WO2015006833A1 - A method for treating type 1 diabetes - Google Patents

A method for treating type 1 diabetes Download PDF

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Publication number
WO2015006833A1
WO2015006833A1 PCT/AU2014/050136 AU2014050136W WO2015006833A1 WO 2015006833 A1 WO2015006833 A1 WO 2015006833A1 AU 2014050136 W AU2014050136 W AU 2014050136W WO 2015006833 A1 WO2015006833 A1 WO 2015006833A1
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Prior art keywords
peptide
amino acid
acid sequence
diabetes
cell
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PCT/AU2014/050136
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French (fr)
Inventor
Stuart MANNERING
Helen Thomas
Thomas Kay
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St. Vincent's Institute Of Medical Research
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Priority claimed from AU2013902683A external-priority patent/AU2013902683A0/en
Application filed by St. Vincent's Institute Of Medical Research filed Critical St. Vincent's Institute Of Medical Research
Publication of WO2015006833A1 publication Critical patent/WO2015006833A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/62—Insulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

  • the present disclosure relates to a method for the treatment or prevention of Type 1 diabetes including a condition associated with Type 1 diabetes and to therapeutic and prophylactic agents useful for same,
  • the present disclosure further teaches diagnostic assays in the detection and monitoring of Type 1 diabetes and its associated conditions,
  • Type 1 diabetes is an autoimmune disease associated with activation of lymphocytes against pancreatic ⁇ -cells. Whilst Type 1 diabetes has been the focus of decades of research, it is still ill defined in human subjects (Faideau t al, (2005) Diabetes 5 f >;S87-S96). The autoimmune disease is highly muitigenic (Todd (2010) Immunity 32(-4):457 ⁇ 467; Rogner et al (2001) Genomics 72f2j:163-171).
  • Type 1 diabetes A feature of human Type 1 diabetes is the long preclinical phase that precedes hyperglycemia (Ziegler and Nepom (2010) Immunity 32(4) 46&-47B), There is also a high level of recurrence of autoimmunity in long-standing patients who have been treated with exogenous insulin (Sible et al. (1985) Laborator Investigation 5J 2j:132 ⁇ 144).
  • p-Cell-speeific CD4 + and CD8 + T-cells maintain immunological memory for years after onset of Type 1 diabetes.
  • Key aiitoantigens associated with Type 1 diabetes are insulin and its precursor forms, proinsulin and preproinsulin (Faideau et al (2005) supra; Homo-Deiarche and Boitard (1996) Immunology Today 17(10) :456-4 ⁇ 0).
  • Type 1 diabetes is a debilitating, chronic disease.
  • Antigen-specific therapies are a promising approach in the treatment and prevention of Type 1 diabetes. This approach requires a knowledge of epitopes recognized by pathogenic T-cells and B-celts, in turn, this would also lead to better diagnostic assays to detect and monitor Type 1 diabetes.
  • progress towards the identification of disease-relevant epitopes using ⁇ -ceils and B-cells isolated from peripheral blood has been slow.
  • Islet-infiltrating T-eells have been used to detect T-cell epitopes on proinsulin which, when modified, are useful in the treatment or prevention of Type 1 diabetes or a condition associated with Type: 1 diabetes in mammalian subjects such as humans.
  • Peptides encompassing the identified t-cell epitope are modified by ait amino acid substitution.
  • the modified peptides result in a more sensitive, efficacious and/or active response from T-cells from diabetic subjects.
  • These epitopes are also useful in diagnostic assays for autoreactive T-celis, B-ceils and antibodies.
  • the amino acid sequence in the proinsulitt-derived peptides is modified by a glutamine (Q) to glutamic acid (E) substitution.
  • the peptides can be made during the chemical synthesis of the peptides, by recombinant means, or by recombinant means followed by posttranslational modification.
  • the peptides ma also comprise chemical analog of, or a non-naturally occurring amino acid substitute of, one or more amino acid residues in the sequence.
  • the instant disclosure teaches a method for the treatment or prophylaxis of Type 1 diabetes or a condition associated with Type 1 diabetes in a mammalian subject comprising the administration to the mammalian subject; a peptide comprising an amino acid sequence of at least seven consecutive amino acid residues encompassing a T-cell epitope derived from insulin, proinsulin or preproinsulin wherein the amino acid sequence comprises a glutamine to glutamic acid substitution. It is proposed that the peptide comprises an epitope which, when modified by the glutamine to glutamic acid substitution, is far more sensitive to recognition by antigen-presenting cells and lymphocytes.
  • an isolated T-cell or antigen-presenting cell derived from a mammalian subject presenting a peptide comprising an amino acid sequence of at least seven consecutive amino acids encompassing a T-cell epitope derived from insulin, proinsulin or preproinsulin wherein the amino acid sequence comprises a glutamine to glutamic acid substitution,
  • the isolated T-cell or antigen-presenting cell is useful for treating or preventing Type 1 diabetes or an associated condition wherein the T-cells or antigen- presenting cells are autologous to the subject being treated.
  • T-cell or "T- lymphocyte” includes a CD4 + T-cell and a CD8 + T-cell.
  • This aspect includes regulatory T- cells (Treg cells) useful to enhance a response to the modified peptide.
  • peptide- HLA Class II tetramers may be prepared and used for inducing tolerance and/or to assess CD4 + T-cell autoreactivity,
  • a condition associated with Type 1 diabetes includes a cardiovascular condition, neuropathy, a retinopathy, a nephropathy, hearing impairment, an infection and a complication in. pregnancy.
  • the antigen-presenting cell is serologically HLA-DQ8, In an embodiment, the antigen-presenting cell comprises the hapiotype HLA-DQ A*03:Q1, DQ B*03;02, In another embodiment, T-cells respond to the peptide presented by another HLA molecule, such as but not limited to HLA DQ A*03:01 , DQ B* 03:02.
  • T-cells respond to the peptide presented by another HLA molecule, such as but not limited to H LA DQ2 and HLA DQ 2/8 transdiraers (DQ A*05:01, DQ B*03:02 and/or DQ A*Q3:01 ; DQ 13*02:01), IILA-DR3 (DRB 1 *03xx) and/or HLA-DR4 (DRB 1 *04xx).
  • HLA DQ2 and HLA DQ 2/8 transdiraers DQ A*05:01, DQ B*03:02 and/or DQ A*Q3:01 ; DQ 13*02:01
  • IILA-DR3 DQ 13*02:01
  • HLA-DR4 DRB 1 *04xx
  • a therapeutic or prophylactic composition comprising a peptide comprising an amino acid sequence of at least seven consecutive amino acids encompassing a T-cell epitope derived from insulin, proinsulin or preproinsulin wherein the amino acid sequence comprises a glutamine to glutamic acid substitution and one or more pharmaceutically acceptable carriers, diluents and/or excipients.
  • the subject peptide may also be complexed with an HLA Class II tctramcr.
  • the present specification is also instructional on the use of a peptide comprising at least seven consecutive amino acids encompassing a T-cell epitope derived from insulin, proinsulin or preproinsulin wherein the amino acid sequence comprises a glutamine to glutamic substitution in the manufacture of a medicament for the treatment of Type 1 diabetes or a condition associated with Type 1 diabetes in a mammalian subject.
  • the mammalian subject is a human male or female of any age.
  • Taught herein is a method of detecting autoreactive T-eells including CD4 + T-eells which recognize an epi tope on pro insulin with a modification comprising a glutamine to glutamic acid substitution with greater responsiveness compared to the same peptide but without the glutamine to glutamic acid substitution.
  • CD8 + T-cells can also be detected.
  • HLA Class II tetramers may also be used to interrogate CD4 1 T-cells, The tetramers are complexed with a. subject peptide such as SEQ ID NO:94.
  • J0017 Enabled herein is a method for detecting the presence of autoreactive T-cells indicative of the presence of, or a predisposition for the development of, Type I diabetes, or a condition associated with Type 1 diabetes in a mammalian subject, the method comprising contacting a sample from the subject comprising immune cells with a peptide comprising an amino acid sequence of at least seven consecutive amino acid residues encompassing a T-cell epitope derived from insulin, proinsulin or preprojnsulm wherein the amino acid sequence comprises a glutamine to glutamic acid substitution for a time and under conditions sufficient for an autoreactive T-cell, if present, to be stimulated into producing an immune effector molecule, and then detecting for the presence or level of the immune effector molecule, wherein the presence or level of the immune effector molecule is indicative of Type 1 diabetes or its state or risk of progression.
  • the level of response in a peptide comprising a glutamine to glutamic acid substitution is greater than the same peptid
  • the peptide comprises an amino acid sequence selected from the list consisting of SEQ ID NOs:2 to 5, 32, 35 to 61, 64, 67, 70, 73, 76, 79, 82 to 94 and 96 to 12L [0019
  • an arginme ma be converted to a citrulline such as set forth in a sequence selected from the list consiSsting of SEQ ID NOs:6 to 1, 33, 34, 62, 63, 65, 66, 68, 69, 71, 72, 74, 75, 77, 78, 80, 81 and 95.
  • the peptide is SEQ ID NO:95.
  • Other amino acid substitutions may also be made including the substitution of a natural amino acid with a non-naturally occurring amino acid residue.
  • the instant disclosure further contemplates detecting autoreactive antibodies to insulin, proinsulin Or preproinsulin as well as generating antibodies to the modified peptides of the instant disclosure.
  • a method for screening for autoreactive antibodies associated with Type 1 diabetes or a condition associated therewith in subject comprising contacting body fluid from the subject with a peptide comprising an amino acid sequence encompassing an epitope derived fro insulin, proinsulin or preproinsulin wherein the amino acid sequence comprises a glutamine to glutamic acid substitution for a time and under conditions sufficient for an autoreactive antibody, if present, to bind to the peptide and then screening for the presence of the peptide-antibody complex.
  • the presence or level of the antibody is indicative of Type 1 diabetes or its state or risk of progression.
  • the glutamine to glutamic acid substitution results in higher sensitivity in antibody binding.
  • a peptide of the present disclosure comprising an amino acid sequence selected from SEQ ID NOs:2 to 5, 32, 35 to 61, 64, 67, 70, 73, 76, 79, 82 to 94 and 96 to 121 may also be conjugated to or embedded in a targeting moiety and/or a carrie molecule.
  • the targeting moiety and the carrier molecule are the same entity,
  • a targeting moiety is useful in facilitating the deployment of the peptide to a particular cell type or tissue type. Examples of cell types include antigen-presenting cells, and CD4 + T-cells or other lymphocytes such as CDS T-celJs.
  • targeting moieties include antibodies or antigen-binding fragments or derivatives thereof specific for an antigen on the target cell or tissue, receptor ligands, a glycosaminoglycan, heparan or heparin or a fragment thereof, a cytokine or other molecule capable of specific binding to a desired cell type.
  • HLA Class 11 tetramers may also be used.
  • Carrier molecules include adjuvants, molecules which increase the half life in the blood stream and the like.
  • Other carrier molecules include HLA-DQ8 complexes such as a complex with HLA-DQ A*03:01 ⁇ DQ B*03:02.
  • ' ⁇ -cells respond to the peptide presented by another HLA molecule, such as but not limited to HLA DQ2 and IILA DQ 2/8 traitsdimers (DQ A*Q5:01 , DQ B*03;02 and DQ A*()3:()I ; DQ B* 02:01), HLA-DR3 (DRB 1 *Q3xx) and/or HLA-DR4 (DRB 1 *04xx).
  • HLA DQ2 and IILA DQ 2/8 traitsdimers DQ A*Q5:01 , DQ B*03;02 and DQ A*()3:()I ; DQ B* 02:01
  • HLA-DR3 DQ B*03
  • HLA-DR4 DRB 1 *04xx
  • amino acid residues encompassing a T-cell epitope derived from insulin, proinsulin or preproinsulin wherein the amino aeid sequence comprises a glutamine to glutamic acid substitution in an amino acid sequence selected from SEQ ID NOs:2 to 5, 32, 35 to 61, 64, 67, 70, 73, 76, 79, 82 to 94 and % to 121, the peptide conjugated at its N- or C-terrninal end to a tiirgeting moiety or carrier molecule,
  • a peptide comprising an amino acid sequence of at least seven consecutive amino acid residues encompassin a T-cell epitope derived from insulin, proinsulin or preproinsulin wherein the amino acid sequence comprises an arginine to citrulline substitution in an amin acid sequence selected from SEQ ID Os:6 to 31 , 33, 34, 62, 63, 65, 66, 68, 69, 71, 72, 74, 75, 77, 78, 80, 81 and 95, the peptide embedded in a targeting moiety or carrier molecule,
  • an agent comprising a peptide comprising an amino acid sequence encompassing a T-cell epitope derived from insulin, proinsulin or preproinsulin having a glutamine to glutamic acid substitution and comprising an amino acid sequence set forth in. SEQ ID NOs;2 to 5, 32, 35 to 61, 64, 67, 70, 73, 76, 79, 82 to 94 and 96 to 121, the peptide conjugated at its N- or C-terminal end to a targeting moiety or carrier molecule, [0027J
  • one particular peptide is as defined by SEQ ID NO:94 or comprises the amino acid sequence of SEQ ID NO:94.
  • the subject specification contemplates a method for detecting the presence of autoreactive T-cells indicative of the presence of, or a predisposition for the development oft Type 1 diabetes, or a condition associated with Type 1 diabetes in a subject, the method comprising contacting a sample from the subject comprising immune cells with a peptide comprising an amino acid sequence of at least seven consecutive amino acid residues encompassing a T-ceTl epitope derived from insulin, proinsuiin or prepro insulin wherein the amino acid sequence comprises a glutaroine to glutamic acid substitution for a time and under conditions sufficient for an autoreactive T- cell, if present, to be stimulated into producing an immune effector molecule, and then detecting the presence of the immune effector molecule, wherein the presence o level of the mtmune effector molecule is indicative of Type 1 diabetes or its state or risk of progression or a condition associated with Type 1 diabetes .
  • the subject specification contemplates a method for detecting the presence of autoreactive B-cells indicative of the presence of. or a predisposition for the development of, Type 1 diabetes, or a condition associated with Type 1 diabetes in a subject, the method comprising contacting a sample from the subject comprising immune cells with a peptide comprising an amino acid sequence encompassing an epitope derived from insulin, proinsuiin or preproinsulin wherein the amino acid sequence comprises a glutamine to glutamic acid substitution for a time and under conditions sufficient for an autoreactive B-cell, if present, to be stimulated into producing an antibody, and then detecting the presence of the antibody, wherein the presence or level of the antibody is indicative of Type 1 diabetes or its state or risk of progression or condition associated with Type 1 diabetes.
  • the subject is a human.
  • the peptide comprises an amino acid sequence selected from the list consisting of SEQ ID NOs:2 to 5, 32, 35 to 61, 64, 67, 70, 73, 76, 79, 82 to 94 and 96 to 121.
  • the peptide comprises an amino acid sequence set forth in SEQ 1D N0:94.
  • the condition associated with Type 1 diabetes is selected from the list consisting of a cardiovascular condition, a neuropathy, a retinopathy, a nephropathy, infection and a complication in pregnancy.
  • the peptide has a minimum length of 13 amino acid residues and encompasses an epitope which is recognized by and binds to an antigen- presenting cell which is serologically HLA-DQ8 such as an antigen-presenting cell which comprises the haplotype HLA-DQA*03:01 » DQ B*03:02.
  • T-cells respond to the peptide presented by another HLA molecule, such as but not limited to HLA DQ2 and HLA DQ 2/8 transdimers (DQ A*05:01 , DQ B*03;Q2 and DQ A*03:01 ; DQ B*02:0.1, IILA-DR3 (DRBl *03xx) and/or HLA-DR4 (DRRl *04xx).
  • HLA DQ2 and HLA DQ 2/8 transdimers DQ A*05:01 , DQ B*03;Q2 and DQ A*03:01 ; DQ B*02:0.1, IILA-DR3 (DRBl *03xx) and/or HLA-DR4 (DRRl *04xx).
  • Reference to a minimum length of 13 includes from 13 to 40 including from 13 to 20.
  • the amino acid sequence comprises at least one glutamine to glutamic acid substitution (i.e. a deamidatlon).
  • HLA Class II tetramer incorporating a peptide ' comprising at least 7 consecutive amino acids comprising a T-cell epitope derived from insulin, proinsulin or prepromsulin, wherein the peptide comprises a glutamine to glutamic acid substitution.
  • the peptide comprises at least 13 amino acid residues.
  • the peptide comprises a sequence selected from SEQ ID NO:2 to 5, 32, 35 to 61, 64, 67, 70, 73, 76, 79 and 82 to 94 or 96 to 121.
  • the peptide comprises SEQ ID NO: 94,
  • TRXEAED modified proinsulin-derived peptide
  • EAEDLEV Amino acid sequence of modified proinsulin-derived peptide
  • ABSLEVG Amino acid sequence of modified proinsulin-derived peptide
  • VGEVELG Amino acid sequence of modified proinsulin-derived peptide
  • EPLALEG modified proinsulin-derived peptide
  • VEECCTS modified proinsul in-derived peptide
  • EECCTSI Amino acid sequence of modified proinsulin-derived peptide
  • Figure 1 is a photographic representation of T-cells emerging from human islets.
  • Figure 2 is a representation of an ovemew of the cloning of human islet infiltrating T-cells.
  • Figure 3 is a diagrammatic representation of an analysis of the antigen specific of T-cell clones.
  • Figure 4 is a graphical representation of clonal expansio of islet-infiltrating T-cells.
  • Figure 5 is a graphical representation of the screening for responses to posttranslational modified peptides
  • Figure 6 is a graphical representation of the response of clone ⁇ 2. ⁇ 1 to an epitope formed by posttranslational modification.
  • Figure 7 is a graphical representation of the titration of Q25H peptides.
  • Figure 8 is a graphical representation showing that Pool E stimulates clone A4.7.
  • Figure 9 is a graphical representation showing that R.65X is the active peptide.
  • Figure 10 is a graphical representation showing that a monoclonal antibody specific for HLA-DQ inhibits a INFy-based response to a peptide derived and modified from promsulm (SEQ ID NO:94).
  • Figure 11 is a graphical representation showing that a T-cell clone's response to a peptide requires an antigen-presenting cell (APC) which expresses HLA DQ8 (HLA- DQ A*03;Q1 S DQ B*03:02).
  • APC antigen-presenting cell
  • figure 12 is a graphical representation of a fine epitope mapping.
  • Panel A these data show that the minimum number of amino acid residues required to stimulate a T-cell clone is 13.
  • Panel B to confirm the results in Panel A, peptides covering the sequence of one particular epitope but with each amino acid in torn substituted with an alanine (A) or lysine (K) were tested. The results showed which residues could be modified without impairing the T-cell response.
  • Figure 13 is a graphical representation of CD4 + T-cell responses measured using a CFSE-based proliferation assay in (A) typical diabetes; and (B) health controls, The results show the ratio of the number of CD4 + T-cells that have proliferated in response to a non-modified peptide (with glutamine [Q]) compared to a glutamic acid (E) substitution,
  • FIG. 1.4 is a graphical representation showing the effects of l Srner peptide covering an epitope in an unmodified (glutamine (Qj) or modified (glutamic acid [E]) on IFNy response from a T-cell clone.
  • the present specification teaches the generation of peptides comprising an amino acid sequence having at least seven consecutive amino acid residues comprising a T-cell epitope derived from insulin, proinsulin or preproinsulin,
  • the amino acid sequence of the peptides is modified with a glutamine to glutamic acid substitution
  • the peptide may contain other modifications to the amino acid residues such as a chemical modification to an amino acid residue or a substitution of an amino acid residue for a non- natural ly occurring amino acid.
  • the peptide may alternatively or in addition comprise an arginine to citmiline substitution.
  • the glutamine to glutamic acid substitution in the amino acid sequence of the epitope results in a higher degree of recognition by antigen-presenting cells and lymphocytes and antibodies.
  • the peptide epitopes derived from insulin, proinsulin or preproinsulin are used to induce tolerance of autoreactive T-cells and/or other immune cells in the treatment or prophylaxis of Type 1 diabetes or an associated condition in a mammalian subject.
  • the modified peptides are also useful in diagnostic assays to detect autoreactive antibodies and the peptides and/or the antibodies are also useful to measure the level of autoreactive T-cells such as CD4 T T-cells or other T-cells such as CD8 + T-eells.
  • Such autoreactive antibodies or T-cells lead to the development or progression of Type 1 diabetes or a condition associated therewith. Hence, their direct detection or indirect detection assists in 5 determining the presence of Type 1 diabetes, such as prior to hyperglycemia and/or the state of progression of diabetes.
  • Antibodies may also be generated specifically to the modified peptides. Such antibodies are useful in diagnostic assays.
  • Conditions associated with Type 1 diabetes include cardiovascular conditions 10 such as angina, heart attack, stroke, atherosclerosis and hypertension; neuropathy; retinopathy; nephropathy; hearing impairment; chronic gum infection; and complications in pregnancy such as miscarriage, preeclampsia and diabetic ketoacidosis,
  • a method for the treatment or prophylaxis of Type 1 diabetes I S or a condition associated with Type I diabetes in a mammalian subject comprising administering to the subject a peptide comprising an amino acid sequence of at least seven consecutive amino acids encompassing a T-cell epitope derived from insulin, proinsulin or preproinsulin wherein the amino acid sequence comprises a glutamine to glutamic acid substitution,
  • the peptides comprise epitopes which are recog mzed by CD4 + T-cells, CDS T-cells and/or antigen-presenting cells as well as B-cells in mammalian subjects sensitized to insulin, proinsulin or preproinsulin.
  • the T-cells are CD4 + T-cells.
  • cognized is meant that the peptide induces a T-cell mediated response statistically 5 significant relative to a T-cell control using the same peptide to which the mammal is not sensitized or an unrelated peptide in a sensitized or non-sensitized mammal or is bound by autoreactive antibodies directed to insulin, proinsulin or preproinsulin.
  • the amino acid sequence is "derived from" insulin, proinsulin or preproinsulin meaning the peptide is a fragment of one of these molecules or is chemically synthesized based on the amino acid 0 sequence of the desired region or is produced by recombinant means alone or by recombinant means followed by posttranslational chemical modification.
  • the peptide with the glutamine to glutamic acid substitution is more strongly recognized by a serologically HLA-DQ8 antigen-presenting cell such as an antigen- presenting cell which comprises the haplotype HLA-DQ A* 03:01, DQ B* 03:02.
  • T-eells respond to th peptide presented by another HLA molecule, such as but not limited to HLA DQ2 and HLA DQ 2/8 transdimers (DQ A* 05 :()! ., DQ B* 03:02 and DQ A* 03:01 ; DQ B* 02:01) or HLA-DR3 (DRBl *Q3xx) and/or HLA-D 4 (DRB l *04xx).
  • the peptide binds to HLA-DQ A* 03:01 ; DQ B*03: ( )2, In another embodiment, the peptide binds to another HLA molecule selected from one or more of HLA DQ2, HLA DQ 2/8 transdimer, DR3 and/or DR4, in a embodiment, the peptide binds to an antigen-presenting cell which comprises the haplotype DQ A*G5:01 , DQ B*03:02 and/or DQ A*Q3:01, DQ B*02:01 , j (M)61
  • the peptides of the instant disclosure comprise from 7 to 40 consecutive amino acid residues meaning 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 amino acid residues.
  • the only requirement is a glutamine to glutamic acid substitution.
  • the "peptide” may contain from 2 to 40 amino acid residues provided it contains this substitution.
  • the seven consecutive amino acids represented in SEQ ID NOs:2 to 93 are comprised within a larger 10 to 20 amino acid sequence, Examples of larger amino acid sequences include SEQ ID NGs:94 to 121.
  • 10 to 20 means 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid residues.
  • the peptides are useful in tolerizing or down-regulating the priming and/or activity of T-cells of a mammal with Type 1 diabetes, The peptides are also useful in diagnostic assays. f0062]
  • the peptide has a minimum of 13 amino acid residues, hence from 13 to 40 including 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 amino acid residues.
  • a method for the treatment or prophylaxis of Type 1 diabetes or a conditio associated with Type 1 diabetes in a mammalian subject comprising administering to the subject a peptide comprising an amino acid sequence of at least 13 consecutive amino acids encompassing a T-cell epitope derived from insulin, proinsulin or preproinsulin wherein the amino acid sequence comprises a glutamiiie to glutamic acid substitution.
  • at least 13 means from “13 to 40” and in particular "13 to 20”
  • the amino acid sequence comprises at least one glutamine to glutamic acid substitution (i.e. a deamidation)
  • a method for the treatment or prophylaxis of Type 1 diabetes or a condition associated with Type 1 diabetes in a mammalian subject comprising administering to the subject a peptide comprising an amino acid sequence of from 13 to 40 consecutive amino acids encompassing a T-cell epitope derived from insulin, proinsulin or preproinsulin wherein the amino acid sequence comprises a glutamine to glutamic acid substitution,
  • the amino acid sequence comprises a glutamine to glutamic acid substitution
  • at least 13 means from “13 to 40” and in particular "13 to 20”.
  • the amino acid sequence comprises at least one glutamine to glutamic acid substitution (i.e. a deamidation).
  • a method for the treatment or prophylaxis of Type 1 diabetes or a condition associated with Type 1 diabetes in a mammalian subject comprising administering to the subject a peptide comprising an amino acid sequence of from 13 to 20 consecutive amino acids encompassing a T-eell epitope derived from insulin, proinsulin or preproinsulin wherein the amino acid sequence comprises a glutamine to glutamic acid substitution.
  • a peptide comprising an amino acid sequence of from 13 to 20 consecutive amino acids encompassing a T-eell epitope derived from insulin, proinsulin or preproinsulin wherein the amino acid sequence comprises a glutamine to glutamic acid substitution.
  • at least 13 means from “13 to 40” and in particular "13 to 20”.
  • the amino acid sequence comprises at least one glutamine to glutamic acid substitution (i.e. a deamidation)
  • the peptide binds to an antigen-presenting cell which comprises the haplotype HLA-DQ A*03:01, DQ B*03:02.
  • the peptide is part of an HLA Class II tetranier.
  • Reference to a "mammalian subject” includes a human or other primate, laboratory test animal such as a mouse, rat, rabbit or guinea pig, a farm animal such as a horse, donkey, pig, cow or sheep, a companion animal such as a dog or cat and a captive wild animal.
  • the mammalian subject is a human.
  • the human may be an infant, child, young adult, adult or elderly person.
  • the human may be male or female, [0068]
  • a method for the treatment or prophylaxis of Type I diabetes or a condition associated with Type 1 diabetes in a human subject comprising administering to the subject a peptide comprising an amino acid sequence of at least seven consecutive amino acids encompassin a T-ceU epitope derived from insulin, proinsulu oi prepro insulin wherein the amino acid sequence comprises a glutamine t glutamic acid substitution.
  • the term "at least seven consecutive amino acids” includes the aspect where a peptide of 10 to 20 amino acid residues including a peptide of at least ⁇ 3 amino acid residues comprises the seven amino acid sequences.
  • An example includes SEQ ID HOs:94 or a peptide selected from SEQ ID ' NO: 96 to 121 ,
  • the present specification is further instructional on a method for the treatment or prophylaxis of Type I diabetes or a condition associated with Type 1 diabetes in a mammalian subject, the method comprising administering to the subject a peptide comprising m amino acid sequence of at, least seven consecutive amino acids encompassing a T-cell epitope derived from insulin, proinsulin or preproinsulin wherei the amino acid sequence comprises a glutamine to glutamic acid substitution wherein the peptide comprises an amino acid sequence selected from SBQ ID NOs:2 to 5.
