type 1 diabetes.

The present invention also provides a peptide comprising, consisting essentially of, or consisting of: PGAGSLQPLALE (SEQ ID NO: 2);

SLQPLALEGSL (SEQ ID NO: 3);

QPLALEGSL (SEQ ID NO: 4);

QPLALEGSLQ (SEQ ID NO: 5);

PGAGSLQPLAL (SEQ ID NO: 6); GQVELGGGPGAG (SEQ ID NO: 7);

AEDLQVGQVEL (SEQ ID NO: 8);

GSLQPLALEGSLQ (SEQ ID NO: 9);

SLQPLALEGS (SEQ ID NO: 10);

AGSLQPLAL (SEQ ID NO: 1 1 ); VELGGGPGAG (SEQ ID NO: 12);

EDLQVGQVELGG (SEQ ID NO: 13);

LQVGQVELGGGPGAGSLQ (SEQ ID NO: 14);

RREAEDLQVGQVELGGGP (SEQ ID NO: 15);

GAGSLQPLALEGSLQKRG (SEQ ID NO: 16); or any peptide as shown in the figures and tables included herein (such as any of the peptides having sequences as shown in SEQ ID NOs: 59 to 145), or functional derivatives or homologues thereof. In any aspect, the peptide is isolated, substantially purified, purified, recombinant or synthetic.

The present invention also provides a pharmaceutical composition comprising a peptide of the invention. Preferably, the composition further comprises a carrier, diluent or excipient. In one embodiment, the only active ingredient present in the composition is a peptide of the invention.

The invention provides a pharmaceutical composition comprising as an active ingredient a peptide of the invention and a pharmaceutically acceptable diluent, excipient or carrier. In one embodiment, the only active ingredient present in the composition is a peptide of the invention.

The invention provides a pharmaceutical composition for treating or preventing type 1 diabetes comprising as a main ingredient a peptide of the invention and a pharmaceutically acceptable diluent, excipient or carrier. In one embodiment, the only active ingredient present in the composition is a peptide of the invention. The present invention also provides a kit for use, or when used, in a method of the invention, the kit comprising, consisting essentially of or consisting of:

- a peptide of the invention; and optionally

- written instructions describing the use of the peptide in a method of the invention. As used herein, except where the context requires otherwise, the term

"comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings. Brief description of the drawings

Figure 1 : CD4+ T-cell responses to C-peptide in PBMC. A) Representative flow cytometry plots. PBMC from individuals with recent-onset (<3 months) T1 D (“RO T1 D”), long-standing (>5 years) T1 D (“LS T1 D”) and without T1 D (“HC”) were analysed for a C-peptide specific CD4+ T-cell response using a CFSE-based proliferation assay. The upper right-hand gate includes CD4+ (CFSE^bnght) cells that did not proliferate in the presence of antigen. The second gate includes the CD4+ (CFSE^dim) cells that did proliferate in the presence of antigen. The number of events in each gate is shown. Nil Antigen refers to the sample incubated without antigen, C-peptide refers to the sample incubated with C-peptide (10mM). Dead cells were excluded by PI (propidium iodide) staining.

B) Summary of responses to C-peptide. CD4+ T-cell responses to C-peptide in peripheral blood were measured using the CFSE-based proliferation assay. The magnitude of the response is expressed as the Cell Division Index (CDI) (Mannering et al 2003). The means of triplicate CDI for responses to C-peptide in individuals with recent-onset T 1 D (n=23), long-standing T 1 D (n=15) and without T 1 D (n=15) are plotted. CDI of 2 is marked as a dotted line. CDI > 2 is traditionally considered a positive proliferative response to an antigen. CDI of 3 is also indicated to demonstrate the improved disease specificity with a higher CDI cut-off. Each point represents an individual. Statistical significance was determined using unpaired Welch’s Two-Tailed t Test was defined at a p<0.05.

Figure 2: Analysis of PBMC-derived C-peptide specific CD4+ T-cell clones.

CD4+ T-cell clones’ responses to antigen were measured by the secretion of IFN-y measured by ELISA. (A) “Coarse” epitope mapping: Clones were tested in triplicate against 18-mer peptides, at a final concentration of 10mM, spanning the length of C- peptide, overlapping by 12 amino acids. A representative clone (H8.5) is shown. (B) “Fine” epitope mapping of clone H8.5. Epitope specificity was further refined using peptides truncated by a single amino acid from either the N- or the C-terminus. The parallel lines in red delineate the sequence of the minimum epitope determined by the results of the experiment. (C) K9.5“coarse” mapping results: representative of clones without a detectable response to the overlapping 18-mer peptides (10mM). (D)“Fine” epitope mapping for clones such as K9.5 was achieved using a series of peptides of the same length, encompassing the known length of “coarse” mapped specificity, but differing by a single amino acid substitution. Statistical significance was determined using unpaired Welch’s two-tailed t test, and defined as p<0.05.

Figure 3. Analysis of T-cell clone’s HLA restriction. The HLA restriction of the C-peptide specific CD4+ T-cell clones was determined in two steps. First, antibodies against HLA-DR, -DP, and -DQ were included in the peptide stimulation assays. HLA restriction analysis of clone H8.5 is shown here. (A) Clone H8.5’s response to C-peptide (10mM) was significantly (p<0.001 ) inhibited by anti-HLA-DQ (clone SPV-L3) at a final concentration of 5.0pg/mL, but not HLA-DP (clone B7/21 ) or HLA-DR (clone L243) at the same concentration. To define the HLA allele HLA-class II negative cells transduced with different HLA alleles were tested. (B) Clone H8.5 responded to C-peptide presented by antigen-presenting cells (APC; T2) that express HLA-DQ8 (A1 ^*0301 , B1 ^*0302), but not to APC expressing other HLA DQ alleles. The EBV line, KJ EBV (HLA-DRB1 ^*03:01 , 04:04; DQB1 ^*02:01 , 03:02) that was used as a positive control APC and PMA/lonomycin was used as a positive control for T cell function. HLA restriction analysis of clone K6.4. (C) Responses to C-peptide were significantly (p<0.01 ) inhibited by anti- HLA-DR (mAb clone L243), but not by anti-HLA DQ (mAb clone SPV-L3), or anti-HLA-DP (mAb clone B7/21 ). (D) Clone K6.4 only responded to C-peptide presented by APC expressing HLA DRB1 ^*04:01 molecule, but not the other HLA-DR molecules indicated. Responses were measured through IFN-g ELISA (pg/mL). A representative of duplicate experiments is shown. Statistical significance was determined using unpaired Welch’s Two-Tailed t Test, and defined as p<0.05.

Figure 4: Comparison of the stimulatory capacity of full-length and 18mer peptides synthetic C peptides. Starting at 100pM, five to seven 1 :1 serial dilutions of full-length C-peptide compared to an 18mer peptide incorporating the clone’s known specificity was used to compare their relative potency. Epstein Barr Virus transformed B-cell line (KJ; HLA-DRB1 ^*03:01 , 04:04; DQB1 ^*02:01 , 03:02) were used as APC for HLA DQ-restricted clones, and a BLS cell line transfected with HLA-DR^*0401 was used as APC for HLA DR^*0401 -restricted clones. The demonstrates the response curves of clones (A) K9.6, (B) D1.1 , (C) E2.3, (D) T17.1 , (E) K9.5, (F) B3.3, (G) H3.3 to full-length (31 amino acids) C-peptide (closed circles), compared to an 18-mer peptide that still encompasses its epitope specificity (open squares). T-cell responses were measured through IFN-g ELISA (pg/mL).

Figure 5: C-peptide specific CD4+ T cell response to human pancreatic islet extract Islet or acinar lysate. Fluman pancreatic islet extract Islet or acinar lysate were diluted in culture media (concentrations as indicated) and incubated with Epstein Barr Virus transformed B-cell line (KJ; HLA-DRB1 ^*03:01 , 04:04; DQB1 ^*02:01 , 03:02) and CD4+ T-cell clones. Responses were detected by IFN-g ELISA. Clone A1.9, a C- peptide specific CD4+ T cell clone derived from donor islets was included as a positive control.

Figure 6: INS--VNTR, HLA genotype and responses to C-peptide. /A/S-VNTR genotype for recent-onset T1 D subjects plotted against the CD4⁺ T-cell response to C- peptide, measured by CFSE dilution and expressed as a cell division index (CDI), and HLA class II allele.

Figure 7: CD4⁺ T-cell clones’ responses to antigen were measured by the secretion of IFN-g measured by ELISA, (i)“Coarse” epitope mapping: Clones were tested in triplicate against 18-mer peptides, at a final concentration of 10 mM, spanning the length of C-peptide, overlapping by 12 amino acids. Where a response was not elicited, peptide concentration was increased to 50 mM (as indicated on the figure), (ii) “Fine” epitope mapping of clone. Epitope specificity was further refined using peptides truncated by a single amino acid from either the N- or the C-terminus. The parallel lines in red delineate the sequence of the minimum epitope determined by the results of the experiment. A) B3.1 , B) B3.3, C) D1.1 , D) D1.4, E) E2.3, F) H3.7, G) H7.4, H) H1 1.5, I) H12.2, J) K4.4, K) K6.2, L) K6.4, M) K9.6, N) T6.1 , O) E2.5

Figure 8: Analysis of T-cell clone’s HLA restriction. The HLA restriction of the C-peptide specific CD4⁺ T-cell clones was determined in two steps. First, (i) antibodies against HLA-DR, -DP, and -DQ were included in the peptide stimulation assays at a final concentration of 5.0pg/mL. To define the HLA allele HLA-class II negative cells transduced with different HLA alleles were tested(ii). The EBV line, KJ EBV (HLA- DRB1 ^*03:01 , 04:04; DQB1 ^*02:01 , 03:02) that was used as a positive control APC and PMA/lonomycin was used as a positive control for T cell function. A representative of duplicate experiments is shown. A) B3.1 , B) B3.3, C) D1.1 , D) D1.4, E) E2.3, F) H3.7,

Figure 9: CD4⁺ T-cell clones’ responses to antigen were measured by the secretion of IFN-g measured by ELISA, i)“Coarse” epitope mapping: Clones were tested in triplicate against 18-mer peptides, at a final concentration of 10mM, spanning the length of C-peptide, overlapping by 12 amino acids, (ii) Alternative“coarse” epitope mapping of clone. These clones did not respond to any of the five 18mer peptides and were mapped using variants of the full-length C-peptide which were sequentially truncated from either the N- or C- terminus by three amino acids, iii)“Fine” epitope mapping: the minimum epitope was further refined by using a panel of peptides sequentially truncated by one amino acid from either the N- or C-terminus. iv) Further “fine” epitope mapping: The minimum epitope was then determined by testing the clones against a panel of peptides with a single amino acid substitution. The substitutions were chosen to disrupt the putative HLA-binding or TCR recognition. A) H 3.3, B) T17.1

Detailed description of the embodiments

It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described. It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

All of the patents and publications referred to herein are incorporated by reference in their entirety. For purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.

The present invention is based on the unexpected identification that the T cells of individuals with type 1 diabetes, in particular those with recent onset type 1 diabetes, respond to the C-peptide of proinsulin and fragments thereof. Further, the FILA molecules of those individuals bind to the C-peptide of proinsulin and fragments thereof. This indicates that the immune system of individuals with type 1 diabetes can respond to the C-peptide of proinsulin and fragments thereof.

An advantage of an aspect of the present invention is that the capacity of the immune system of individuals at risk of type 1 diabetes to respond to the C-peptide of proinsulin and fragments thereof can provide a mechanism to monitor disease progression or treatment response. Further, it also provides the ability to provide peptide-based immunotherapy to reduce the autoimmune response to beta cell autoantigens such as insulin and proinsulin.

Methods of the invention In a first embodiment, the present invention provides a method of treating type 1 diabetes in an individual in need thereof, the method comprising administering to the individual an effective amount of a polypeptide comprising, consisting essentially of, or consisting of the C-peptide of proinsulin or a fragment thereof, thereby treating type 1 diabetes in the individual. The terms‘treatment’ or‘treating’ of an individual includes the application or administration of a composition, or combination therapy, as described herein, to an individual, with the purpose of delaying, slowing, stabilizing, curing, healing, alleviating, relieving, altering, remedying, less worsening, ameliorating, improving, or affecting the disease or condition, the symptom of T1 D. The term "treating" refers to any indication of success in the treatment or amelioration of T1 D including any objective or subjective parameter such as abatement; remission; lessening of the rate of worsening; lessening severity of the disease; stabilization, diminishing of symptoms or making the injury, pathology or condition more tolerable to the subject; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; or improving a subject's physical or mental well-being.

As used herein,‘preventing’ or‘prevention’ is intended to refer to at least the reduction of likelihood of the risk of (or susceptibility to) developing T1 D (i.e., causing at least one of the clinical symptoms of the disease not to develop in a patient that may be predisposed to T1 D but does not yet experience or display symptoms of T 1 D).

The present invention also provides a method of inhibiting progression of type 1 diabetes in an individual in need thereof, the method comprising administering to the individual an effective amount of a polypeptide comprising, consisting essentially of, or consisting of the C-peptide of proinsulin or a fragment thereof, thereby inhibiting progression of type 1 diabetes in the individual.

As used herein,‘inhibiting the progression’ of type 1 diabetes includes inhibiting, halting or reversing pancreatic islet infiltration thereby preventing further generation of memory T cells and thereby preventing the progress of type 1 diabetes. Inhibiting the progress of type 1 diabetes may include causing at least one of the clinical symptoms of the disease not to develop in a patient that may be predisposed to T1 D but does not yet experience or display symptoms of T1 D. Further it may include delaying the onset of a clinical symptom of T1 D in the individual.

