WO2001030378A1 - Procede de prophylaxie et de traitement - Google Patents

Procede de prophylaxie et de traitement Download PDF

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Publication number
WO2001030378A1
WO2001030378A1 PCT/AU2000/001299 AU0001299W WO0130378A1 WO 2001030378 A1 WO2001030378 A1 WO 2001030378A1 AU 0001299 W AU0001299 W AU 0001299W WO 0130378 A1 WO0130378 A1 WO 0130378A1
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WIPO (PCT)
Prior art keywords
cells
antigen
ctl
agent
insulin
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PCT/AU2000/001299
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English (en)
Inventor
Leonard C Harrison
Arno Hanninen
Nathan R Martinez
David Kramer
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The Walter And Eliza Hall Institute Of Medical Research
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Priority claimed from AUPQ3627A external-priority patent/AUPQ362799A0/en
Priority claimed from AUPQ4347A external-priority patent/AUPQ434799A0/en
Priority claimed from AUPQ7621A external-priority patent/AUPQ762100A0/en
Application filed by The Walter And Eliza Hall Institute Of Medical Research filed Critical The Walter And Eliza Hall Institute Of Medical Research
Priority to CA002387652A priority Critical patent/CA2387652A1/fr
Priority to JP2001532795A priority patent/JP2003512435A/ja
Priority to AU11153/01A priority patent/AU767688B2/en
Priority to EP00972430A priority patent/EP1225912A4/fr
Publication of WO2001030378A1 publication Critical patent/WO2001030378A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2875—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route

Definitions

  • the present invention relates generally to a method of prophylaxis and treatment of autoimmune disease conditions and agents useful for same.
  • the present invention contemplates the use of mucosal antigens and/or agents capable of blocking or otherwise delaying cytotoxic T-lymphocyte (CTL) induction and/or maturation to prevent or at least reduce the likelihood or risk of CTL-mediated autoimmune disease. More particularly, the present invention contemplates mucosa-mediated tolerance to protect against or ameliorate the symptoms associated with autoimmune pathology.
  • CTL cytotoxic T-lymphocyte
  • the present invention provides a method for preventing clinical insulin- dependent diabetes mellitus (IDDM) or preventing or reducing or ameliorating the effects of clinical IDDM by the aerosol administration of IDDM-associated autoantigens to mucosal surfaces or agents which block CTL induction and/or maturation.
  • IDDM clinical insulin- dependent diabetes mellitus
  • IDDM insulin-dependent diabetes mellitus
  • BB Bio-Breeding
  • NOD non- obese diabetic
  • islet/ ⁇ -cell autoantigens have been identified by their reactivity with circulating antibodies or T cells in rodents and humans with sub-clinical or clinical IDDM, in particular insulin, glutamic acid decarboxylase (GAD) and tyrosine phosphatases of the IA-2 family (13).
  • IDDM insulin, glutamic acid decarboxylase (GAD) and tyrosine phosphatases of the IA-2 family (13).
  • GAD glutamic acid decarboxylase
  • tyrosine phosphatases of the IA-2 family 13
  • insulin and its precursor, pre-proinsulin are the only IDDM autoantigens that are ⁇ -cell specific.
  • IAN insulin autoantibodies
  • the present invention further provides for blocking or otherwise delaying CTL induction and/or maturation by, for example, blocking interaction between CD40 and CD40 ligand (CD40L).
  • One aspect of the present invention contemplates a method of inducing immune tolerance, including cytotoxic T-lymphocyte (CTL) tolerance, while substantially avoiding CTL immunity in response to a mucosal antigen, said method comprising selecting said mucosal antigen or modifying a mucosal antigen to disable the function of an MHC class I restricted epitope and then administering said selected or modified antigen for a time and under conditions sufficient to prevent or reduce CTL immunity to said mucosal antigen.