  • the peptide comprises at least 13 amino acid residues.
  • the peptide binds to an antigen-presenting cell which comprises the haplotype HLA-DQ A* 03:01, DQ B*03:02.
  • T-cells respond to the peptide presented by another HLA molecule, such as but not limited to HLA DQ2 and HLA DQ 2/g transdimers (DQ A* 05:01, DQ B* 03:02 and/or DQ A* 03:01 ; DQ B* 02:01 ), HLA-D 3 (DRBl *03xx) and/or HLA-OR4 (DRB1 *04xx).
  • HLA DQ2 and HLA DQ 2/g transdimers DQ A* 05:01, DQ B* 03:02 and/or DQ A* 03:01 ; DQ B* 02:01
  • HLA-D 3 DQ B* 02:01
  • DQ B* 02 HLA-OR4
  • the peptide is or comprises SEQ ID NO:94, [0070)
  • the present specification is further instructional on a method for the treatment or prophylaxis of Type 1 diabetes or a condition associated with Type 1 diabetes in a mammalian subject, the method comprising administering to the subject a peptide having a T-cell epitope derived from insulin, proinsulin or preproinsulin wherein the peptide comprises an amino acid sequence set forth in SEQ I ' D NO:94.
  • the peptide consists of SEQ ID NO:94.
  • the peptide comprises the sequence selected from SEQ ID NOs:96 to 123.
  • the peptide may contain further chemical modifications such as the chemical addition of amino acid residues in a synthetic reaction designed to generate peptides of a defined amino acid sequence, chemically modifying an amino acid residue, Another form of chemical modification is substituting an existing amino acid with a non-naturally occurring amino acid residue.
  • the peptides defined by reference to SEQ ID NQs:2 to 5 , 32, 35 to 61, 64, 67, 70, 73, 76, 79, 82 to 94 and 96 to 121 and SEQ ID NOs:6 to 31 , 33, 34, 62, 63, 65, 66, 68, 69, 71, 72, 74, 75, 77, 78, 80, 81 and 95 and SEQ ID M3 ⁇ 4:96 to 12.1 includes those having the glutamine to glutamic acid/or arginine to eitmiline substitution but may also comprise a chemically modified amino acid or contain a non-naturali occurring amino acid substitution or another natural amino acid substitution,
  • the present specification is further instructional on a method for the treatment or prophylaxis of Type 1 diabete or a condition associated with Type 1 diabetes in a human subject, the method comprising administering to the subject a peptide comprising at least seven consecutive amino acids encompassing a T-cell epitope derived from insulin, proinsulin or wherein the amino acid sequence comprises a glutamine to glutamic acid substitution wherein the peptide is chemically synthesized and comprises an amino acid sequence selected from SEQ ID NOs:2 to 5, 32, 35 to 61, 64, 67, 70, 73, 76, 79, 82 to 94 or 96 to 121.
  • the present specification is further instructional on a method for the treatment or prophylaxis of Type 1 diabetes or a condition associated with Type 1 diabetes in a human subject, the method comprising administering to the subject a chemically synthesized peptide encompassing a T-cell epitope derived from insulin, proinsulin or preproinsulin comprises or consists of an amino acid sequence set forth in SEQ ID NG:94.
  • the present specification is further instructional on a method for the treatment or prophylaxis of Type 1 diabetes or a condition associated with Type 1 diabetes m a human subject, the method comprising administering to the subject a chemically synthesized peptide encompassing a T-cell epitope derived from insulin, proinsulin or preproinsulin comprises an amino acid sequence set forth in SEQ ID NGs:96 to 121 ,
  • the present specification teaches a method for the treatment or prophylaxis of Type 1 diabetes or a condition associated with Type 1 diabetes in a human subject, the method comprising administering to the subject a peptide comprising at least seven consecutive amino acids having a T-cell epitope derived from insulin, proinsulin or wherein the amino acid sequence comprises a arginine to citruUme substitution wherein the peptide is chemically synthesized and comprises an amino acid sequence selected from SEQ ID NC)s: t 31 , 33, 34, 62, 63, 65, 66, 68, 69, 71 , 72, 74, 75, 77, 78, 80, 81 and 95.
  • the present specification is further instructional on a method fo the treatment or prophylaxis of Type 1 diabetes or a condition associated with Type 1 diabetes in a human subject, the method comprising administering to the subject a chemically synthesized peptide encompassing a T-cell epitope derived from insulin, proinsulin or preproinsulin wherein the peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:95.
  • Reference to "tolerance” includes a reduction in a T-cell response and/or a B- ceil response which is specific for insulin, proinsulin or preproinsulin. Tolerance may occur by any of a number of mechanisms including reducing autoreactive T-cells or 13- cells, inducing ignorance of insulin, proinsulin or preproinsulin, mechanisms that imprint an intrinsic status on tolerant lymphocytes in the form of anergy and immunedeviation and on extrinsic mechanisms involving regulating cells. Molecular interactions in the presentation of autoantigen in the periphery are central to the tolerance process and in strategies aiming at restoring or inducing immune tolerance in autoimmunity.
  • the peptides facilitate removal, elimination or reprogramming of deleterious autoreactive T-cells and antigen-presenting cells and activation of regulatory cells to control autoimmune effectors. Tolerance may also be induced using complexes between peptides such as SEQ ID NO:95 and HLA Class II tetramers.
  • the peptides or nucleic acid molecules encoding same are useful i the treatment or prophylaxis of Type 1 diabetes or an associated condition in a mammalian subject as well as a condition associated with Type 1 diabetes.
  • the treatment of Type 1 diabetes includes the amelioration of symptoms of diabetes.
  • the peptides are recognized by CD4 + T-cells, CD8 + T-cells and/or or antigen-presenting cells
  • the T-cells are CD4* T-cells.
  • the peptide is at least 13 amino acids in length such as from 1 to 40, and it binds to HLA-DQ A* 03:01 » DQ B* 03:02,
  • T-cells respond to the peptide presented by another HLA molecule, such as but not limited to HLA DQ2 and HLA DQ 2/8 transdimers (DQ A*
  • at least 1 amino acids in length means 13 to 40 such as 13 to 20.
  • the present specification is instructional on a method of tolerizing or down- 0 regulating the priming or activity of T-cells in mammalian subjects with or at risk of developing Type 1 diabetes by the administration of a peptide selected to comprise an amino acid sequence set forth in SEQ ID NOs:2 to 5, 32, 35 to 61, 64, 67, 70, 73, 76, 79 and 82 to 94 wherein the peptide comprises a glutaniine to glutamic acid substitution.
  • the present specification is instructional on a method of tolerizing or down- regulating the priming or activity of T-cells in mammalia subjects with or at risk of developing Type 1 diabetes by the administration of a peptide selected to comprise an amino acid sequence set forth in SEQ ID NOs:96 to 121 wherein the peptide comprises a glutamine to glutamic acid substitution.
  • the present specification is instructional on a method of tolerizing or down- regulatin the priming or inactivity of T-cells in. mammalian subjects with of at risk of developing Type 1 diabetes by the administration of a peptide comprising the amino acid sequence set forth in SEQ ID NO:95.
  • the present specification teaches a method of tolerizing or down-regulating the priming or activity of T-cells in mammalian subjects with or at risk of developing Type 3 diabetes by the administration of a peptide selected to comprise an amino acid sequence set forth in SEQ ID NOs:6 to 31 , 33, 34, 62, 63, 65, 66, 68, 69, 71, 72, 74, 75, 77, 78, 80, 81 and 95 wherein the peptide comprises an arginine to citrulline substitution and/or an arginine to citrulline substitution.
  • the present specification is instructional on a method of tolerizing or down- regulating the priming or inactivity of T-cells in mammalian subjects with or at risk of developing Type 1 diabetes by the administration of a peptide comprising the amino acid sequence set forth in SEQ ID NQ:95.
  • the present specification is instructional on a method of tolerizing or down- regulating the priming or activity of B-cells in mammalian subjects with or at risk of developing Type 1 diabetes by the administration of a peptide selected to comprise an amino acid sequence set forth in SEQ ID NQs:2 to 5, 32, 35 to 61, 64, 67, 70, 73, 76, 79 and 82 to 94 wherein the peptide comprises a glutamine to glutamic acid substitution.
  • the present specification is instructional on a method of tolerizing or down- regulating the priming or inactivity of B-cells in mammalian subjects with or at risk of developing Type 1 diabetes by the administration of a peptide comprising the amino acid sequence set forth in SEQ ID NO:94.
  • the present specification is instructional on a method of tolerizing or down- regulating, the priming or inactivit of B-cells in mammalian subjects with or at risk of developing Type 1 diabetes by the administration of a peptide comprising the amino acid sequence set forth in SEQ ID NO:96 to 121, [0087]
  • the present specification is instructional on a method of tolerizing or down- regulating the priming or activity of B-cells in mammalian subjects with or at risk of developing Type 1 diabetes by the administration of a peptide selected to comprise an amino acid sequence set forth in SEQ ID NGs; 6 to 31, 33, 34, 62, 63, 65, 66, 68, 69, 71, 5 72, 74, 75, 77, 78, 80, 81 and 96 wherein the peptide comprises an arginme to citxullme substitution *
  • tolerization may employ the use of an HLA Class II tetramer comprising a peptide of the present inventio such as s but not limited to, SEQ ID NO:94.
  • the peptides may further comprise other modifications such as to an amino acid or by the substitution of a non-naturally occurring amino acid residue.
  • the present specification is instructional on a method of tolerizing or down- 0 regulating the priming or inactivity of T-cells in human subjects with or at risk of developing Type 1 diabetes by the administration of a chemically synthesized peptide selected to comprise an amino acid sequence set forth in SEQ ID NOs:2 to 5, 32, 35 to 61 , 64, 67, 70, 73, 76, 79 and 82 to 94 wherein the peptide comprises a glutamine to glutamic acid substitution.
  • the present specification is instructional on a method of tolerizing or down- regulating the priming or inactivity of T-cells in human subjects with or at risk of developing Type 1 diabetes by the administration of a chemically synthesized peptide comprising the amino acid sequence set forth in SEQ ID NO: 94,
  • the present specification is instructional on a method of tolerizing or down- regulating the priming or inactivity of T-ceMs in human subjects with or at risk of developing Type 1 diabetes by the administration of a chemically synthesized peptide selected to comprise an amino acid sequence set forth in SEQ IP NOs;96 to 121 wherein the peptide comprises an arginme to citrulline substitution.
  • the present specification is instructional on a method of tolerizing or down- regulating the priming or inactivity of T-ceilS: in hum n subjects with or at risk, of developing Type 1 diabetes by the administration of a chemically synthesized peptide selected to comprise an amino acid sequence set forth in SEQ ID NOs:6 to 31, 33, 34, 62, 63, 65, 66, 68, 69, 71, 72, 74, 75 5 77 5 78, 80, 81 and 95 wherein the peptide comprises an arginme to citrulline substitution,
  • the present specification is instructional on a method of tolerizing or down- regulating the priming or inactivity of T-ce!Is in human subjects with or at risk of developing Type 1 diabetes by the- administration of a chemically ' synthesized peptide comprising the amino acid sequence set forth in SEQ ID NO:95.
  • the present specification is instructional on a method of tolerizing or down- regulating the priming or inactivity of B-cells in huinan subjects with or at risk of developing Type. 1 diabetes by the administration of a chemically synthesized peptide selected to comprise an amino acid sequence set forth in SEQ ID N ' Os:2 to 5, 32, 35 to 61, 64, 67, 70, 73, 76.
  • the present specification is instructional on a method of tolerizing or down- regulating the priming or inactivity of B-cells i human subjects with or at risk of developing Type 1 diabetes by the administration of a chemically synthesized peptide seieeted to comprise an amino acid sequence set forth in SEQ ID MOs:96 to 121 wherein the peptide comprises a glutamine to glutamic acid substitution.
  • the present specification is instructional on a method of tolerizing or down- regulating the primin or inactivity of B-cells in human subjects with or at risk of developing Type 1 diabetes b the administration of a chemically synthesized peptide comprising the amino acid sequence set forth in SEQ ID NO; 4,
  • the present specification is instructional on a method o tolerizing or down- regulating the priming or inactivity of B-cells in human subjects with or at risk of developing Type 1 diabetes by the administration of a chemically synthesized peptide selected to comprise an amino ae d sequence set forth in SEQ ID NOs:6 to 31 , .33, 34, 62, 63, 65, 66, 68, 69 .» 71 , 72, 74, 75, 77, 78, 80, 81 and 95 wherein the peptide comprises an arginrae to citrulline substitution.
  • the present specification is instructional on a method of toleri3 ⁇ 4ing or down- reguiatmg the priming or inactivity of B-cells in human subjects with or at risk of developing Type 1 diabetes by the administration of a chemically synthesized peptide comprising the amino acid sequence set forth in SEQ ID NO:93»
  • the peptide may further comprise one or more chemical modifications to one or more amino acid residues including an amino acid additio and/or deletion as well as a substitution of a naturally occurring or non-naturally occurring amino acid for one or more of the amino acid residues,
  • amides of the peptide or peptide variants of the present invention may also be prepared by techniques well known in the art for converting a carbo ylic acid group or precursor to an amide.
  • One preferred method for amide formation at the C-terminal carboxyl group is to cleave the peptide from a solid support with an appropriate amine, or to cleave in the presence of an alcohol, yielding an ester, followed by aminoiysis with the desired amine.
  • Salts of carboxyl groups of a peptide or peptide variant may be prepared in the usual mamier by contacting the peptide with one or more equivalents of a desired base such as, for example, a metallic hydroxide base, e.g., sodium hydroxide; a metal carbonate or bicarbonate base such as, for example, sodium carbonate or sodium bicarbonate; or an amine base such as, for example, triethylamine, triethanolamine, and the like,
  • a desired base such as, for example, a metallic hydroxide base, e.g., sodium hydroxide
  • a metal carbonate or bicarbonate base such as, for example, sodium carbonate or sodium bicarbonate
  • an amine base such as, for example, triethylamine, triethanolamine, and the like
  • N-acyl derivatives of an amino group of the peptide or peptide variants may be prepared by utilizing an N-aeyl protected amino acid for the final condensation, or by aeylating a protected or unprotected peptide.
  • O-acyl derivatives may be prepared, for example, by acylation of a free hydrox peptide or peptide resin. Either acylation may he carried out using standard aeylating reagents such as acyl halides, anhydrides, acyl imidazoles, and the like. Both N- and O-aeylation may be carried out together, if desired.
  • Formyl-metbionine, pyroglulamine and trimethyl- lanine may be substituted at the N-terminal residue of the peptide or peptide variant.
  • Other amino-terminal modifications include ammooxypentane modifications (see Simmons et at. (1997) Science 276:276),
  • amino acid sequence of a peptide can be modified so as to result in a peptide variant (see above).
  • the modification includes the substitution of at least one amino acid residue in the peptide for another amino acid residue, including substitutions which utilize the D rather than L form, as well as other well known amino aeid analogs.
  • analogs include phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, gamma-carboxyglutamate; hippuric aeid, octahydroindole-2-carboxylie acid, statine, l,2,3,4 tetrahydroisoquinoline-3-earboxylic acid, penicillamine, ornithine, citnilline, cx-methyl-alanine, -benzoyl-phenylalanine, phenylglycine, propargyl glycine, sarcosine, and tert-butylglycine.
  • the amino aeid sequence of the peptide will comprise on or more of a glutamine to glutamic aeid substitution and/or an arginine to citrulline substitution.
  • One or mor of the residues of the peptide can be altered, so long as the peptide variant is biologically active in the sense it retains its epitopic function.
  • Conservative amino acid substitutions include, for example, aspartio-glutamic as acidic amino acids; lysine/arginine/histidine as basic amino acids; leucine/isoletieine, methionine/valine, alanine/valine as hydrophobic amino acids; serine/glycine/alanine/threonine as hydrophilic amino acids.
  • Amino acid substitutions falling within the scope of the invention are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobic! ty of the molecule at the target site, or (c) the hulk of the side chain.
  • Naturally occurring residues are divided into groups based on. common side-chain properties:
  • Acid addition salts of the peptide or variant peptide, or of amino residues of the peptid or variant peptide may be prepared by contacting the peptide or amine with one or more equivalents of the desired inorganic or organic acid, such as, for example, hydrocliloric acid.
  • Esters of carboxyl groups of the peptides may also be prepared by any of the usual methods known in the art.
  • Crosslinkers can be used, for example, to stabilize 3D conformations, using homo-hifunctional crosslinkers such as the bifimctional. imido esters having (C3 ⁇ 4)n spacer groups with n - 1 to n TM 6, glutaraldehyde, N-hydroxysuccinirnide- esters and hetero- bifunctional reagents which usually contain an amino-reactive moiety such as N- hydroxysuccinimide and another group specific-reactive moiety such as raaleimido or dithio moiety (SH) or carbodiimide (COOH).
  • homo-hifunctional crosslinkers such as the bifimctional. imido esters having (C3 ⁇ 4)n spacer groups with n - 1 to n TM 6, glutaraldehyde, N-hydroxysuccinirnide- esters and hetero- bifunctional reagents which usually contain an amino-reactive moiety such as N- hydroxysuccinimide and another group specific-
  • peptides can be conformationaiiy constrained by, for example, incorporation of C « and N a -methylamino acids, introduction of double bonds between C a and Cp atoms of amino acids and the formation of cyclic peptides or analogs by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.
  • the peptides may be produced by the stepwise addition of amino acid residues by chemical synthetic means or by recombinant means or by recombinant means followed by posttranslational modification.
  • any number of methods may be employed including using 9-fluoronyImethox -carbonyl (Fmoc) synthesis. Purity can be readily determined by HPLC and peptide masses determined by s tor example, mass spectrometry.
  • the peptides may be synthesised by standard solution phase methodology, as described in Hruby et al. (1998) Chemical synthesi qf peptides. University of Arizona, USA.
  • Linear peptides may also be synthesised by solid phase methodology using Boc chemistry, as described by Schnolzer et al (1992) Int J Pept Protein Res 0: 180-1 3, Following deproteetion and cleavage from the solid support the reduced peptides are purified using preparative chromatography, [0116] As indicated above, peptides may also be synthesised by solid phase methodology using Fmoc chemistry, as described below:
  • Peptide is synthesized b Fmoc solid-phase peptide synthesis using an automatic synthesizer.
  • Peptide is synthesized from its C -terminus by stepwise addition of amino acids.
  • the first Fmoe-ammo acid is attached to an insoluble support resin via an acid labile linker.
  • the second Ffnoc- amino acid is coupled utilizing a pre-activated species or in situ activation.
  • sources of nucleotide sequences from which a nucleic acid molecule encoding a peptide or variant thereof include total or polyA + RNA from a mammalian (e,g, human) source from which eDNAs ean be derived by methods known in the art.
  • Other sources of DNA molecules include genomic libraries derived from a mammalian (e.g. human) source,
  • DNA molecules may be prepared in vitro, e.g., by synthesizing an oligonucleotide or by subeloning a portion of a DNA segment that encodes a particular peptide.
  • isolated and/or purified refer to in vitro isolation of a DNA, peptide or polypeptide molecule from its natural eellular o biological environment, and from association with other components of the cell, such as nucleic acid or polypeptide, so that it can be sequenced, replicated, and/or expressed.
  • an "isolated, preselected nucleic acid” is RNA or DNA that encode at least a portion of a peptide selected from SEQ ID Os: l through 121 , or a RNA or DNA complementary thereto, that is complementary or hybridizes, respectively, to RNA or DNA encoding the peptide, or polypeptide comprising the peptide, and remains stably bound under stringent conditions, as defined by methods well known in the art, e.g. in Sambrook ei l, (1989) Molecular Cloning; A Laboratory Manual, Cold Spring Harbor, N.Y. Once expressed and a peptide produced, it then undergoes posttranslational modification.
  • the RNA or DNA is “isolated” in that it is free from at least one contaminating nucleic acid with which it is normally associated in the natural source of the RNA or DNA and is substantially free of any other mammalian RNA or DNA.
  • the phrase "free from at least one contaminating source nucleic acid with which it is normally associated” includes the case where the nucleic acid is reintroduced into the source or natural cell but is in a different chromosomal location or is otherwise flanked by nucleic acid sequences not normally found in the source cell.
  • the term "recombinant nucleic acid” or "preselected nucleic acid,” e.g., “recombinant DNA sequence or segment” or “preselected DNA sequence or segment” refers to a nucleic acid, e.g., to DNA, that has been derived or isolated from any appropriate tissue source, that may be subsequently chemically altered in vitro, so that its sequence is not naturally occurring,, or corresponds to naturally occurring sequences that are not positioned as they would be positioned in a genome which has not been transformed with exogenous DNA,
  • An example of preselected DNA "derived” from a source would be a DNA sequence that is identified as a useful fragment withi a given organism, and which is then chemically synthesized in essentially pure form.
  • DNA "isolated" from a source would be a useful DNA sequence that is excised or removed from the source by chemical means, e.g., by the use of restriction endonucleases, so that it can be further manipulated, e.g., amplified, for use in the invention, by the methodology of genetic engineering.
  • Nucleic acid molecules encoding amino mid sequence variants of a. peptide of the invention are prepared by a variety of methods known in the art. These methods include, but are not limited to, preparation by oligonucleoti de-mediated (or site-directed) mutagenesis, PGR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the preselected peptide, Posttranslalional modification ma also be used to alter the amino acid sequence.
  • Oligonucleotide-mediated mutagenesis is a method for preparing amino acid substitution variants of a peptide. This technique is described by Adelman et ai,.
  • DNA 2:1 S3 (1983) DNA 2:1 S3, Briefly, DNA is altered by hybridizing an oligonucleotide encoding the desired mutation to a DNA template, where the template is the single-stranded form of a plasmid or bacteriophage coniaining the unaltered or native DNA sequence. After hybridization, a DNA polymerase is used to synthesize an entire second complementary strand of the template that will thus incorporate the oligonucleotide primer, and will code for the selected alteration in the preselected DNA.
  • a DNA polymerase is used to synthesize an entire second complementary strand of the template that will thus incorporate the oligonucleotide primer, and will code for the selected alteration in the preselected DNA.
  • the recombinant or preselected DNA sequence or segment may be circular or linear, double-stranded or single- stranded.
  • the preselected DNA sequence or segment is in the form of chimeric DNA, such as plasmid DNA, that can also contain coding regions flanked by control sequences which promote the expression of the preselected DNA present in the resuItanT- cell line.
  • the peptides including their salts are generally administered so as to achieve a reduction in activity of autoreactive T-cells to thereby ameliorate at least one symptom associated with Type 1 diabetes.
  • the peptide or a valiant thereof is administered at dosages of at least about 0,001 to about 100 mg/kg including about 0,01 to about .10 mg/kg including about 0.1 to about 5 mg/kg, of body weight, although other dosages may provide beneficial results.
  • the amoun administered will vary depending on various factors including, but not limited to, the peptide selected, the stage of the disease, the weight, the physical condition, and the age of the mammal, whether prevention or treatment is to be achieved, and whether the peptide is chemically modified. Such factors can be readily determined by the clinician employing animal models or other test systems which are well known to the ait.
  • the mammal is a human.
  • Formulations suitable for peptides and for adaptive immunity are well known in the art.
  • the subject peptides or peptide-all complexes or peptide-MLA-DQ A*03:01, DQ B*03;02 complexes can be formulated using standard techniques.
  • Other complexes contemplated herein include peptide complexes with HLA DQ 2 and DQ 2/8 transdimers (DQ A* 05:01 , DQ B* 03:02 and/or DQ A* 03:01; DQ B* 02:01), HLA-DR3 (DRBl *03xx and/or HLA- DR4 (DRBl*04xx).
  • compositions, dosages and routes of administration for nucleic acids are generally disclosed, for example, in Feigner et al. supra.
  • G127f Administration of the therapeutic or prophylactic agents in accordance with the instant disclosure may be continuous or intermittent, depending, for example, upon the recipient's physiological condition, whether the purpose of the .administration is therapeutic or prophylactic, and other factors known to skilled practitioners.
  • the administration of the peptides may be essentially continuous over a preselected period of time or may be in a series of spaced doses. Both local and systemic administration is contemplated.
  • composition s peptides are synthesized or otherwise obtained, purified and then lyophilized and stabilized, The peptide can then be adjusted to the appropriate concentration, and optionally combined with other agents.
  • the absolute weight of a given peptide included in a unit dose of a tolerogen can vary widely, For example, about 0.01 to about 10 mg, including about 0.5 to about 5 mg, of at least one peptide comprising an epitope sequence from insulin, proinsulin or prepromsulin, can be administered.
  • a unit dose of the tolerogen may be administered either via a mucous membrane, e.g., by respiratory, e.g.,.
  • one or more suitable unit dosage forms comprising the therapeutic or prophylactic peptides, which, as discussed below, may optionally he formulated for sustained release (for example using microencapsulation, see WO 94/07529, and U.S. Pat. No. 4,962,0 1), can be administered by a variety of routes including oral, or parenteral, including by rectal, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, intrathoracic, intrapulmonary and intranasal (respiratory) routes.
  • the formulations may, where appropriate, be conveniently presented in discrete unit dosage forms and may be prepared b any of the methods well known to pharmacy. Such methods may include the step of bringing into association the therapeutic or prophylactic peptide with liquid carriers, solid matrices, semi-solid carriers, finely divided solid caniers or combinations thereof, and then, if necessary, introducing or shaping the product into the desired delivery system.
  • the peptides are prepared for oral administration, they are combined with a pharmaceutically acceptable carrier, diluent or excipient to form a. pharmaceutical formulation, or unit dosage form.
  • a pharmaceutical formulation, or unit dosage form Conveniently, orally administered peptides are formulated for sustained release, e.g., the agents are microencapsulated.
  • the total active ingredients in such formulations comprise from 0.1 to 99,9% by weight of the formulation.
  • pharmaceutically acceptable it is meant the carrier, diluent excipient, arid or salt must be compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof.
  • the active ingredient for oral administration may be present as a powder or as granules; as a solution, a suspension or an emulsion; or in achievable base such as a synthetic resin for ingestion of the active ingredients from a chewing gum.
  • the active ingredient may also be presented as a bolus, electuary or paste.
  • compositions containing the therapeutic or prophylactic peptide can be prepared by procedures known in the art using well known and readil available ingredients.
  • the agent can be formulated with common excipients, diluents, or carriers, and formed into tablets, capsules, suspensions, powders, and the like.
  • excipients, diluents, and carriers that are suitable for such formulations include the following fillers and extenders such as starch, sugars, mannitol, and silicic derivatives; binding agents such as carboxymethyl cellulose, HPMC and other cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone; moisturizing agents such as glycerol; disintegrating agents such as calcium carbonate and sodium bicarbonate; agents for ⁇ retarding dissolution such as paraffin; resorption accelerators such as quaternary ammonium compounds; surface active agents such as cetyl alcohol, glycerol monostearate; adsorptive carriers such as kaolin and bentonite; and lubricants such as talc, calcium and magnesium stearate, and solid polyethyl glycols.