Still further, the present invention provides a method of inducing regulatory T cells in an individual having type 1 diabetes, the method comprising administering to the individual an effective amount of a polypeptide comprising, consisting essentially of, or consisting of the C-peptide of proinsulin or a fragment thereof, thereby inducing regulatory T cells in the individual.

As used herein, measuring for the induction of regulatory T cells can be accomplished by measuring the suppression of a bystander T cell response. More specifically, induced regulatory T cells could be cultured with effector T cells stimulated by antigen or a monoclonal antibody that is specific for CD3. C-peptide-specific regulatory T cell function would be detected by the decrease, or inhibition of the effector T cells’ response, when the regulatory T cell is activated by the peptide. This type of experiment is well within the purview of the person skilled in the art. In alternative methods, the induction of regulatory T cells can be confirmed by detecting an increase in the expression of the transcription factor FoxP3 (which is a regulatory T cell transcription factor), or by measuring for the increased levels of cytokines secreted by regulatory T cells (such as, for example, IL-10 and/or TGF-b). Again, methods for detecting changes in transcription factor expression or cytokine secretion are well within the purview of the person skilled in the art.

The present invention provides a method of inducing deletion of pathogenic CD4+ T cells in an individual having type 1 diabetes, or an individual having early signs of type 1 diabetes, the method comprising administering to the individual an effective amount of a polypeptide comprising, consisting essentially of, or consisting of the C- peptide of proinsulin or a fragment thereof, thereby inducing deletion of pathogenic CD4+ T cells in an individual. (As explained further herein, an individual having early signs of type 1 diabetes, e.g., an individual in“stage 1 or stage 2” includes an individual having evidence of beta cell autoimmunity).

Deletion of pathogenic CD4+ T cells can be monitored in an individual by utilising a 5,6- carboxylfluorescein diacetate succinimidyl ester (CFSE)-based proliferation assay, as further described herein and previously described in Mannering et al., (2003), J. Immunol. Methods, 283: 173-183, the entire contents of which are hereby incorporated by reference in their entirety. Other methods include ELIspot, Tetramer staining and TCR analysis, as further described herein. The invention also provides methods of inducing anergy or exhaustion of CD4+ T cells in an individual having type 1 diabetes. The method includes administering to the individual an effective amount of a polypeptide comprising, consisting essentially of, or consisting of the C-peptide of proinsulin or a fragment thereof, thereby inducing anergy or exhaustion of pathogenic CD4+ T cells in an individual. The skilled person will be familiar with methods for determining whether CD4+ T cells have become exhausted (hyporesponsive). For example, the response of CD4+ T cells to a stimulus can also be determined using the methods described herein, for example, by detecting a response (or change in response, including a reduction in response) to C-peptide, or a fragment thereof.

The skilled person will be familiar with methods for determining whether or not the levels of insulin produced by an individual have changed, including whether insulin production has increased to levels seen before treatment was commenced. For example, for an individual with type 1 diabetes, increased insulin production can be determined by measuring the beta cell mass produced by the pancreas of an individual. Insulin production can also be determined using standard methods known in the art.

As such, the present invention also provides a method for restoring insulin production in an individual, the method comprising, consisting essentially of, or consisting of administering to the individual an effective amount of a polypeptide comprising, consisting essentially of, or consisting of the C-peptide of proinsulin or a fragment thereof, thereby restoring insulin production in an individual.

The methods of the present invention find particular application in individuals having particular T cell receptors. As such, in any method of the invention, the method further comprises determining whether the individual has T cells expressing the T cell receptor genes as shown in Table 2. For example, in one embodiment, the method includes determining whether the CD4+ T cells of the individual have a T-cell receptor (TCR) with a CDR3 sequence, TCR alpha variable sequence, or TCR beta variable sequence as shown in Table 2. Although not so limited, the methods described herein may find particular application in individuals having HLA alleles DQ8, DQ2, DR3 and/or D4. The HLA-DQ8 (serological nomenclature) comprises an alpha chain and a beta chain. The molecule designations for this molecule are: DQA^*03:01 (for the alpha chain) and DQB*03:02 (for the beta chain). For the DQ2 molecule, the designations are DQA*05:01 (for the alpha chain) and DQB*02:01 (for the beta chain). In preferred embodiments of the invention, the individuals receiving treatment or preventative therapy or diagnosis according to the present invention have a DQ8 allele that is DQA*03:01 or DQB^*03:02 and DQ2 allele is DQA^*05:01 or DQB^*02:01. The individual may have alleles HLA-DQ8frans ( HLA - DQA 1 *05:01, HLA-DQB1 *03:02), HLA-DQ2 trans (HLA-DQA 1 *03:01, HLA- DQB 1 ^*02:01).

In alternative embodiments, the individuals receiving treatment or preventative therapy or diagnosis according to the present invention have a HLA-DR allele, including for example, wherein the allele is HLA-DRB^*04:01 (HLA-DRA^*01 :01, HLA-DRB1 ^*04:0X, wherein ΌC’ indicates any allele of this class, and thus includes DRB1 ^*04:01 , -02, -03, - 04, -05 etc).

The skilled person will be familiar with methods for determining the genotype of an individual, including determining the T cell receptor (TCR) gene sequence expressed by the T cells of the individual. The skilled person will also be familiar with methods for determining the HLA alleles expressed by the individual.

Risk and progression of Type 1 Diabetes

The methods of the present invention have application for a number of patient groups. For example, in any aspect of the invention, the individual may be at risk of type 1 diabetes, have recent-onset type 1 diabetes or long-standing type 1 diabetes. Alternatively, the individual may have stage 1 , 2 or 3 type 1 diabetes. It will be understood that the treatment or therapeutic outcome will vary depending on the individual: for example, if the individual is one who is determined to be at risk of type 1 diabetes, the present methods can be utilised to prevent or delay onset of type 1 diabetes in that individual. Without being bound by theory, this may be accomplished by ensuring that autoimmunity to C-peptide is not established or is reversed. In alternative embodiments, for example, if the individual is one who has recent onset type 1 diabetes, or established disease, the methods described herein may be useful for preventing or reversing the further progression of the disease, by inducing the production of Treg cells which respond to C-peptide.

In certain embodiments, the present invention provides methods for determining whether an individual at risk of type 1 diabetes is developing early signs or symptoms of the disease, by monitoring beta-cell specific T cell response in an individual.

More specifically, the invention provides a method of determining whether an individual at risk of type 1 diabetes is developing early signs or symptoms of the disease, the method comprising - providing a population of T cells derived from the individual in whom the betacell specific T cell response is to be determined;

- contacting the population of T cells with a polypeptide comprising, consisting essentially of, or consisting of the C-peptide of proinsulin or a fragment thereof

- determining the response of the population of T cells to the population wherein an increase in the activation or proliferation of T cells upon contact with the polypeptide indicates the individual is developing early signs or symptoms of type 1 diabetes.

The skilled person will be familiar with methods for obtaining a population of T cells for use in such methods. For example, in a preferred embodiment, the population of T cells can be obtained from the peripheral blood mononuclear cell (PBMC) preparation obtained from a sample of the individual’s blood. Methods for obtaining PBMCs are well known to the person skilled in the art.

The development of T1 D and progression of the disease is a multi-stage process. In 2015, the JDRF (Juvenile Diabetes Research Foundation), the Endocrine Society and the American Diabetes association, released a scientific statement, establishing the adoption of a staging classification for the development of T1 D (Insel et al., 2015, Diabetes Care, 38:1964-1974, the entire contents of which are herein incorporated in their entirety). Briefly, the stages are: Pre-stage 1: genetic susceptibility and genetic risk of T1D

Stage 1: autoimmunity/presymptomatic/normoglycaemia;

Stage 2: autoimmunity+/presymptomatic/dysglycaemia; and

Stage 3: autoimmunity+/dysglycaemia/symptomatic T1D The skilled person will be familiar with methods for determining whether an individual is at risk of type 1 diabetes (i.e., pre-stage 1 ), or whether the individual has symptoms of early onset diabetes (e.g., stage 1 and/or 2).

For example, the HLA region on chromosome 6 accounts for about 30-50% of the genetic risk of T1 D, with the greatest association with HLA class II haplotypes DRB1 ^*0301 -DQB1 ^*0201 (HLA-DR3-DQ2) and DRBV0401-

DQB1 ^*0302 (HLA-DR4-DQ8). The genotype associated with the highest risk for T1 D is the heterozygous DR3-DQ2/DR4-DQ8 genotype. HLA class II DRB1 ^*1501 and DQA 1 ^*0102-DQB1 ^*0602 confer disease resistance, at least in children younger than 12 years of age. The remaining genetic risk for T1 D can be attributed to the approximately 50 non-

HLA genes or loci identified via candidate gene and genome-wide association study approaches, each with modest to small effects on disease risk. The highest non-HLA genetic contribution arises from the INS, PTPN22, CTLA4, and IL2RA genes, with the latter three genes also contributing to susceptibility to other autoimmune diseases. Stage 1 represents individuals who have developed two or more T1 D-associated islet autoantibodies but are normoglycemic. Islet autoantibodies can be measured with standardized, sensitive, and high-throughput assays.

The number of detectable islet autoantibodies correlates with risk and the presence of two or more autoantibodies is used as the major criterion for stage 1. The majority of individuals (85%) with a single autoantibody do not progress to overt symptomatic type 1 diabetes within 10 years. However, some single autoantibody subjects can progress, and progression appears to occur more frequently in children aged <5 years, if the single autoantibody is directed to IA-2 or if the single autoantibody displays higher affinity. Stage 2 is defined as the presence of b-cell autoimmunity with dysglycemia and is presymptomatic. Stage 2, like stage 1 , includes individuals with islet autoantibodies but whose disease has now progressed to the development of glucose intolerance, or dysglycemia, that arises from loss of functional b-cell mass. Dysglycemia in this stage of T1 D has been defined in several studies by impaired fasting plasma glucose of >100 mg/dL (>5.6 mmol/L) or >110 mg/dL (>6.2 mmol/L), impaired glucose tolerance with 2-h plasma glucose with a 75-g oral glucose tolerance test (OGTT) of >140 mg/dL (>7.8 mmol/L), high glucose levels at intermediate time points on OGTT (30, 60, 90 min levels of >200 mg/dL [>11.1 mmol/L]), and/or HbA_1c >5.7% (>39 mmol/mol).

Stage 3 represents manifestations of the typical clinical symptoms and signs of diabetes, which may include polyuria, polydipsia, weight loss, fatigue, diabetic ketoacidosis (DKA), and others.

In any embodiment of the invention, the individual may have residual endogenous insulin production although may be classified as falling within Stage 3 (i.e., having clinical symptoms of T1 D in addition to dysglycaemia and autoimmunity). The present invention also contemplates methods for treating T1 D in such individuals, including for example, to reduce or reverse symptoms of active disease, or enable the individual to be considered“in remission” with respect to symptomatic T1 D.

Thus, in any embodiment of the present invention, an individual who is considered at risk of T1 D, or who will benefit from the treatments and therapies described herein includes an individual who is classified according to any of the stages 1 -3 outlined above, or in“pre-stage 1”.

It will be well within the purview of the skilled person to be able to perform the relevant clinical tests to determine whether an individual is at risk of the development of T1 D (e.g. using the classification system described herein), and is therefore an individual for whom delay or prevention of T1 D may be achieved using the methods and compositions of the present invention.

In order to determine the presence and level of the autoantibodies herein described in an individual (for example, to determine risk of T1 D), assays may be performed on plasma or serum samples from individuals using standard techniques and commercially available reagents. For example, in any embodiment of the present invention, the antibodies may be measured using standard radiobinding assays (for example, from DLD Diagnostika, Germany) or electrochemiluminescence (ECL) assays (for example, as described in Steck et al., (2016), Diabetes Technol Ther, 18:410-414, the entire contents of which are herein incorporated in their entirety). Alternatively, standard immunoassay methods including enzyme-linked immunosorbent assay (ELISA) may also be used. Commercially produced ELISA kits and reagents for determining autoantibody levels in patient samples can be obtained, for example, from RSR Ltd, (Cardiff, UK) or Launch Diagnostics (Longfield, UK).

The skilled person will also be familiar with the threshold levels of seropositivity for each autoantibody indicative of risk of T1 D and conventions for the measurement of islet autoantibodies (for example, as described in The Diabetes Antibody Standardisation Program, DASP, see Torn et al., (2008) Diabetalogia, 51 : 846-52 and Schlosser et al., (201 1 ) Diabetes Care, 34: 2410-2412, the entire contents of which are hereby incorporated in their entirety).

The skilled person will further be familiar with methods for genetic screening to identify whether the individual has a“high risk HLA genotype” including, for example, any of the HLA genotypes described herein and which are associated with risk of T1 D. For example, methods for screening for at risk HLA genotypes, including those defined herein, will be well within the purview of the skilled person.

The skilled person will also be familiar with methods for determining whether an individual is“normoglycaemic” or has symptoms of “dysglycaemia”, which may assist in determining the stage of T1 D development, or whether the methods of treatment are successful and whether the individual is displaying symptoms of T1 D. Methods for determining blood glucose levels, including after oral glucose challenge, will be familiar to the skilled person.

As used herein, “normoglycaemia” (or euglycaemia) refers to a normal blood glucose level (i.e., one which is not dysglycaemic or hyperglycaemic as herein defined).