  • CTL cytotoxic T-lymphocyte
  • Another aspect of the present invention contemplates a method of suppressing a cell- mediated autoimmune disease while substantially avoiding CTL immunity in response to a mucosal autoantigen, said method comprising selecting an autoantigen or modifying said mucosal autoantigen to disable the function of an MHC class I restricted epitope and then administering said selected or modified autoantigen for a time and under conditions sufficient to induce tolerance but prevent or reduce CTL immunity to said autoantigen.
  • a further aspect of the present invention contemplates a method of suppressing a cell- mediated autoimmune disease in a subject, said method comprising the administration as an aerosol of an effective amount of an antigen associated with said autoimmune disease for a time and under conditions sufficient to prevent, reduce or otherwise ameliorate autoimmune pathology wherein said antigen substantially lacks a functional MHC class I interacting region.
  • Yet another aspect of the present invention provides a method of preventing, reducing or otherwise ameliorating an autoimmune disease condition in a subject, said method comprising the aerosol administration to said subject of an effective amount of an antigen associated with said autoimmune disease for a time and under conditions sufficient to induce or stimulate immunoregulatory mechanisms which are protective against cell- mediated autoimmune pathology wherein said antigen substantially lacks a MHC class I interacting region.
  • Still yet another aspect of the present invention contemplates a method of preventing, reducing or otherwise ameliorating IDDM, slowly progressive (SP) IDDM or gestational type 1 diabetes in a subject, said method comprising the administration, as an aerosol or other functionally equivalent means, to said subject of an effective amount of an autoantigen associated with IDDM for a time and under conditions sufficient for induction of regulatory T cells and/or other suitable mechanisms sufficient to suppress cell-mediated autoimmune pathology associated with IDDM wherein said autoantigen substantially lacks a functional MHC class I interacting epitope.
  • SP slowly progressive
  • Still another aspect of the present invention contemplates a method of inducing, suppressing or otherwise ameliorating or preventing IDDM in a subject, said method comprising administering proinsulin peptide truncated at its C-terminal antigen end to disable the function of any MHC class I restricted epitope for a time and under conditions sufficient to prevent or reduce CTL immunity and otherwise induce immune tolerance, including CTL tolerance.
  • Still yet another aspect of the present invention provides a composition comprising an antigen associated with an autoimmune disease in an aerosol formulation including one or more pharmaceutically acceptable carriers and/or diluents.
  • Another aspect of the present invention contemplates a method of inducing CTL tolerance while substantially avoiding CTL immunity in response to a mucosal antigen, said method comprising administering to a subject a nucleic acid molecule or analogue thereof encoding said mucosal antigen but wherein said antigen substantially lacks a functional MHC class I restricted epitope for a time and under conditions sufficient to prevent or reduce induction of CTL immunity.
  • a further aspect of the present invention contemplates a method of preventing or suppressing IDDM while substantially avoiding CTL immunity, said method comprising administering to a subject a nucleic acid molecule or analogue thereof encoding an IDDM- associated mucosal antigen which substantially lacks a functional MHC class I restricted epitope for a time and under conditions sufficient to prevent or reduce the effects of IDDM.
  • Another aspect of the present invention contemplates a method of inducing tolerance to a mucosal antigen while substantially avoiding CTL immunity to said antigen, said method comprising administering said mucosal antigen or a nucleic acid encoding same for a time and under conditions sufficient to prevent or reduce CTL immunity, before, simultaneously or sequentially with the administration of an antagonist of CTL induction and/or maturation.
  • the present invention contemplates a method of inducing tolerance to a mucosal antigen while substantially avoiding CTL immunity to said antigen, said method comprising administering said mucosal antigen or a nucleic acid encoding same for a time and under conditions sufficient to prevent or reduce CTL immunity, before, simultaneously or sequentially with the administration of an antagonist of CD40L-CD4 interaction.
  • Yet another aspect of the present invention provides a method of inducing tolerance to a mucosal antigen while substantially avoiding CTL immunity to said antigen, said method comprising administering an agent for a time and under conditions sufficient to block or otherwise delay CTL induction and/or maturation.