  • fillers and extenders such as starch, sugars, mannitol, and silicic derivatives
  • binding agents such as carboxymethyl cellulose, HPMC and other cellulose derivative
  • tablets or caplets containing the peptides can include buffering agents such as calcium carbonate, magnesium oxide and magnesium carbonate.
  • Caplets and tablets can also include inactive ingredients such as cellulose, pregelatini3 ⁇ 4ed starch, silicon dioxide, hydroxy propyl methyl cellulose, magnesium stearate, microcrystalime cellulose, starch, talc, titanium dioxide, benzoic acid, citric acid, corn starch, mineral oil, polypropylene glycol, sodium phosphate, and zinc stearate, and the like.
  • Hard or soft- gelatin capsules comprising a therapeutic or prophylactic peptide can contain inactive ingredients such as gelatin, macrocrystalline cellulose, sodium lauryl sulfate,: starch, talc, and titanium dioxide, and the like, as well as liquid vehicles such as polyethylene glycols (PEGs) and vegetable oil, Moreover, enteric coated caplets or tablets of an agent of the invention are designed to resist disintegration in the stomach and dissolve in the more neutral to alkaline environment of the duodenum.
  • the therapeutic or prophylactic peptides can also be formulated as elixirs or solutions for convenient oral administration or as solutions appropriate for parenteral administration, for instance by intramuscular, subcutaneous or intravenous routes.
  • the pharmaceutical formulations of the peptide can also take the form of an aqueous or anhydrous solution or dispersion, or alternatively the form of an emulsion or suspension.
  • the therapeutic or prophylactic peptide may be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dose form in ampules, pre-filled syringes, small volume infusion containers or in multi-dose containers with an added preservative.
  • the active ingredients may take such forms as suspensions, solutions, or emulsions in oil or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredients may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.
  • formulations can contain pharmaceutically acceptable vehicles and adjuvants which are well known in the art. It is possible, for example, to prepare solutions using one or more organic solvent(s) that is/are acceptable from the physiological standpoint, chosen, in addition to water, from solvents such as acetone, ethanol, isopropyl alcoho!, glycol ethers such as the products sold under the name "Dowanol", polyglycols and polyethylene glycols, C1 -C4 alkyl esters of short-chain acids, preferably ethyl or tsopropyl lactate, fatty acid triglycerides suc as the products marketed under the name " iglyol 11 , isopropyl mynstate, animal mineral and vegetable oils and polysi!oxanes,
  • compositions described herein can also contain thickening agents such as cellulose and/or cellulose derivatives. They can also contain gyms such as xanthan, guar or carho gum or gum arabie, or alternatively polyethylene glycols, bentones and mont orillonites,. and the like,
  • antioxidants chosen from antioxidants . , surfactants, other preservatives, film-forming, keratolytic or eornedolytie agents, perfumes and colorings.
  • other active ingredients may be added, whether for the conditions described or some other condition.
  • the agents are well suited to formulation as sustained release dosage forms and the like,
  • the formulations can be so constituted that the release the active inpedient only or preferably in a particular part of the intestinal or respiratory tract, possibly over a period of time.
  • the coatings, envelopes, and protective matrices ma be made, for example, from polymeric substances, such as polylaetide -glycol ates : liposomes, microemulsions, microparticles, nanoparticles, or waxes. These coatings, envelopes, and protective matrices are useful to coat indwelling devices, e.g., stents, catheters, peritoneal dialysis tubing, and the like.
  • the therapeutic peptides disclosed herein can be delivered via patches for transdermal administration. See U.S. Pat. No. 5,560,922 for examples of patches suitable for transdermal delivery of a therapeutic agent.
  • Patches for transdermal deliver can comprise a backing layer and a polymer matri which has dispersed or dissolved therein a therapeutic agent, along with one or more skin permeation enhancers.
  • the backing layer can be made of any suitable material which is impermeable to the peptide.
  • the backing layer serves as a protective cover for the matrix layer and provides also a support function.
  • the backing can be formed so that it is essentially the same size layer as the polymer matrix or it can be of larger dimension so that it can extend beyond the side of the polymer matrix or overlay the side or sides of the polymer matrix and then can extend outwardly in a manner that the surface of the extension of the backing layer can be the base for an adhesive means.
  • the polymer matrix can contain, or be formulated of, an adhesive polymer, such as polyacrylate or acrylate/vinyl acetate copolymer.
  • an adhesive polymer such as polyacrylate or acrylate/vinyl acetate copolymer.
  • Therapeutic or prophylactic peptides released from a transdermal delivery- system must be capable of penetrating each layer of skin. I order to increase the rate of permeation of a therapeutic agent, a transdermal drag delivery system must be able in particular to increase the permeability of the outermost layer of skin, the stratum eorneum, which provides the most resistance to the penetration of molecules.
  • the fabrication of patches for transdermal delivery of therapeutic agents is well known to the art.
  • the therapeutic or prophylactic peptides may be formulated as is known in the art for direct application to a target area.
  • Conventional forms for this purpose include wound dressings, coated bandages or other polymer coverings, ointments, creams, lotions, pastes, jellies, sprays, and aerosols.
  • Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents.
  • Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents.
  • the active ingredients can also be delivered via iontophoresis, e.g., as disclosed in U.S. Pat, os. 4,140,122; 4,383,529; or 4,051,842.
  • the percent by weight of a therapeutic agent of the invention present in a topical forimilation will depend on various factors, but generally will be from 0,01% to 95% of the total weight of the formulation, and typically 0,1-25% by weight.
  • Drops such as eye drops or nose drops, may be formulated with an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilizing agents or suspending agents.
  • Liquid sprays are conveniently delivered from pressurized packs. Drops can be delivered via a simple eye dropper-capped bottle, or via a plastic bottle adapted to deliver liquid contents dropwise, via a specially shaped closure,
  • the therapeutic or prophylactic peptide may further be formulated for topical administration in the mouth or throat.
  • the active ingredients may be formulated as a lozenge further comprising a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the composition in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the peptide composition in a suitable liquid carrier.
  • the pharmaceutical formulations of the present invention may include, as optional ingredients, pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, and salts of the type that are well-known in the art. Examples of such substances include normal saline solutions such as physiologically buffered saline solutions and water.
  • a peptide of the present disclosure comprising an amino acid sequence selected from SEQ ID NOs;2 through 121 inclusive may be conjugated to or embedded in a targeting moiety and/or a carrier molecule.
  • the targetin moiety and the carrier molecule are the same entity.
  • a targeting moiety is useful in facilitating the deployment of the peptide to a particular cell type or tissue type. Examples of cell types include antigen-presenting cells and CD4' T-cells or other lymphocytes such as CD8 + T- cells.
  • targeting moieties include antibodies or antigen-binding fragments or derivatives thereof specific for an antigen on the target cell or tissue, receptor Bgands, a glycosaminoglycan, heparan or heparin or a fragment thereof, a cytokine or other molecule capable of specific binding to a desired cell type.
  • Carrier molecules include adjuvants, molecules which increase the half life in the blood stream and the like.
  • an agent comprising a peptide comprising an amino acid sequence having a T-cell epitope derived from insulin, proinsulin or preproinsulin having a glutamine to glutamic acid substitution and comprising an amino acid sequence set forth in SEQ ID NOs:2 to 5, 32, 35 to 61 5 64, 67, 70, 73, 76, 79 and 82 to 94 inclusive, the peptide conjugated at its N- or C-terminal end to a targeting moiety or carrier molecule,
  • a peptide comprising amino acid sequence having a T-cell epitope derived from insulin, proinsulin or preproinsulin having a glutamine to glutamic acid substitution and comprising an amino acid sequence set forth in SEQ ID NOs:96 to 121 , the peptide embedded in a targeting moiety or carrier molecule.
  • a peptide comprising art amino acid sequence derived comprising a T-cell epitope from insulin, proinsulin o preproinsulin having a glutamine to glutamic acid substitution and comprising an amino acid sequence set forth in SEQ ID NOs:2 to 5, 32, 35 to 61, 64, 67, 70, 73, 76, 79 and 82 to 94, the peptide embedded in a targeting moiety or carrier molecule.
  • an agent comprising a peptide comprising an amino acid sequence having a T-cell epitope derived from insulin, proinsulin or preproinsulin having an arginine to citrulline substitution and comprising an amino acid sequence set forth in SEQ ID NOs:6 to 31, 33, 34, 62, 63, 65, 66, 68, 69, 71, 72, 74, 75, 77, 78, 80, 81 and 95 inclusive, the peptide conjugated at its N- or C-terminal end to a targeting moiety or carrier molecule.
  • a peptide comprising an amino acid sequence having an epitope derived from insulin, proinsulin or preproinsulin having an arginine to citrulline acid substitution and comprising an amino acid sequence set forth in SEQ ID NOs:6 to 31, 33, 34, 62, 63, 65, 66, 68, 69, 71, 72, 74, 75, 77, 78, 80, 81 and 95, the peptide embedded in a targeting moiety or carrier molecule, 10153 ⁇
  • the peptide comprises or consists of SEQ ID NO:94.
  • the peptide comprises or consists of SEQ ID NO: 95.
  • the targeting moiety is a moiety which specifically targets an antigen-presenting cell or a T-cell such as a CD4 ' T-cell or CD8 ! T-cell, Examples include an antibody, receptor ligand, cytokine or a glycosaminoglycan,
  • the carrier molecule is human serum albumin.
  • the carrier molecule is a cyclic peptide.
  • the peptide ma be embedded in the cyclic peptide to facilitate increasing its blood half-life and/or to enhance stability,
  • the peptide is conjugated to an antigen-presenting cell or T-cell such as a CD4 + T-cell, cell or other cell of the immune system
  • the conjugated peptide or peptide-cell complex is useful in a method for the treatment or prophylaxis of Type 1 diabetes or a condition associated with Type 1 diabetes in a human subject, the method comprising administering to the subject a peptide having an amino acid sequence comprising a T-cell epitope derived from insulin, proinsulin or preproinsulm wherein the amino acid sequence comprises a gliitamine to glutamic acid substitution and comprising an amino acid sequence selected from SEQ ID NOs:2 to 5, 32, 35 to 61, 64, 67, 70, 73, 76, 79 and 82 to 94 wherein the peptide is conjugated to or embedded in a targeting moiety, carrier molecule or immune cell.
  • the conjugated peptide or peptide-cell complex is useful in a method for the treatment or prophylaxis of Type 1 diabetes or a condition associated with Type 1 diabetes in a human subject, the method comprising administering to the subject a peptide having an amino acid sequence comprising a T-cell epitope derived from insulin, proinsulin or preproinsulin wherein the amino acid sequence comprises an arginine to eitmlline substitution and comprising an amino acid sequence selected from SEQ ID NOs;6 to 31, 33, 34, 62, 63, 65, 66, 68, 69, 71 , 72, 74, 75, 77, 78, 80, 81 and 95 wherein the peptide is conjugated to or embedded in a targeting moiety, carrier molecule or immune cell,
  • compositions described in relation to the peptide equally apply to a. conjugated or embedded peptide.
  • the peptides may comprise other chemical modifications to the amino acid residue.
  • the peptides described herein including those having an amino acid sequence selected from SEQ ID NOs:2 to 5, 32, 35 to 61 , 64, 67, 70, 73, 76, 79 and 82 to 94 are also useful in diagnostic assays to detect or monitor the progression of Type 1 diabetes. In an embodiment, Type 1 diabetes is detected prior to onset of hyperglycemia. The peptides are also useful in monitoring T-eell responses in a patient's blood, monitoring reactivity to pancreatic islet cells and to detect autoreactive antibodies.
  • the peptides described herein including those having an amino acid sequence selected from SEQ ID NOs:6 to 31, 33, 34, 62, 63, 65, 66, 68, 69, 71, 72, 74, 75, 77, 78, 80, 81 and 95 are also useful in diagnostic assays to detect or monitor the progression of Type 1 diabetes.
  • Type 1 diabetes is detected prior to onset of hyperglycemia.
  • the peptides are also useful in monitoring T-cell responses in a patient's blood, monitoring reactivity to pancreatic islet cells and to detect autoreactive antibodies.
  • the peptides described herein including those having an amino acid sequence selected from SEQ ID NOs:96 to 121 are also useful in diagnostic assays to detect or monitor the progression ol ' Type 1 diabetes. In an embodiment, Type 1 diabetes is detected prior to onset of hyperglycemia. The peptides are also useful in monitoring T-cell responses in a patient's blood, monitoring reactivity to pancreatic islet cells and to detect autoreactive antibodies.
  • the assay may be conducted in any . number of ways including incubating the peptides with a sample of peripheral blood mononuclear cells or a cellular fraction thereof and screening for T-cell activation. This can be determined, for example, by T-cell proliferation or the presence of immune, effector molecules.
  • a method for measuring a cell-mediated immune response in a subject comprising collecting a sample from the subject wherein, the sample comprises cells of the immune system which are capable of producing immune effector molecules following stimulation by an autoaniigen selected from a modified peptide comprising a T-cell epitope derived from insulin, proinsulin or preproinsulhi, incubating the sample with the antigen and then measurin fee presence of or elevation in the level of an immune effector molecule wherein the presence or level of the immune effector molecule is indicative of the capacity of the subject, to mount an autoreactive a cell- mediated immune response,
  • the subject specification contemplates a method for detecting the presence of autoreactive T-ce!ls indicative of the presence of, or a predisposition for the development of, Type 1 diabetes, or a condition associated with Type I diabetes in a subject, the method comprising contacting a sample from the subject comprising immune cells with a peptide comprising an amino acid sequence of at least seven consecutive amino acid residues comprising a T-cell epitope derived from insulin, proinsulin or prepro insulin or a chemical analog of, or a non-natural ly occurring amino acid substitute of, one or more amino acid residues in the secjuenee wherein the amino acid sequence comprises a glutaniiiie to glutamic acid substitution and/or an arginine to eitruttme substitution for a time and under conditions sufficient for an autoreactive T-cell, if present, to be stimulated into producing an immune effector molecule, and then detecting the presence of the immune effector molecule, wherein the presence or level of the immune
  • a method for screening for autoreactive antibodies associated with Type 1 diabetes or a condition associated therewith in a subject comprising contacting body fluid from the subject with a peptide comprising an amino acid sequence having a T-cell epitope derived from insulin, proinsulin or preproinsulra or a chemical analog of, or a non-naturally occurring amino acid substitute of, one or more amino acid residues in the sequence wherein the amino acid sequence comprises an arginine to citriiUine substitution for a time and under conditions sufficient for an autoreactive antibody, if present, to bind to the peptide and then screening for the presence of the peptide-antibod complex.
  • the presence or level of the antibody is indicative of Type 1 diabetes or its state or risk of progression.
  • body fluid include serum, whole blood, tissue fluid, sputum, urine, and the like.
  • the subject is a human.
  • the peptide comprises an amino acid sequence selected from the list consisting of SEQ ID NO: SEQ ID NOs:2 to 5, 32, 35 to 61 , 64, 67, 70 ? 73, 76, 79 and 82 to 94,
  • the peptide comprises an amino acid sequence set forth in SEQ ID O;94, In an aspect, the peptide consists of SEQ ID NO;94.
  • the peptide comprises an amino acid sequence selected from the list consisting of SEQ ID NOs:6 to 31 , 33, 34, 62, 63, 65, 66, 68, 69, 71, 72, 74, 75, 77, 78, 80, 81 and 95.
  • the peptide comprises an amino acid sequence set forth in. SEQ ID NO;95.
  • the peptide comprises an amino acid sequence set forth in SEQ ID NOs:96 to 121.
  • the condition associated with Type 1 diabetes is selected from the list consisting of a cardiovascular condition, a neuropathy, a retinopathy, a nephropathy, infection and a complication in pregnancy, [0174 ⁇
  • the immune effector molecule is detected by an antibody labeled with a reporter molecule, capable of providing a detectable signal,
  • lymphocytes including natural killer (NK) ceils, T-cells, (CD4 ⁇ and/or CD8 + cells), B-cells, macrophages and monocytes, dendritic cells or any other cell which is capable of producing an effector molecule in response to direct or indirect antigen stimulation.
  • the immune cells are lymphocytes and includes CD4* T-celis.
  • the immune effector molecules for T-cell assays may be any of a range of molecules which are produced in response to cell activation or stimulation by the peptide antigen.
  • Immune effector molecules include interferon (e.g.. IFN-y), a range of cytokines such as interleukins (US), e.g. IL-2, IL-4, IL-10 or IL-12, tumor necrosis factor alpha (TNF-ot), a colony stimulating factor (CSF) such as granulocyte (G)-CSF or granulocyte macrophage (GM)-CSF amongst many others such as complement or components in the complement pathway.
  • interferon e.g.. IFN-y
  • cytokines such as interleukins (US), e.g. IL-2, IL-4, IL-10 or IL-12
  • TNF-ot tumor necrosis factor alpha
  • CSF colony stimulating factor
  • G granulocyte
  • GM granulocyte macrophag
  • the sample collected from the subject is generally deposited into a collection tube.
  • a collection tube includes a blood draw tube or other similar vessel.
  • the blood collection tube is heparinized.
  • heparin is added to the tube after the blood is collected.
  • whole blood is the most convenient sample, the present disclosure extends to other samples containing immune cells such as lymph fluid, cerebral fluid, tissue fluid and respiratory fluid including nasal and pulmonary fluid.
  • the use of blood collection tubes is useful since they are compatible with standard automated laboratory systems and these are amenable to analysis in large-scale and random access sampling. Blood collectio tubes also minimize handling costs and reduce laboratory exposure to whole blood and plasma and, hence, reduce the risk of laboratory personnel from contracting a pathogenic agent such as HIV or hepatitis B virus (HBV).
  • HBV hepatitis B virus
  • the incubation step with the peptide antigen may be from 5 to 50 hours, including 5 to 40 hours and 8 to 24 hours or a time period in between,
  • Another aspect enabled herein contemplates a method for measuring a cell- mediated immune response in a subject including a human subject, the method comprising collecting a sample of whole blood from the subject, incubating the whole blood sample with a peptide comprising an amino acid sequence selected from SEQ ID NOs:2 to 5, 32, 35 to 61 , 64, 67, 70, 73, 76, 79 and 82 to 94 and then measuring the presence or elevation in level of an immune effector molecule such as IFN- ⁇ wherein the presence or level of the immune effector molecule is indicative of the capacity of the subject to mount a ⁇ $11- mediated immune response to insulin, proinsulin or preproinsulin leading to Type 1 diabetes.
  • a peptide comprising an amino acid sequence selected from SEQ ID NOs:2 to 5, 32, 35 to 61 , 64, 67, 70, 73, 76, 79 and 82 to 94 and then measuring the presence or elevation in level of an immune effector molecule such as IFN- ⁇ where
  • Another aspect enabled herein contemplates a method for measuring a cel.1- mediated immune response in a subject including a human subject, the method comprising collecting a sample of whole blood from the subject, incubating the whole blood sample with a peptide comprising a amino acid sequence selected from SEQ ID NOs:96 to 121 and then measuring the presence or elevation in level of an immune effector molecule such as IFN-y wherein the presence or level of the immune effector molecule is indicative of the capacity of the subject to mount a cell-mediated immune response to insulin, proinsulin or preproinsulin leading to Type 1 diabetes.
  • an immune effector molecule such as IFN-y
  • Another aspect enabled herein contemplates a method for measuring a cell- mediated immune response in a subject including a human subject, the method comprising collecting a sample of whole blood from the subject, incubating the whole blood sample with a peptide comprising an amino acid sequence selected from SEQ ID NOs:6 to 31, 33, 34, 62, 63, 65, 66, 68, 69, 71 , 72, 74, 75, 77, 78, 80, 81 and 95 and then measuring the presence or elevation in level of an immune effector molecule such as IFN-y wherein the presence or level of the immune effector molecule is indicative of the capacity of the subjec to mount a cell-mediated immune response to insulin, proinsulin or preproinsulin leading to Type 1 diabetes.
  • a peptide comprising an amino acid sequence selected from SEQ ID NOs:6 to 31, 33, 34, 62, 63, 65, 66, 68, 69, 71 , 72, 74, 75, 77, 78, 80
  • the peptide emploj'ed is or comprises SEQ ID NG;94.
  • the ability to measure an autoreactive cell-mediated immune response is important for assessing a subject's likelihood of mounting an autoimmune response to insulin, proinsuHn or preproinsulin and developing Type 1 diabetes and its associated conditions.
  • Detection of the immune effector molecules may be measured at the protein or nucleic acid levels. Consequently,., reference to "presence or level of the immune effector molecule" includes direct and indirect data. For example, high levels of IF - ⁇ mRNA is indirect data showing increased levels of TFN-y,
  • Ligands such as antibodies to the immune effectors are useful in detecting and/or quantitating these molecules.
  • autoreactive antibodies may be detected such as by anti-IgOr antibodies, labeled with a reporter molecule.
  • Techniques for the assays contemplated herein are known in. the art and include, for example, sandwich assays, ELISA and ELISpot.
  • Reference to "antibodies” includes parts of antibodies, mammalianized (e.g. humanized) antibodies, recombinant or synthetic antibodies and hybrid and single chain antibodies.
  • Both polyclonal and monoclonal antibodies to the effector molecule or autoreactive antibodies are obtainable by immunization with the immune effectors or antigenic fragments thereof and either type is u&Ikable for immunoassays.
  • the methods of obtaining both types of sera are well known in the art.
  • Polyclonal sera are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of the immune effector, or antigenic part thereof, collecting serum from the animal and isolating - 35 - specific sera by any of the known itnaiunoadsorbent techniques, Although antibodies produced by this method are utilizable in virtually any type of immunoassay, they are generally less favored because of the potential heterogeneity of the product, [0188]
  • the use of monoclonal antibodies in an immunoassay is useful due to the ability to produce them in large quantities and the homogeneity of the product.
  • the preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art.
  • Another aspect of the present invention contemplates, therefore, a method for detecting an immune effector m response to autoreactive T-cells to a peptide derived from insulin, pro insulin or preproinsulin and comprising an amnio acid sequence as set forth in SEQ ID NOs:2 through 121 or a chemically modified form of the amino acid sequence in a sample comprisin immune cells from a subject, the method comprising incubating the sample with the peptide and capturing an immune effector molecules produced by stimulated T-cells with an antibody specific for the immune effector or antigenic fragment and then detecting the capture immune effector.
  • Another aspect of the present invention contemplates, therefore, a method for detecting an autoreactive antibody in response to autoreactive B-cells to a peptide derived from insulin, proinsulin or preproinsuiin and comprising an amino acid sequence as set forth in SEQ ID NGs:2 through 121 or a chemically modified form of the amino acid sequence in a sample comprising immune cells from a subject, the method comprising incubating the sample with the peptide and capturing any autoreactive antibodies produced by stimulated B-cells with an antibody specific for the autoreactive antibody or antigenic fragment and then detecting the capture immune effector.
  • a sample includes whole blood. This method includes micro-arrays and macro- arrays on planar or spherical solid supports. A micro- or macro-array is useful. [0192] A wide range of immunoassay techniques are available as can be seen by reference to U.S. Patent Mos. 4,016,043, 4,424,279 and 4,018,653.
  • a monoclonal or polyclonal capture antibody is employed which is coated asepti ally onto the surface of well. These antibodies have specificity for the immune effector molecule.
  • the plate is blocked, usually with a serum protein that is non-reactive with, any of the antibodies in the assay. After this, immune ceils (e.g.
  • ⁇ a blood sample are plated out at varying densities, along with a peptide comprising an amino acid sequence selected from SEQ ID Os:2 through 121 , or a modified form of the peptide comprising amino acid substitution, addition or deletion or comprising a non-naturally occurring amino acid residue, and then placed in a humidified 37°C C ⁇ 3 ⁇ 4 incubator for a specified period of time.
  • the immune effector molecule secreted by activated cells is captured locally by the coated antibody on the high surface area microplate.
  • synthetic peptides comprising an arginine to eitruihne substitution are coated to the surface of a reaction vessel.
  • an antibody labeled with a reporter molecule and specific for the immune effector or autoreactive antibody is added to the wells. This antibody is reactive wit a distinct epitope of the immune effector or autoreactive antibod and i used to detect the captured immune effector or autoreactive antibody.
  • the detected immune effector or autoreactive antibody is then visualized using a detection system.
  • the spots can be counted manually (e.g. with a dissecting microscope) or using an automated reader to capture the mlcrowell images and to analyse spot number size.
  • Such an assay can also be used to quantitate the number of T-cells producing immune effectors or to quantitate or quantitate the presence of autoreactive antibodies.
  • the production of immune effectors means the presence of autoreactive T- cells and/or the preparation of autoreactiv antibodies means the potential for, or state of, Type 1 diabetes, (01 51 Ja t3 ⁇ 4ese assays, the immobilized antibody having specificity for the instant ⁇ immune effector or the synthetic peptide is either covalently or passively bound to a solid surface.
  • the solid surface is typically glass or a polymer, the most commonl used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, pol vinyiidene fluoride or polypropylene.
  • the solid supports may be in the form of tubes, beads, spheres, discs of microplates, or any other surface suitable for conducting an immunoassay.
  • the binding processes are well known m the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g.
  • the antibod subunit solid phase is washed and dried and incubated with a second antibod specific for a portion of the antigen.
  • the second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to the antibody-immune effector complex or peptide-autoreactive antibody complex.
  • reporter molecule as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of immune effector-bound antibody. Detection may be either qualitative or quantitative.
  • the most commonly used reporter molecules in this type of assay are either enzymes, fiuorophores or radionuclide containing molecules (i.e. radioisotopes) and chemilurainescent molecules.
  • an enzyme immunoassay an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan.
  • Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase, amongst others.
  • the substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable color change.
  • suitable enzymes include alkaline phosphatase and horse radish peroxidase. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above.
  • the enzyme-labeled antibody is added to the first antibody-immune effector comple or peptide-autoreactive antibody complex, allowed to bind, and then the excess reagent is washed away, A solution containing the appropriate substrate is then added to the complex of antibody-immune effector-antibody or peptide-autoreactive antibody-antibody.
  • the substrate reacts with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually speclrophotometrically, to give an indication of the amount of immune effector or autoreactive antibody which was present in the sample.
  • the present invention extends to a substantially simultaneous assay.
  • fluorescent compounds such as fluorescein and rhodamine
  • fluorescein and rhodamine may be chemically coupled to antibodies without altering their binding capacity.
  • the fluorochrome-labeled antibody When activated by illumination with light of a particvilar wavelength, the fluorochrome-labeled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic color visually detectable with a light microscope.
  • the fluorescent labeled antibody is allowed to bind to the ' first antibody- immune effector complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength the fluorescence observed indicates the presence of the antigen of interest.
  • Immunofliiorescene and EI A techniques are both well established in the art, Other reporter molecules, such as radioisotope, cbemilurninescent or bioluminesccnt molecules, may also be employed.
  • the present disclosure also contemplates genetic assays such as involving PGR analysis to detect R.NA expression products of a genetic sequence encoding an immune effector or autoreactive antibody.
  • PGR is conducted using pairs of prirners t one or both of which are generally labeled with the same or a different reporter molecule capable of givtag a distinguishable signal.
  • the use of fluorophores is. useful in the practice of this aspect of the invention.