As used herein, dysglycemia refers to impaired fasting plasma glucose of >100 mg/dL (>5.6 mmol/L) or >110 mg/dL (>6.2 mmol/L), impaired glucose tolerance with 2-h plasma glucose with a 75 fg oral glucose tolerance test (OGTT) of >140 mg/dL (>7.8 mmol/L), high glucose levels at intermediate time points on OGTT (30, 60, 90 min levels of >200 mg/dL [>11.1 mmol/L]), and/or HbAi_c ³5.7% (>39 mmol/mol).

The term "hyperglycaemia" as used herein refers generally to blood glucose levels that are above normal. Hyperglycaemia can be determined by any measure accepted and utilized by those of skill in the art. Currently, in humans, normal blood glucose is considered to be between about 70 and 120 mg/dl (3.9 - 6.6 mmol/L), but varies depending on the fasting state. Before a meal, blood glucose can range from about 80 to 120 mg/dl (4.4 - 6.6 mmol/L), whereas two hours after a meal, blood glucose can be at or below about 180 mg/dl (10 mmol/L). Additionally, in fasted individuals, normal blood glucose is below about 110 mg/dl (6.1 mmol/L). A subject having a blood glucose value of about 126 mg/dl (7 mmol/L) or greater is generally considered hyperglycaemic, and a subject whose blood glucose is above about 200 mg/dl (1 1.1 mmol/L) is generally considered diabetic.

The skilled person will also be able to determine whether an individual is“pre- symptomatic” or has commenced exhibiting symptoms of clinical manifestations of T1 D. For example, symptoms of polyuria, polydipsia, weight loss, fatigue and diabetic ketoacidosis are symptoms of T1 D. As used herein, polyuria refers to excessive or abnormally large production or passage of urine (greater than 2.5 or 3 L over 24 hours in adults). Frequent urination is sometimes included by definition but is nonetheless usually an accompanying symptom.

As used herein, polydipsia refers to excessive thirst, and may also be accompanied by dry mouth.

Diabetic ketoacidosis (DKA) is related to hyperglycaemia, is associated with illness or very high blood glucose levels in type 1 diabetes and can be a sign of insufficient insulin production. In the absence of sufficient insulin, the body burns fat for energy instead, which may lead to accumulation of ketones in the blood (and which also may appear in the urine). DKA generally refers to high blood glucose levels and moderate to heavy ketones in the urine. Other symptoms or indicators of DKA include rapid breathing, flushed cheeks, abdominal pain, sweet acetone (similar to paint thinner or nail polish remover) smell on the breath, vomiting and dehydration. As used herein, recent-onset diabetes will be taken to refer to an approximate onset of diabetes of less than a year, preferably 6 months or less.

As used herein, long-standing type 1 diabetes will be taken to refer to type 1 diabetes for which onset occurred approximately more than 1 year ago.

Immune tolerance to insulin (including proinsulin), as an indicator of the success of the methods of the present invention, may be determined by conventional assays as herein described, for determining levels of insulin or proinsulin autoantibody in samples obtained from the individual. Other means for determining the success of treatment (and provision of immunotolerance to insulin including proinsulin) include determining the frequency of CD4+ T-cells in peripheral lymphoid tissue of the individual (whereby a decline in antigen-experienced insulin specific CD4+ and CD8+ T cells is indicative of the development of immune tolerance to insulin). The skilled person will be familiar with such methods, including as described in Jhala et al., (2016) JCI Insight, 1 : e86065.

Methods of monitoring disease progression

The present invention also provides methods of monitoring beta-cell specific T cell response in an individual. These methods may find application in an individual who has early markers of type 1 diabetes, has a family history of type 1 diabetes but does not yet display overt clinical symptoms. Still further, these methods may find application in cases where an individual with symptomatic type 1 diabetes is receiving treatment for disease.

In particular, the proposed methods are based on the finding by the inventors that the T cells of individuals who are healthy or who have long-standing type 1 diabetes do not respond to contact with C-peptide. Conversely, individuals in the early stages of disease, have significant T-cell responses to C-peptide. Accordingly, the response of a population of beta-cell specific T cells in an individual can be used as a measure of whether the individual is in the early stages of type 1 diabetes, or, in cases where the individual was previously diagnosed with type 1 diabetes, provide a measure of whether the disease has further progressed, or whose progress has been attenuated.

The invention provides a method for monitoring beta-cell specific T cell response in an individual, the method comprising: - providing a population of T cells derived from the individual in whom the betacell specific T cell response is to be determined;

- contacting the population of T cells with a polypeptide comprising, consisting essentially of, or consisting of the C-peptide of proinsulin or a fragment thereof and

- determining if there is a response of the T cells in the population to contact with the polypeptide.

An increase in the activation or proliferation of T cells upon contact with the polypeptide is indicative that there is progression of type 1 diabetes in the individual. Thus, this result in an individual receiving treatment for type 1 diabetes, would be indicative that the treatment is not successful. Alternatively, if the individual is one who is not receiving treatment for type 1 diabetes, and has not previously been diagnosed with type 1 diabetes, this result would suggest a need for initiation of treatment for type 1 diabetes.

Moreover, a decrease in the activation or proliferation of T cells upon contact with the polypeptide, where the individual has previously been diagnosed with type 1 diabetes and displays an improvement in clinical symptoms of type 1 diabetes indicates that the treatment for type 1 diabetes has been successful. Conversely, a decrease in the activation or proliferation of T cells upon contact with the polypeptide in an individual who still displays clinical symptoms of type 1 diabetes indicates that the treatment for type 1 diabetes has not been successful.

The skilled person will be familiar with methods for obtaining a population of T cells for use in any of the methods described herein. For example, in a preferred embodiment, T cells can be obtained from the peripheral blood mononuclear cell (PBMC) preparation obtained from a sample of the individual’s blood. Methods for obtaining PBMCs are well known to the person skilled in the art.

Further, the skilled person will be familiar with methods for measuring the response of a T cell population as described above. For example, in one embodiment, proliferation of CD4+ T cells can be measured using standard methods, including the CFSE staining methods described herein. Further, activation of CD4+ T cells can be determined by a number of parameters, including by measuring for cytokine production or secretion in response to exposure to a peptide of the invention (for example, IFNy secretion as indicative of the activation of effector cells and IL10 secretion, as indicative of the activation of regulatory cells, measured using standard methods including ELIspot). In alternative embodiments, the activation of the CD4+ T cells may be determined by measuring changed in gene expression in the CD4+ T cells. Genes for which altered expression may be determined include those genes encoding cytokines or associated with cytokine secretion. Again, the skilled person will be familiar with standard techniques for determining alternations in gene expression in a population of T cells. It will be appreciated that it is also possible to monitor the success of treatments for type 1 diabetes, by determining the beta-cell specific T cell response of the individual receiving treatment. More specifically, the present invention provides: a method of monitoring the response of an individual to a treatment for type 1 diabetes, the method comprising providing a population of T cells derived from an individual who has received a treatment for type 1 diabetes. contacting the population of T cells with a polypeptide comprising, consisting essentially of, or consisting of the C-peptide of proinsulin or a fragment thereof determining the response of the population of T cells to the polypeptide wherein an increase in the activation or proliferation of T cells upon contact with the polypeptide indicates the treatment has not been successful.

The present invention also provides a method for determining the strength of an individual’s autoimmune response to proinsulin, the method comprising:

- contacting the population of T cells with a polypeptide comprising, consisting essentially of, or consisting of the C-peptide of proinsulin or a fragment thereof; - determining the response of the T cells in the population to contact with the polypeptide;

It will be appreciated that the above methods can also be utilised to determine changes or the relative strength of an individual’s autoimmune response to pancreatic beta cells in an individual, given that proinsulin and insulin are produced by these cells. In certain embodiments, the control sample obtained from the individual may include a population of T cells from the individual at an earlier time point. This may be useful, for example, in comparing the strength of an individual’s autoimmune response to proinsulin, before and after the individual has received a treatment for type 1 diabetes. Alternatively, the control sample may be a population of T cells from a healthy individual, for which the response to the polypeptide has previously been determined. The skilled person will be familiar with various methods for determining and comparing the level of proliferation or activation of T cells, including by reference to various standard methods described herein.

The skilled person will also appreciate that in any embodiment of the invention, the step of contacting a population of T cells with a polypeptide may include contacting the cells with “free” polypeptide (i.e., polypeptide that is not bound to any other molecule). Alternatively, the T cells may be contacted with a polypeptide that is presented by an HLA molecule. This may include the polypeptide presented by an HLA molecule that is bound to an antigen presenting cell. Alternatively, the polypeptide may be provided in the form of an HLA-peptide complex (such as an HLA-peptide tetramer, wherein the tetramer is derived from an HLA molecule as described herein).

In certain embodiments, the treatment for type 1 diabetes may be a treatment as described herein (i.e., a treatment comprising administration of C-peptide, or a fragment or homolog thereof). In this circumstance, it will be appreciated that a period of washout may be required between administration of the treatment and determination of betacell specific T cell response as described above.

The immune status generally, and specifically levels of regulatory T cells and cytokine profiles, may be readily determined throughout any treatment regime using conventional methods known to those skilled in the art. For example, regulatory T cell levels may be monitored by cytometric analysis following labelling with commercially available antibodies specific to T cell subsets. Other examples of methods suitable for determining the status of the subject include purification of peripheral blood mononuclear cells by density centrifugation followed by stimulation by incubation with well-known antigens such GAD, IA-2 family members, insulin or proinsulin. Resulting proliferation may be quantified by using the CFSE methodology described herein, or alternatively, by assaying for incorporation of FI³ thymidine. Said cytokines can be detected using, for example, specific cytokine antibodies. 24 hours after stimulation with antigen, stimulated cells can be phenotypically characterised by, for example, flow cytometric analysis of activation marker expression (for example CD69, CD44, CTLA4, CD25). Following cell surface labelling of activated cells, said cells may be further fixed and incubated with fluorochrome labelled antibodies to specific cytokines to determine intracellular cytokine levels. In particular, for example, cells may be further assessed by double labelling assays. The double labelled cells may be analysed utilising flow cytometric analysis or fluorescence spectroscopy.

C-peptide

As used, herein“C-peptide” (also referred to as“connecting peptide) refers to the short 31 -amino-acid polypeptide that connects insulin's A-chain to its B-chain in the proinsulin molecule. During the synthesis of insulin, the preproinsulin molecule is translocated into the endoplasmic reticulum of beta cells of the pancreas with an A-chain, a C-peptide, a B- chain, and a signal sequence. The signal sequence is cleaved from the N-terminus of the peptide by a signal peptidase, resulting in the production of the “proinsulin” molecule. After proinsulin is packaged into vesicles in the Golgi apparatus (beta- granules), the C-peptide is excised, leaving the A-chain B-chain, bound together by disulfide bonds that constitute the insulin molecule.

EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ (SEQ ID NO: 1 ). In any aspect of the invention, a fragment of a C-peptide comprises a peptide that can stimulate T cell proliferation. Preferably, the fragment is presented by an HLA molecule DQ8, DQ2, DR3 or DR4. The HLA-DQ8 molecule may be DQA1 ^*03:01 and DQB1 ^*03:02. The HLA-DQ2 molecule may be DQA1 ^*05:01 or DQB1 ^*02:01. More preferably, the fragment comprises any one of the following amino acid sequences, or functional derivatives or homologues thereof:

PGAGSLQPLALE (SEQ ID NO: 2);

SLQPLALEGSL (SEQ ID NO: 3);

QPLALEGSL (SEQ ID NO: 4);

QPLALEGSLQ (SEQ ID NO: 5); PGAGSLQPLAL (SEQ ID NO: 6);

GQVELGGGPGAG (SEQ ID NO: 7); AEDLQVGQVEL (SEQ ID NO: 8); GSLQPLALEGSLQ (SEQ ID NO: 9); SLQPLALEGS (SEQ ID NO: 10);

AGSLQPLAL (SEQ ID NO: 1 1 );

VELGGGPGAG (SEQ ID NO: 12); EDLQVGQVELGG (SEQ ID NO: 13); LQVGQVELGGGPGAGSLQ (SEQ ID NO: 14); RREAEDLQVGQVELGGGP (SEQ ID NO: 15);

GAGSLQPLALEGSLQKRG (SEQ ID NO: 16); or any peptide as shown in the figures and tables included herein.

The C-peptide or fragments for use in any of the methods and compositions described herein, can be isolated, partially purified, purified, recombinant or synthetic. The term "isolated" in relation to a protein or polypeptide means that by virtue of its origin or source of derivation is not associated with naturally-associated components that accompany it in its native state; is substantially free of other proteins from the same source.

A protein may be rendered substantially free of naturally associated components or substantially purified by isolation, using protein purification techniques known in the art.

By “substantially purified” is meant the protein is substantially free of contaminating agents, e.g., at least about 70% or 75% or 80% or 85% or 90% or 95% or 96% or 97% or 98% or 99% free of contaminating agents. As used herein, the term“recombinant” shall be understood to mean the product of artificial genetic recombination. Accordingly, in the context of a recombinant protein comprising or consisting of C-peptide or a fragment thereof, this term does not encompass a C-peptide naturally-occurring within a subject’s body. However, if such a protein is isolated, it is to be considered an isolated protein comprising or consisting of proinsulin. Similarly, if nucleic acid encoding the protein is isolated and expressed using recombinant means, the resulting protein is a recombinant protein comprising or consisting of C-peptide. A recombinant protein also encompasses a protein expressed by artificial recombinant means when it is within a cell, tissue or subject, e.g., in which it is expressed.