  • the present invention provides a method of inducing tolerance to a mucosal antigen while substantially avoiding CTL immunity to said antigen, said method comprising administering an agent for a time and under conditions sufficient to block or otherwise delay CTL induction and/or maturation wherein said agent blocks or otherwise disrupts CD40-CD40L interaction.
  • Another aspect of the present invention contemplates a use of a mucosal antigen with an inactive MHC class I epitope in the manufacture of a medicament for the treatment or prophylaxis of a disease condition in a subject.
  • Yet another aspect of the present invention contemplates the use of an agent in the manufacture of a medicament for the treatment or prophylaxis of a disease condition, said agent capable of blocking CTL induction and/or maturation.
  • the present invention further provides for the use in combination of any of the methodologies contemplated above.
  • Figure 1 is a graphical representation showing that intranasal human insulin (80 ⁇ g) at 56 days of age delays diabetes onset in female NOD mice.
  • Figure 2 is a graphical representation showing that intranasal human proinsulin (40 ⁇ g) at 56 days of age delays diabetes onset.
  • Figure 3 is a graphical representation showing that intranasal human proinsulin aa24-36 (40 ⁇ g at 56 days of age) delays diabetes onset.
  • Figure 4 is a graphical representation showing that aerosol insulin induces CD8 T cells that suppress transfer of diabetes.
  • B-E fractionated splenocytes from aerosol insulin- or ovalbumin-treated NOD females, and their incidence of diabetes subsequently monitored.
  • aerosol donor mice had been treated for 10 consecutive days and then weekly from 49 days of age and were normoglycemic when sacrificed at 156 days of age.
  • Figure 5 is a graphical representation showing that aerosol insulin induces CD8 ⁇ T cells that suppress transfer of diabetes.
  • Young male NOD mice were co-injected with "diabetic" splenocytes (2 x 10 7 ) and total or fractionated splenic T cells from aerosol-treated mice, as in the legend to Figure 4.
  • the numbers of fractionated cells injected were, in A) ⁇ 10 7 total T cells and, from aerosol insulin mice, ⁇ 10 7 ⁇ -depleted T cells or 1.4 x 10 5 ⁇ T cells and, in B), from aerosol insulin mice, ⁇ 10 7 total T cells, 2 x 10 6 CD8 T cells, 2 x 10 6 ⁇ - depleted CD8 T cells or 1.5 x 10 5 CD8 ⁇ +ve T cells.
  • Figure 6 is a diagrammatical representation showing that adoptive transfer of diabetes is suppressed by CD8 ⁇ T cells induced by aerosol insulin: summary of 11 experiments.
  • Figure 7 is a graphical representation showing that intranasal human proinsulin aa24-36 (40 ⁇ g at 56 days of age) induces CD4 T cells that suppress adoptive transfer of diabetes, in NOD mice.
  • Figure 8 is a graphical representation showing that mouse proinsulin aa26-34 and to a greater extent mouse proinsulin aa25-34 bind to the MHC class I molecule K d .
  • expression of K d on the surface of RMA-S cells is monitored by binding of a monoclonal anti-K d antibody detected by fluorescence in a flow cytometer.
  • Addition of a K d -binding peptide to the cells stabilizes K and increases its expression on the cell surface, indicated by a right shift in the signal response.
  • Isotype control control monoclonal antibody
  • no peptide constitutive K d expression
  • HAP and LLO peptides from 'flu hemagglutinin and Listeria known to bind to K d and used as positive controls.
  • Figure 9 is a graphical representation showing that mouse proinsulin aa25-34 induces K d - restricted cytotoxic T lymphocytes (CTL) in NOD mice.
  • CTL cytotoxic T lymphocytes
  • Six week old female mice were immunised subcutaneously with 50 ⁇ g of peptide in Complete Freund's Adjuvant. After 14 days their spleens were removed and splenocytes re-stimulated in vitro with 10 ⁇ g/ml peptide for 6 days. Splenocytes were then tested for CTL activity against 51 Cr and pep tide- loaded RMA-S target cells.