  • any suitable method of analyzing fluorescence emissio is encompassed herein, in this regard, the instant disclosure contemplates techniques including but not restricted to 2-phcton and 3 -photon time resolved fluorescence spectroscop as, for example, disclosed by Lakowic? et al (1997) Btoph s, J, 72:567, fluorescence lifetime imaging as, for example, disclosed by Eriksson et al (1993) Biophys. I J' 64 and fluorescence resonance energy transfer as 5 for example, disclosed by Youvan et al (1 97) 8 lot ecknohgy et elia 3. ⁇ - 18.
  • Luminescence and phosphorescence may result respectively from a suitable luminescent or phosphorescent label as is known in the art, Any optical meaos of identifying such label may be used in this regard,
  • Infrared radiation may result from a suitable infrared dye
  • exemplary infrared dyes that may be employed in. the invention include but are not limited to those disclosed in Lewis et al (1999) Dyes Pigm. 42(2) ⁇ 97; Tawa et al Mater. Res. Soe, Symp. Proc. 488 (Electrical, Optical and Magnetic Properties of Organic S-olid-State Materials IV], 885- 890; Daneshvar et al (1999) J. Immunol Methods 226(1-2);) ⁇ 9 ⁇ ) 2& Rapaport et al (1999) Appl P ys. Lett.
  • any suitable infrared spectroscopic method may be employed to interrogate the infrared dye.
  • foarier transform infrared spectroscopy as, for example, described by Rahman ei al, (1 98) J. Org. Chem, 63:6196 may be used in this regard.
  • electromagnetic scattering may result from diffraction, reflection, polarization or refraction of the incident electromagnetic radiation including light and X- rays. Such scattering can be used to quantltate the level of mRNA or level of protein.
  • Flow cytometry is also useful in analyzin fluorophore emission.
  • flow cytometry is a high throughput technique which involves rapidly analyzing the physical and chemical characteristics of particles (e,g, labeled mRNA, DNA or proteins) as they pass through the path of one Of more laser beams while suspended in a fluid stream. As each particle intercepts the laser beam, the scattered light and fluorescent light emitted by each cell or particle is detected and recorded using any suitable tracking algorithm.
  • particles e,g, labeled mRNA, DNA or proteins
  • a modern flow cytometer is able to perform these tasks up to 100,000 cells/particles s '1 .
  • a number of lasers with, different excitation wavelengths may be used.
  • fiuorophores can be used to target and examine, for example, different immune effectors within a sample or immune effectors from multiple subjects,
  • Suitable flow cytometers which may be used in the methods of the present invention include those which measure five to nine optical parameters using a single excitation laser, commonly an argon ion air-cooled laser operating at 15 mW on its 488 ran spectral line. More advanced flow cytometers are capable of using multiple excitation lasers such as a IleNe laser (633 nm) or a HeCd laser (325 nm) in addition to the argon ion laser (488 or 514 nm).
  • a single excitation laser commonly an argon ion air-cooled laser operating at 15 mW on its 488 ran spectral line.
  • More advanced flow cytometers are capable of using multiple excitation lasers such as a IleNe laser (633 nm) or a HeCd laser (325 nm) in addition to the argon ion laser (488 or 514 nm).
  • the instant disclosure further contemplates autoreactive antibodies which have a binding affinity to the modified peptides derived from insulin* proinsulin o preproinsuii wherein the modified peptides comprise an arginine to citraliine substitution.
  • Antibodies may also be generated to such modified peptides tor use inter alia for diagnostic purposes,
  • an assay to screen for autoreactive antibodies to insulin, proinsulin or preproinsuiin comprising contacting a biological sample such as serum ftom a subject to be tested with a peptide derived insulin, proinsulin or preproinsuiin wherein the peptide comprises an arginine to citruliine substitution, so as to form a complex between an autoreactive antibody if present in the sample and the peptide and then detecting the presence of the antibody-peptide complex,
  • the sample is selected from serum, whole blood, sputum, urine or .tissue fluid.
  • the presence of an autoreactive antibody or the level or presence of the antibody is an indicator of Type 1 diabetes or a stage of developed Type 1 diabetes such as early stage Type 1 diabetes
  • the present specification further teaches animal models such as mouse, rat, rabbit, guinea pig, hamster or non-human primate models for Type 1 diabetes. These animal models may comprise humanized insulin or humanized human components in a diabetic model. These animal models are useful for testing the peptides in treatment, protocols and in diagnostic assays. Existing animal models may be used or readily adapted. Examples of existing animal models are disclosed in Example 1 1.
  • the present specification further teaches immunotherapies- and immunodiagnostics based on HLA Class II tetramers.
  • peptide-labeled HLA Class II tetramers are used to elucidate the role of antigen-specific Tregs in diabetes autoimmunity and to monitor insulin-specific CD4 + T-cells
  • Peptidc-HLA Class II tetramers display the antigen peptide in a form which is a surrogate for the recognition events which occur between T-cells and antigen-presenting cells, They are useful to interrogate CD4 + T-cells and in desensitization protocols (Nepon (2012) J. Immunol i f3 ⁇ 4);2477-2482).
  • [0216 J Enabled herein is an isolated HLA Class II tetraraer incorporating a peptide comprising at least 7 consecutive amino acids comprising a T ⁇ cell epitope derived from insulin, proinsulin or preproinsulin, wherein the peptide comprises a glutamine to glutamic acid substitution, [02l7j In an embodiment the peptide comprises at least 13 amino acid residues,
  • the peptide comprises a sequence selected from SEQ ID X():2 to 5, 32, 35 to 6i 5 64, 67, 70, 73, 76, 79 and 82 to 94 or 96 to 121, (0219] In an embodiment the peptide comprises SEQ ID NO:.94.
  • T-cells are isolated from within pancreatic islets due to the fact that this constitutes evidence that a T-cell contributes to beta-cell autoimmunity and destruction. Analysis of islet-infiltrating T-cells leads to the identification of disease-relevant epitopes. These epitopes are prime candidates for developing T-cell assays and antigen-specific therapies for Type 1 diabetes. Islet T-cells are shown in Figure 1 , The cloning of human islet T-cells is shown in Figure 2,
  • Table 6 is a summary of epitope mapping-epitope formed by posttransiational modification. See also Figure 6.
  • Table 7 provides a summary of epitope mapping-Epitope formed by posttranslational modification. See also Figures 8 and 9.
  • the T-cell clone* s response to peptide [SEQ ID NO:94] requires an antigen-presenting cell that expresses HLA DQ8 (HLA-DQ A*03;0 L DQ B*03:02) [ Figure 11].
  • the peptide binds to HLA DQ2 and/or HLA DQ 2/8 transdimer such as DQ A*05:01, DQ B*03:02 and/or DQ A*03;01, DQ B*02:0l, HLA- DR3 (DR.B1 *03xx) and/or HLA-DR4 (DRBl*04xx).
  • Fine epitope mapping [0230] The minimum sequence of amino acids required to stimulate the T-cell clone was determined by tested and panel of peptide of differing length (Panel A), To confirm this result, a panel of peptides covering the sequence of the epitope set forth in SEQ ID NO:94, but with each amino acid in turn subst tuted by alanine or lysine, whichever is most different from the native amino acid was tested. This, confirmed the epitope mapping and showed which residues could be modified without impairing the T-cell response.
  • Type I diabetes [0231 ] CD4 + T-cell responses were measured using a carboxy fluorescein succinimidyl ester .(CFSE)-based proliferation assay.
  • Peripheral blood mononuclear cells PBMC
  • the results show the ratio of the number of CD4 'T cells that have proliferated in response to the peptides relative to control samples cultured without peptide. The ratio is known as a "Cell Division Index" or CD1 (Mannering et al (2003) J. Immunol Methods 283: 173-183), A CDI> 2.0 is considered, to be a positive response ( Figure 13).
  • Figure 14 provides the results of an INF-y-based assay detected from sensitive T-cells in response to the modified peptide of SEQ ID NO;94 (with the glutamme to glutamic acid substitution) compared to a non-modified peptide.
  • the modified peptide was significantly more active in stimulating a response.
  • CD4 + T-cells are isolated and characterized specific for the deamidated epitopes derived from human proinsuiin. The epitopes are subjected to assays to determine specificity, HLA restriction and binding to T-cell receptors determination.
  • the SEQ ID NO.94 peptide is specific for HLA-DQ A*03:01 , DQ B*03:02.
  • a transgenic NOD mouse model of human CD4* T-cell responses against deamidated proinsulin epitopes is developed.
  • standard mouse models can be employed such as described by Niens et ai, (201 1 ) Diabetes 60:1229- 1236; Daniel et al. (2011) I Exp. Med. 10.1084/jcm.201 10574; Unger et al (2012) 7 iij;e49213.doi.:10. l 37l/Jounal.Pone,0049213; and Scotto ei al (2012) Di beMogia 55:2026-2031.

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Abstract

The present disclosure relates to a method for the treatment or prevention of Type 1 diabetes including a condition associated with Type 1 diabetes and to therapeutic and prophylactic agents useful for same. The present disclosure further teaches diagnostic assays in the detection and monitoring of Type 1 diabetes and its associated conditions.

Description

A METHOD FOR TREATING TYPE 1 DIABETES
FILING DATA [0001] This application is associated with and claims priority from Australian Provisional Patent Application No, 2013902683, filed on 19 July 20135 entitled "Method of treating Type I diabetes", the entire contents of which, are incorporated herein by reference. BACKGROUND
FIELD
[0002] The present disclosure relates to a method for the treatment or prevention of Type 1 diabetes including a condition associated with Type 1 diabetes and to therapeutic and prophylactic agents useful for same, The present disclosure further teaches diagnostic assays in the detection and monitoring of Type 1 diabetes and its associated conditions,
DESCRIPTION OF PRIOR ART
[0003] Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.
[0004] Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part, of the common general knowledge in any country.
[0005] Type 1 diabetes is an autoimmune disease associated with activation of lymphocytes against pancreatic β-cells. Whilst Type 1 diabetes has been the focus of decades of research, it is still ill defined in human subjects (Faideau t al, (2005) Diabetes 5 f >;S87-S96). The autoimmune disease is highly muitigenic (Todd (2010) Immunity 32(-4):457~467; Rogner et al (2001) Genomics 72f2j:163-171). A feature of human Type 1 diabetes is the long preclinical phase that precedes hyperglycemia (Ziegler and Nepom (2010) Immunity 32(4) 46&-47B), There is also a high level of recurrence of autoimmunity in long-standing patients who have been treated with exogenous insulin (Sible et al. (1985) Laborator Investigation 5J 2j:132~144).
[0006] p-Cell-speeific CD4+ and CD8+ T-cells maintain immunological memory for years after onset of Type 1 diabetes. Key aiitoantigens associated with Type 1 diabetes are insulin and its precursor forms, proinsulin and preproinsulin (Faideau et al (2005) supra; Homo-Deiarche and Boitard (1996) Immunology Today 17(10) :456-4ό0).
[0007J Type 1 diabetes is a debilitating, chronic disease. Antigen-specific therapies are a promising approach in the treatment and prevention of Type 1 diabetes. This approach requires a knowledge of epitopes recognized by pathogenic T-cells and B-celts, in turn, this would also lead to better diagnostic assays to detect and monitor Type 1 diabetes. However, progress towards the identification of disease-relevant epitopes using Ί -ceils and B-cells isolated from peripheral blood has been slow.
SUMMARY
[0008] A summary of sequence identifiers used throughout the subject specification is provided in Table 1.
[0009] Islet-infiltrating T-eells have been used to detect T-cell epitopes on proinsulin which, when modified, are useful in the treatment or prevention of Type 1 diabetes or a condition associated with Type: 1 diabetes in mammalian subjects such as humans. Peptides encompassing the identified t-cell epitope are modified by ait amino acid substitution. The modified peptides result in a more sensitive, efficacious and/or active response from T-cells from diabetic subjects. These epitopes are also useful in diagnostic assays for autoreactive T-celis, B-ceils and antibodies. The amino acid sequence in the proinsulitt-derived peptides is modified by a glutamine (Q) to glutamic acid (E) substitution. This substitution can be made during the chemical synthesis of the peptides, by recombinant means, or by recombinant means followed by posttranslational modification. The peptides ma also comprise chemical analog of, or a non-naturally occurring amino acid substitute of, one or more amino acid residues in the sequence.
[0010) Hence, the instant disclosure teaches a method for the treatment or prophylaxis of Type 1 diabetes or a condition associated with Type 1 diabetes in a mammalian subject comprising the administration to the mammalian subject; a peptide comprising an amino acid sequence of at least seven consecutive amino acid residues encompassing a T-cell epitope derived from insulin, proinsulin or preproinsulin wherein the amino acid sequence comprises a glutamine to glutamic acid substitution. It is proposed that the peptide comprises an epitope which, when modified by the glutamine to glutamic acid substitution, is far more sensitive to recognition by antigen-presenting cells and lymphocytes. This is indicated by the level of response by activated T-cells in the presence of the peptide compared to a peptide comprising a non-modified sequence. [0011] Further enabled herein is an isolated T-cell or antigen-presenting cell derived from a mammalian subject presenting a peptide comprising an amino acid sequence of at least seven consecutive amino acids encompassing a T-cell epitope derived from insulin, proinsulin or preproinsulin wherein the amino acid sequence comprises a glutamine to glutamic acid substitution, [0012] The isolated T-cell or antigen-presenting cell is useful for treating or preventing Type 1 diabetes or an associated condition wherein the T-cells or antigen- presenting cells are autologous to the subject being treated. Reference to a "T-cell" or "T- lymphocyte" includes a CD4+ T-cell and a CD8+ T-cell. This aspect includes regulatory T- cells (Treg cells) useful to enhance a response to the modified peptide. Similarly, peptide- HLA Class II tetramers may be prepared and used for inducing tolerance and/or to assess CD4+ T-cell autoreactivity, A condition associated with Type 1 diabetes includes a cardiovascular condition, neuropathy, a retinopathy, a nephropathy, hearing impairment, an infection and a complication in. pregnancy. In an embodiment, the antigen-presenting cell is serologically HLA-DQ8, In an embodiment, the antigen-presenting cell comprises the hapiotype HLA-DQ A*03:Q1, DQ B*03;02, In another embodiment, T-cells respond to the peptide presented by another HLA molecule, such as but not limited to HLA DQ A*03:01 , DQ B* 03:02. In another embodiment, T-cells respond to the peptide presented by another HLA molecule, such as but not limited to H LA DQ2 and HLA DQ 2/8 transdiraers (DQ A*05:01, DQ B*03:02 and/or DQ A*Q3:01 ; DQ 13*02:01), IILA-DR3 (DRB 1 *03xx) and/or HLA-DR4 (DRB 1 *04xx).
[0013] Further taught is a therapeutic or prophylactic composition comprising a peptide comprising an amino acid sequence of at least seven consecutive amino acids encompassing a T-cell epitope derived from insulin, proinsulin or preproinsulin wherein the amino acid sequence comprises a glutamine to glutamic acid substitution and one or more pharmaceutically acceptable carriers, diluents and/or excipients. The subject peptide may also be complexed with an HLA Class II tctramcr.
[0014] The present specification is also instructional on the use of a peptide comprising at least seven consecutive amino acids encompassing a T-cell epitope derived from insulin, proinsulin or preproinsulin wherein the amino acid sequence comprises a glutamine to glutamic substitution in the manufacture of a medicament for the treatment of Type 1 diabetes or a condition associated with Type 1 diabetes in a mammalian subject.
[0015] In an embodiment, the mammalian subject is a human male or female of any age.
{00161 Diagnostic assays are also enabled by the present disclosure, Taught herein is a method of detecting autoreactive T-eells including CD4+ T-eells which recognize an epi tope on pro insulin with a modification comprising a glutamine to glutamic acid substitution with greater responsiveness compared to the same peptide but without the glutamine to glutamic acid substitution. CD8+ T-cells can also be detected. HLA Class II tetramers may also be used to interrogate CD41 T-cells, The tetramers are complexed with a. subject peptide such as SEQ ID NO:94. J0017] Enabled herein is a method for detecting the presence of autoreactive T-cells indicative of the presence of, or a predisposition for the development of, Type I diabetes, or a condition associated with Type 1 diabetes in a mammalian subject, the method comprising contacting a sample from the subject comprising immune cells with a peptide comprising an amino acid sequence of at least seven consecutive amino acid residues encompassing a T-cell epitope derived from insulin, proinsulin or preprojnsulm wherein the amino acid sequence comprises a glutamine to glutamic acid substitution for a time and under conditions sufficient for an autoreactive T-cell, if present, to be stimulated into producing an immune effector molecule, and then detecting for the presence or level of the immune effector molecule, wherein the presence or level of the immune effector molecule is indicative of Type 1 diabetes or its state or risk of progression. The level of response in a peptide comprising a glutamine to glutamic acid substitution is greater than the same peptide without this substitution.
[0018] In an embodiment, the peptide comprises an amino acid sequence selected from the list consisting of SEQ ID NOs:2 to 5, 32, 35 to 61, 64, 67, 70, 73, 76, 79, 82 to 94 and 96 to 12L [0019| In an embodiment, the peptide comprises an amino acid sequence set forth in SEQ ID NO:94. [0020] In an embodiment, the peptide may be further modified to include a non- naturally occurring amino acid in place of or in addition to an existing amino acid residue. For example, an arginme ma be converted to a citrulline such as set forth in a sequence selected from the list consiSsting of SEQ ID NOs:6 to 1, 33, 34, 62, 63, 65, 66, 68, 69, 71, 72, 74, 75, 77, 78, 80, 81 and 95. In an embodiment, the peptide is SEQ ID NO:95, Other amino acid substitutions may also be made including the substitution of a natural amino acid with a non-naturally occurring amino acid residue.
[0021 J The instant disclosure further contemplates detecting autoreactive antibodies to insulin, proinsulin Or preproinsulin as well as generating antibodies to the modified peptides of the instant disclosure.
[0022] Further enabled herein is a method for screening for autoreactive antibodies associated with Type 1 diabetes or a condition associated therewith in subject, comprising contacting body fluid from the subject with a peptide comprising an amino acid sequence encompassing an epitope derived fro insulin, proinsulin or preproinsulin wherein the amino acid sequence comprises a glutamine to glutamic acid substitution for a time and under conditions sufficient for an autoreactive antibody, if present, to bind to the peptide and then screening for the presence of the peptide-antibody complex. The presence or level of the antibody is indicative of Type 1 diabetes or its state or risk of progression. The glutamine to glutamic acid substitution results in higher sensitivity in antibody binding.
[0023] A peptide of the present disclosure comprising an amino acid sequence selected from SEQ ID NOs:2 to 5, 32, 35 to 61, 64, 67, 70, 73, 76, 79, 82 to 94 and 96 to 121 may also be conjugated to or embedded in a targeting moiety and/or a carrie molecule. In an embodiment, the targeting moiety and the carrier molecule are the same entity, A targeting moiety is useful in facilitating the deployment of the peptide to a particular cell type or tissue type. Examples of cell types include antigen-presenting cells, and CD4+ T-cells or other lymphocytes such as CDS T-celJs. Examples of targeting moieties include antibodies or antigen-binding fragments or derivatives thereof specific for an antigen on the target cell or tissue, receptor ligands, a glycosaminoglycan, heparan or heparin or a fragment thereof, a cytokine or other molecule capable of specific binding to a desired cell type. HLA Class 11 tetramers may also be used. Carrier molecules include adjuvants, molecules which increase the half life in the blood stream and the like. Other carrier molecules include HLA-DQ8 complexes such as a complex with HLA-DQ A*03:01} DQ B*03:02. In another embodiment, 'Γ-cells respond to the peptide presented by another HLA molecule, such as but not limited to HLA DQ2 and IILA DQ 2/8 traitsdimers (DQ A*Q5:01 , DQ B*03;02 and DQ A*()3:()I ; DQ B* 02:01), HLA-DR3 (DRB 1 *Q3xx) and/or HLA-DR4 (DRB 1 *04xx). [0024] Enabled herein is an agent comprising a peptide comprising an amino acid sequence of at. least seven consecutive amino acid residues encompassing a T-cell epitope derived from insulin, proinsulin or preproinsulin wherein the amino aeid sequence comprises a glutamine to glutamic acid substitution in an amino acid sequence selected from SEQ ID NOs:2 to 5, 32, 35 to 61, 64, 67, 70, 73, 76, 79, 82 to 94 and % to 121, the peptide conjugated at its N- or C-terrninal end to a tiirgeting moiety or carrier molecule,
[0025] In an alternative embodiment, a peptide is provided comprising an amino acid sequence of at least seven consecutive amino acid residues encompassin a T-cell epitope derived from insulin, proinsulin or preproinsulin wherein the amino acid sequence comprises an arginine to citrulline substitution in an amin acid sequence selected from SEQ ID Os:6 to 31 , 33, 34, 62, 63, 65, 66, 68, 69, 71, 72, 74, 75, 77, 78, 80, 81 and 95, the peptide embedded in a targeting moiety or carrier molecule,
[0026] Enabled herein is an agent comprising a peptide comprising an amino acid sequence encompassing a T-cell epitope derived from insulin, proinsulin or preproinsulin having a glutamine to glutamic acid substitution and comprising an amino acid sequence set forth in. SEQ ID NOs;2 to 5, 32, 35 to 61, 64, 67, 70, 73, 76, 79, 82 to 94 and 96 to 121, the peptide conjugated at its N- or C-terminal end to a targeting moiety or carrier molecule, [0027J In accordance with these embodiments, one particular peptide is as defined by SEQ ID NO:94 or comprises the amino acid sequence of SEQ ID NO:94.
[0028] In an embodiment, the subject specification contemplates a method for detecting the presence of autoreactive T-cells indicative of the presence of, or a predisposition for the development oft Type 1 diabetes, or a condition associated with Type 1 diabetes in a subject, the method comprising contacting a sample from the subject comprising immune cells with a peptide comprising an amino acid sequence of at least seven consecutive amino acid residues encompassing a T-ceTl epitope derived from insulin, proinsuiin or prepro insulin wherein the amino acid sequence comprises a glutaroine to glutamic acid substitution for a time and under conditions sufficient for an autoreactive T- cell, if present, to be stimulated into producing an immune effector molecule, and then detecting the presence of the immune effector molecule, wherein the presence o level of the mtmune effector molecule is indicative of Type 1 diabetes or its state or risk of progression or a condition associated with Type 1 diabetes .
(0029J In an embodiment, the subject specification contemplates a method for detecting the presence of autoreactive B-cells indicative of the presence of. or a predisposition for the development of, Type 1 diabetes, or a condition associated with Type 1 diabetes in a subject, the method comprising contacting a sample from the subject comprising immune cells with a peptide comprising an amino acid sequence encompassing an epitope derived from insulin, proinsuiin or preproinsulin wherein the amino acid sequence comprises a glutamine to glutamic acid substitution for a time and under conditions sufficient for an autoreactive B-cell, if present, to be stimulated into producing an antibody, and then detecting the presence of the antibody, wherein the presence or level of the antibody is indicative of Type 1 diabetes or its state or risk of progression or condition associated with Type 1 diabetes. [0030] In an embodiment, the subject is a human.
[0031] In an embodiment, the peptide comprises an amino acid sequence selected from the list consisting of SEQ ID NOs:2 to 5, 32, 35 to 61, 64, 67, 70, 73, 76, 79, 82 to 94 and 96 to 121.
[0032] In an embodiment,, the peptide comprises an amino acid sequence set forth in SEQ 1D N0:94.
[0033] In an embodiment, the condition associated with Type 1 diabetes is selected from the list consisting of a cardiovascular condition, a neuropathy, a retinopathy, a nephropathy, infection and a complication in pregnancy. [0034] In an embodiment, the peptide has a minimum length of 13 amino acid residues and encompasses an epitope which is recognized by and binds to an antigen- presenting cell which is serologically HLA-DQ8 such as an antigen-presenting cell which comprises the haplotype HLA-DQA*03:01 » DQ B*03:02. In another embodiment, T-cells respond to the peptide presented by another HLA molecule, such as but not limited to HLA DQ2 and HLA DQ 2/8 transdimers (DQ A*05:01 , DQ B*03;Q2 and DQ A*03:01 ; DQ B*02:0.1, IILA-DR3 (DRBl *03xx) and/or HLA-DR4 (DRRl *04xx). Reference to a minimum length of 13 includes from 13 to 40 including from 13 to 20. There is a proviso that the amino acid sequence comprises at least one glutamine to glutamic acid substitution (i.e. a deamidatlon).
[0035] Enabled herein is an isolated HLA Class II tetramer incorporating a peptide' comprising at least 7 consecutive amino acids comprising a T-cell epitope derived from insulin, proinsulin or prepromsulin, wherein the peptide comprises a glutamine to glutamic acid substitution.
[0036] In an embodiment the peptide comprises at least 13 amino acid residues. [0037] In an embodiment the peptide comprises a sequence selected from SEQ ID NO:2 to 5, 32, 35 to 61, 64, 67, 70, 73, 76, 79 and 82 to 94 or 96 to 121.