The term“protein” shall be taken to include a single polypeptide chain, i.e., a series of contiguous amino acids linked by peptide bonds or a series of polypeptide chains covalently or non-covalently linked to one another (i.e., a polypeptide complex). For example, the series of polypeptide chains can be covalently linked using a suitable chemical or a disulphide bond. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions.

The term “polypeptide” or “polypeptide chain” will be understood from the foregoing paragraph to mean a series of contiguous amino acids linked by peptide bonds.

The term "derivatives" includes fragments, parts, portions, chemical equivalents, mutants, homologs and analogs of C-peptide. Analogs may be derived from natural synthetic or recombinant sources and include fusion proteins. Chemical equivalents of proinsulin can act as a functional analog of C-peptide. Chemical equivalents may not necessarily be derived from proinsulin but may share certain conformational similarities. Alternatively chemical equivalents may be specifically designed to mimic certain physiochemical properties of C-peptide. Chemical equivalents may be chemically synthesised or may be detected following, for example, natural product screenings.

Derivatives include one or more insertions, deletions or substitutions of amino acids. Amino acid insertional derivatives include amino and/or carboxylic terminal fusions as well as intrasequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site although random insertion is also possible with suitable screening of the resulting product. Deletion variants are characterised by the removal of one or more amino acids from the sequence. Substitution amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. Additions to amino acid sequences include fusions with other peptides or polypeptides.

Also contemplated for use in the invention is a biologically active variant or analog of C-peptide, preferably human, that is a polypeptide or peptidomimetic that may have, for example, at least 70%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any one of SEQ ID NOs: 1 to 7, which also retains the biological activity described herein. The biologically active variant or analog may contain one or more conservative amino acid substitutions, or non-native amino acid substitutions.

“Percent (%) amino acid sequence identity” or“percent (%) identical” with respect to a polypeptide sequence, i.e. a polypeptide, protein or fusion protein of the invention defined herein, is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific polypeptide of the invention, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.

Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms (non-limiting examples described below) needed to achieve maximal alignment over the full-length of the sequences being compared. When amino acid sequences are aligned, the percent amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain percent amino acid sequence identity to, with, or against a given amino acid sequence B) can be calculated as: percent amino acid sequence identity = X/Y x100, where X is the number of amino acid residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and Y is the total number of amino acid residues in B. If the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percent amino acid sequence identity of A to B will not equal the percent amino acid sequence identity of B to A.

In calculating percent identity, typically exact matches are counted. The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTN and BLASTX programs of Altschul et al. (1990) J. Mol. Biol. 215:403. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., BLASTX and BLASTN) can be used. Alignment may also be performed manually by inspection. Another non- limiting example of a mathematical algorithm utilized for the comparison of sequences is the ClustalW algorithm (Higgins et al. (1994) Nucleic Acids Res. 22:4673-4680). ClustalW compares sequences and aligns the entirety of the amino acid or DNA sequence, and thus can provide data about the sequence conservation of the entire amino acid sequence. The ClustalW algorithm is used in several commercially available DNA/amino acid analysis software packages, such as the ALIGNX module of the Vector NTI Program Suite (Invitrogen Corporation, Carlsbad, CA). After alignment of amino acid sequences with ClustalW, the percent amino acid identity can be assessed. A non-limiting examples of a software program useful for analysis of ClustalW alignments is GENEDOC™ or JalView (http://www.jalview.org/). GENEDOC™ allows assessment of amino acid (or DNA) similarity and identity between multiple proteins. Another non- limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988) CABIOS 4:1 1 - 17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys, Inc., 9685 Scranton Rd., San Diego, CA, USA). When utilizing the ALIGN program for comparing amino acid sequences, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

The polypeptide desirably comprises an amino end and a carboxyl end. The polypeptide can comprise D-amino acids, L-amino acids or a mixture of D- and L-amino acids. The D-form of the amino acids, however, is particularly preferred since a polypeptide comprised of D-amino acids is expected to have a greater retention of its biological activity in vivo.

The polypeptide can be prepared by any of a number of conventional techniques. The polypeptide can be isolated or purified from a naturally occurring source or from a recombinant source. Recombinant production is preferred. For instance, in the case of recombinant polypeptides, a DNA fragment encoding a desired peptide can be subcloned into an appropriate vector using well-known molecular genetic techniques (see, e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory, 1982); Sambrook et al., Molecular Cloning A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory, 1989). The fragment can be transcribed and the polypeptide subsequently translated in vitro. Commercially available kits also can be employed (e.g., such as manufactured by Clontech, Palo Alto, Calif.; Amersham Pharmacia Biotech Inc., Piscataway, N.J.; InVitrogen, Carlsbad, Calif., and the like). The polymerase chain reaction optionally can be employed in the manipulation of nucleic acids.

The term "conservative substitution" as used herein, refers to the replacement of an amino acid present in the native sequence in the peptide or polypeptide with a naturally or non- naturally occurring amino acid or a peptidomimetic having similar steric properties. Where the side-chain of the native amino acid to be replaced is either polar or hydrophobic, the conservative substitution should be with a naturally occurring amino acid, a non- naturally occurring amino acid or with a peptidomimetic moiety which is also polar or hydrophobic (in addition to having the same steric properties as the side- chain of the replaced amino acid).

Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art. The following six groups are examples of amino acids that may be considered to be conservative substitutions for one another:

1 ) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

As naturally occurring amino acids are typically grouped according to their properties, conservative substitutions by naturally occurring amino acids can be determined bearing in mind the fact that replacement of charged amino acids by sterically similar non-charged amino acids are considered as conservative substitutions. For producing conservative substitutions by non-naturally occurring amino acids it is also possible to use amino acid analogs (synthetic amino acids) well known in the art. A peptidomimetic of the naturally occurring amino acid is well documented in the literature known to the skilled person and non-natural or unnatural amino acids are described further below. When affecting conservative substitutions the substituting amino acid should have the same or a similar functional group in the side chain as the original amino acid. Alterations of the native amino acid sequence to produce mutant polypeptides, such as by insertion, deletion and/or substitution, can be done by a variety of means known to those skilled in the art. For instance, site-specific mutations can be introduced by ligating into an expression vector a synthesized oligonucleotide comprising the modified site. Alternately, oligonucleotide-directed site-specific mutagenesis procedures can be used, such as disclosed in Walder et al., Gene 42: 133 (1986); Bauer et al., Gene 37: 73 (1985); Craik, Biotechniques, 12-19 (January 1995); and U.S. Pat. Nos. 4,518,584 and 4,737,462. A preferred means for introducing mutations is the QuikChange Site-Directed Mutagenesis Kit (Stratagene, LaJolla, Calif.). Any appropriate expression vector (e.g., as described in Pouwels et al., Cloning Vectors: A Laboratory Manual (Elsevier, N.Y.: 1985)) and corresponding suitable host can be employed for production of recombinant polypeptides of C-peptide, biologically active variants or analogs thereof. Expression hosts include, but are not limited to, bacterial species within the genera Escherichia, Bacillus, Pseudomonas, Salmonella, mammalian or insect host cell systems including baculovirus systems (e.g., as described by Luckow et al., Bio/Technology 6: 47 (1988)), and established cell lines such as the COS-7, C127, 3T3, CHO, HeLa, and BHK cell lines, and the like. The skilled person is aware that the choice of expression host has ramifications for the type of polypeptide produced. For instance, the glycosylation of polypeptides produced in yeast or mammalian cells (e.g., COS-7 cells) will differ from that of polypeptides produced in bacterial cells, such as Escherichia coli.

Alternately, a polypeptide of the invention, i.e. C-peptide or a fragment thereof as described herein, biologically active variants or analogs thereof, can be synthesized using standard peptide synthesizing techniques well-known to those of ordinary skill in the art (e.g., as summarized in Bodanszky, Principles of Peptide Synthesis (Springer- Verlag, Heidelberg: 1984)). In particular, the polypeptide can be synthesized using the procedure of solid-phase synthesis (see, e.g., Merrifield, J. Am. Chem. Soc. 85: 2149- 54 (1963); Barany et al., Int. J. Peptide Protein Res. 30: 705-739 (1987); and U.S. Pat. No. 5,424,398). If desired, this can be done using an automated peptide synthesizer. Removal of the t-butyloxycarbonyl (t-BOC) or 9-fluorenylmethyloxycarbonyl (Fmoc) amino acid blocking groups and separation of the polypeptide from the resin can be accomplished by, for example, acid treatment at reduced temperature. The polypeptide- containing mixture can then be extracted, for instance, with dimethyl ether, to remove non-peptidic organic compounds, and the synthesized polypeptide can be extracted from the resin powder (e.g., with about 25% w/v acetic acid). Following the synthesis of the polypeptide, further purification (e.g., using high performance liquid chromatography (HPLC)) optionally can be done in order to eliminate any incomplete polypeptides or free amino acids. Amino acid and/or HPLC analysis can be performed on the synthesized polypeptide to validate its identity. For other applications according to the invention, it may be preferable to produce the polypeptide as part of a larger fusion protein, such as by the methods described herein or other genetic means, or as part of a larger conjugate, such as through physical or chemical conjugation, as known to those of ordinary skill in the art and described herein.

A“peptidomimetic” is a synthetic chemical compound that has substantially the same structure and/or functional characteristics of a polypeptide of the invention, the latter being described further herein. Typically, a peptidomimetic has the same or similar structure as a polypeptide of the invention, for example the same or similar sequence of any one of SEQ ID NOs: 1 to 7. A peptidomimetic generally contains at least one residue that is not naturally synthesised. Non-natural components of peptidomimetic compounds may be according to one or more of: a) residue linkage groups other than the natural amide bond ('peptide bond') linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce secondary structural mimicry, i.e , to induce or stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like.

Peptidomimetics can be synthesized using a variety of procedures and methodologies described in the scientific and patent literatures, e.g., Organic Syntheses Collective Volumes, Gilman et al. (Eds) John Wiley & Sons, Inc., NY, al-Obeidi (1998) Mol. Biotechnol. 9:205-223; Flruby (1997) Curr. Opin. Chem. Biol. 1 :1 14-1 19; Ostergaard (1997) Mol. Divers. 3:17-27; Ostresh (1996) Methods Enzymot.267:220- 234.

In still further embodiments, the polypeptides as described herein can be modified wherein the modification results in an increased half-life of the molecule when administered to an individual requiring treatment. In alternative embodiments, modification may be utilised to reduce the likelihood of degradation at the N’ and C’ terminal ends of the peptide. The skilled person will be familiar with various means for modifying a polypeptide as herein described, for example, by generating a fusion protein comprised of a C-peptide moiety (or fragment of a C-peptide) fused to an antibody fragment and/or PEGylation. The antibody fragment may include a Fab or an Fc. Alternatively, the C-peptide or fragment thereof may be PEGylated to increase the half-life. Still further, the antibody fragment to which the C-peptide or fragment thereof is fused may be PEGylated (i.e., may include a polyethylene glycol or PEG moiety) to further increase the half-life of the molecule. The skilled person will be familiar with various means for PEGylating a fusion protein. Examples of such methods are described in Jevsevar et al., (2012) Methods Mol Biol, 901 : 233-46, the entire contents of which are herein incorporated in their entirety.

Other methods for modifying the polypeptides described herein for the purpose of increasing their half-life will be known to the skilled person. For example, the polypeptides may be also be modified with carbohydrate molecules or other moiety which facilitates an increase in half-life, or targeting of the polypeptide to a particular cellular or subcellular location.

Compositions, formulations and kits, and administration thereof The present invention also provides pharmaceutical compositions comprising a

C-peptide, or fragment thereof, as described herein, and a pharmaceutically acceptable carrier, diluent or excipient.

By the term“excipient” herein is meant a pharmaceutically acceptable material that is employed together with peptide for the proper and successful administration of the peptide to a patient. Suitable excipients are well known in the art, and are described, for example, in the Physicians’ Desk Reference, the Merck Index, and Remington's Pharmaceutical Sciences.

The phrase‘therapeutically effective amount’ generally refers to an amount of C- peptide or fragment thereof that (i) treats the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein.

Generally, daily oral doses of antigen will be from about 0.01 mg/kg per day to 1000 mg/kg per day. Small doses (0.01 -1 mg) may be administered initially, followed by increasing doses up to about 1000 mg/kg per day. In the event that the response in a subject is insufficient at such doses, even higher doses (or effective higher doses by a different, more localised delivery route) may be employed to the extent patient tolerance permits. A single dose may be administered or multiple doses may be required on an hourly, daily, weekly or monthly basis. Effective amounts of antigen vary depending on the individual but may range from about 0.1 pg to about 20 mg, preferably from about 1 pg to about 10 mg and more preferably from about 1 pg to 5 mg per dose.

In any embodiment of the present invention, the C-peptide, fragment or derivative or variant thereof is provided in the individual by administration of C-peptide or fragment directly to the individual. For example, the C-peptide or fragment thereof may be administered intravenously, intranasally, by inhalation, intradermally, intramuscularly or subcutaneously.