  • Figures 10A and , 10B are diagrammatic representations showing that C-terminal truncations enhance the effect of intranasal proinsulin B-C peptide to suppress diabetes.
  • Figure 11 is a diagrammatic representation showing postulated mechanisms of ⁇ -cell destruction in type 1 diabetes, including the role of CD40L-CD40 interaction in activating CD8 T cells to become CTLs.
  • Figure 12 is a graphical representation showing (A) systemic and (B) oral priming of CTL require CD40L signalling.
  • A A single i.p. injection of 250 ⁇ g of control mAb 6C8 (open squares) or anti-CD40L mAb MRl (open circles) was given to C57B1/6 mice one day before challenge with 20 x 10 6 i.v. OVA-coated H-2K bm" ' splenocytes to prime CTL. After 14 days mice were killed and their splenocytes tested for CTL activity, expressed as OVA- specific lysis for representative individual mice.
  • Figure 13 is a graphical representation showing activation and expansion of OVA-specific CTL by oral OVA requires CD40L.
  • C57B1/6 recipient mice congenic for Ly5.1 were adoptively transferred with 3 x 10 6 transgenic OT-I cells (Ly5.2) and then given control mAb 6C8 or anti-CD40L mAb MRl, 250 ⁇ g i.p.
  • Mice from each treatment group were then divided into two groups and fed either PBS or 20 mg OVA in PBS on three alternate days. mAb treatment was repeated before the third feeding. Mice were killed 14 days from the start of feeding and the numbers and phenotype of OT-I cells in their spleens analyzed by flow cytometry.
  • A Dot-plots of individual mice show CD44 (left) and CD62L (L- selectin) (right) expression on OT-I cells. The % of cells expressing a high level of CD44 or a low level of CD62L is shown in the corresponding quadrant. The number of OT-I cells per spleen (B) CD44 expression (C) and % CD62L 10 (D) OT-I cells in individual recipient mice treated with 6C8 or MRl and then fed PBS or OVA are shown for a single experiment, but similar results were obtained three experiments.
  • Figure 14 is a graphical representation showing anti-CD40L treatment prevents induction of diabetes by oral OVA in RIP-OVA 10 mice.
  • PJP-OVA 10 mice bearing OT-I and OT-II cells were injected with control mAb 6C8 or anti-CD40L mAb MRl, 250 ⁇ g i.p.
  • mice were then fed OVA, 0.5 mg on five alternate days. Blood glucose was measured 12 days after the start of feeding and values above 13 mmol/1 were considered diagnostic of diabetes. Data are pooled from two experiments.
  • FIG. 15 is a graphical representation showing anti-CD40L treatment does not limit oral tolerance to systemic priming of CTL.
  • CTL activity in response to i.v. priming with OVA- coated splenocytes (A) or to s.c. priming with OVA in CFA (B) is similarly attenuated by oral OVA in mice treated with control mAb 6C8 and anti-CD40L mAb MRl .
  • Mice were injected with 6C8 or MRl, 250 ⁇ g i.p. and then fed either PBS (black squares) or 20 mg OVA in PBS (open circles) on three alternate days.
  • mice were primed systemically and seven days later killed and their splenocytes recovered for a standard in vitro 51 Cr release assay of CTL activity.
  • Primed splenocytes as effectors (E) were tested against 51 Cr-loaded cells as targets (T).
  • T 51 Cr-loaded cells as targets
  • Each plot represents an individual mouse.
  • CTL activity plots for individual mice were converted into lytic units from four experiments (C) after priming as in (A) and from two experiments (D) after priming as in (B), in which mice received either PBS or 20 mg oral OVA on three alternate days (C) or PBS or 0.5 mg oral OVA on five alternate days (D).
  • Figure 16 is a graphical representation showing anti-CD40L treatment does not limit oral tolerance to systemic priming of T-cell proliferation (A) and IFN- ⁇ (B) responses, or antibody production (C).
  • spleens and inguinal lymph nodes and sera were harvested for measurement of T-cell proliferation and cytokine production in the absence (solid) or presence (hatched) of O.lmg/ml OVA (mean and standard deviation shown for spleen), and anti-OVA antibodies, as described in Methods.