[0038] embodiment the peptide comprises SEQ ID NO: 94,
Table 1
Summary of sequence identifiers
SEQUENCE DESCRIPTION ID NO; ■ *X ~ ciirulliiie
1 Amino add sequence of human proi nsulm
2 Amino acid sequence of modified pramsulin-derived peptide (FVMEHLC)
3 Amino acid sequence ofmodified proinsul in- derived peptide (VNEHLCG)
4 Amino acid sequence ofmodified proinsulin-derived peptide (NEHLCGS)
5 Amino acid sequence of modified proinsulin-derived peptide (EHLCGSH)
6 Amino acid sequence of modified proinsulin-derived peptide (YLVCGEX)
7 Amino acid sequence of modified proinsulin-derived pepti de (LVCGEXG)
8 Amino acid sequence ofmodified proinsulin-derived peptide (VCGEXGF)
9 Amino acid sequence of modified proinsulin-derived peptide (CGEXGFF)
10 Amino acid sequence of modified proinsulin-derived peptide (GEXGFFY)
1 1 Amino acid sequence of modified proinsulin-derived peptide (EXGFFYT)
12 Amino acid sequence of modified proinsulin-derived peptide (XGFFYTP)
13 Amino acid sequence of modified proinsulin-derived peptide (FYTPKTX)
14 Amino acid sequence of modified proinsulin-derived peptide (YTPKTXR) Ϊ5 Amino acid sequence of modified proinsulin-derived peptide (YTPKTRX)
16 Amino acid sequence of modified proinsulin-derived peptide (YTPKTXX)
17 Amino acid sequence of modified proinsulin-derived peptide (TPKTXRE)
18 Amino acid sequence of modified proinsulin-derived peptide (TPKTRXE)
19 Amino acid sequence of modified proinsulin-derived peptide (TP TXXE)
20 Amino acid sequence of modified proinsulin-derived peptide (P TXREA)
21 Amino acid sequence of modified proinsulin-derived peptide (PKTRXEA)
2 Amino acid sequence of modified proinsulin-derived peptide (P TXXEA)
23 Amino acid sequence of modified proinsulin-derived peptide (KTXREAE)
24 Amino acid sequence ofmodified proinsulin-derived peptide (KTRXEAE) '
25 Amino acid sequence of modified proinsulin-derived peptide (KTXXEAE) SEQUENCE DESCRIPTION
ID NO: *X ~ citruJJiiie
26 Amino acid sequence of modified proinsulin-derived peptide (TXREAED)
27 Amino acid sequence of modified proinsulin-derived peptide (TRXEAED)
28 Amino acid sequence of modified proinsulin-derived peptide (TXXEAED)
29 Amino acid sequence of modified proinsulin-derived peptide (XREAEDL)
30 Amino acid sequence of modified proinsulin-derived peptide (RXEABDL)
31 Amino acid sequence of modified proinsulin-derived peptide (XXEAEDL)
32 * Amino acid sequence of modified proinsulin-derived peptide (REAEDLE)
33 Amino acid sequence of modified proinsulin-derived peptide (XEAEDLQ) 34 Amino acid sequence of modified proinsulin-derived peptide (XB AEDLE)
35 Amino acid sequence of modified proinsulin-derived peptide (EAEDLEV) 36 Amino acid sequence of modified proinsulin-derived peptide (ABDLEVG)
37 Amino acid sequence of modified proinsulin-derived peptide (EDLEVGQ)
3.8 Amino acid sequence of modified proinsulin-derived peptide (EDLQVGE)
39 Amino acid sequence of modified proinsulin-derived peptide (EDLEVGE)
40 Amino acid sequence of modified proinsulin-deri ved peptide (DLEVGQV)
41 Amino acid sequence of modified proinsulin-derived peptide (DLQYGEV) 42 Amino acid sequence of modified proinsulin-derived' peptide (DLEYGEV)
43 Amino acid sequence of modified proinsulin-derived peptide (LEVGQVE)
44 Amino acid sequence of modified proinsulin-derived peptide (LQVGEVE)
45 Amino acid sequence of modified proinsulin-derived peptide (LEVGEVE)
46 Amino acid sequence of modified proinsulin-derived peptide (EVGQVEL)
47 Amino acid sequence of modified proinsulin-derived peptide (QVGEVEL)
48 Amino acid sequence of modified proinsulin-derived peptide (EVGEVEL)
49 Amino acid sequence of modified proinsulin-derived peptide (VGEVELG)
50 Amino acid sequence of modified proinsulin-derived peptide (GEVELGG)
51 Amino acid sequence of modified proinsulin-derived peptide (EVELGGG)
52 Amino acid sequence of modified proinsulin-derived peptide (PGAGSLE)
53 Amino acid sequence of modified proinsulin-derived peptide (G AG.SLEP) SEQUENCE DESCRIPTION
ID NO; *X = atruHine
54 Amino acid sequence of modified pro insulin-derived peptide (AGSLEPL)
55 Amino acid sequence of modified proinsulin-derived peptide (GSLEPLA)
56 Amino acid sequence of modified promsulin-derived peptide (SLEPLAL)
57 Amino acid sequence of modified proinsulin-derived peptide (LEPLALE)
58 Amino acid sequence of modified proinsulin-derived peptide (EPLALEG)
5 Amino acid sequence of modified proinsuiin-derived peptide (ALEGSLE)
60 Amino acid sequence of modified proinsulin-derived peptide (LEGSLEK)
61 Amino acid sequence of modified proinsulin-derived peptide (EGSLE R)
62 Amino acid sequence of modified proinsulin-derived peptide (EGSLQKX)
63 Amino acid sequence of modified proinsulin-deri ved peptide (EGSLEKX)
64 Amino acid sequence of modified proinsulin-derived peptide (GSLEKRG)
65 Amino acid sequence of modified promsulin-derived peptide (GSLQiCXG)
66 Amino acid sequence of modified proinsulin-derived peptide (GSLEKXG)
67 Amino acid sequence of modified proinsulin-derived peptide (SLE RG1)
68 Amino acid sequence of modified proinsulin-derived peptide (SLQKXGI)
69 Amino acid sequence of modified proinsulin-derived peptide (SLEKXGI)
70 Amino acid sequence of modified proinsulin-derived peptide (LEKRG1V)
71 Amino acid sequence of modified proinsulin-derived peptide (LQKXGIV)
72 Amino acid sequence of modified proinsulin-derived peptide (LEKXGIV)
73 Amino acid sequence of modified proinsulin-derived peptide (EKRGIVE)
74 Amino acid sequence of modified proinsulin-derived peptide (QKXGIVE)
75 Amino acid sequence of modified proinsulin-derived peptide (EKXGIVE)
76 Amino acid sequence of modified proinsulin-derived peptide (KRGIVEE)
77 Amino acid sequence of modified proinsuiin-derived peptide (KXGIVEQ)
78 Amino acid sequence of modified proinsulin-derived peptide (KXGIVEE)
79 Amino acid sequence of modified proinsulin-derived peptide (RGIVEEC)
80 Amino acid sequence of modified proinsulin-derived peptide (XGIVEQC)
81 Amino acid sequence of modified proinsulin-derived peptide (XGIVEXC) SEQUENCE DESCRIPTION
II> NO: * 53 citrulline
82 Amino acid sequence of modified promsu!in-derived peptide (GIVEECC)
83 Amino acid sequence of modified pro insulin-derived peptide (IVEECCT)
84 Amino acid sequence of modified proinsul in-derived peptide (VEECCTS) 8 Amino acid sequence of modified proinsulin-derived peptide (EECCTSI)
86 Amino acid sequence of modified proinsulin-derived peptide (ECCTSIC)
87 Amino acid sequence of modified proinsulin-derived peptide (SICSLYE)
88 Amino acid sequence of modified proinsulin-derived peptide (ICSLYEL) 89 Amino acid sequence of modified proinsulin-derived peptide (CSLYELE)
90 Amino acid sequence of modified proinsulin-derived peptide (SLYELEN) 1 Amino acid sequence of modified proinsulin-derived peptide (LYELENY)
92 Amino acid sequence of modified proinsulin-derived peptide (YELENYC)
93 Amino acid sequence of modified proinsulin-derived peptide (ELENYCN)
94 Amino acid sequence of modified proinsulin-derived peptide
(VELGGGPGAGSLEPL)
95 Amino acid sequence of modified proinsulin-derived peptide
(QPLALEGSLQ XGI)
96 Amino acid sequence of modified proinsulin-deri ved peptide
(GQVELGGGPGAGSLEPL)
97 Amino acid sequence of modified proinsulin-derived peptide
(VELGGGPGAGSLQPL)
98 Amino acid sequence of modified proinsulin-derived peptide
(VELGGGPGAGSLEP)
99 Amino acid sequence of modified proinsulin-derived peptide
(VELGGGPGAGSLE) SEQUENCE DESCRIPTION
ID NO: *X = citrulline
too Amino acid sequence of modified proinsulin-derived peptide
(VELGGGPGAGSL)
101 Amino acid sequence of modified proinsulin-derived peptide
(ELGGGPGAGSLEPLAL)
102 Amino acid sequence of modified proinsulin-derived peptide
(ELGGGPGAGSLEPL)
103 Amino acid sequence of modified proinsulin-derived peptide
(ELGGGPGAGSLEPL)
104 Amino acid sequence of modified proinsulin-deri ved peptide
(VELGGGPGAGSLEPL)
105 Amino acid sequence of modified proinsulin-deri ved peptide
(GQVELGGGPGAGSLEPL)
106 Amino acid sequence of modified proinsulin-derived peptide
(QVELGGGPGAGSLEPA)
1 7 Amino acid sequence of modified proinsulin-deri ved peptide
(QVELGGGPGAGSLEAL)
108 Amino acid sequence of modified proinsulin-derived pept ide
(QVELGGGPGAGSLAPL)
109 Amino acid sequence of modified proinsulin-derived peptide
(QVELGGGPGAGSAEPL)
110 Amino acid sequence of modified proinsulin-derived peptide
(QVELGGGPGAGKLEPL)
1 11 Amino acid sequence of modified proinsulin-deri ved peptide
(QVELGGGPGA SLEPL)
112 Amino acid sequence of modified proinsulin-derived peptid
(QVELGGGPGKGSLEPL)
1 13 Amino acid sequence of modified proinsulin-derived peptide
(QVELGGGPKAGSLEPL) SEQUENCE DESCRIPTION
ID NO: * —dtrulliiie
114 Amino acid sequence of modified proinsulin-derived peptide
(QVELGGGAGAGSLEPL)
1 15 Amino acid sequence of modified proinsulin-derived peptide
(QVELGGKPGAGSLEPL)
116 Amino acid sequence of modified proinsulin-derived peptide
(QVELGKGPGAGSJLEPL)
117 Amino acid sequence of modified proinsulin-derived peptide
(QVELKGGPGAGSLBPE)
1 18 Amino acid sequence of modified proinsulin-derived peptide
(QVEAGGGPGAGSLEPL)
11 Amino acid sequence of modified proinsulin-deri ved peptide
(QVALGGGPGAGSLEPL)
120 Amino acid sequence of modified proinsulin-deri ved peptide
(QKELGGGPGAGSLEPL)
121 Amino acid sequence of modified proinsulin-derived peptide
(AVELGGGPGAGSLEPE)
[0039] Single and three letter abbreviations are used in the subject specification are defined in Table 2.
Table 2
Amino acid single and three letter designations
Amino Acid Three-letter One-letter
Abbreviatiori Symbol
Alanine Ala A
Arginme Arg R
As aragine Asn N
Aspartic acid Asp D
Cysteine Cys C
Glutamine Gin Q
Glutamic acid Glu E
Glycine Gly G
Histidine His H
Isoleucine He I
Leucine Leu L
Lysine Lys
Methionine Met M
Phenylalanine Phe F
Proline Pro P
Serine Ser S
Threonine Thr T
Tryptophan Trp w
Tyrosine Tyr Y
Valine Val V
Pyrro!ysine Pyl 0
Selenocysteine Sec u
Citrulline Cit X (or R*J BRIEF DESCRIPTION OF THE FIGURES
(0040] Some figures contain color representations or entities. Color photographs are available from the Patentee upon request or from an appropriate Patent Office. A fee may be imposed if obtained from a Patent Office.
[0041] Figure 1 is a photographic representation of T-cells emerging from human islets. [0042] Figure 2 is a representation of an ovemew of the cloning of human islet infiltrating T-cells.
[0043] Figure 3 is a diagrammatic representation of an analysis of the antigen specific of T-cell clones.
[00441 Figure 4 is a graphical representation of clonal expansio of islet-infiltrating T-cells.
[0045] Figure 5 is a graphical representation of the screening for responses to posttranslational modified peptides,
[0046] Figure 6 is a graphical representation of the response of clone Α2.Γ1 to an epitope formed by posttranslational modification. [0047] Figure 7 is a graphical representation of the titration of Q25H peptides.
[0048] Figure 8 is a graphical representation showing that Pool E stimulates clone A4.7. (0049] Figure 9 is a graphical representation showing that R.65X is the active peptide. [0050] Figure 10 is a graphical representation showing that a monoclonal antibody specific for HLA-DQ inhibits a INFy-based response to a peptide derived and modified from promsulm (SEQ ID NO:94). [00511 Figure 11 is a graphical representation showing that a T-cell clone's response to a peptide requires an antigen-presenting cell (APC) which expresses HLA DQ8 (HLA- DQ A*03;Q1 S DQ B*03:02).
[0052] figure 12 is a graphical representation of a fine epitope mapping. Panel A: these data show that the minimum number of amino acid residues required to stimulate a T-cell clone is 13. Panel B: to confirm the results in Panel A, peptides covering the sequence of one particular epitope but with each amino acid in torn substituted with an alanine (A) or lysine (K) were tested. The results showed which residues could be modified without impairing the T-cell response.
[0053] Figure 13 is a graphical representation of CD4+ T-cell responses measured using a CFSE-based proliferation assay in (A) typical diabetes; and (B) health controls, The results show the ratio of the number of CD4+ T-cells that have proliferated in response to a non-modified peptide (with glutamine [Q]) compared to a glutamic acid (E) substitution,
J0054} Figure 1.4 is a graphical representation showing the effects of l Srner peptide covering an epitope in an unmodified (glutamine (Qj) or modified (glutamic acid [E]) on IFNy response from a T-cell clone.
DETAILED DESCRIPTION
{0055] Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or method step or group of elements or integers or method steps but not the exclusion of any element or integer or method step or group of elements or integers or method steps.
[0056] As used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a peptide" includes a single peptide, as well as two or more peptides; reference to "an epitope" includes a single epitope, as well as two or more epitopes; reference to "the disclosure" includes a single and multiple aspects taught by the disclosure; and so forth. Aspects taught and enabled herein are encompassed by the term "invention". All such aspects are enabled within the width of the claimed invention.
[0057] The present specification teaches the generation of peptides comprising an amino acid sequence having at least seven consecutive amino acid residues comprising a T-cell epitope derived from insulin, proinsulin or preproinsulin, The amino acid sequence of the peptides is modified with a glutamine to glutamic acid substitution, The peptide may contain other modifications to the amino acid residues such as a chemical modification to an amino acid residue or a substitution of an amino acid residue for a non- natural ly occurring amino acid. The peptide may alternatively or in addition comprise an arginine to citmiline substitution. The glutamine to glutamic acid substitution in the amino acid sequence of the epitope results in a higher degree of recognition by antigen-presenting cells and lymphocytes and antibodies. This can be detected by a greater response by sensitized T-cells to the peptides compared to the same peptides without the modification. The peptide epitopes derived from insulin, proinsulin or preproinsulin are used to induce tolerance of autoreactive T-cells and/or other immune cells in the treatment or prophylaxis of Type 1 diabetes or an associated condition in a mammalian subject. The modified peptides are also useful in diagnostic assays to detect autoreactive antibodies and the peptides and/or the antibodies are also useful to measure the level of autoreactive T-cells such as CD4T T-cells or other T-cells such as CD8+ T-eells. Such autoreactive antibodies or T-cells lead to the development or progression of Type 1 diabetes or a condition associated therewith. Hence, their direct detection or indirect detection assists in 5 determining the presence of Type 1 diabetes, such as prior to hyperglycemia and/or the state of progression of diabetes. Antibodies may also be generated specifically to the modified peptides. Such antibodies are useful in diagnostic assays.
[0058] Conditions associated with Type 1 diabetes include cardiovascular conditions 10 such as angina, heart attack, stroke, atherosclerosis and hypertension; neuropathy; retinopathy; nephropathy; hearing impairment; chronic gum infection; and complications in pregnancy such as miscarriage, preeclampsia and diabetic ketoacidosis,
[0059] Enabled herein is a method for the treatment or prophylaxis of Type 1 diabetes I S or a condition associated with Type I diabetes in a mammalian subject, the method comprising administering to the subject a peptide comprising an amino acid sequence of at least seven consecutive amino acids encompassing a T-cell epitope derived from insulin, proinsulin or preproinsulin wherein the amino acid sequence comprises a glutamine to glutamic acid substitution,
0
The peptides comprise epitopes which are recog mzed by CD4+ T-cells, CDS T-cells and/or antigen-presenting cells as well as B-cells in mammalian subjects sensitized to insulin, proinsulin or preproinsulin. in an embodiment, the T-cells are CD4+ T-cells. By "recognized" is meant that the peptide induces a T-cell mediated response statistically 5 significant relative to a T-cell control using the same peptide to which the mammal is not sensitized or an unrelated peptide in a sensitized or non-sensitized mammal or is bound by autoreactive antibodies directed to insulin, proinsulin or preproinsulin. The amino acid sequence is "derived from" insulin, proinsulin or preproinsulin meaning the peptide is a fragment of one of these molecules or is chemically synthesized based on the amino acid 0 sequence of the desired region or is produced by recombinant means alone or by recombinant means followed by posttranslational chemical modification. In an embodiment, the peptide with the glutamine to glutamic acid substitution is more strongly recognized by a serologically HLA-DQ8 antigen-presenting cell such as an antigen- presenting cell which comprises the haplotype HLA-DQ A* 03:01, DQ B* 03:02. In another embodiment, T-eells respond to th peptide presented by another HLA molecule, such as but not limited to HLA DQ2 and HLA DQ 2/8 transdimers (DQ A* 05 :()! ., DQ B* 03:02 and DQ A* 03:01 ; DQ B* 02:01) or HLA-DR3 (DRBl *Q3xx) and/or HLA-D 4 (DRB l *04xx).
[0060] Hence, in an embodiment, the peptide binds to HLA-DQ A* 03:01 ; DQ B*03:()2, In another embodiment, the peptide binds to another HLA molecule selected from one or more of HLA DQ2, HLA DQ 2/8 transdimer, DR3 and/or DR4, in a embodiment, the peptide binds to an antigen-presenting cell which comprises the haplotype DQ A*G5:01 , DQ B*03:02 and/or DQ A*Q3:01, DQ B*02:01 , j (M)61| The peptides of the instant disclosure comprise from 7 to 40 consecutive amino acid residues meaning 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 amino acid residues. In relation to a peptide fo use in an antibody assay, the only requirement is a glutamine to glutamic acid substitution. In this case, the "peptide" may contain from 2 to 40 amino acid residues provided it contains this substitution. In an embodiment, the seven consecutive amino acids represented in SEQ ID NOs:2 to 93 are comprised within a larger 10 to 20 amino acid sequence, Examples of larger amino acid sequences include SEQ ID NGs:94 to 121. By "10 to 20" means 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid residues. It is taught herei that the peptides are useful in tolerizing or down-regulating the priming and/or activity of T-cells of a mammal with Type 1 diabetes, The peptides are also useful in diagnostic assays. f0062] In an embodiment, the peptide has a minimum of 13 amino acid residues, hence from 13 to 40 including 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 amino acid residues.
[0063J Accordingly, enabled herein is a method for the treatment or prophylaxis of Type 1 diabetes or a conditio associated with Type 1 diabetes in a mammalian subject, the method comprising administering to the subject a peptide comprising an amino acid sequence of at least 13 consecutive amino acids encompassing a T-cell epitope derived from insulin, proinsulin or preproinsulin wherein the amino acid sequence comprises a glutamiiie to glutamic acid substitution. By "at least 13" means from "13 to 40" and in particular "13 to 20", There is a proviso that the amino acid sequence comprises at least one glutamine to glutamic acid substitution (i.e. a deamidation),
10064] Hence, enabled herein is a method for the treatment or prophylaxis of Type 1 diabetes or a condition associated with Type 1 diabetes in a mammalian subject, the method comprising administering to the subject a peptide comprising an amino acid sequence of from 13 to 40 consecutive amino acids encompassing a T-cell epitope derived from insulin, proinsulin or preproinsulin wherein the amino acid sequence comprises a glutamine to glutamic acid substitution, By "at least 13" means from "13 to 40" and in particular "13 to 20". There is a proviso that the amino acid sequence comprises at least one glutamine to glutamic acid substitution (i.e. a deamidation).
[0065] Further enabled herein is a method for the treatment or prophylaxis of Type 1 diabetes or a condition associated with Type 1 diabetes in a mammalian subject, the method comprising administering to the subject a peptide comprising an amino acid sequence of from 13 to 20 consecutive amino acids encompassing a T-eell epitope derived from insulin, proinsulin or preproinsulin wherein the amino acid sequence comprises a glutamine to glutamic acid substitution. By "at least 13" means from "13 to 40" and in particular "13 to 20". There is a proviso that the amino acid sequence comprises at least one glutamine to glutamic acid substitution (i.e. a deamidation),
[0066] In an embodiment, the peptide binds to an antigen-presenting cell which comprises the haplotype HLA-DQ A*03:01, DQ B*03:02. In an embodiment the peptide is part of an HLA Class II tetranier.
[0067] Reference to a "mammalian subject" includes a human or other primate, laboratory test animal such as a mouse, rat, rabbit or guinea pig, a farm animal such as a horse, donkey, pig, cow or sheep, a companion animal such as a dog or cat and a captive wild animal. In an embodiment, the mammalian subject is a human. The human may be an infant, child, young adult, adult or elderly person. The human may be male or female, [0068] Hence, enabled herein is a method for the treatment or prophylaxis of Type I diabetes or a condition associated with Type 1 diabetes in a human subject, the method comprising administering to the subject a peptide comprising an amino acid sequence of at least seven consecutive amino acids encompassin a T-ceU epitope derived from insulin, proinsulu oi prepro insulin wherein the amino acid sequence comprises a glutamine t glutamic acid substitution. As indicated above, the term "at least seven consecutive amino acids" includes the aspect where a peptide of 10 to 20 amino acid residues including a peptide of at least Ϊ3 amino acid residues comprises the seven amino acid sequences. An example includes SEQ ID HOs:94 or a peptide selected from SEQ ID 'NO: 96 to 121 , [0069] The present specification is further instructional on a method for the treatment or prophylaxis of Type I diabetes or a condition associated with Type 1 diabetes in a mammalian subject, the method comprising administering to the subject a peptide comprising m amino acid sequence of at, least seven consecutive amino acids encompassing a T-cell epitope derived from insulin, proinsulin or preproinsulin wherei the amino acid sequence comprises a glutamine to glutamic acid substitution wherein the peptide comprises an amino acid sequence selected from SBQ ID NOs:2 to 5. 32, 35 to 61 , 64, 67, 70, 73, 76, 79s 82 to 94 and 96 to 121 , in an embodiment, the peptide comprises at least 13 amino acid residues. In an embodiment, the peptide binds to an antigen-presenting cell which comprises the haplotype HLA-DQ A* 03:01, DQ B*03:02. In another embodiment, T-cells respond to the peptide presented by another HLA molecule, such as but not limited to HLA DQ2 and HLA DQ 2/g transdimers (DQ A* 05:01, DQ B* 03:02 and/or DQ A* 03:01 ; DQ B* 02:01 ), HLA-D 3 (DRBl *03xx) and/or HLA-OR4 (DRB1 *04xx). In an embodiment, the peptide is or comprises SEQ ID NO:94, [0070) The present specification is further instructional on a method for the treatment or prophylaxis of Type 1 diabetes or a condition associated with Type 1 diabetes in a mammalian subject, the method comprising administering to the subject a peptide having a T-cell epitope derived from insulin, proinsulin or preproinsulin wherein the peptide comprises an amino acid sequence set forth in SEQ I'D NO:94. In an embodiment, the peptide consists of SEQ ID NO:94. In another embodiment, the peptide comprises the sequence selected from SEQ ID NOs:96 to 123.
{0071 j The peptide may contain further chemical modifications such as the chemical addition of amino acid residues in a synthetic reaction designed to generate peptides of a defined amino acid sequence, chemically modifying an amino acid residue, Another form of chemical modification is substituting an existing amino acid with a non-naturally occurring amino acid residue. The peptides defined by reference to SEQ ID NQs:2 to 5 , 32, 35 to 61, 64, 67, 70, 73, 76, 79, 82 to 94 and 96 to 121 and SEQ ID NOs:6 to 31 , 33, 34, 62, 63, 65, 66, 68, 69, 71, 72, 74, 75, 77, 78, 80, 81 and 95 and SEQ ID M¾:96 to 12.1 includes those having the glutamine to glutamic acid/or arginine to eitmiline substitution but may also comprise a chemically modified amino acid or contain a non-naturali occurring amino acid substitution or another natural amino acid substitution,
[0072] The present specification is further instructional on a method for the treatment or prophylaxis of Type 1 diabete or a condition associated with Type 1 diabetes in a human subject, the method comprising administering to the subject a peptide comprising at least seven consecutive amino acids encompassing a T-cell epitope derived from insulin, proinsulin or wherein the amino acid sequence comprises a glutamine to glutamic acid substitution wherein the peptide is chemically synthesized and comprises an amino acid sequence selected from SEQ ID NOs:2 to 5, 32, 35 to 61, 64, 67, 70, 73, 76, 79, 82 to 94 or 96 to 121.
}0073j The present specification is further instructional on a method for the treatment or prophylaxis of Type 1 diabetes or a condition associated with Type 1 diabetes in a human subject, the method comprising administering to the subject a chemically synthesized peptide encompassing a T-cell epitope derived from insulin, proinsulin or preproinsulin comprises or consists of an amino acid sequence set forth in SEQ ID NG:94. [0074] The present specification is further instructional on a method for the treatment or prophylaxis of Type 1 diabetes or a condition associated with Type 1 diabetes m a human subject, the method comprising administering to the subject a chemically synthesized peptide encompassing a T-cell epitope derived from insulin, proinsulin or preproinsulin comprises an amino acid sequence set forth in SEQ ID NGs:96 to 121 ,
[0075] In another embodiment, the present specification teaches a method for the treatment or prophylaxis of Type 1 diabetes or a condition associated with Type 1 diabetes in a human subject, the method comprising administering to the subject a peptide comprising at least seven consecutive amino acids having a T-cell epitope derived from insulin, proinsulin or wherein the amino acid sequence comprises a arginine to citruUme substitution wherein the peptide is chemically synthesized and comprises an amino acid sequence selected from SEQ ID NC)s: t 31 , 33, 34, 62, 63, 65, 66, 68, 69, 71 , 72, 74, 75, 77, 78, 80, 81 and 95.
[0076] The present specification is further instructional on a method fo the treatment or prophylaxis of Type 1 diabetes or a condition associated with Type 1 diabetes in a human subject, the method comprising administering to the subject a chemically synthesized peptide encompassing a T-cell epitope derived from insulin, proinsulin or preproinsulin wherein the peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:95.
[0077] Reference to "tolerance" includes a reduction in a T-cell response and/or a B- ceil response which is specific for insulin, proinsulin or preproinsulin. Tolerance may occur by any of a number of mechanisms including reducing autoreactive T-cells or 13- cells, inducing ignorance of insulin, proinsulin or preproinsulin, mechanisms that imprint an intrinsic status on tolerant lymphocytes in the form of anergy and immunedeviation and on extrinsic mechanisms involving regulating cells. Molecular interactions in the presentation of autoantigen in the periphery are central to the tolerance process and in strategies aiming at restoring or inducing immune tolerance in autoimmunity. It is proposed herein that the peptides facilitate removal, elimination or reprogramming of deleterious autoreactive T-cells and antigen-presenting cells and activation of regulatory cells to control autoimmune effectors. Tolerance may also be induced using complexes between peptides such as SEQ ID NO:95 and HLA Class II tetramers.
5 J007S] In an embodiment, the peptides or nucleic acid molecules encoding same are useful i the treatment or prophylaxis of Type 1 diabetes or an associated condition in a mammalian subject as well as a condition associated with Type 1 diabetes. The treatment of Type 1 diabetes includes the amelioration of symptoms of diabetes. In general, the peptides are recognized by CD4+ T-cells, CD8+ T-cells and/or or antigen-presenting cells
10 and/or B-cells from a mammalian subject having or at risk of developing Type I diabetes.
In an embodiment, the T-cells are CD4* T-cells. In an embodiment, the peptide is at least 13 amino acids in length such as from 1 to 40, and it binds to HLA-DQ A* 03:01 » DQ B* 03:02, In another embodiment, T-cells respond to the peptide presented by another HLA molecule, such as but not limited to HLA DQ2 and HLA DQ 2/8 transdimers (DQ A*
I S 05:01 , DQ B* 03:02 and/or DQ A* 03:01 ; DQ B* 02:01), HLA-DR3 (DRBl *03xx) and/or HLA-DR4 (DRBl *04xx). By "at least 1 amino acids in length" means 13 to 40 such as 13 to 20.