Pharmaceutical compositions may be formulated for any appropriate route of administration including, for example, topical (for example, transdermal or ocular), oral, buccal, nasal, vaginal, rectal or parenteral administration. The term parenteral as used herein includes subcutaneous, intradermal, intravascular (for example, intravenous), intramuscular, spinal, intracranial, intrathecal, intraocular, periocular, intraorbital, intrasynovial and intraperitoneal injection, as well as any similar injection or infusion technique. In certain embodiments, compositions in a form suitable for oral use or parenteral use are preferred. Suitable oral forms include, for example, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Within yet other embodiments, compositions provided herein may be formulated as a lyophilizate.

The various dosage units are each preferably provided as a discrete dosage tablet, capsules, lozenge, dragee, gum, or other type of solid formulation. Capsules may encapsulate a powder, liquid, or gel. The solid formulation may be swallowed, or may be of a suckable or chewable type (either frangible or gum-like). The present invention contemplates dosage unit retaining devices other than blister packs; for example, packages such as bottles, tubes, canisters, packets. The dosage units may further include conventional excipients well-known in pharmaceutical formulation practice, such as binding agents, gellants, fillers, tableting lubricants, disintegrants, surfactants, and colorants; and for suckable or chewable formulations.

Compositions intended for oral use may further comprise one or more components such as sweetening agents, flavouring agents, colouring agents and/or preserving agents in order to provide appealing and palatable preparations. Tablets contain the active ingredient in admixture with physiologically acceptable excipients that are suitable for the manufacture of tablets. Such excipients include, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate, granulating and disintegrating agents such as corn starch or alginic acid, binding agents such as starch, gelatine or acacia, and lubricating agents such as magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate may be employed.

Formulations for oral use may also be presented as hard gelatine capsules wherein the active ingredient is mixed with an inert solid diluent such as calcium carbonate, calcium phosphate or kaolin, or as soft gelatine capsules wherein the active ingredient is mixed with water or an oil medium such as peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active ingredient(s) in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as naturally-occurring phosphatides (for example, lecithin), condensation products of an alkylene oxide with fatty acids such as polyoxyethylene stearate, condensation products of ethylene oxide with long chain aliphatic alcohols such as heptadecaethyleneoxycetanol, condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol mono-oleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides such as polyethylene sorbitan monooleate. Aqueous suspensions may also comprise one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more colouring agents, one or more flavouring agents, and one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredients in a vegetable oil such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and/or flavouring agents may be added to provide palatable oral preparations. Such suspensions may be preserved by the addition of an antioxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, such as sweetening, flavouring and colouring agents, may also be present.

Pharmaceutical compositions may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil such as olive oil or arachis oil, a mineral oil such as liquid paraffin, or a mixture thereof. Suitable emulsifying agents include naturally- occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soy bean lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides such as sorbitan monoleate, and condensation products of partial esters derived from fatty acids and hexitol with ethylene oxide such as polyoxyethylene sorbitan monoleate. An emulsion may also comprise one or more sweetening and/or flavouring agents.

Syrups and elixirs may be formulated with sweetening agents, such as glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also comprise one or more demulcents, preservatives, flavouring agents and/or colouring agents.

Compounds may be formulated for local or topical administration, such as for topical application to the skin. Formulations for topical administration typically comprise a topical vehicle combined with active agent(s), with or without additional optional components.

Suitable topical vehicles and additional components are well known in the art, and it will be apparent that the choice of a vehicle will depend on the particular physical form and mode of delivery. Topical vehicles include organic solvents such as alcohols (for example, ethanol, iso-propyl alcohol or glycerine), glycols such as butylene, isoprene or propylene glycol, aliphatic alcohols such as lanolin, mixtures of water and organic solvents and mixtures of organic solvents such as alcohol and glycerine, lipid- based materials such as fatty acids, acylglycerols including oils such as mineral oil, and fats of natural or synthetic origin, phosphoglycerides, sphingolipids and waxes, protein- based materials such as collagen and gelatine, silicone-based materials (both nonvolatile and volatile), and hydrocarbon-based materials such as microsponges and polymer matrices.

A composition may further include one or more components adapted to improve the stability or effectiveness of the applied formulation, such as stabilizing agents, suspending agents, emulsifying agents, viscosity adjusters, gelling agents, preservatives, antioxidants, skin penetration enhancers, moisturizers and sustained release materials. Examples of such components are described in Martindale - The Extra Pharmacopoeia (Pharmaceutical Press, London 1993) and Martin (ed.), Remington's Pharmaceutical Sciences. Formulations may comprise microcapsules, such as hydroxymethylcellulose or gelatine-microcapsules, liposomes, albumin microspheres, microemulsions, nanoparticles or nanocapsules.

A topical formulation may be prepared in a variety of physical forms including, for example, solids, pastes, creams, foams, lotions, gels, powders, aqueous liquids, emulsions, sprays and skin patches. The physical appearance and viscosity of such forms can be governed by the presence and amount of emulsifier(s) and viscosity adjuster(s) present in the formulation. Solids are generally firm and non-pourable and commonly are formulated as bars or sticks, or in particulate form. Solids can be opaque or transparent, and optionally can contain solvents, emulsifiers, moisturizers, emollients, fragrances, dyes/colorants, preservatives and other active ingredients that increase or enhance the efficacy of the final product. Creams and lotions are often similar to one another, differing mainly in their viscosity. Both lotions and creams may be opaque, translucent or clear and often contain emulsifiers, solvents, and viscosity adjusting agents, as well as moisturizers, emollients, fragrances, dyes/colorants, preservatives and other active ingredients that increase or enhance the efficacy of the final product. Gels can be prepared with a range of viscosities, from thick or high viscosity to thin or low viscosity. These formulations, like those of lotions and creams, may also contain solvents, emulsifiers, moisturizers, emollients, fragrances, dyes/colorants, preservatives and other active ingredients that increase or enhance the efficacy of the final product. Liquids are thinner than creams, lotions, or gels, and often do not contain emulsifiers. Liquid topical products often contain solvents, emulsifiers, moisturizers, emollients, fragrances, dyes/colorants, preservatives and other active ingredients that increase or enhance the efficacy of the final product.

Emulsifiers for use in topical formulations include, but are not limited to, ionic emulsifiers, cetearyl alcohol, non-ionic emulsifiers like polyoxyethylene oleyl ether, PEG-40 stearate, ceteareth-12, ceteareth-20, ceteareth-30, ceteareth alcohol, PEG-100 stearate and glyceryl stearate. Suitable viscosity adjusting agents include, but are not limited to, protective colloids or nonionic gums such as hydroxyethylcellulose, xanthan gum, magnesium aluminum silicate, silica, microcrystalline wax, beeswax, paraffin, and cetyl palmitate. A gel composition may be formed by the addition of a gelling agent such as chitosan, methyl cellulose, ethyl cellulose, polyvinyl alcohol, polyquaterniums, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carbomer or ammoniated glycyrrhizinate. Suitable surfactants include, but are not limited to, nonionic, amphoteric, ionic and anionic surfactants. For example, one or more of dimethicone copolyol, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, lauramide DEA, cocamide DEA, and cocamide MEA, oleyl betaine, cocamidopropyl phosphatidyl PG-dimonium chloride, and ammonium laureth sulfate may be used within topical formulations.

Preservatives include, but are not limited to, antimicrobials such as methylparaben, propylparaben, sorbic acid, benzoic acid, and formaldehyde, as well as physical stabilizers and antioxidants such as vitamin E, sodium ascorbate/ascorbic acid and propyl gallate. Suitable moisturizers include, but are not limited to, lactic acid and other hydroxy acids and their salts, glycerine, propylene glycol, and butylene glycol. Suitable emollients include lanolin alcohol, lanolin, lanolin derivatives, cholesterol, petrolatum, isostearyl neopentanoate and mineral oils. Suitable fragrances and colours include, but are not limited to, FD&C Red No. 40 and FD&C Yellow No. 5. Other suitable additional ingredients that may be included in a topical formulation include, but are not limited to, abrasives, absorbents, anticaking agents, antifoaming agents, antistatic agents, astringents (such as witch hazel), alcohol and herbal extracts such as chamomile extract, binders/excipients, buffering agents, chelating agents, film forming agents, conditioning agents, propellants, opacifying agents, pH adjusters and protectants.

Typical modes of delivery for topical compositions include application using the fingers, application using a physical applicator such as a cloth, tissue, swab, stick or brush, spraying including mist, aerosol or foam spraying, dropper application, sprinkling, soaking, and rinsing. Controlled release vehicles can also be used, and compositions may be formulated for transdermal administration (for example, as a transdermal patch).

Pharmaceutical compositions may also be prepared in the form of suppositories such as for rectal administration. Such compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Suitable excipients include, for example, cocoa butter and polyethylene glycols.

Pharmaceutical compositions may be formulated as sustained release formulations such as a capsule that creates a slow release of modulator following administration. Such formulations may generally be prepared using well-known technology and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site. Carriers for use within such formulations are biocompatible, and may also be biodegradable. Preferably, the formulation provides a relatively constant level of modulator release. The amount of modulator contained within a sustained release formulation depends upon, for example, the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.

A pharmaceutical composition comprising C-peptide, or a fragment of derivative thereof, may be formulated as inhaled or intranasal formulations, including sprays, mists, or aerosols. The inhaled formulation may be for application to the upper (including the nasal cavity, pharynx and larynx) and/or lower respiratory tract (including trachea, bronchi and lungs). For inhalation formulations, the composition or combination provided herein may be delivered via any inhalation methods known to a person skilled in the art. Such inhalation or intranasal methods and devices include, but are not limited to, metered dose inhalers with propellants such as HFA or propellants that are physiologically and environmentally acceptable.

A particularly preferred form of administration of C-peptide, fragments or derivative thereof is intranasal administration via an aerosol spray, drip or vapour.

Other suitable devices are breath operated inhalers, multidose dry powder inhalers and aerosol nebulizers. Aerosol formulations for use in the subject method typically include propellants, surfactants and co-solvents and may be filled into conventional aerosol containers that are closed by a suitable metering valve. Different devices and excipients can be used depending on whether the application is to the upper (including the nasal cavity, pharynx and larynx) or lower respiratory tract (including trachea, bronchi and lungs) and can be determined by those skilled in the art. Further, processes for micronisation and nanoparticle formation for the preparation of compounds described herein for use in an inhaler, such as a dry powder inhaler, are also known by those skilled in the art.

Inhalant compositions may comprise liquid or powdered compositions containing the active ingredient(s) that are suitable for nebulization and intrabronchial use, or aerosol compositions administered via an aerosol unit dispensing metered doses. Suitable liquid compositions comprise the active ingredient in an aqueous, pharmaceutically acceptable inhalant solvent such as isotonic saline or bacteriostatic water. The solutions are administered by means of a pump or squeeze-actuated nebulized spray dispenser, or by any other conventional means for causing or enabling the requisite dosage amount of the liquid composition to be inhaled into the patient's nose or lungs. Suitable formulations, wherein the carrier is a liquid, for administration, as for example, a nasal spray or as nasal drops, include aqueous or oily solutions of the active ingredient. Examples of inhalation drug delivery devices are described in Ibrahim et al. Medical Devices: Evidence and Research 2015:8 131-139, are contemplated for use in the present invention.

The term‘administered’ means administration of a therapeutically effective dose of the aforementioned C-peptide or fragments thereof. By ‘therapeutically effective amount’ is meant a dose that produces the effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. As is known in the art and described above, adjustments for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.

The present invention also includes a kit or“article of manufacture” which may comprise a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, blister pack, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a therapeutic composition which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label or package insert indicates that the therapeutic composition is used for treating the condition of choice. In one embodiment, the label or package insert includes instructions for use.

The kit may comprise the C-peptide, or fragment or variant thereof as herein described, or a pharmaceutical composition as described herein comprising C-peptide, fragment or variant thereof. The kit in this embodiment of the invention may further comprise a package insert indicating the composition and other active principle can be used to treat or prevent a disorder described herein. Alternatively, or additionally, the kit may further comprise a second (or third) container comprising a pharmaceutically- acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

In certain embodiments a therapeutic composition as described herein may be provided in the form of a device, disposable or reusable, including a receptacle for holding the therapeutic, prophylactic or pharmaceutical composition. In one embodiment, the device is a syringe. The device may hold 1 -2 ml_ of the therapeutic composition. The therapeutic or prophylactic composition may be provided in the device in a state that is ready for use or in a state requiring mixing or addition of further components.

It will be understood that these examples are intended to demonstrate these and other aspects of the invention and although the examples describe certain embodiments of the invention, it will be understood that the examples do not limit these embodiments to these things. Various changes can be made and equivalents can be substituted and modifications made without departing from the aspects and/or principles of the invention mentioned above. All such changes, equivalents and modifications are intended to be within the scope of the claims set forth herein.

Examples Example 1

Research design and methods

Subjects.

Participants (aged 3-45 years) without type 1 diabetes (“healthy controls”), diagnosed with type 1 diabetes within 100 days (“recent-onset”) or greater than 100 days (“long-standing”) were recruited. Type 1 diabetes was diagnosed according to American Diabetes Association criteria (American Diabetes, A (2017)“2. Classification and Diagnosis of Diabetes.” Diabetes Care, 40 (SuppM ): S1 1 -S24). The majority of recent-onset participants had been screened for diabetes-associated autoantibodies prior to recruitment for this study (see Table 4 and 5). All healthy controls were screened for diabetes-related autoantibodies (see Table 6). All participants’ HLA-DQ and -DR alleles were determined by the Australian Red Cross Blood Services (ARCBS) Victorian Transplantation and Immunogenetics Service (VITS). Healthy controls were defined as individuals without diabetes and autoantibodies against insulin, IA-2 and GAD-65 who had either an HLA-DQ2 or HLA-DQ8 allele. Subjects on steroids or immunomodulatory drugs were excluded from the study.