[0079] The present specification is instructional on a method of tolerizing or down- 0 regulating the priming or activity of T-cells in mammalian subjects with or at risk of developing Type 1 diabetes by the administration of a peptide selected to comprise an amino acid sequence set forth in SEQ ID NOs:2 to 5, 32, 35 to 61, 64, 67, 70, 73, 76, 79 and 82 to 94 wherein the peptide comprises a glutaniine to glutamic acid substitution. 5 (00801 The present specification is instructional on a method of tolerizing or down- regulating the priming or activity of T-cells in mammalia subjects with or at risk of developing Type 1 diabetes by the administration of a peptide selected to comprise an amino acid sequence set forth in SEQ ID NOs:96 to 121 wherein the peptide comprises a glutamine to glutamic acid substitution.
0
[0081] The present specification is instructional on a method of tolerizing or down- regulatin the priming or inactivity of T-cells in. mammalian subjects with of at risk of developing Type 1 diabetes by the administration of a peptide comprising the amino acid sequence set forth in SEQ ID NO:95. [0082] The present specification teaches a method of tolerizing or down-regulating the priming or activity of T-cells in mammalian subjects with or at risk of developing Type 3 diabetes by the administration of a peptide selected to comprise an amino acid sequence set forth in SEQ ID NOs:6 to 31 , 33, 34, 62, 63, 65, 66, 68, 69, 71, 72, 74, 75, 77, 78, 80, 81 and 95 wherein the peptide comprises an arginine to citrulline substitution and/or an arginine to citrulline substitution.
[0083] The present specification is instructional on a method of tolerizing or down- regulating the priming or inactivity of T-cells in mammalian subjects with or at risk of developing Type 1 diabetes by the administration of a peptide comprising the amino acid sequence set forth in SEQ ID NQ:95.
[00.84] The present specification is instructional on a method of tolerizing or down- regulating the priming or activity of B-cells in mammalian subjects with or at risk of developing Type 1 diabetes by the administration of a peptide selected to comprise an amino acid sequence set forth in SEQ ID NQs:2 to 5, 32, 35 to 61, 64, 67, 70, 73, 76, 79 and 82 to 94 wherein the peptide comprises a glutamine to glutamic acid substitution.
[0085] The present specification is instructional on a method of tolerizing or down- regulating the priming or inactivity of B-cells in mammalian subjects with or at risk of developing Type 1 diabetes by the administration of a peptide comprising the amino acid sequence set forth in SEQ ID NO:94.
[0086] The present specification is instructional on a method of tolerizing or down- regulating, the priming or inactivit of B-cells in mammalian subjects with or at risk of developing Type 1 diabetes by the administration of a peptide comprising the amino acid sequence set forth in SEQ ID NO:96 to 121, [0087] The present specification is instructional on a method of tolerizing or down- regulating the priming or activity of B-cells in mammalian subjects with or at risk of developing Type 1 diabetes by the administration of a peptide selected to comprise an amino acid sequence set forth in SEQ ID NGs; 6 to 31, 33, 34, 62, 63, 65, 66, 68, 69, 71, 5 72, 74, 75, 77, 78, 80, 81 and 96 wherein the peptide comprises an arginme to citxullme substitution*
(0088] The present specification is instructional on a method of tolerizing or down- regulating the priming or inactivity of B-cells in mammalian subjects with or at risk of 10 developing Type 1 diabetes by the administration of a peptide comprising the amino acid sequence set forth in SEQ ID NO: 95, In addition, tolerization may employ the use of an HLA Class II tetramer comprising a peptide of the present inventio such ass but not limited to, SEQ ID NO:94.
I S [0089] In relation to these embodiments, the peptides may further comprise other modifications such as to an amino acid or by the substitution of a non-naturally occurring amino acid residue.
[0090] The present specification is instructional on a method of tolerizing or down- 0 regulating the priming or inactivity of T-cells in human subjects with or at risk of developing Type 1 diabetes by the administration of a chemically synthesized peptide selected to comprise an amino acid sequence set forth in SEQ ID NOs:2 to 5, 32, 35 to 61 , 64, 67, 70, 73, 76, 79 and 82 to 94 wherein the peptide comprises a glutamine to glutamic acid substitution.
5
[0091] The present specification is instructional on a method of tolerizing or down- regulating the priming or inactivity of T-cells in human subjects with or at risk of developing Type 1 diabetes by the administration of a chemically synthesized peptide comprising the amino acid sequence set forth in SEQ ID NO: 94,
0
[0092] The present specification is instructional on a method of tolerizing or down- regulating the priming or inactivity of T-ceMs in human subjects with or at risk of developing Type 1 diabetes by the administration of a chemically synthesized peptide selected to comprise an amino acid sequence set forth in SEQ IP NOs;96 to 121 wherein the peptide comprises an arginme to citrulline substitution.
[0093 J The present specification is instructional on a method of tolerizing or down- regulating the priming or inactivity of T-ceilS: in hum n subjects with or at risk, of developing Type 1 diabetes by the administration of a chemically synthesized peptide selected to comprise an amino acid sequence set forth in SEQ ID NOs:6 to 31, 33, 34, 62, 63, 65, 66, 68, 69, 71, 72, 74, 755 775 78, 80, 81 and 95 wherein the peptide comprises an arginme to citrulline substitution,
(0094] The present specification is instructional on a method of tolerizing or down- regulating the priming or inactivity of T-ce!Is in human subjects with or at risk of developing Type 1 diabetes by the- administration of a chemically' synthesized peptide comprising the amino acid sequence set forth in SEQ ID NO:95. f0095] The present specification is instructional on a method of tolerizing or down- regulating the priming or inactivity of B-cells in huinan subjects with or at risk of developing Type. 1 diabetes by the administration of a chemically synthesized peptide selected to comprise an amino acid sequence set forth in SEQ ID N'Os:2 to 5, 32, 35 to 61, 64, 67, 70, 73, 76. 79 and 82 to 94 wherein the peptide comprises a glutamine to glutamic acid substitution. [0096] The present specification is instructional on a method of tolerizing or down- regulating the priming or inactivity of B-cells i human subjects with or at risk of developing Type 1 diabetes by the administration of a chemically synthesized peptide seieeted to comprise an amino acid sequence set forth in SEQ ID MOs:96 to 121 wherein the peptide comprises a glutamine to glutamic acid substitution. l'0O97] The present specification is instructional on a method of tolerizing or down- regulating the primin or inactivity of B-cells in human subjects with or at risk of developing Type 1 diabetes b the administration of a chemically synthesized peptide comprising the amino acid sequence set forth in SEQ ID NO; 4,
[0098] The present specification is instructional on a method o tolerizing or down- regulating the priming or inactivity of B-cells in human subjects with or at risk of developing Type 1 diabetes by the administration of a chemically synthesized peptide selected to comprise an amino ae d sequence set forth in SEQ ID NOs:6 to 31 , .33, 34, 62, 63, 65, 66, 68, 69.» 71 , 72, 74, 75, 77, 78, 80, 81 and 95 wherein the peptide comprises an arginrae to citrulline substitution.
(0100! The present specification is instructional on a method of toleri¾ing or down- reguiatmg the priming or inactivity of B-cells in human subjects with or at risk of developing Type 1 diabetes by the administration of a chemically synthesized peptide comprising the amino acid sequence set forth in SEQ ID NO:93»
[0101] As indicated above, the peptide may further comprise one or more chemical modifications to one or more amino acid residues including an amino acid additio and/or deletion as well as a substitution of a naturally occurring or non-naturally occurring amino acid for one or more of the amino acid residues,
[0102] Once isolated and characterized, derivatives, e.g., chemically derived derivatives, of a given peptide can be readily prepared. For example, amides of the peptide or peptide variants of the present invention may also be prepared by techniques well known in the art for converting a carbo ylic acid group or precursor to an amide. One preferred method for amide formation at the C-terminal carboxyl group is to cleave the peptide from a solid support with an appropriate amine, or to cleave in the presence of an alcohol, yielding an ester, followed by aminoiysis with the desired amine.
[01 3] Salts of carboxyl groups of a peptide or peptide variant may be prepared in the usual mamier by contacting the peptide with one or more equivalents of a desired base such as, for example, a metallic hydroxide base, e.g., sodium hydroxide; a metal carbonate or bicarbonate base such as, for example, sodium carbonate or sodium bicarbonate; or an amine base such as, for example, triethylamine, triethanolamine, and the like,
[0104] N-acyl derivatives of an amino group of the peptide or peptide variants may be prepared by utilizing an N-aeyl protected amino acid for the final condensation, or by aeylating a protected or unprotected peptide. O-acyl derivatives may be prepared, for example, by acylation of a free hydrox peptide or peptide resin. Either acylation may he carried out using standard aeylating reagents such as acyl halides, anhydrides, acyl imidazoles, and the like. Both N- and O-aeylation may be carried out together, if desired.
(0105) Formyl-metbionine, pyroglulamine and trimethyl- lanine may be substituted at the N-terminal residue of the peptide or peptide variant. Other amino-terminal modifications include ammooxypentane modifications (see Simmons et at. (1997) Science 276:276),
[0106] In addition, the amino acid sequence of a peptide can be modified so as to result in a peptide variant (see above). The modification includes the substitution of at least one amino acid residue in the peptide for another amino acid residue, including substitutions which utilize the D rather than L form, as well as other well known amino aeid analogs. These analogs include phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, gamma-carboxyglutamate; hippuric aeid, octahydroindole-2-carboxylie acid, statine, l,2,3,4 tetrahydroisoquinoline-3-earboxylic acid, penicillamine, ornithine, citnilline, cx-methyl-alanine, -benzoyl-phenylalanine, phenylglycine, propargyl glycine, sarcosine, and tert-butylglycine.
(O1 7J Regardless of the presence of an other modifications, the amino aeid sequence of the peptide will comprise on or more of a glutamine to glutamic aeid substitution and/or an arginine to citrulline substitution. [0108] One or mor of the residues of the peptide can be altered, so long as the peptide variant is biologically active in the sense it retains its epitopic function. Conservative amino acid substitutions include, for example, aspartio-glutamic as acidic amino acids; lysine/arginine/histidine as basic amino acids; leucine/isoletieine, methionine/valine, alanine/valine as hydrophobic amino acids; serine/glycine/alanine/threonine as hydrophilic amino acids.
[0109] Amino acid substitutions falling within the scope of the invention, are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobic! ty of the molecule at the target site, or (c) the hulk of the side chain. Naturally occurring residues are divided into groups based on. common side-chain properties:
(1) hydrophobic; norleucine, met, ala, vak leu, ile;
(2) neutral hydrophilic: cys, ser, thr;
(3) acidic: asp, glu;
(4) basic; asn, gin, Ms, lys, arg;
(5) residues that influence chain orientatio : gly, pro; and
(6) aromatic; trp, tyr, phe.
[0110] Acid addition salts of the peptide or variant peptide, or of amino residues of the peptid or variant peptide, may be prepared by contacting the peptide or amine with one or more equivalents of the desired inorganic or organic acid, such as, for example, hydrocliloric acid. Esters of carboxyl groups of the peptides may also be prepared by any of the usual methods known in the art.
[0111 ] A list of unnatural amino acids, contemplated herein and which may be incorporated into the peptide is shown in Table 3. Table 3
Non-conventional amino acids
Non-conventional Code Non -conventi onal Code amino acid amino acid -aminobutyr acid Abu L~N-methylalanine Nmala a-amino-a-methyi butyrate Mgabu L-N-m thylarginine Nrnarg ammocyclopropane- Cpro I..-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys ammonorbornyl- Norb L-N-methylglutamine Nmgln carbox laie L-N -methyl glutamic acid Nmglu eyelohexylalanine Chexa L-Nniethylhistidine Nmhis cyeiopentylalanine Cpen L-N-methylisolleucine Nmile
D-alanine Dal L-N-methylleucine mleu
D-arginine Darg L-N-methyllysine Nmlys
D-aspartic acid Dasp L-W-meraylmethionine Nmmet
D-eysteine Dcys L-N-methylnorleucine Nmnle
D-glutamine Dgln L-N-methylnorvaline Nmnva
D-glutamic acid Dglu L-N-methyiorni thine Nmorn
D-histidine Dhis L-N-methylphenylalanine Nmphe
D-isoleucine Dile L-N-methylproline Nmpro
D-leiicme Dleu L-N-methylserine Nmser
D -lysine Dlys L-N-metliylthreonine Nmthr
D-methionine Dmet L-N-memyltryptophan Nmtrp
D-omithine Dom L-N-methyltyrosine Nnityr
D-phenylalanme Dphe L-N-methylvaline Nmval
D-proline Dpro L-N-methylethylglycine Nmetg
D-serine Dser L-N-methyl-t-butylglycine Nmtbug
D-threonine Dthr L-norleucine Nle
D-tryptophan Dtr L-norvaline Nva
D~tyrosine Dtyr -methyl-aminoisobutyrate Maib
D-valine Dval a-methyl-y-aminobutyrate Mgabu
D- -methylalanine Dmala -methylcyclohcxylalanine Mchexa
D- x-methylarginine Dmarg a-methylcyleopentylalanine Mcpen
D-a-methylasparagine Dmasn a-methyl-ot-napthylalanine Manap
D-a-methyl aspartate Dmasp a-memylpenicillarnine Mpen
D-a-methyleysteine Dmcys N-(4- aminobutyl) glycine Nglu
D-a-methy glutan ine Dmgl N-(2-aminoethyl)glycine Naeg Non-conventional Code Non-conventional Code amino acid amino acid
D-a-methy i i s tidine Dmhis N"(3 -aminopropyl)glycine Nom
D- -methyii soleucine Dmile N-am ino-a-ra ethy Ibutyrate Nraaabu
D-a-methylleucine Dmleu a-napthylalanine Anap -a-methyllysine Dmlys N-benzylglyeine Nphe -a-raelhylmethi onine Dmrnet N*(2~carb amy lethyl)gly c iiie Ngln
D-a-methylomithine Dmorn N-(carbamylmethyl)glycine Hasn r a-methy.lphenyialanine Dmphe N-(2-carboxyethyl)glycine Nglu
D-a-methylproiine Dmpro N- (carb oxymethy l)glyci ne Nasp
D -a -methyl serine Dmser N-cyclobutyiglycine Ncbut
D-a-me&ylthreanine Dmthr N-cyeloheptylglycine Nchep
D~«~m eihy I try p tophan Dmtrp N-cyclohexylglycine Nchex
D-a-methy Ityrosine Dmty N-cyclodecylglycine Ncdec
D-a-methyl valine Dmval N-cylcododecylglycine Ncdod
D-N-methylalanine Dnmala N-cycl ooctylglycine Mcoct
D-N-raethylarginine Dnmarg N -cyclopr opyl glycine Ncpro
D-N-methylasparagine Dnmasn N-eycloundecylglycine Ncund
D-N-methylaspartate Dnm sp N-(2,2-diphenylethyl)glyc.me Nbhm
D-N-methylcysteme Dnmcys N-(3 ,3 -diphcnylpropy I )g lycine Nbhe
D-N-methy 1 g ί utamine Dnmgln N - (3 - guanidinopropy l)gly cine Narg
D-N-methy 1 gl iamate Dnmglu N-( 1 -hydroxyethy l)g1ycme Nthr
D-N-methylhistidme Dnmhis N-(hydi'oxyethy )glycine Nser
D-N-methyii soleucine Dnmile N-(.imidazolylethyl))glycine Nhis
B-N-methyileueine Dntnleu N*(3 -mdoIylyethyi)gIycine Nhtrp
D-N-rnethyllysine Drmilys N-methyl -γ-aminobutyrate Nmgabu
N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet
D-N-methylomithine Dnmorn N -methyl cycl openly lalanine Nmcpen
N-methylglyeine Nala D-N-methylphenylalanine Dnmphe
N-methylammoisobutyrate Nmaib D-N-methylproline Dnm ro
N-(l-niethylpropyl)glyrcine Nile D-N-methylserine Dnmser
N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr
D-N-methyltryptophan Dnmtrp N-(l -methyletbyl)glyeine Nval
D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Ntnanap
D-N-methylvaline Dnrnval N-methylpenicillamine Nmpen γ-aminobutyric acid Gabu N-(/?-hydroxyphenyl)glycine Nhtyr
L-/-butylglycine Tbug N-(thiomethyl)glycine Ncvs
L-ethylgiycine Etg penicillamine Pen
Γ ,-homopheny lalanine Hphe L-a-methylalanine Mala
L-a-methylarginine Marg L-a-methylasparagine Masn
L-a-methy 1 aspartate Masp L- -methyl -i-buty 1 gl cine Mtbug Non-conventional Code Non-conventional Code amino acid amino acid
L-a-methyl cysteine Mcys L-methylethylglycine Metg
L-a-methylglutamine Mgln L-a-methylglutamate Mgiu
L- -methylhisti dine Mhis L- a-methylhomophen l alani e Mhphe
L-a-raethyl isoleucine Mile N-(2-methy).thiQethy1)glycine Nmet
L-a-methylleucine Mleu L~o methyllysine Mlys
L-a-methylmethionine Mmet L-a-methylnorleiicine Mule
L-a-methy InorvaJine Mnva L-(x-methylormthine Mom
L- -methylphenylal anin e Mphe L-a-methylproline Mpro
L-cx-methylserine Mser L- -methyl threonine Mthr
L-a-merayi tryptophan Mtrp L-a-methyltyrosine Mtyr
L-ct-methylvaline Mval L~N -methy lhomopheny lal ant ne Nmhphe
N-(N-{2,2-diphenylethyl) Nnbhm N-(N-(3 ,3 -diphenylpropyl) Nnbhe carbmny!methyl)glycine carbamylmethy])g]ycine
l-earboxy- 1 -(2,2-diphenyl- Nmbc
emylarnino)cyelopropane
[0112] Crosslinkers can be used, for example, to stabilize 3D conformations, using homo-hifunctional crosslinkers such as the bifimctional. imido esters having (C¾)n spacer groups with n - 1 to n ™ 6, glutaraldehyde, N-hydroxysuccinirnide- esters and hetero- bifunctional reagents which usually contain an amino-reactive moiety such as N- hydroxysuccinimide and another group specific-reactive moiety such as raaleimido or dithio moiety (SH) or carbodiimide (COOH). In addition, peptides can be conformationaiiy constrained by, for example, incorporation of C« and N a-methylamino acids, introduction of double bonds between Ca and Cp atoms of amino acids and the formation of cyclic peptides or analogs by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.
[0113] The peptides may be produced by the stepwise addition of amino acid residues by chemical synthetic means or by recombinant means or by recombinant means followed by posttranslational modification. In relation to the chemical synthesis of peptides, any number of methods may be employed including using 9-fluoronyImethox -carbonyl (Fmoc) synthesis. Purity can be readily determined by HPLC and peptide masses determined bys tor example, mass spectrometry. [0114] For example, the peptides may be synthesised by standard solution phase methodology, as described in Hruby et al. (1998) Chemical synthesi qf peptides. University of Arizona, USA. Editor(s): Hecht, Sidney, M. Bioorganic Chemistry: Peptides and Proteins :27-64, Oxford University Press, New York, N. Y, [0115] Linear peptides may also be synthesised by solid phase methodology using Boc chemistry, as described by Schnolzer et al (1992) Int J Pept Protein Res 0: 180-1 3, Following deproteetion and cleavage from the solid support the reduced peptides are purified using preparative chromatography, [0116] As indicated above, peptides may also be synthesised by solid phase methodology using Fmoc chemistry, as described below:
1) Peptide is synthesized b Fmoc solid-phase peptide synthesis using an automatic synthesizer.
2) Peptide is synthesized from its C -terminus by stepwise addition of amino acids. 3) The first Fmoe-ammo acid is attached to an insoluble support resin via an acid labile linker.
4) After deproteetion of Fmoc by treatment with pipcridine. the second Ffnoc- amino acid is coupled utilizing a pre-activated species or in situ activation.
5) After the desired peptide is synthesized, the resin bound peptide is deprotected and detached from the resin via TFA cleavage.
6) Following deproteetion and cleavage from the solid support the reduced peptides are purified using preparative chromatography.
[0117] Following deproteetion and cleavage from the solid support the reduced peptides are purified using preparative chromatography. [0118] In terras of production of peptides by recombinant means, sources of nucleotide sequences from which a nucleic acid molecule encoding a peptide or variant thereof include total or polyA+ RNA from a mammalian (e,g, human) source from which eDNAs ean be derived by methods known in the art. Other sources of DNA molecules include genomic libraries derived from a mammalian (e.g. human) source,
[0119] Moreover, DNA molecules may be prepared in vitro, e.g., by synthesizing an oligonucleotide or by subeloning a portion of a DNA segment that encodes a particular peptide.
[0120] As used herein, the terms "isolated and/or purified" refer to in vitro isolation of a DNA, peptide or polypeptide molecule from its natural eellular o biological environment, and from association with other components of the cell, such as nucleic acid or polypeptide, so that it can be sequenced, replicated, and/or expressed. For example, an "isolated, preselected nucleic acid" is RNA or DNA that encode at least a portion of a peptide selected from SEQ ID Os: l through 121 , or a RNA or DNA complementary thereto, that is complementary or hybridizes, respectively, to RNA or DNA encoding the peptide, or polypeptide comprising the peptide, and remains stably bound under stringent conditions, as defined by methods well known in the art, e.g. in Sambrook ei l, (1989) Molecular Cloning; A Laboratory Manual, Cold Spring Harbor, N.Y. Once expressed and a peptide produced, it then undergoes posttranslational modification. Thus, the RNA or DNA is "isolated" in that it is free from at least one contaminating nucleic acid with which it is normally associated in the natural source of the RNA or DNA and is substantially free of any other mammalian RNA or DNA. The phrase "free from at least one contaminating source nucleic acid with which it is normally associated" includes the case where the nucleic acid is reintroduced into the source or natural cell but is in a different chromosomal location or is otherwise flanked by nucleic acid sequences not normally found in the source cell. [0121] As used herein, the term "recombinant nucleic acid" or "preselected nucleic acid," e.g., "recombinant DNA sequence or segment" or "preselected DNA sequence or segment" refers to a nucleic acid, e.g., to DNA, that has been derived or isolated from any appropriate tissue source,, that may be subsequently chemically altered in vitro, so that its sequence is not naturally occurring,, or corresponds to naturally occurring sequences that are not positioned as they would be positioned in a genome which has not been transformed with exogenous DNA, An example of preselected DNA "derived" from a source, would be a DNA sequence that is identified as a useful fragment withi a given organism, and which is then chemically synthesized in essentially pure form. An example of such DNA "isolated" from a source would be a useful DNA sequence that is excised or removed from the source by chemical means, e.g., by the use of restriction endonucleases, so that it can be further manipulated, e.g., amplified, for use in the invention, by the methodology of genetic engineering.
[0122] Nucleic acid molecules encoding amino mid sequence variants of a. peptide of the invention are prepared by a variety of methods known in the art. These methods include, but are not limited to, preparation by oligonucleoti de-mediated (or site-directed) mutagenesis, PGR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the preselected peptide, Posttranslalional modification ma also be used to alter the amino acid sequence. [0123] Oligonucleotide-mediated mutagenesis is a method for preparing amino acid substitution variants of a peptide. This technique is described by Adelman et ai,.: (1983) DNA 2:1 S3, Briefly, DNA is altered by hybridizing an oligonucleotide encoding the desired mutation to a DNA template, where the template is the single-stranded form of a plasmid or bacteriophage coniaining the unaltered or native DNA sequence. After hybridization, a DNA polymerase is used to synthesize an entire second complementary strand of the template that will thus incorporate the oligonucleotide primer, and will code for the selected alteration in the preselected DNA.
[0124] To prepare expression cassettes for transformation herein, the recombinant or preselected DNA sequence or segment may be circular or linear, double-stranded or single- stranded. Generally, the preselected DNA sequence or segment is in the form of chimeric DNA, such as plasmid DNA, that can also contain coding regions flanked by control sequences which promote the expression of the preselected DNA present in the resuItanT- cell line. [0125] The peptides including their salts, are generally administered so as to achieve a reduction in activity of autoreactive T-cells to thereby ameliorate at least one symptom associated with Type 1 diabetes. To achieve this effect, the peptide or a valiant thereof is administered at dosages of at least about 0,001 to about 100 mg/kg including about 0,01 to about .10 mg/kg including about 0.1 to about 5 mg/kg, of body weight, although other dosages may provide beneficial results. The amoun administered will vary depending on various factors including, but not limited to, the peptide selected, the stage of the disease, the weight, the physical condition, and the age of the mammal, whether prevention or treatment is to be achieved, and whether the peptide is chemically modified. Such factors can be readily determined by the clinician employing animal models or other test systems which are well known to the ait. In an embodiment, the mammal is a human. Formulations suitable for peptides and for adaptive immunity are well known in the art. The subject peptides or peptide-all complexes or peptide-MLA-DQ A*03:01, DQ B*03;02 complexes can be formulated using standard techniques. Other complexes contemplated herein include peptide complexes with HLA DQ 2 and DQ 2/8 transdimers (DQ A* 05:01 , DQ B* 03:02 and/or DQ A* 03:01; DQ B* 02:01), HLA-DR3 (DRBl *03xx and/or HLA- DR4 (DRBl*04xx).
[0126] Administration of sense nucleic acid molecules is also contemplated herein. accomplished through the introduction of cells transformed with an expression cassette comprising the nucleic acid molecule (see, for example, WO 93/02556) or the administration of the nucleic acid molecule (see, for example, Feigner et al U.S. Pat. No.
5,580.859, Pardoll et al (1995) Immunity 3:165; Stevenson et al (1995) Immunol Rev.
145:211 ; Moiling (1997) J, Mol, Med. 75:242; Donnelly et al (1 95) Ann, N. Y. Acad. Sci
772:40; Yang et al. (1996) Mol. Med, Today 2;476; Abdallah et al (1995) Biol, Cell 85; ] ), Pharmaceutical formulations, dosages and routes of administration for nucleic acids are generally disclosed, for example, in Feigner et al. supra. |G127f Administration of the therapeutic or prophylactic agents in accordance with the instant disclosure may be continuous or intermittent, depending, for example, upon the recipient's physiological condition, whether the purpose of the .administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of the peptides may be essentially continuous over a preselected period of time or may be in a series of spaced doses. Both local and systemic administration is contemplated.
[0128] To prepare the compositions peptides are synthesized or otherwise obtained, purified and then lyophilized and stabilized, The peptide can then be adjusted to the appropriate concentration, and optionally combined with other agents, The absolute weight of a given peptide included in a unit dose of a tolerogen can vary widely, For example, about 0.01 to about 10 mg, including about 0.5 to about 5 mg, of at least one peptide comprising an epitope sequence from insulin, proinsulin or prepromsulin, can be administered. A unit dose of the tolerogen may be administered either via a mucous membrane, e.g., by respiratory, e.g.,. nasal (e.g., instill or inhale aerosol) or genitourinary tract administration, or orally, although other routes, such as subcutaneous and intraperitoneal are envisioned to be useful to induce tolerance. [0129] Thus, one or more suitable unit dosage forms comprising the therapeutic or prophylactic peptides, which, as discussed below, may optionally he formulated for sustained release (for example using microencapsulation, see WO 94/07529, and U.S. Pat. No. 4,962,0 1), can be administered by a variety of routes including oral, or parenteral, including by rectal, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, intrathoracic, intrapulmonary and intranasal (respiratory) routes. The formulations may, where appropriate, be conveniently presented in discrete unit dosage forms and may be prepared b any of the methods well known to pharmacy. Such methods may include the step of bringing into association the therapeutic or prophylactic peptide with liquid carriers, solid matrices, semi-solid carriers, finely divided solid caniers or combinations thereof, and then, if necessary, introducing or shaping the product into the desired delivery system. [0130] When the peptides are prepared for oral administration, they are combined with a pharmaceutically acceptable carrier, diluent or excipient to form a. pharmaceutical formulation, or unit dosage form. Conveniently, orally administered peptides are formulated for sustained release, e.g., the agents are microencapsulated. The total active ingredients in such formulations comprise from 0.1 to 99,9% by weight of the formulation. By "pharmaceuticall acceptable" it is meant the carrier, diluent excipient, arid or salt must be compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof. The active ingredient for oral administration may be present as a powder or as granules; as a solution, a suspension or an emulsion; or in achievable base such as a synthetic resin for ingestion of the active ingredients from a chewing gum. The active ingredient may also be presented as a bolus, electuary or paste.