The protocol and consent documents were approved by St Vincent’s Hospital, Royal Melbourne Hospital and Southern Health Ethics committees (HREC-A 135/08, 2009.026 and 12185B). All participants provided written informed consent.

Synthetic peptides

Peptides were synthesized by Purar Chemicals, Doncaster East, VIC, Australia. Peptides were reconstituted in 40% acetonitrile, 0.5% acetic acid and water, or DMSO, to 5mM and stored at -80°C. CFSE-based proliferation assay and CD4+ T cell cloning

Blood was obtained by venepuncture. Peripheral blood mononuclear cells (PBMC) were isolated over Ficoll-paque (GE Healthcare, Sweden) and washed twice in phosphate buffered saline (PBS) as described in Mannering et al., (2003) A sensitive method for detecting proliferation of rare autoantigen-specific human T cell, J. Immunol. Methods, 283: 173-183.

The CFSE (5,6- carboxylfluorescein diacetate succinimidyl ester) proliferation assays were performed as described previously in Mannering et al., (2005) An efficient method for cloning human autoantigen-specific T cells, J. Immunol. Methods 298: 83-92 and Mannering et al., (2003. Briefly, PBMC were labeled with 0.1 mM CFSE (Life Technologies, Carlsbad, CA) and cultured (0.1 x10⁶ cells in 0.1 ml/well) in sterile 96-well U-bottom plates. CFSE-labeled PBMC were cultured with either: no antigen, C-peptide (31 amino acids, EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ, 10mM), or tetanus toxoid (10LfU/ml, Statens Serum Institut). After 7 days of culture the cells were washed in PBS and stained on ice with anti-human CD4-AlexaFluor-647 (clone OKT4, conjugated in-house). Optimal compensation and gain settings for the flow cytometer were determined for each experiment based on unstained and single stained samples. Propidium iodide (PI) was used to exclude dead cells. CD4+ T-cell proliferation was measured by determining the number of CD4+, CFSEdim cells for every 5,000 CD4+ CFSEbright cells. The results are presented as a cell division index (CDI) which is the ratio of the number of CD4+ cells that have proliferated in the presence of antigen: without antigen.

For cloning C-peptide specific CD4+ T cells, a single, viable (propidium iodide negative), CD4+, CFSEdim cell was sorted into each well of a 96-well plate, the full protocol detailed in Mannering et al., (2005). Each well contained 2x10⁵ gamma- irradiated (6,000 rad) thawed feeder cells, cytokines, and Fungizone (2.5 pg/mL, Amphotericin B, Apothecan, Princeton, NJ). The cytokines (Peprotech, Rocky Hill, NJ) were used at the final concentrations of IL-2 (20U/ml_), IL-4 (10ng/ml_) and anti-CD3 (clone OKT-3, 30ng/ml_). Clones were cultured in RPMI (Sigma, UK) supplemented with 5% pooled human serum, 2mM L-glutamine (Glutamax, Gibco, USA), penicillin 100U/mL , streptomycin (100 pg/mL) and 100 pM non-essential amino acids (Gibco, USA), referred to as culture medium. The total volume of each well was 100pL. Cells were fed at day 7 with fresh cytokines (IL-2 and IL-4) in 50pL of culture medium, at the same final concentrations indicated. At day 10-14, proliferating clones were identified by visual inspection and confirmed using reverse-phase microscopy. Clones were expanded into 48-well plates and antigen-specificity was tested for by 3H-thymidine incorporation assay. Antigen-specific clones were expanded with anti-CD3, IL-2+IL-4 and feeder cells. Antigen-specificity was confirmed at the end of expansion by repeat 3H-thymidine incorporation assay. Clones that retained antigen-specificity after expansion were stored in 10% dimethyl sulfoxide (DMSO) and foetal calf serum (FCS) (Bovogen, Victoria, Australia) at 5x10⁶ cells/mL at -80°C.

Screening antigen-specific clones

Clones were tested for antigen specificity using the 3H-thymidine incorporation assay (see Mannering et al., 2005). APC were either autologous PBMC that had been stored in liquid nitrogen, or class II HLA matched Epstein Barr Virus transformed B-cell line (KJ; HLA-DRB1 ^*03:01 , 04:04; DQB1 ^*02:01 , 03:02). APCs were gamma- irradiated (2,000 rad for autologous PBMC and 10,000 rad for EBV KJ line). Each clone was tested in duplicate against APC (20,000 cells/well) with and without antigen. Cultures were set up in 96-well round-bottom plates, in complete culture media. T-cell clones were re-suspended and half the volume (usually 200pL) was transferred to a 10 ml tube. The T cells were washed, first in PBS and then in 0.5 ml of culture medium and finally re-suspended in 400mI_ of culture medium. Cells were not counted, but 100mI_ of washed, cloned, T cells were added to wells containing APC with and without antigen. After 2 days, ³H-thymidine (0.5 pCi/ well) was added for 12-18h after which the cells were harvested and incorporated radioactivity measured by h-scintillation counting. Clones that had a stimulation index (SI, cpm with antigen/cpm without antigen) of >3.0 were considered to be antigen specific.

Functional Analysis of CD4+ T-cell clones

Cloned CD4+ T cells were thawed and used directly in functional assays as described previously in Pathiraja et al., (2015) Pro-insulin-specific, HLA-DQ8 and HLA- DQ8-Transdimer-restricted CD4+ T cells infiltrate islets in Type 1 Diabetes, Diabetes, 64: 172-182. To identify the epitopes recognized by the cloned CD4+ T cells, they were incubated with a class II HLA matched Epstein Barr Virus transformed B-cell line (KJ; HLA-DRB1 ^*03:01 , 04:04; DQB1 ^*02:01 , 03:02) (Mannering et al., (2005)) and synthetic peptides indicated in the figure legends.

Cloned CD4+ T cells (50,000/well) were cultured with 20,000 antigen presenting cells (APC) with and without peptides as indicated in the figures. A T cell’s response to antigen was measured as interferon gamma (IFNy secretion into the culture media. The IFNy concentration in the culture media was determined by ELISA (Biolegend, San Francisco, CA).

Determining a clone’s HLA restriction

The FILA restriction of the C-peptide specific CD4+ T-cell clones was determined in two steps as described previously (Mannering et al., (1997) FILA-DR1 -restricted bcr- abl (b3a2)-specific CD4+ T lymphocytes respond to dendritic cells pulsed with b3a2 peptide and antigen-presenting cells exposed to b3a2 containing cell lysates. Blood, 90: 290-297; Mannering, et al., (2005) and Mannering, et al., (2009) The A-chain of insulin is a hot spot for CD4+ T cell epitopes in human type 1 diabetes, Clin. Exp. Immunol., 156: 226-231 ). First monoclonal antibodies specific for FILA-DR (mAb clone L243), FILA-DP (mAb clone B7/21 ) and HLA-DQ (mAb clone SPV-L3) were added to the peptide stimulated T-cell cultures to a final concentration of 5.0 pg/ml. Second the FILA alleles was determined using a panel of T2 cells transfected with FILA-DQA and FILA- DQB alleles indicated in the figure legends. Where the HLA blocking assay indicated HLA-DR restriction, HLA alleles were determined using a panel of BLS cells transfected with HLA-DR alleles indicated in the figure legends.

Sequencing the TCR genes from C-peptide specific clones Five cells per well in 0.5% FCS/PBS were sorted into 96-well PCR plates. Plates were stored at -80°C overnight. Reverse transcription mix was dispensed directly into wells, converting to cDNA using Superscript VILO Reverse Transcriptase (Invitrogen, CA). TRA and TRB genes were amplified using pools of reverse primers using Taq polymerase (Qiagen, Germany) as described byWang et al., (2012) T cell receptor alphabeta diversity inversely correlates with pathogen-specific antibody levels in human cytomegalovirus infection, Sci. Transl. Med., 4: 128ra142. PCR product was sequenced and the TCR genes identified by alignment with the IMGT database (http://www.imgt.org/IMGT_vquest) (Brochet et al., (2008) IMGT/V-QUEST: the highly customized and integrated system for IG and TR standarised V-J and V-D-J sequence analysis, Nucleic Acids Res, 36: W503-508.) In some cases, RNA was extracted using Bioline RNA Isolate II Mini extraction kit from 0.5-2.0x106 cells and converted to cDNA using Superscript III Reverse transcriptase kit (Invitrogen, CA). The TRA or TRB gene was amplified and its presence confirmed using only reverse primers to suspected TCR genes. Sanger sequencing was performed by Australian Genome Research Facility (AGRF), Melbourne.

Insulin VNTR genotyping. Genomic DNA was purified using Genomic DNA purification NucleoBond kit (Macherey-Nagel, Germany) from thawed 0.5mL of whole blood that had been snap frozen and stored at -80oC at the time of venepuncture. Genotyping of the single nucleotide polymorphism, rs689 was performed using an inventoried Taqman SNP Genotyping Assay obtained from Applied Biosystems (Life Technologies, Carlsbad, California). The assay was run on a Roche LightCycler 480 II using Roche Probe Master Mix (Roche Applied Science, Penzberg, Germany) according to manufacturer’s instructions. Preparation of human pancreatic islet and acinar extract

Snap frozen human islets, supplied by the Tom Mandel Islet Transplantation Program, from five donors was pooled and homogenized in a pre-prepared buffer (60% Tris 20mM/NaCI 50mM, sucrose 0.3M, methionine 1 mM, protease inhibitor 1 :200 (Sigma-Aldrich); 30% acetonitrile, 10% butanol) optimised to reduce oxidation and degradation of protein whilst not interfering with T-cell proliferation. Acinar tissue from the same donors was prepared in the same way to use as a negative control. Protein quantity was estimated using the Pierce bicinchoninic acid (BCA) protein assay (Thermo Scientific, Rockford, USA). Aliquots were stored in -80°C. Islet and acinar lysate were lyophilised and diluted in culture media to the required concentration.

Statistical analysis

Prism 7 was used to analyse the data and perform statistical analysis. Statistical analysis of comparisons of CD4+ T-cell responses as measured by CFSE between groups, and the comparison of measured IFN-g by ELISA for clone specificity and HLA restriction was made using unpaired Welch’s two-tailed t test. Statistical significance was defined as p<0.05.

Results

Response of peripheral CD4⁺ T-cells to C-peptide

The CFSE-based proliferation assay was used to measure CD4⁺ T-cell responses to full-length C-peptide (PI32-63) in peripheral blood mononuclear cells (PBMCs) Mannering et al., (2005). CD4⁺ T-cell responses to full-length C-peptide were detected (CDI > 3.0) in fourteen of 23 subjects (61 %) with recent-onset diabetes, one of 13 (8%) healthy subjects, and two of 15 (13%) subjects with long-standing diabetes (Figure 1 ). The magnitude of the CD4⁺ T-cell responses to C-peptide were significantly greater in PBMC from individuals with recent onset T1 D 10.8 (SEM ±3.9) compared to healthy controls 2.0 (SEM ±2.5) (p = 0.03, Welsh’s t test).

A cell division index (CDI) (Mannering et al., (2003)) of > 3.0 was found to give the greatest disease specificity and sensitivity, determined using the Receiver-Operator Curve (ROC) (Table 6). To determine if a subject’s insulin VNTR genotype correlated with their responses to C-peptide the Insulin VNTR alleles were determined for 18 of the 23 recent onset

T1 D subjects. Eleven of 18 (61 %) subjects were homozygous for the high T1 D risk, class I insulin VNTR. Six of 18 (33%) were heterozygous for the allele (class I/Ill VNTR). There was no correlation between an individual’s CD4⁺ T-cell response to C- peptide and their insulin VNTR genotype (Figure 6).

Mapping the C-peptide derived epitopes recognized by peripheral blood CD4 T cells

To further characterize the C-peptide derived epitopes recognized by peripheral blood, CD4⁺ T-cells clones were isolated using the CFSE-based cloning method (see Mannering et al., (2005)). A total of 32 CD4⁺ T-cell clones were isolated from the peripheral blood of six subjects with recent-onset T1 D. Of the 32, five were excluded: two failed to expand sufficiently for further characterisation, and three clones could not be characterised. Therefore, 27 had epitope specificity and FILA-restriction characterised. To determine the epitope specificity, the clones were tested against a panel of five overlapping 18mer peptides that spanned the length of C-peptide. As an example, epitope mapping for clone H8.5 is shown in Figure 2A. Fifteen of the 27 clones (56%) responded to one of the five 18mer peptides (Table 5). For these clones, the minimum epitopes required to stimulate the CD4⁺ T-cell clones were determined using a panel of peptides sequentially truncated by one amino acid from either the N- or C-terminus (Figure 2B, clone H8.5). The epitopes recognized by the remaining 12 clones that did not respond to any of the five 18mer peptides, were mapped using variants of the full-length C-peptide which were sequentially truncated from either the N- or C- terminus by three amino acids (Figure 6). One clone, clone K9.5, is shown as an example in Figure 2C. The minimum epitope was then determined by testing the clones against a panel of peptides with a single amino acid substitution (Figure 2D, clone K9.5). The substitutions were chosen to disrupt the putative HLA-binding or TCR recognition. Using this approach, the epitope specificity for seven of the 12 clones were determined, but the epitope(s) recognized by five of the clones could not be determined. Epitopes were identified across the entire sequence of proinsulin, but the C-terminal end of proinsulin was the most commonly recognized by C-peptide specific CD4⁺-T-cell clones. A summary of the epitope mapping is shown in Table 1 .