{0131] Pharmaceutical formulations containing the therapeutic or prophylactic peptide can be prepared by procedures known in the art using well known and readil available ingredients. For example, the agent can be formulated with common excipients, diluents, or carriers, and formed into tablets, capsules, suspensions, powders, and the like. Examples of excipients, diluents, and carriers that are suitable for such formulations include the following fillers and extenders such as starch, sugars, mannitol, and silicic derivatives; binding agents such as carboxymethyl cellulose, HPMC and other cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone; moisturizing agents such as glycerol; disintegrating agents such as calcium carbonate and sodium bicarbonate; agents for ■retarding dissolution such as paraffin; resorption accelerators such as quaternary ammonium compounds; surface active agents such as cetyl alcohol, glycerol monostearate; adsorptive carriers such as kaolin and bentonite; and lubricants such as talc, calcium and magnesium stearate, and solid polyethyl glycols.
[0132] For example, tablets or caplets containing the peptides can include buffering agents such as calcium carbonate, magnesium oxide and magnesium carbonate. Caplets and tablets can also include inactive ingredients such as cellulose, pregelatini¾ed starch, silicon dioxide, hydroxy propyl methyl cellulose, magnesium stearate, microcrystalime cellulose, starch, talc, titanium dioxide, benzoic acid, citric acid, corn starch, mineral oil, polypropylene glycol, sodium phosphate, and zinc stearate, and the like. Hard or soft- gelatin capsules comprising a therapeutic or prophylactic peptide can contain inactive ingredients such as gelatin, macrocrystalline cellulose, sodium lauryl sulfate,: starch, talc, and titanium dioxide, and the like, as well as liquid vehicles such as polyethylene glycols (PEGs) and vegetable oil, Moreover, enteric coated caplets or tablets of an agent of the invention are designed to resist disintegration in the stomach and dissolve in the more neutral to alkaline environment of the duodenum. [0133] The therapeutic or prophylactic peptides can also be formulated as elixirs or solutions for convenient oral administration or as solutions appropriate for parenteral administration, for instance by intramuscular, subcutaneous or intravenous routes.
[0134] The pharmaceutical formulations of the peptide can also take the form of an aqueous or anhydrous solution or dispersion, or alternatively the form of an emulsion or suspension.
[0135] The therapeutic or prophylactic peptide may be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dose form in ampules, pre-filled syringes, small volume infusion containers or in multi-dose containers with an added preservative. The active ingredients may take such forms as suspensions, solutions, or emulsions in oil or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredients may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.
[0136] These formulations can contain pharmaceutically acceptable vehicles and adjuvants which are well known in the art. It is possible, for example, to prepare solutions using one or more organic solvent(s) that is/are acceptable from the physiological standpoint, chosen, in addition to water, from solvents such as acetone, ethanol, isopropyl alcoho!, glycol ethers such as the products sold under the name "Dowanol", polyglycols and polyethylene glycols, C1 -C4 alkyl esters of short-chain acids, preferably ethyl or tsopropyl lactate, fatty acid triglycerides suc as the products marketed under the name " iglyol11, isopropyl mynstate, animal mineral and vegetable oils and polysi!oxanes,
}0137] The compositions described herein can also contain thickening agents such as cellulose and/or cellulose derivatives. They can also contain gyms such as xanthan, guar or carho gum or gum arabie, or alternatively polyethylene glycols, bentones and mont orillonites,. and the like,
[0138] It is possible to add, if necessary; an adjuvant chosen from antioxidants., surfactants, other preservatives, film-forming, keratolytic or eornedolytie agents, perfumes and colorings. Also, other active ingredients may be added, whether for the conditions described or some other condition.
[0139] For example, among antioxidants, t-butylhydroquinone, butylated h droxyanisole, butylated hydroxytoluene and .alpha. -tocopherol and its derivatives may be mentioned. The galenical forms chiefly conditioned for topical application take the form of creams, milks, gels, dispersion or microemulsions, lotions thickened to a greater or lesser extent, impregnated pads, ointments or sticks, or alternatively the form of aerosol formulations in spray or foam form or alternatively in the form of a cake of soap.
J0140] Additionally, the agents are well suited to formulation as sustained release dosage forms and the like, The formulations can be so constituted that the release the active inpedient only or preferably in a particular part of the intestinal or respiratory tract, possibly over a period of time. The coatings, envelopes, and protective matrices ma be made, for example, from polymeric substances, such as polylaetide -glycol ates: liposomes, microemulsions, microparticles, nanoparticles, or waxes. These coatings, envelopes, and protective matrices are useful to coat indwelling devices, e.g., stents, catheters, peritoneal dialysis tubing, and the like. [0141] The therapeutic peptides disclosed herein can be delivered via patches for transdermal administration. See U.S. Pat. No. 5,560,922 for examples of patches suitable for transdermal delivery of a therapeutic agent. Patches for transdermal deliver can comprise a backing layer and a polymer matri which has dispersed or dissolved therein a therapeutic agent, along with one or more skin permeation enhancers. The backing layer can be made of any suitable material which is impermeable to the peptide. The backing layer serves as a protective cover for the matrix layer and provides also a support function. The backing can be formed so that it is essentially the same size layer as the polymer matrix or it can be of larger dimension so that it can extend beyond the side of the polymer matrix or overlay the side or sides of the polymer matrix and then can extend outwardly in a manner that the surface of the extension of the backing layer can be the base for an adhesive means. Alternatively, the polymer matrix can contain, or be formulated of, an adhesive polymer, such as polyacrylate or acrylate/vinyl acetate copolymer. For long-term applications it might be desirable to use mlcroporous and/or breathable backing laminates, so hydration or maceration of the skin can be minimized,
[0142] Therapeutic or prophylactic peptides released from a transdermal delivery- system must be capable of penetrating each layer of skin. I order to increase the rate of permeation of a therapeutic agent, a transdermal drag delivery system must be able in particular to increase the permeability of the outermost layer of skin, the stratum eorneum, which provides the most resistance to the penetration of molecules. The fabrication of patches for transdermal delivery of therapeutic agents is well known to the art.
[0143] For topical administration, the therapeutic or prophylactic peptides may be formulated as is known in the art for direct application to a target area. Conventional forms for this purpose include wound dressings, coated bandages or other polymer coverings, ointments, creams, lotions, pastes, jellies, sprays, and aerosols. Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents. The active ingredients can also be delivered via iontophoresis, e.g., as disclosed in U.S. Pat, os. 4,140,122; 4,383,529; or 4,051,842. The percent by weight of a therapeutic agent of the invention present in a topical forimilation will depend on various factors, but generally will be from 0,01% to 95% of the total weight of the formulation, and typically 0,1-25% by weight.
{0144] Drops, such as eye drops or nose drops, may be formulated with an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilizing agents or suspending agents. Liquid sprays are conveniently delivered from pressurized packs. Drops can be delivered via a simple eye dropper-capped bottle, or via a plastic bottle adapted to deliver liquid contents dropwise, via a specially shaped closure,
[0145] The therapeutic or prophylactic peptide may further be formulated for topical administration in the mouth or throat. For example, the active ingredients may be formulated as a lozenge further comprising a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the composition in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the peptide composition in a suitable liquid carrier. [0.146] The pharmaceutical formulations of the present invention may include, as optional ingredients, pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, and salts of the type that are well-known in the art. Examples of such substances include normal saline solutions such as physiologically buffered saline solutions and water.
[0147] A peptide of the present disclosure comprising an amino acid sequence selected from SEQ ID NOs;2 through 121 inclusive may be conjugated to or embedded in a targeting moiety and/or a carrier molecule. In an embodiment, the targetin moiety and the carrier molecule are the same entity. A targeting moiety is useful in facilitating the deployment of the peptide to a particular cell type or tissue type. Examples of cell types include antigen-presenting cells and CD4' T-cells or other lymphocytes such as CD8+ T- cells. Examples of targeting moieties include antibodies or antigen-binding fragments or derivatives thereof specific for an antigen on the target cell or tissue, receptor Bgands, a glycosaminoglycan, heparan or heparin or a fragment thereof, a cytokine or other molecule capable of specific binding to a desired cell type. Carrier molecules include adjuvants, molecules which increase the half life in the blood stream and the like.
[0148] Enabled herein is an agent comprising a peptide comprising an amino acid sequence having a T-cell epitope derived from insulin, proinsulin or preproinsulin having a glutamine to glutamic acid substitution and comprising an amino acid sequence set forth in SEQ ID NOs:2 to 5, 32, 35 to 615 64, 67, 70, 73, 76, 79 and 82 to 94 inclusive, the peptide conjugated at its N- or C-terminal end to a targeting moiety or carrier molecule,
(0149] In another embodiment, a peptide is provided comprising amino acid sequence having a T-cell epitope derived from insulin, proinsulin or preproinsulin having a glutamine to glutamic acid substitution and comprising an amino acid sequence set forth in SEQ ID NOs:96 to 121 , the peptide embedded in a targeting moiety or carrier molecule.
[0150] in an alternative embodiment, a peptide is provided comprising art amino acid sequence derived comprising a T-cell epitope from insulin, proinsulin o preproinsulin having a glutamine to glutamic acid substitution and comprising an amino acid sequence set forth in SEQ ID NOs:2 to 5, 32, 35 to 61, 64, 67, 70, 73, 76, 79 and 82 to 94, the peptide embedded in a targeting moiety or carrier molecule.
[0151] Enabled herein is an agent comprising a peptide comprising an amino acid sequence having a T-cell epitope derived from insulin, proinsulin or preproinsulin having an arginine to citrulline substitution and comprising an amino acid sequence set forth in SEQ ID NOs:6 to 31, 33, 34, 62, 63, 65, 66, 68, 69, 71, 72, 74, 75, 77, 78, 80, 81 and 95 inclusive, the peptide conjugated at its N- or C-terminal end to a targeting moiety or carrier molecule.
[0152] in a alternative embodiment, a peptide is provided comprising an amino acid sequence having an epitope derived from insulin, proinsulin or preproinsulin having an arginine to citrulline acid substitution and comprising an amino acid sequence set forth in SEQ ID NOs:6 to 31, 33, 34, 62, 63, 65, 66, 68, 69, 71, 72, 74, 75, 77, 78, 80, 81 and 95, the peptide embedded in a targeting moiety or carrier molecule, 10153} In an embodiment, the peptide comprises or consists of SEQ ID NO:94. In another embodiment, the peptide comprises or consists of SEQ ID NO: 95. f0154] In an embodiment, the targeting moiety is a moiety which specifically targets an antigen-presenting cell or a T-cell such as a CD4 ' T-cell or CD8! T-cell, Examples include an antibody, receptor ligand, cytokine or a glycosaminoglycan,
[0155} In an embodiment, the carrier molecule is human serum albumin. In an embodiment, the carrier molecule is a cyclic peptide. In the case of tire latter, the peptide ma be embedded in the cyclic peptide to facilitate increasing its blood half-life and/or to enhance stability,
(0156] In another embodiment, the peptide is conjugated to an antigen-presenting cell or T-cell such as a CD4+ T-cell, cell or other cell of the immune system, [0157] The conjugated peptide or peptide-cell complex is useful in a method for the treatment or prophylaxis of Type 1 diabetes or a condition associated with Type 1 diabetes in a human subject, the method comprising administering to the subject a peptide having an amino acid sequence comprising a T-cell epitope derived from insulin, proinsulin or preproinsulm wherein the amino acid sequence comprises a gliitamine to glutamic acid substitution and comprising an amino acid sequence selected from SEQ ID NOs:2 to 5, 32, 35 to 61, 64, 67, 70, 73, 76, 79 and 82 to 94 wherein the peptide is conjugated to or embedded in a targeting moiety, carrier molecule or immune cell.
[0158] The conjugated peptide or peptide-cell complex is useful in a method for the treatment or prophylaxis of Type 1 diabetes or a condition associated with Type 1 diabetes in a human subject, the method comprising administering to the subject a peptide having an amino acid sequence comprising a T-cell epitope derived from insulin, proinsulin or preproinsulin wherein the amino acid sequence comprises an arginine to eitmlline substitution and comprising an amino acid sequence selected from SEQ ID NOs;6 to 31, 33, 34, 62, 63, 65, 66, 68, 69, 71 , 72, 74, 75, 77, 78, 80, 81 and 95 wherein the peptide is conjugated to or embedded in a targeting moiety, carrier molecule or immune cell,
{0159} It is proposed herein that such a conjugated entity is useful in inducing tolerance as hereinbefore defined, Pharmaceutical compositions described in relation to the peptide equally apply to a. conjugated or embedded peptide. As indicated above, the peptides may comprise other chemical modifications to the amino acid residue.
{0160] The peptides described herein including those having an amino acid sequence selected from SEQ ID NOs:2 to 5, 32, 35 to 61 , 64, 67, 70, 73, 76, 79 and 82 to 94 are also useful in diagnostic assays to detect or monitor the progression of Type 1 diabetes. In an embodiment, Type 1 diabetes is detected prior to onset of hyperglycemia. The peptides are also useful in monitoring T-eell responses in a patient's blood, monitoring reactivity to pancreatic islet cells and to detect autoreactive antibodies.
[0161] The peptides described herein including those having an amino acid sequence selected from SEQ ID NOs:6 to 31, 33, 34, 62, 63, 65, 66, 68, 69, 71, 72, 74, 75, 77, 78, 80, 81 and 95 are also useful in diagnostic assays to detect or monitor the progression of Type 1 diabetes. In an embodiment. Type 1 diabetes is detected prior to onset of hyperglycemia. The peptides are also useful in monitoring T-cell responses in a patient's blood, monitoring reactivity to pancreatic islet cells and to detect autoreactive antibodies. [0162] The peptides described herein including those having an amino acid sequence selected from SEQ ID NOs:96 to 121 are also useful in diagnostic assays to detect or monitor the progression ol'Type 1 diabetes. In an embodiment, Type 1 diabetes is detected prior to onset of hyperglycemia. The peptides are also useful in monitoring T-cell responses in a patient's blood, monitoring reactivity to pancreatic islet cells and to detect autoreactive antibodies. [0163] The assay may be conducted in any .number of ways including incubating the peptides with a sample of peripheral blood mononuclear cells or a cellular fraction thereof and screening for T-cell activation. This can be determined, for example, by T-cell proliferation or the presence of immune, effector molecules.
[0164] Enabled herein is a method for measuring a cell-mediated immune response in a subject, the method comprising collecting a sample from the subject wherein, the sample comprises cells of the immune system which are capable of producing immune effector molecules following stimulation by an autoaniigen selected from a modified peptide comprising a T-cell epitope derived from insulin, proinsulin or preproinsulhi, incubating the sample with the antigen and then measurin fee presence of or elevation in the level of an immune effector molecule wherein the presence or level of the immune effector molecule is indicative of the capacity of the subject, to mount an autoreactive a cell- mediated immune response,
[0165] In an embodiment, the subject specification contemplates a method for detecting the presence of autoreactive T-ce!ls indicative of the presence of, or a predisposition for the development of, Type 1 diabetes, or a condition associated with Type I diabetes in a subject, the method comprising contacting a sample from the subject comprising immune cells with a peptide comprising an amino acid sequence of at least seven consecutive amino acid residues comprising a T-cell epitope derived from insulin, proinsulin or prepro insulin or a chemical analog of, or a non-natural ly occurring amino acid substitute of, one or more amino acid residues in the secjuenee wherein the amino acid sequence comprises a glutaniiiie to glutamic acid substitution and/or an arginine to eitruttme substitution for a time and under conditions sufficient for an autoreactive T-cell, if present, to be stimulated into producing an immune effector molecule, and then detecting the presence of the immune effector molecule, wherein the presence or level of the immune effector molecule is indicative of Type 1 diabetes or its state or risk of progression. {Θ1.66] Further enabled herein is a method for screening for autoreactive antibodies associated with Type 1 diabetes or a condition associated therewith in a subject, comprising contacting body fluid from the subject with a peptide comprising an amino acid sequence having a T-cell epitope derived from insulin, proinsulin or preproinsulra or a chemical analog of, or a non-naturally occurring amino acid substitute of, one or more amino acid residues in the sequence wherein the amino acid sequence comprises an arginine to citriiUine substitution for a time and under conditions sufficient for an autoreactive antibody, if present, to bind to the peptide and then screening for the presence of the peptide-antibod complex. The presence or level of the antibody is indicative of Type 1 diabetes or its state or risk of progression. Examples of body fluid include serum, whole blood, tissue fluid, sputum, urine, and the like.
10167] In an embodiment, the subject is a human. [0168] In an embodiment, the peptide comprises an amino acid sequence selected from the list consisting of SEQ ID NO: SEQ ID NOs:2 to 5, 32, 35 to 61 , 64, 67, 70? 73, 76, 79 and 82 to 94,
[0169] In an embodiment, the peptide comprises an amino acid sequence set forth in SEQ ID O;94, In an aspect, the peptide consists of SEQ ID NO;94.
[0170] In an embodiment, the peptide comprises an amino acid sequence selected from the list consisting of SEQ ID NOs:6 to 31 , 33, 34, 62, 63, 65, 66, 68, 69, 71, 72, 74, 75, 77, 78, 80, 81 and 95.
[0171] In an embodiment, the peptide comprises an amino acid sequence set forth in. SEQ ID NO;95.
[0172] In an embodiment, the peptide comprises an amino acid sequence set forth in SEQ ID NOs:96 to 121. [0173] In an embodiment, the condition associated with Type 1 diabetes is selected from the list consisting of a cardiovascular condition, a neuropathy, a retinopathy, a nephropathy, infection and a complication in pregnancy, [0174} In an embodiment, the immune effector molecule is detected by an antibody labeled with a reporter molecule, capable of providing a detectable signal,
[0175} Reference to "immune cells" includes ceils such as lymphocytes including natural killer (NK) ceils, T-cells, (CD4÷ and/or CD8+ cells), B-cells, macrophages and monocytes, dendritic cells or any other cell which is capable of producing an effector molecule in response to direct or indirect antigen stimulation. Conveniently, the immune cells are lymphocytes and includes CD4* T-celis.
[0176] The immune effector molecules for T-cell assays may be any of a range of molecules which are produced in response to cell activation or stimulation by the peptide antigen. Immune effector molecules include interferon (e.g.. IFN-y), a range of cytokines such as interleukins (US), e.g. IL-2, IL-4, IL-10 or IL-12, tumor necrosis factor alpha (TNF-ot), a colony stimulating factor (CSF) such as granulocyte (G)-CSF or granulocyte macrophage (GM)-CSF amongst many others such as complement or components in the complement pathway.
[0177] The sample collected from the subject is generally deposited into a collection tube. A collection tube includes a blood draw tube or other similar vessel. Conveniently, when the sample is whole blood, the blood collection tube is heparinized. Alternatively, heparin is added to the tube after the blood is collected. Notwithstanding that whole blood is the most convenient sample, the present disclosure extends to other samples containing immune cells such as lymph fluid, cerebral fluid, tissue fluid and respiratory fluid including nasal and pulmonary fluid. [0178] The use of blood collection tubes is useful since they are compatible with standard automated laboratory systems and these are amenable to analysis in large-scale and random access sampling. Blood collectio tubes also minimize handling costs and reduce laboratory exposure to whole blood and plasma and, hence, reduce the risk of laboratory personnel from contracting a pathogenic agent such as HIV or hepatitis B virus (HBV).
[0179] The incubation step with the peptide antigen may be from 5 to 50 hours, including 5 to 40 hours and 8 to 24 hours or a time period in between,
10180) Another aspect enabled herein contemplates a method for measuring a cell- mediated immune response in a subject including a human subject, the method comprising collecting a sample of whole blood from the subject, incubating the whole blood sample with a peptide comprising an amino acid sequence selected from SEQ ID NOs:2 to 5, 32, 35 to 61 , 64, 67, 70, 73, 76, 79 and 82 to 94 and then measuring the presence or elevation in level of an immune effector molecule such as IFN-γ wherein the presence or level of the immune effector molecule is indicative of the capacity of the subject to mount a ©$11- mediated immune response to insulin, proinsulin or preproinsulin leading to Type 1 diabetes. jOlSl] Another aspect enabled herein contemplates a method for measuring a cel.1- mediated immune response in a subject including a human subject, the method comprising collecting a sample of whole blood from the subject, incubating the whole blood sample with a peptide comprising a amino acid sequence selected from SEQ ID NOs:96 to 121 and then measuring the presence or elevation in level of an immune effector molecule such as IFN-y wherein the presence or level of the immune effector molecule is indicative of the capacity of the subject to mount a cell-mediated immune response to insulin, proinsulin or preproinsulin leading to Type 1 diabetes.
[0182] Another aspect enabled herein contemplates a method for measuring a cell- mediated immune response in a subject including a human subject, the method comprising collecting a sample of whole blood from the subject, incubating the whole blood sample with a peptide comprising an amino acid sequence selected from SEQ ID NOs:6 to 31, 33, 34, 62, 63, 65, 66, 68, 69, 71 , 72, 74, 75, 77, 78, 80, 81 and 95 and then measuring the presence or elevation in level of an immune effector molecule such as IFN-y wherein the presence or level of the immune effector molecule is indicative of the capacity of the subjec to mount a cell-mediated immune response to insulin, proinsulin or preproinsulin leading to Type 1 diabetes.
[0183] In an embodiment, the peptide emploj'ed is or comprises SEQ ID NG;94.
[0184] The ability to measure an autoreactive cell-mediated immune response is important for assessing a subject's likelihood of mounting an autoimmune response to insulin, proinsuHn or preproinsulin and developing Type 1 diabetes and its associated conditions.
[0185] Detection of the immune effector molecules may be measured at the protein or nucleic acid levels. Consequently,., reference to "presence or level of the immune effector molecule" includes direct and indirect data. For example, high levels of IF -γ mRNA is indirect data showing increased levels of TFN-y,
J01S6] Ligands such as antibodies to the immune effectors are useful in detecting and/or quantitating these molecules. Similarly., autoreactive antibodies may be detected such as by anti-IgOr antibodies, labeled with a reporter molecule. Techniques for the assays contemplated herein are known in. the art and include, for example, sandwich assays, ELISA and ELISpot. Reference to "antibodies" includes parts of antibodies, mammalianized (e.g. humanized) antibodies, recombinant or synthetic antibodies and hybrid and single chain antibodies.
[0187] Both polyclonal and monoclonal antibodies to the effector molecule or autoreactive antibodies are obtainable by immunization with the immune effectors or antigenic fragments thereof and either type is u&Ikable for immunoassays. The methods of obtaining both types of sera are well known in the art. Polyclonal sera are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of the immune effector, or antigenic part thereof, collecting serum from the animal and isolating - 35 - specific sera by any of the known itnaiunoadsorbent techniques, Although antibodies produced by this method are utilizable in virtually any type of immunoassay, they are generally less favored because of the potential heterogeneity of the product, [0188] The use of monoclonal antibodies in an immunoassay is useful due to the ability to produce them in large quantities and the homogeneity of the product. The preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art.
[01β | Another aspect of the present invention contemplates, therefore, a method for detecting an immune effector m response to autoreactive T-cells to a peptide derived from insulin, pro insulin or preproinsulin and comprising an amnio acid sequence as set forth in SEQ ID NOs:2 through 121 or a chemically modified form of the amino acid sequence in a sample comprisin immune cells from a subject, the method comprising incubating the sample with the peptide and capturing an immune effector molecules produced by stimulated T-cells with an antibody specific for the immune effector or antigenic fragment and then detecting the capture immune effector. (0190] Another aspect of the present invention contemplates, therefore, a method for detecting an autoreactive antibody in response to autoreactive B-cells to a peptide derived from insulin, proinsulin or preproinsuiin and comprising an amino acid sequence as set forth in SEQ ID NGs:2 through 121 or a chemically modified form of the amino acid sequence in a sample comprising immune cells from a subject, the method comprising incubating the sample with the peptide and capturing any autoreactive antibodies produced by stimulated B-cells with an antibody specific for the autoreactive antibody or antigenic fragment and then detecting the capture immune effector.
[0191] A sample includes whole blood. This method includes micro-arrays and macro- arrays on planar or spherical solid supports. A micro- or macro-array is useful. [0192] A wide range of immunoassay techniques are available as can be seen by reference to U.S. Patent Mos. 4,016,043, 4,424,279 and 4,018,653.
[0193] In one type of assay, either a monoclonal or polyclonal capture antibody is employed which is coated asepti ally onto the surface of well. These antibodies have specificity for the immune effector molecule. The plate is blocked, usually with a serum protein that is non-reactive with, any of the antibodies in the assay. After this, immune ceils (e.g. ίτοτη a blood sample) are plated out at varying densities, along with a peptide comprising an amino acid sequence selected from SEQ ID Os:2 through 121 , or a modified form of the peptide comprising amino acid substitution, addition or deletion or comprising a non-naturally occurring amino acid residue, and then placed in a humidified 37°C C<¾ incubator for a specified period of time.
|0194] The immune effector molecule secreted by activated cells is captured locally by the coated antibody on the high surface area microplate. Alternatively, synthetic peptides comprising an arginine to eitruihne substitution are coated to the surface of a reaction vessel. After washing the wells to remove cells, debris, and media components, an antibody labeled with a reporter molecule and specific for the immune effector or autoreactive antibody is added to the wells. This antibody is reactive wit a distinct epitope of the immune effector or autoreactive antibod and i used to detect the captured immune effector or autoreactive antibody. Following a wash to remove any unbound labeled antibody, the detected immune effector or autoreactive antibody is then visualized using a detection system. The spots can be counted manually (e.g. with a dissecting microscope) or using an automated reader to capture the mlcrowell images and to analyse spot number size. Such an assay can also be used to quantitate the number of T-cells producing immune effectors or to quantitate or quantitate the presence of autoreactive antibodies. The production of immune effectors means the presence of autoreactive T- cells and/or the preparation of autoreactiv antibodies means the potential for, or state of, Type 1 diabetes, (01 51 Ja t¾ese assays, the immobilized antibody having specificity for the instant ■immune effector or the synthetic peptide is either covalently or passively bound to a solid surface. The solid surface is typically glass or a polymer, the most commonl used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, pol vinyiidene fluoride or polypropylene. The solid supports may be in the form of tubes, beads, spheres, discs of microplates, or any other surface suitable for conducting an immunoassay. The binding processes are well known m the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g. 2-120 minutes or where more convenient, overnight) and under suitable conditions (e.g. for about 20°C to about 40°C) to allow binding of any subunit present in the antibody. Following the incubation period, the antibod subunit solid phase is washed and dried and incubated with a second antibod specific for a portion of the antigen. The second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to the antibody-immune effector complex or peptide-autoreactive antibody complex.
|0196j By "reporter molecule" as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of immune effector-bound antibody. Detection may be either qualitative or quantitative. The most commonly used reporter molecules in this type of assay are either enzymes, fiuorophores or radionuclide containing molecules (i.e. radioisotopes) and chemilurainescent molecules. In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase, amongst others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable color change. Examples of suitable enzymes include alkaline phosphatase and horse radish peroxidase. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In ail cases, the enzyme-labeled antibody is added to the first antibody-immune effector comple or peptide-autoreactive antibody complex, allowed to bind, and then the excess reagent is washed away, A solution containing the appropriate substrate is then added to the complex of antibody-immune effector-antibody or peptide-autoreactive antibody-antibody. The substrate reacts with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually speclrophotometrically, to give an indication of the amount of immune effector or autoreactive antibody which was present in the sample. Again, the present invention extends to a substantially simultaneous assay.