Most clones are HLA-D8 restricted

The HLA restriction of the CD4⁺ T-cell responses to proinsulin was determined in two steps. First, blocking of T-cell recognition in the presence of antibodies to HLA-DP, - DQ, and -DR. The C-peptide specific responses of 20 of 27 clones (74%) were inhibited by the antibody specific for HLA-DQ (SPV-L3), five of 27 (19%) were blocked by an anti- HLA-DR antibody (L243). Representative results for two clones, H8.5 and K6.4 are shown in Figure 3A, C. For two clones, the FILA Class II alleles could not be determined. The second step was to define the restricting FILA alleles using a panel of T2 (Riberdy and Cresswell (1992) The antigen-processing mutant T2 suggests a role for MFIC-linked genes in class II antigen presentation, J. Immunol., 148: 2586-2590) or BLS (Bare lymphocyte syndrome) (Kovats et al., (1995) Deficient antigen-presenting cell function in multiple genetic complementation groups of type II bare lymphocyte syndrome, J. Clin. Invest., 96: 217-223) lines transduced with individual FILA-DQ or FILA-DR genes, respectively. All five of the FILA-DR restricted clones recognized C- peptide presented by FILA-DR 04:01 . Of the 22 FILA-DQ restricted clones, 10 (41 %) responded to C-peptide presented by FILA-DQ8 (DQA1 ^*03:01 , DQB1 ^*03:02), five (23%) were restricted by FILA-DQ2 (DQA1 ^*05:01 , DQB1 ^*02:01 ) and three (14%) were restricted by FILA-DQ8-transdimer (DQA1 ^*05:01 , DQB1 ^*03:02). Some clones exhibited promiscuous recognition; one clone (5%) responded to both FILA-DQ8 and FILA-DQ8 transdimer expressing APCs, and one (5%) responded to both FILA-DQ2 and -DQ2 transdimer. Results for clone FI8.5 and K6.4 (Fig 3B and 3D respectively) are shown as examples. Data for all clones are shown in Figure 6. Table 1 summarizes the FILA- restriction for all the clones analyzed. Flence, with the exception of FILA-DR3, C-peptide specific clones were restricted by all the FILA alleles strongly associated with risk of T1 D.

TCR sequences expressed by C-peptide specific T cells

To examine the clonal diversity of the peripherally-derived C-peptide specific CD4⁺ T-cells, TCR genes expressed by the clones were sequenced. The clones used a range of TRAV and TRBV genes (Table 2). TCR a and b genes were sequenced from all 20 clones for which an epitope and HLA restriction could be determined. Fifteen distinct TRA/TRB combinations were found (Table 3). Four of the six (67%) subjects from whom more than one clone was isolated, had identical T-cell receptors in two or more of their clones. This is suggestive of clonal expansion within one donor. As expected, clones with identical TCRs had matching epitope specificity and FILA restriction. TRAV 12-1 ^*01 was identified in clones from two of six donors, and TRBV 20- 1 ^*01 -05 was identified in four of the six donors. Flowever, there was no evidence of a ‘public’ TCR.

Full-length C-peptide is a more potent agonist for some CD4⁺ T-cell clones The data indicated that FILA-DQ responses to full-length C-peptide could be relatively easily detected by stimulating PBMC with full length C-peptide (PI33-63 SEQ ID NO:1 ).. To determine if full length C-peptide was a more potent agonist for CD4⁺ T cells we used our clones of known epitope specificity to compare the potency of full-length (31 aa) C-peptide to 18mer peptides incorporating the same epitope. In each case the epitope was flanked by at least two amino acids at both the N- and C-terminii. Remarkably, for some clones the full-length C-peptide was >100x more potent than the 18-mer peptide (Figure 4 and Table 2). In contrast, some clones responded equally to both full-length C-peptide and an overlapping 18mer. The clones that were more sensitive to the full-length C-peptide recognized epitopes towards the C-terminus of the C-peptide. The full-length C-peptide is a much stronger agonist for some, but not all, C- peptide specific CD4⁺ T-cell clones.

Peripheral C-peptide specific CD4⁺ T cells respond to pancreatic islet extract

Finally, a representative clone from each of the six subjects was tested for their response to human pancreatic islet extract presented by the Epstein Barr Virus transformed B-cell line (KJ; HLA-DRB1 ^*03:01 , 04:04; DQB1 ^*02:01 , 03:02). Five of the six clones responded to human pancreatic islet extract (Figure 5). No response was seen to acinar extract from the same organ donors. Discussion

This work shows that CD4⁺ T-cell responses against the full-length C-peptide can be detected in the peripheral blood of >60% of individuals recently diagnosed with T1 D. Cloning and analysis of these C-peptide specific T cells revealed that the epitopes recognized by these clones were found throughout C-peptide, but most epitopes fell between PI residues 48 (P) and 63(Q). Most clones were restricted by HLA-DQ8, or DQ2. Dose-response experiments revealed that for some clones, full length C-peptide was >100x more potent stimulator of CD4⁺ T-cell responses than 18mer peptides. These data suggest that full length C-peptide (PI33-63), may be particularly useful for detecting T-cell responses in the peripheral blood of subjects with recent-onset T1 D, and useful in antigen-specific therapies.

It was found that >60% of people within 100 days of diagnosis of T1 D had a detectable CD4⁺ T-cell response to C-peptide in their peripheral blood. This is in comparison to 8% of healthy controls.

In contrast to recent onset subjects, C-peptide specific responses in this study could be detected in very few of the individuals with long-standing T1 D (median 14 years from diagnosis, range 1 -32 years). Although longitudinal data was not collected in this study, this observation suggests that T cell responses to C-peptide decline after the onset of T1 D. This could be due to migration of C-peptide specific CD4⁺ T cells away from the peripheral blood in the context of a relative lack or absence of circulating C- peptide.

No correlation was evident with an individual’s /A/S-VNTR genotype and the presence of a peripheral CD4⁺ T-cell response to C-peptide. The genetic control of proinsulin transcription levels is determined by the polymorphic INS promotor region. This region is classified according to the number of tandem repeats of a 14-15 bp consensus element, i.e. VNTR I (short, 26-63 repeats), VNTR II (63-140 repeats) or VNTR III (long, 141 -209 repeats). VNTR I/I homozygosity confers the highest risk of T1 D as it is associated with a higher transcription level of insulin in thymic medullar epithelial cells. The converse applies to the VNTR lll/lll or I/Ill genotype, which is associated with a higher transcription level of insulin in the thymus and thus a lower risk of T1 D. Overall the /A/S-VNTR genotype is associated with an odds ratio for T1 D susceptibility of 2.2. Given the sensitivity of the CFSE assay and the potency demonstrated of C-peptide as an antigen, it is possible that the lack of VNTR correlation seen in this study is due to the detection of low-avidity proinsulin-specific T cells.

Characterisation of peripheral C-peptide specific CD4⁺ T-cell clones from patients with recent-onset T1 D revealed that the majority (81 %) were HLA-DQ restricted, with nearly half of these (41 %) restricted by HLA-DQ8. These findings are particularly significant given the paucity of studies identifying HLA-DQ restricted T cells in the periphery, despite the known importance of HLA-DQ2 and HLA-DQ8 in the risk of developing T1 D. This may be partly due to the techniques used, the advantage of the CFSE-based proliferation assay is its sensitivity to detect rare circulating T cells and the ability to clone and characterise the responding cells. The majority of studies in the past have used the less sensitive ³H-thymidine proliferation assays, or ELISpot assays which do not offer the ability to clone and characterise the responding cells. Finally, pHLA- tetramers have been used to identify antigen specific T cells. However, the application of tetramers is limited to known peptide/HLA combinations. All studies investigating CD4⁺ T cells in human peripheral blood in T1 D to-date have used HLA-DR4 tetramers due to the lack of identified HLA-DQ2 and DQ8 restricted epitopes and the instability of these HLA molecules.

Analysis of the TCR genes used by C-peptide specific CD4⁺ T cells revealed unique TRAV and TRBV gene usage between subjects. Four of the seven (57%) subjects had two or three clones with identical T-cell receptors. This implies that the C- peptide specific clones isolated from the same donor, may be derived from a single progenitor. While there was narrow TCR repertoire within an individual, there was a very wide range of TCRs used between individuals. Interestingly K9.6 had an identical alpha chain to islet-infiltrating clone A5.5. Their specificity spanned the same region of C- peptide (GQVELGGGPGAG vs. QVELGGGPG) and they were both restricted by HLA- DQ8. This shows that, using our approach, T cell responses to C-peptide occurring in the pancreas may be detected in a sample of peripheral blood. Further supporting this is the response seen of the peripherally derived C-peptide specific CD4⁺ T cells to human pancreatic islet extract. Full-length C-peptide has greater potency than 18-mer peptides spanning the relevant epitope for a particular clone. For 12 of the 27 clones isolated (44%), full-length C-peptide had a greater than 70-fold increased potency compared to an 18-mer peptide. Interestingly, all the clones for which full-length C-peptide had greater potency targeted the C-terminal end of C-peptide. This C-terminal end is thought to contain a biologically active penta-peptide that interacts with cell membranes. We suggest that the full-length of C-peptide is a clinically relevant beta-cell antigen in T1 D that has been overlooked.

We have demonstrated that C-peptide stimulates a disease-specific peripheral CD4⁺ T-cell response in a cohort of individuals with recent-onset T1 D, and these responses are restricted by the high-risk alleles FILA-DQ2/8. We believe this is related to the use of the full-length C-peptide which we have demonstrated to be a more potent antigen compared to shorter epitopes spanning its length. This is the first study that has investigated C-peptide specific CD4⁺ T-cell responses and characterised the responding cells to this detail. Responses to full-length C-peptide may form the basis, along with other beta-cell antigens, of a T-cell assay capable of monitoring changes to beta-cell specific T-cell responses over the stages of progression of T1 D, and/or during diseasemodifying treatment. These results indicate that antigen-specific therapy using full- length C-peptide is likely to be an effective approach to preventing T1 D.

Table 1 : Summary of epitope mapping and HLA restriction analysis of C-peptide specific CD4+ T cells

Table 2. Summary of TCR gene sequencing of C-peptide specific CD4⁺ T-cell clones

Table 3: Responses to long and short peptides by epitope specificity and HLA restrictions.

Table 5: Baseline characteristics of“long-standing” subjects.

Table 7 ROC analysis of C-peptide specific CD4⁺ T-cell responses

1. Cell Division Index (CDI) calculated from CFSE-based proliferation assay.

2. All subjects were included in the analysis (recent-onset T1 D subjects n=23, healthy control subjects n=13)

Table 8: Summary of C-peptide specific CD4⁺ T-cell clones’ TCR usage

Table 9: Differences in potency of full length C-peptide and 18mer peptides

Claims

1. A method of treating type 1 diabetes in an individual in need thereof, the method comprising administering to the individual an effective amount of a polypeptide comprising, consisting essentially of, or consisting of the C-peptide of proinsulin or a fragment thereof, thereby treating type 1 diabetes in the individual.

2. A method of inhibiting progression of type 1 diabetes, in an individual in need thereof, the method comprising administering to the individual an effective amount of a polypeptide comprising, consisting essentially of, or consisting of the C-peptide of proinsulin or a fragment thereof, thereby inhibiting progression of type 1 diabetes in the individual.

3. A method of inducing regulatory T cells in an individual having type 1 diabetes, the method comprising administering to the individual an effective amount of a polypeptide comprising, consisting essentially of, or consisting of the C-peptide of proinsulin or a fragment thereof, thereby inducing regulatory T cells in the individual.

4. A method of maintaining insulin production in an individual having type 1 diabetes, the method comprising, consisting essentially of, or consisting of administering to the individual an effective amount of a polypeptide comprising, consisting essentially of, or consisting of the C-peptide of proinsulin or a fragment thereof, thereby maintaining insulin production in the individual.

5. The method of any one of the preceding claims, wherein the polypeptide comprises or consists essentially of, or consists of the sequence as set out in EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ (SEQ ID NO: 1 ).

6. The method of any one of the preceding claims, wherein the polypeptide consists or consists essentially of the sequence as set out in EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ (SEQ ID NO: 1 ).

7. The method of any one of the preceding claims, wherein the polypeptide comprises, or consists essentially of, or consists of the sequence as set out in any one of SEQ ID NOs 2 to 16 or 59 to 145.

8. The method of any one of the preceding claims, wherein the polypeptide consists or consists essentially of the sequence as set out in any one of SEQ ID NOs: 2 to 16 or 59 to 145.

9. The method of claim 8, wherein the polypeptide consists or consists essentially of the sequence PGAGSLQPLALE (SEQ ID NO: 2).

10. The method of claim 8, wherein the polypeptide consists or consists essentially of the sequence SLQPLALEGSL (SEQ ID NO: 3).

1 1. The method of claim 8, wherein the polypeptide consists or consists essentially of the sequence QPLALEGSL (SEQ ID NO: 4).

12. The method of claim 8, wherein the polypeptide consists or consists essentially of the sequence QPLALEGSLQ (SEQ ID NO: 5).

13. The method of claim 8, wherein the polypeptide consists or consists essentially of the sequence PGAGSLQPLAL (SEQ ID NO: 6).

14. The method of claim 8, wherein the polypeptide consists or consists essentially of the sequence GQVELGGGPGAG (SEQ ID NO: 7).

15. The method of claim 8, wherein the polypeptide consists or consists essentially of the sequence AEDLQVGQVEL (SEQ ID NO: 8).

16. The method of claim 8, wherein the polypeptide consists or consists essentially of the sequence GSLQPLALEGSLQ (SEQ ID NO: 9).

17. The method of claim 8, wherein the polypeptide consists or consists essentially of the sequence SLQPLALEGS (SEQ ID NO: 10).

18. The method of claim 8, wherein the polypeptide consists or consists essentially of the sequence AGSLQPLAL (SEQ ID NO: 1 1 ).

19. The method of claim 8, wherein the polypeptide consists or consists essentially of the sequence VELGGGPGAG (SEQ ID NO: 12).

20. The method of claim 8, wherein the polypeptide consists or consists essentially of the sequence EDLQVGQVELGG (SEQ ID NO: 13).

21. The method of claim 8, wherein the polypeptide consists or consists essentially of the sequence LQVGQVELGGGPGAGSLQ (SEQ ID NO: 14).

22. The method of claim 8, wherein the polypeptide consists or consists essentially of the sequence RREAEDLQVGQVELGGGP (SEQ ID NO: 15).

23. The method of claim 8, wherein the polypeptide consists or consists essentially of the sequence GAGSLQPLALEGSLQKRG (SEQ ID NO: 16).

24. The method of any one of the preceding claims, wherein the polypeptide is isolated, substantially purified, purified, recombinant or synthetic.

25. The method of any one of the preceding claims, wherein the method further comprises determining whether the individual is at risk of type 1 diabetes, or has recent-onset type 1 diabetes.

26. The method of any one of the preceding claims, wherein the individual expresses the HLA alleles DQ8, DQ2 DR3 and/or DR4.

27. The method of claim 26, wherein the DQ8 allele is DQA1 ^*03:01 or DQB1 ^*93:02.

28. The method of claim 26, wherein the DQ2 allele is DQA1 ^*05:01 or DQB1 ^*02:01.

29. The method of claim 26, wherein the DRB1 allele is DRB1 ^*04:0X.

30. The method of any one of the preceding claims, wherein the individual has CD4+ T cells which have a T-cell receptor with a CDR3 sequence as shown in Table

2 (SEQ ID NOs: 17 to 58).

31. A method of determining whether an individual at risk of developing type 1 diabetes is displaying early signs or symptoms of the disease, the method comprising: providing a population of T cells derived from an individual who is at risk of developing type 1 diabetes; contacting the population of T cells with a polypeptide comprising, consisting essentially of, or consisting of the C-peptide of proinsulin or a fragment thereof; determining if there is a response of the T cells in the population to contact with the polypeptide; wherein an increase in the activation or proliferation of T cells upon contact with the polypeptide indicates the individual is displaying early signs or symptoms of type 1 diabetes.

32. A method of monitoring beta-cell specific T cell response in an individual who has early markers of type 1 diabetes, the method comprising: providing a population of T cells derived from an individual in whom the beta-cell specific T cell response is to be determined; contacting the population of T cells with a polypeptide comprising, consisting essentially of, or consisting of the C-peptide of proinsulin or a fragment thereof; and determining if there is a response of the T cells in the population to contact with the polypeptide.

33. The method of claims 32, wherein an increase in the activation or proliferation of T cells upon contact of the cells with the polypeptide indicates the progression of type 1 diabetes in the individual.

34. The method of claim 33, wherein the individual has stage 1 type 1 diabetes, has family history of type 1 diabetes, or other risk factors for type 1 diabetes.

35. A method of determining the success of a treatment for type 1 diabetes in an individual, the method comprising: providing a population of T cells derived from an individual in whom the success of a treatment for type 1 diabetes is to be determined; contacting the population of T cells with a polypeptide comprising, consisting essentially of, or consisting of the C-peptide of proinsulin or a fragment thereof; and determining if there is a response of the T cells in the population to contact with the polypeptide, wherein an increase in the activation or proliferation of T cells upon contact of the cells with the polypeptide indicates the progression of type 1 diabetes in the individual; wherein a decrease in the activation or proliferation of T cells upon contact with the polypeptide and an absence of clinical symptoms of type 1 diabetes indicates that the treatment for type 1 diabetes has been successful; and wherein a decrease in the activation or proliferation of T cells upon contact with the polypeptide and the presence of clinical symptoms of type 1 diabetes indicates that the treatment for type 1 diabetes has not been successful.

36. The method of any one of claims 31 to 35, wherein the individual expresses the

HLA alleles DQ8, DQ2, DR3 and/or DR4.

37. The method of claim 36, wherein the DQ8 allele is HLA-DQA1 ^*03:01 or HLA- DQB1 ^*03:02.

38. The method of claim 36, wherein the DQ2 allele is DQA1 ^*05:01 or DQB1 ^*02:01.

39. The method of claim 36, wherein the DRB1 allele is DRB1 ^*04:0X.

40. The method of any one of claims 31 to 39, wherein the individual has T cells expressing the T cell receptor genes having a sequence as shown in Table 2.

41. The method of claim 40, wherein the individual has CD4+ T cells which have a T-cell receptor with a CDR3 sequence as shown in Table 2 (SEQ ID NOs: 17 to 58).

42. The method of any one of claims 31 to 41 , wherein the polypeptide comprises or consists essentially of, or consists of the sequence as set out in any one of SEQ ID NOs: 1 to 16 or 59 to 145.

43. The method of any one of claims 31 to 42, wherein the polypeptide consists essentially of, or consists of the sequence as set out in any one of SEQ ID NOs: 1 to 16 or 59 to 145.

44. The method of claim 43, wherein the polypeptide consists or consists essentially of the sequence EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ (SEQ ID NO: 1 ).

45. The method of claim 43, wherein the polypeptide consists or consists essentially of the sequence PGAGSLQPLALE (SEQ ID NO: 2).

46. The method of claim 43, wherein the polypeptide consists or consists essentially of the sequence SLQPLALEGSL (SEQ ID NO: 3).

47. The method of claim 43, wherein the polypeptide consists or consists essentially of the sequence QPLALEGSL (SEQ ID NO: 4).

48. The method of claim 43, wherein the polypeptide consists or consists essentially of the sequence QPLALEGSLQ (SEQ ID NO: 5).

49. The method of claim 43, wherein the polypeptide consists or consists essentially of the sequence PGAGSLQPLAL (SEQ ID NO: 6).

50. The method of claim 43, wherein the polypeptide consists or consists essentially of the sequence GQVELGGGPGAG (SEQ ID NO: 7).

51. The method of claim 43, wherein the polypeptide consists or consists essentially of the sequence AEDLQVGQVEL (SEQ ID NO: 8).

52. The method of claim 43, wherein the polypeptide consists or consists essentially of the sequence GSLQPLALEGSLQ (SEQ ID NO: 9).

53. The method of claim 43, wherein the polypeptide consists or consists essentially of the sequence SLQPLALEGS (SEQ ID NO: 10).

54. The method of claim 43, wherein the polypeptide consists or consists essentially of the sequence AGSLQPLAL (SEQ ID NO: 1 1 ).

55. The method of claim 43, wherein the polypeptide consists or consists essentially of the sequence VELGGGPGAG (SEQ ID NO: 12).

56. The method of claim 43, wherein the polypeptide consists or consists essentially of the sequence EDLQVGQVELGG (SEQ ID NO: 13).

57. The method of claim 43, wherein the polypeptide consists or consists essentially of the sequence LQVGQVELGGGPGAGSLQ (SEQ ID NO: 14).

58. The method of claim 43, wherein the polypeptide consists or consists essentially of the sequence RREAEDLQVGQVELGGGP (SEQ ID NO: 15).

59. The method of claim 43, wherein the polypeptide consists or consists essentially of the sequence GAGSLQPLALEGSLQKRG (SEQ ID NO: 16).

60. Use of a polypeptide comprising, consisting essentially of, or consisting of the C- peptide of proinsulin or a fragment thereof in the manufacture of a medicament for (a) treating type 1 diabetes, (b) inhibiting progression of type 1 diabetes, (c) inducing regulatory T cells, (d) inducing deletion, anergy or exhaustion of pathogenic CD4+ T cells, or (e) restoring insulin production, in an individual having type 1 diabetes.

61. The use of claim 60, wherein the polypeptide comprises, or consists essentially of, or consists of the sequence as set out in any one of SEQ ID NOs 1 to 16 or 59 to 145.

62. The use of claim 60 or 61 , wherein the polypeptide consists essentially of, or consists of the sequence as set out in any one of SEQ ID NOs 1 to 16 or 59 to 145.

63. The use of claim 62, wherein the polypeptide consists or consists essentially of the sequence EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ (SEQ ID NO: 1 ).

64. A polypeptide comprising, consisting essentially of, or consisting of the C-peptide of proinsulin or a fragment thereof for use in (a) treating type 1 diabetes, (b) inhibiting progression of type 1 diabetes, (c) inducing regulatory T cells, (d) inducing deletion, anergy or exhaustion of pathogenic CD4+ T cells, or (e) restoring insulin production, in an individual having type 1 diabetes.

65. The polypeptide for use of claim 64, wherein the polypeptide comprises, or consists essentially of, or consists of the sequence as set out in any one of SEQ ID NOs 1 to 16 or 59 to 145.

66. The polypeptide for the use of claim 64 or 65, wherein the polypeptide consists essentially of, or consists of any one of the sequences set forth in SEQ ID NOs: 1 to

16 or 59 to 145.

67. The polypeptide for use of any one of claims 64 to 66, wherein the polypeptide of peptide is isolated, substantially purified, purified, recombinant or synthetic.

68. A peptide comprising, consisting essentially of, or consisting of PGAGSLQPLALE (SEQ ID NO: 2);

SLQPLALEGSL (SEQ ID NO: 3);

QPLALEGSL (SEQ ID NO: 4);

QPLALEGSLQ (SEQ ID NO: 5);

PGAGSLQPLAL (SEQ ID NO: 6); GQVELGGGPGAG (SEQ ID NO: 7);

AEDLQVGQVEL (SEQ ID NO: 8);

GSLQPLALEGSLQ (SEQ ID NO: 9);

SLQPLALEGS (SEQ ID NO: 10);

AGSLQPLAL (SEQ ID NO: 11 ); VELGGGPGAG (SEQ ID NO: 12);

EDLQVGQVELGG (SEQ ID NO: 13);

LQVGQVELGGGPGAGSLQ (SEQ ID NO: 14);

RREAEDLQVGQVELGGGP (SEQ ID NO: 15); GAGSLQPLALEGSLQKRG (SEQ ID NO: 16); or any peptide as shown in SEQ ID NOs: 59 to 145 or functional derivatives or homologues thereof.

69. A peptide according to claim 68, wherein the peptide consists or consists essentially of the sequence EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ (SEQ

ID NO: 1 ).

70. A peptide according to claim 68, wherein the peptide consists or consists essentially of the sequence PGAGSLQPLALE (SEQ ID NO: 2).

71. A peptide according to claim 68, wherein the peptide consists or consists essentially of the sequence SLQPLALEGSL (SEQ ID NO: 3).

72. A peptide according to claim 68, wherein the peptide consists or consists essentially of the sequence QPLALEGSL (SEQ ID NO: 4).

73. A peptide according to claim 68, wherein the peptide consists or consists essentially of the sequence QPLALEGSLQ (SEQ ID NO: 5).

74. A peptide according to claim 68, wherein the peptide consists or consists essentially of the sequence PGAGSLQPLAL (SEQ ID NO: 6).

75. A peptide according to claim 68, wherein the peptide consists or consists essentially of the sequence GQVELGGGPGAG (SEQ ID NO: 7).

76. A peptide according to claim 68, wherein the peptide consists or consists essentially of the sequence AEDLQVGQVEL (SEQ ID NO: 8).

77. A peptide according to claim 68, wherein the peptide consists or consists essentially of the sequence GSLQPLALEGSLQ (SEQ ID NO: 9).

78. A peptide according to claim 68, wherein the peptide consists or consists essentially of the sequence SLQPLALEGS (SEQ ID NO: 10).

79. A peptide according to claim 68, wherein the peptide consists or consists essentially of the sequence AGSLQPLAL (SEQ ID NO: 1 1 ).

80. A peptide according to claim 68, wherein the peptide consists or consists essentially of the sequence VELGGGPGAG (SEQ ID NO: 12).

81. A peptide according to claim 68, wherein the peptide consists or consists essentially of the sequence EDLQVGQVELGG (SEQ ID NO: 13).

82. A peptide according to claim 68, wherein the peptide consists or consists essentially of the sequence LQVGQVELGGGPGAGSLQ (SEQ ID NO: 14).

83. A peptide according to claim 68, wherein the peptide consists or consists essentially of the sequence RREAEDLQVGQVELGGGP (SEQ ID NO: 15).

84. A peptide according to claim 68, wherein the peptide consists or consists essentially of the sequence GAGSLQPLALEGSLQKRG (SEQ ID NO: 16).

85. The peptide of claim 68, wherein the peptide consists or consists essentially of any one of the amino acid sequences set forth in SEQ ID NOs: 59 to 145.

86. The peptide of any one of claims 68 to 85, wherein the polypeptide of peptide is isolated, substantially purified, purified, recombinant or synthetic.

87. A pharmaceutical composition comprising a peptide of any one of claims 68 to

86 and a pharmaceutically acceptable carrier, diluent or excipient.

88. A kit comprising a peptide of any one of claims 68 to 85, optionally with written instructions for performing a method according to any one of claims 1 to 59.