(0197) Alternately, fluorescent compounds, such as fluorescein and rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particvilar wavelength, the fluorochrome-labeled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic color visually detectable with a light microscope. The fluorescent labeled antibody is allowed to bind to the 'first antibody- immune effector complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength the fluorescence observed indicates the presence of the antigen of interest. Immunofliiorescene and EI A techniques are both well established in the art, Other reporter molecules, such as radioisotope, cbemilurninescent or bioluminesccnt molecules, may also be employed.
[0198] There are a range of other detection systems which may be employed including colloidal gold and all such detection systems are encompassed herein,
[0199] The present disclosure also contemplates genetic assays such as involving PGR analysis to detect R.NA expression products of a genetic sequence encoding an immune effector or autoreactive antibody. [0200] In on embodiment, PGR is conducted using pairs of prirnerst one or both of which are generally labeled with the same or a different reporter molecule capable of givtag a distinguishable signal. The use of fluorophores is. useful in the practice of this aspect of the invention.
|020l] Any suitable method of analyzing fluorescence emissio is encompassed herein, in this regard, the instant disclosure contemplates techniques including but not restricted to 2-phcton and 3 -photon time resolved fluorescence spectroscop as, for example, disclosed by Lakowic? et al (1997) Btoph s, J, 72:567, fluorescence lifetime imaging as, for example, disclosed by Eriksson et al (1993) Biophys. I J' 64 and fluorescence resonance energy transfer as5 for example, disclosed by Youvan et al (1 97) 8 lot ecknohgy et elia 3.Ί - 18.
}0202] Luminescence and phosphorescence may result respectively from a suitable luminescent or phosphorescent label as is known in the art, Any optical meaos of identifying such label may be used in this regard,
[0203J Infrared radiation may result from a suitable infrared dye, Exemplary infrared dyes that may be employed in. the invention include but are not limited to those disclosed in Lewis et al (1999) Dyes Pigm. 42(2) \97; Tawa et al Mater. Res. Soe, Symp. Proc. 488 (Electrical, Optical and Magnetic Properties of Organic S-olid-State Materials IV], 885- 890; Daneshvar et al (1999) J. Immunol Methods 226(1-2);) \9~) 2& Rapaport et al (1999) Appl P ys. Lett. 7^:329-331 and Durig et al (1993) J, Raman Spectr&sc. 24(5) :2 l-2$5, Any suitable infrared spectroscopic method may be employed to interrogate the infrared dye. For instance, foarier transform infrared spectroscopy as, for example, described by Rahman ei al, (1 98) J. Org. Chem, 63:6196 may be used in this regard.
{0204] Suitably, electromagnetic scattering may result from diffraction, reflection, polarization or refraction of the incident electromagnetic radiation including light and X- rays. Such scattering can be used to quantltate the level of mRNA or level of protein.
[0205] Flow cytometry is also useful in analyzin fluorophore emission. [0206] As is known in the art, flow cytometry is a high throughput technique which involves rapidly analyzing the physical and chemical characteristics of particles (e,g, labeled mRNA, DNA or proteins) as they pass through the path of one Of more laser beams while suspended in a fluid stream. As each particle intercepts the laser beam, the scattered light and fluorescent light emitted by each cell or particle is detected and recorded using any suitable tracking algorithm.
[0207] A modern flow cytometer is able to perform these tasks up to 100,000 cells/particles s'1. Through the use of an optical array of filters and dic-hroic mirrors, different wavelengths of fluorescent light can be separated and simultaneously detected. In addition, a number of lasers with, different excitation wavelengths may be used. Hence, a variety of fiuorophores can be used to target and examine, for example, different immune effectors within a sample or immune effectors from multiple subjects,
[0208] Suitable flow cytometers which may be used in the methods of the present invention include those which measure five to nine optical parameters using a single excitation laser, commonly an argon ion air-cooled laser operating at 15 mW on its 488 ran spectral line. More advanced flow cytometers are capable of using multiple excitation lasers such as a IleNe laser (633 nm) or a HeCd laser (325 nm) in addition to the argon ion laser (488 or 514 nm).
[0209] For example, Biggs et al. (1999) Cytometry 3<5;36-45 have constructed an 1 1- parametcr flow cytometer using three excitation lasers and have demonstrated the use of nine distinguishable- fiuorophores in addition to forward and side scatter measurements for purposes of immunophenotyping (i.e. classifying) particles. The maximum number of parameters commercially available currently is 17; forward scatter, side scatter and three excitation lasers each with five fluorescence detectors. Whether all of the parameters can be adequately used depends heavily on the extinction coefficients, quantum yields and amount of spectral overlap between all fiuorophores (Malemed et al (1990) '-Flow cytometry and sorting", 2nd Ed„ New York, Wiley-Liss). However, it will be understood that the present invention is not restricted to any particular flow cytometer or any particular set of parameters. In this regard, the invention also contemplates use in place of a conventional flow cytometer, a microfahricated flow cytometer as, for example, disclosed by Fu el al, (1999) Nature Biotechnology 17; 1109-1 H I .
(0210] The instant disclosure further contemplates autoreactive antibodies which have a binding affinity to the modified peptides derived from insulin* proinsulin o preproinsuii wherein the modified peptides comprise an arginine to citraliine substitution. Antibodies may also be generated to such modified peptides tor use inter alia for diagnostic purposes,
10211) Hence, in an embodiment, enabled herein is an assay to screen for autoreactive antibodies to insulin, proinsulin or preproinsuiin, the assay comprising contacting a biological sample such as serum ftom a subject to be tested with a peptide derived insulin, proinsulin or preproinsuiin wherein the peptide comprises an arginine to citruliine substitution, so as to form a complex between an autoreactive antibody if present in the sample and the peptide and then detecting the presence of the antibody-peptide complex,
10212] In an embodiment the sample is selected from serum, whole blood, sputum, urine or .tissue fluid.
[02.13] in an embodiment, the presence of an autoreactive antibody or the level or presence of the antibody is an indicator of Type 1 diabetes or a stage of developed Type 1 diabetes such as early stage Type 1 diabetes, ]0214] The present specification further teaches animal models such as mouse, rat, rabbit, guinea pig, hamster or non-human primate models for Type 1 diabetes. These animal models may comprise humanized insulin or humanized human components in a diabetic model. These animal models are useful for testing the peptides in treatment, protocols and in diagnostic assays. Existing animal models may be used or readily adapted. Examples of existing animal models are disclosed in Example 1 1. [0215] The present specification further teaches immunotherapies- and immunodiagnostics based on HLA Class II tetramers. In particular, peptide-labeled HLA Class II tetramers are used to elucidate the role of antigen-specific Tregs in diabetes autoimmunity and to monitor insulin-specific CD4+ T-cells, Peptidc-HLA Class II tetramers display the antigen peptide in a form which is a surrogate for the recognition events which occur between T-cells and antigen-presenting cells, They are useful to interrogate CD4+ T-cells and in desensitization protocols (Nepon (2012) J. Immunol i f¾);2477-2482).: [0216 J Enabled herein is an isolated HLA Class II tetraraer incorporating a peptide comprising at least 7 consecutive amino acids comprising a T~cell epitope derived from insulin, proinsulin or preproinsulin, wherein the peptide comprises a glutamine to glutamic acid substitution, [02l7j In an embodiment the peptide comprises at least 13 amino acid residues,
[0218] In an embodiment the peptide comprises a sequence selected from SEQ ID X():2 to 5, 32, 35 to 6i5 64, 67, 70, 73, 76, 79 and 82 to 94 or 96 to 121, (0219] In an embodiment the peptide comprises SEQ ID NO:.94.
EXAMPLES
[0220] Embodiments contemplated herein are now described by the following non- limiting Examples.
EXAMPLE 1
Selection of islet-infiltrating T-cells
[022,13 T-cells are isolated from within pancreatic islets due to the fact that this constitutes evidence that a T-cell contributes to beta-cell autoimmunity and destruction. Analysis of islet-infiltrating T-cells leads to the identification of disease-relevant epitopes. These epitopes are prime candidates for developing T-cell assays and antigen-specific therapies for Type 1 diabetes. Islet T-cells are shown in Figure 1 , The cloning of human islet T-cells is shown in Figure 2,
EXA PLE 2
Instituting islet-mfiltMtmg T-cells
\Q222] A summary of -ce-ll clones from Type 1 diabetic organ donors is provided in Table 4. The nietliodology is shown in Figure 3.
Tabie 4
Donor Disease HLA Class I HLA Class II CD4+ CD8+ diration clones clones
A 3 years A1, 2, B6, 51 D B1*0¾01, 04:04 62 27
D0B1TE;Q1; 03:02
B >20 years A2, 30, DRB1*01:.0i, 03:01 153 128
Β 8:01, 44:02 DCS1O201, 05:01
C 19 years A*02:01 -, DRB1*03:01, 04:01 29 58
8*18:01, 39:01/46 DQB1*02;01, 03:02
D 8 years A1.A23. DRS1O3:G1:01G,- 60 30
B8, SO
E Syears A*G2-0V, DRB1*01:01, 04:01 85 1?
6*15:01,4001 DQBn&iS, 05:01
F 15 years A*02;01, 23:01 Df¾B1*0301v 0401 103
B*15:01,50:01 001*0201,0302
EXMAPLE 3
Searching for modifications that fit the p9 pocket
[0223] Pools of two peptides (Pools A-E) are made and screened against Donor A's clones (Figure 4).
|t)224] A summary of epitope mapping-epitope formed by posttranslational modification is provided in Table 5 and Figures 5 to 9, A glutamine to glutamic acid substitution is referred to herein as a deamidation,
Table 5
I 8-chain C-peptide A-chain
I FVNQHLCGSHLVEALrtVCGERGFF
j Unmodified contra! 1 ELGGGPGAGSLQPLALEG
j Unmodified control 2 GAGSLGPLALEGSLQ RG
I Q2SE short peptide VELGGGPGAGSLEPL
I Q25E long peptide TRREAEDLQVGQVELGGGPGAGSLEPLALEGSLQ
Table 6 is a summary of epitope mapping-epitope formed by posttransiational modification. See also Figure 6.
ς
O
o
Figure imgf000068_0001
Table 7 provides a summary of epitope mapping-Epitope formed by posttranslational modification. See also Figures 8 and 9.
TaMe 7
Donor A
B"chain C" ePtide A-Chain clonel
FVNQHLCGSHLVEALYLVCGE GFFYTPKTRREAEDLQV(3aVELGGGPGA<3SUQPLALEGStQKRGiVE CCTSlCSLYQLeNYCN
QPLALEGSLQKR*G1 1
R*= citrulline
[0227] The data show that eitruiline (Cit or *) is recognised by autoreactive antibodies generated in Type i diabetes. EXAMPLE 4
HLA blocking
[0228 J To determine which HLA molecules present the peptide to T-celis mAb specific for HLA-DR. -DP or DQ are added to the peptides stimulation assay. The results are shown in Figure 10. The data show that only the mAB specific for HLA-DQ inhibits the response to the peptide [SEQ ID NO: 94] (Figure 10).
EXAMPLE 5
HLA restriction
[0229] sho that the T-cell clone* s response to peptide [SEQ ID NO:94] requires an antigen-presenting cell that expresses HLA DQ8 (HLA-DQ A*03;0 L DQ B*03:02) [Figure 11]. In another embodiment, the peptide binds to HLA DQ2 and/or HLA DQ 2/8 transdimer such as DQ A*05:01, DQ B*03:02 and/or DQ A*03;01, DQ B*02:0l, HLA- DR3 (DR.B1 *03xx) and/or HLA-DR4 (DRBl*04xx).
EXAMPLE 6
Fine epitope mapping [0230] The minimum sequence of amino acids required to stimulate the T-cell clone was determined by tested and panel of peptide of differing length (Panel A), To confirm this result, a panel of peptides covering the sequence of the epitope set forth in SEQ ID NO:94, but with each amino acid in turn subst tuted by alanine or lysine, whichever is most different from the native amino acid was tested. This, confirmed the epitope mapping and showed which residues could be modified without impairing the T-cell response. EXAMPLE 7
Response to deamidated peptide in the peripheral bloo from people with and without
Type I diabetes [0231 ] CD4+ T-cell responses were measured using a carboxy fluorescein succinimidyl ester .(CFSE)-based proliferation assay. Peripheral blood mononuclear cells (PBMC) were isolated from venous blood samples from individuals with Typ 1 diabetes, or those without Type 1 diabetes 'healthy controls"). The results show the ratio of the number of CD4'T cells that have proliferated in response to the peptides relative to control samples cultured without peptide. The ratio is known as a "Cell Division Index" or CD1 (Mannering et al (2003) J. Immunol Methods 283: 173-183), A CDI> 2.0 is considered, to be a positive response (Figure 13).
EXAMPLE 8
T-cell assay
[0232] Figure 14 provides the results of an INF-y-based assay detected from sensitive T-cells in response to the modified peptide of SEQ ID NO;94 (with the glutamme to glutamic acid substitution) compared to a non-modified peptide. The modified peptide was significantly more active in stimulating a response.
EXAMPLE
Epitope characterization [0233] CD4+ T-cells are isolated and characterized specific for the deamidated epitopes derived from human proinsuiin. The epitopes are subjected to assays to determine specificity, HLA restriction and binding to T-cell receptors determination. The SEQ ID NO.94 peptide is specific for HLA-DQ A*03:01 , DQ B*03:02. EXAMPLE 16
Role of regulatory T-cells
[0234] The role of regulatory T (Tre ) cell responses against the deamidated epitopes and their unmodified counterparts is Investigated using standard procedures. Therapies combining peptide delivery and Treg-ceil modification or regulation is contemplated, Fmtheniiore, Treg-eells and effector T-eell responses, against modified and unmodified peptides, in people with Type 1 diabetes and healthy subject, EXAMPLE 11
Animal model
J0235] A transgenic NOD mouse model of human CD4* T-cell responses against deamidated proinsulin epitopes is developed. During development, standard mouse models can be employed such as described by Niens et ai, (201 1 ) Diabetes 60:1229- 1236; Daniel et al. (2011) I Exp. Med. 10.1084/jcm.201 10574; Unger et al (2012) 7 iij;e49213.doi.:10. l 37l/Jounal.Pone,0049213; and Scotto ei al (2012) Di beMogia 55:2026-2031. EXAMPLE 12
Effects of physiological stress agents
10236] Deamidated proinsulin peptides are analyzed in human beta cells when the islets are exposed, to different physiological stressors (e.g. cytokines, elevated glucose concentrations). This leads to combination therapies and protocols to augmentation therapies. I 2373 Those skilled in the art will appreciate that the disclosure described herein is susceptible to variations and modifications other than those specifically described, it is to be understood that the disclosure contemplates all such variations and modifications, The disclosure also enables all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any arid all combinations of any two or more of the steps or features or compositions or compounds.
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Claims

CLAIMS:
1. A method for the treatment or prophylaxis of Type 1 diabetes or a condition associated with Type 1 diabetes i a mammalian subject, said method comprising administering to said subject a peptide comprising an amino acid sequence of at least seven consecutive amino acid residues comprising a T-cell epitope derived from insulin, proinsulin or preproinsulin wherein the amino acid sequence comprises a glutamine to glutamic acid substitution,
2. The method of Claim 1 wherein the mammalian subject is a human.
3. The method of Claim 1 or 2 wherein the peptide comprises from 13 to 40 amino acid residues with the proviso that it contains at least one glutamine to glutamic acid substitution.
4. The method of Claim 1 or 2 wherein the peptide comprises from 33 to 20 amino acid residues with the proviso that it contains at least one glutamine to glutamic acid substitution.
5. The method of Claim 3 or 2 wherein the peptide comprises an amino acid sequence selected from the list consisting of SEQ ID NOs;2 to 5, 32, 35 to 61, 64, 67, 70, 73, 76, 79, 82 to 94 and 96 to ] 21.
6. The method of Claim 1 or 2 wherein the peptide comprises an amino acid sequence set forth in SEQ JD O:94.
7. The method of Claim 1 wherein the peptide alternativel comprises or contains in addition an arginine to citrulline substitution.
8. The method of Claim 7 wherein the peptide comprises an amino acid sequence selected from the list consisti g of SEQ ID NGs:6 to 31 , 33, 34, 62, 63, 65, 66, 68, 69, 71 , 72, 74, 75, 77, 78, 80, 81 and 95.
9. The method of Claim 8 wherein the peptide com prises an amino acid sequence set forth in SEQ ID NQ:9S,
10. The method of any one of Claims 1 to 9 wherein the peptide binds to an antigen- presenting cell which comprises the haplotype HLA-DQ A*03:01 ; DQ B*03;02.
1 1. The method of any one of Claims 1 to 9 wherein the peptide binds to an HLA molecule selected from one or more of HLA DQ 2, HLA DQ 2/8 transdimer, HLA-DR3 {DRBl*03xx) and/or HLA-D 4 (DRBl*04xx).
12. The method of Claim 1 1 wherein the haplotype is DQ A*0S:01, DQ B*03:02 and/or DQ A*03:0L DQ B*01:01.
13. The method of Clai 1 wherein the condition associated with Type 1 diabetes is selected from the list consisting of a cardi vascular condition, a neuropathy, a retinopathy, a nephropathy, infection and a complication in pregnancy,
14. The method of Claim .1 wherein the peptide is conjugated to or embedded in a targeting moiety, carrier molecule or a immune cell,
15. An isolated T-cell or antigen-presenting cell derived from a mammalian subject presenting a peptide comprising an amino acid sequence of at least seven consecutive amino acid residues comprising a T-cell epitope derived from insulin, promsulm or preproinsulin wherein the amino acid sequence comprises a glutamine or glutamic acid substitution.
16. The isolated T-cell or antigen-presenting cell of Claim 15 wherein the mammalian subject is human.
17. The isolated T-cell or antigen-presenting cell of Claim 16 wherei the peptide is from 13 to 40 amino acid residues in length.
18. The isolated T-cell or antigen-presenting cell of Claim 17 wherein the peptides is from 13 to 20 amino acids in length.
19. The isolated T-cell or antigen-presenting cell of Claim 18 wherein the peptide comprises an amino acid sequence selected from the list consisting of SEQ ID NOs:2 to 5, 32, 35 to 61, 64, 67, 70, 73, 76, 79 and 82 to 94 or 96 to 121 ,
20. The isolated T-cell or antigen-presenting cell of Claim 19 wherein the peptide comprises an amino acid sequence set forth in SEQ ID NO:94.
21. The isolated T-cell or antigen-presenting cell of any one of Claims 15 to 20 for treating or preventing Type 1 diabetes or a condition associated with Type 1 diabetes wherein the T-cell s or antigen-presenting cells are autologous to the subject being treated.
21. The isolated T-cell or antigen-presenting cell of Claim 21 wherein the condition associated with Type 1 diabetes is selected from the list consisting of a cardiovascular condition, a neuropathy, a retinopathy, a nephropathy, infection and a complication in pregnancy.
23. A therapeutic or prophylactic composition comprising a peptide comprising an amino acid sequence of at least seven consecutive amino acid residues comprising a T-cell epitope derived from insulin, proinsulin or preproinsulin wherein the amino acid sequence comprises a glutamine to glutamic acid substitution and one or more pharmaceutically acceptable carriers, diluents and/or excipients.
24. The therapeutic or prophylactic composition of Claims 1 to 23 wherein the peptide is from 13 to 40 amino acids in length.
25. The therapeutic or prophylactic compositi on of Claim 23 wherein the peptide is from 13 to 20 amino acids in length.
26. The therapeutic or prophylactic composition of Claim 23 wherein peptide comprises an amino acid sequence selected from the list consisting of SEQ ID NOs:2 to 5, 32, 35 to 61, 64, 67, 70, 73, 76, 79 and 82 to 94 or 96 to 121.
27. The therapeutic or prophylactic composition of Claim 26 wherein the peptide comprises an amino acid sequence set forth in SEQ ID NO: 94.
28. The therapeutic or prophylactic composition of Claim 23 wherein the peptide hinds to an antigen -presenting ceil which comprises the haplotype HLA-DQ A*03:.Q1 ; DQ B*03 ;02,
29. The therapeutic or prophylactic composition of Claim 28 wherein the peptide binds to an HLA molecule selected from one or more of HLA DQ 2, HL.A DQ 2/8 transdimer, HLA-DR3 (DRBl*03xx) and/or HLA.-DR4 (DRB1 *04xx).
30. The therapeutic or prophylactic composition of Claim 23 is in the form of an HLA Class Π tetramer.
31. The therapeutic or prophylactic composition of Claim 23 wherein the peptide is conjugated to or embedded in targeting moiety, carrier molecule or an immune cell.
32. Use of a peptide comprising an amino acid sequence of at least seven consecutive amino acid residues comprising an epitope derived from insulin, promsulin or preproinsuh wherein the amino acid sequence comprises a gkitamine to glutamic acid substitution in the manufacture of a medicament for the treatment of Type 1 diabetes or a condition associated with Type 1 diabetes in a mammalian subject.
33. Use of CI aim 22 wherein the mammalian subject is a human,
34. Use of Claim 32 or 33 wherein the peptide is from 13 to 40 amino acids in length.
35, Use of Claim 34 wherein the peptide is from 13 to 20 amino acids in length.
36, Use of Claim 35 wherein the peptide comprises an amino acid sequence selected from the list consisting of SEQ ID NOs:2 to 5, 32, 3.5 to 61, 64, 67, 70, 73, 76, 79, 82 to 94 and 96 to 121 ,
37, Use of Claim 35 wherein the peptide comprises an amino acid sequence set fort in SEQ ID N0:94.
38, Use of Claim 32 wherein the peptide binds to an antigen -presenting cell which comprises the hapiotype HLA-DQ A*03:01, DQ B*03 :02.
39, Use of Claim 33 wherein the peptide binds to an HLA molecule selected from one or more of HLA DQ 2, HLA DQ 2/8 transdimer, HLA-DR3 (DRBl.*03xx) and/or HLA-DR4 (DRBl *04xx).
40, The method of Claim 39 wherein the hapiotype is DQ A*05:Q1, DQ B*03:0 and/or DQ A*03:01 , DQ 8*01 :01 .
4.1. Use of Claim 32 wherein a condition associated with Type 1 diabetes is selected from selected from the list consisting of a cardiovascular condition, a neuropathy, a retinopathy, a nephropathy, infection and a complication in pregnancy.
42. Use of Claim 32 wherein the peptide is conjugated to or embedded in a targeting moiety, carrier molecule or an immune cell.
43. A method for detecting the presence of autoreactive T-cells indicative of the presence of, or a predisposition for the development of, Type 1 diabetes, or a condition associated with Type 1 diabetes in a subject, said method comprising contacting a sample from said subject comprising immune cells with a peptide comprising an amino acid sequence of at least seven consecutive amino acid residues comprising a t-cell epitope derived from insulin, proinsulin or preproinsulin wherein the amino acid sequence comprises a glutamine to glutamic acid substitution for a time and under conditions sufficient for an autoreactive T-cell, if present, to be stimulated into producing an immune effector molecule, and then detecting the presence of said immune effector molecule, wherein the presence or level of the immune effector molecule is indicative of Type 1 diabetes or its state or risk of progression or a condition associated with Type 1 diabetes.
44. An assay to screen for autoreactive antibodies to insulin, proinsulin or preproinsulin, the assay comprising contacting a biological sample such as serum from a subject to be tested with peptide comprising a T-cell epitope derived insulin, proinsulin or preproinsuli wherein the peptide comprises a glutamine to glutamic acid substitution, so as to form a complex between an autoreactive antibody if present in the sample and the peptide and then detecting the presence of the antibody-peptide complex.
45. A method for screening for autoreactive antibodies associated with Type 1 diabetes or a condition associated therewith in a subject, comprising contacting body fluid from the subject with a peptide comprising an amino acid sequence comprising a T-cell epitope derived from insulin, proinsulin or preproinsulin wherein the amino acid sequence comprises an glutamine to glutamic acid substitution for a time and under conditions sufficient for an autoreactive antibody, if present, to bind to the peptide and then screening for the presence of the peptide-antibody complex.
46. A method for detecting the presence of autoreactive B-cells indicative of the presence of, or a predisposition for the development of, Type 1 diabetes, or a condition associated with Type 1 diabetes in a subject, the method comprising contactin a sample from the subject comprising immune cells with a peptide comprising an amino acid sequence comprising a T-cell epitope derived from insulin, proinsuiin or preproinsulin wherei the amino acid sequence comprises a glutamine to glutamic acid substitution for a time and under conditions sufficient for an autoreactive B-eeli, if present, to be stimulated into producing an antibody, and then detecting the presence of th antibody, wherein the presence or level of the antibody is indicative of Type 1 diabetes or its state or risk of progression or a condition associated with Type I diabetes.
47. The method of any one of Claims 43 to 46 wherein the subject is a human.
48. The method of Claim 47 wherein the presence of autoreactive antibodies is an indication of Type 1 diabetes or its stage of development.
49. The method of Claim 47 wherein the peptides is from 13 to 40 amino acids in length.
50. the method of Claim 47 wherein the peptide i s from 13 to 20 amino acids in length.
51. The method of Claim 50 wherein the peptid comprises an amino acid sequence selected from the list consisting of SEQ ID NOs:2 to 5, 32, 35 to 61, 64, 67, 70, 73, 76, 79 and 82 to 94 or 96 to 121.
52. The method of Claim 50 wherein the peptide comprises an amino acid sequence set forth in SEQ ID NO:94.
53. The method of any one of Claims 43 to 46 wherein the condition associated with Type 1 diabetes is selected, from the list consisting of a cardiovascular condition, a neuropathy, a retinopathy, a nephropathy, infection and a complication in pregnancy.
54. An isolated HLA Class II tetramer incorporating a peptide comprising at least 7 consecutive amino acids comprising a T-cell epitope derived from insulin, proinsulin or preproinsulin, wherein the peptide comprises a glutamine to glutamic acid substitution.
55. The isolated HLA Class II tetramer of Claim 54 wherein the peptide comprises at least 13 amino acid residues.
56. The insulin HLA Class II tetramer of Claim 55 wherein the peptide comprises a sequence selected from SEQ ID NO:2 to 5, 32, 35 to 61 , 4, 67. 70, 73, 76, 79 and 82 to 94 or 96 to 121.
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