  • Figure 17 is a graphical representation showing the effect of treatment with anti-CD40L monoclonal antibody (MR-1) on diabetes incidence in NOD mice given aerosol insulin.
  • the present invention is predicated on the surprising discovery that insulin or its precursor could be used to induce immune tolerance.
  • one aspect of the present invention contemplates a method of inducing immune tolerance while substantially avoiding CTL immunity in response to a mucosal antigen in a subject, said method comprising selecting said mucosal antigen or modifying a mucosal antigen to disable the function of an MHC class I restricted epitope and then administering said selected or modified antigen for a time and under conditions sufficient to prevent or reduce CTL immunity to said mucosal antigen.
  • the mucosal antigen is used for preventing a CTL-mediated autoimmune disease such as but not limited to diabetes and in particular, IDDM.
  • another aspect of the present invention contemplates a method of suppressing a cell-mediated autoimmune disease while substantially avoiding CTL immunity in response to a mucosal autoantigen, said method comprising selecting an autoantigen or modifying said mucosal autoantigen to disable the function of an MHC class I-restricted epitope and then administering said selected or modified autoantigen for a time and under conditions sufficient to induce tolerance but prevent or reduce CTL immunity to said autoantigen.
  • Administration of the autoantigen may be by DNA or polypeptide/peptide delivery and may be by any appropriate means but the preferred route of administration is via mucosal surfaces including via oral, nasal, pharyngeal, or bronchial passages and via aerosol including intranasal aerosol.
  • Yet another aspect of the present invention contemplates a method of suppressing a cell- mediated autoimmune disease in a subject, said method comprising the administration as an aerosol of an effective amount of an antigen associated with said autoimmune disease for a time and under conditions sufficient to prevent, reduce or otherwise ameliorate autoimmune pathology wherein said antigen substantially lacks a functional MHC class I interacting region.
  • the present invention provides a method of preventing, reducing or otherwise ameliorating an autoimmune disease condition in a subject, said method comprising the aerosol administration to said subject of an effective amount of an antigen associated with said autoimmune disease for a time and under conditions sufficient to induce or stimulate immunoregulatory mechanisms which are protective against cell- mediated autoimmune pathology wherein said antigen substantially lacks a MHC class I interacting region.
  • immunodegulatory mechanisms should be understood as a reference to all mechanisms which regulate cell-mediated immune responses including, but not limited to, regulation of T-cell functional activity, for example, regulation by one or more of suppressor CD4 T cells, Thl, Th2 or CD8 T cells including ⁇ T cells (referred to herein as “regulatory T cells”), or via regulation of cytokine production by lymphoid, myeloid or stromal cells.
  • the present invention is predicated in part on the recognition by the inventors that some mucosal autoantigens contain epitopes for MHC class I-restricted CTLs. As a result, administration of these antigens may result in CTL immunity and CTL tolerance. Accordingly, the present invention requires the selection of- antigens which lack or to modify the antigens to remove functional MHC class I interacting epitopes.
  • the MHC class I epitope is an MHC class I (K )- restricted epitope.
  • the present invention is hereinafter described with respect to preventing, reducing or otherwise ameliorating IDDM, slowly progressive IDDM (SPIDDM) also referred to as latent autoimmune diabetes in adults [LADN] and gestational diabetes due to underlying IDDM. This is done, however, with the understanding that the present invention extends to a range of cell-mediated autoimmune conditions.
  • SPIDDM slowly progressive IDDM
  • another aspect of the present invention contemplates a method of preventing, reducing or otherwise ameliorating IDDM, SPIDDM or gestational diabetes in a subject, said method comprising the administration, as an aerosol or other functionally equivalent means, to said subject of an effective amount of an autoantigen associated with IDDM for a time and under conditions sufficient for induction of regulatory T cells and/or other suitable mechanisms sufficient to suppress cell-mediated autoimmune pathology associated with IDDM wherein said autoantigen substantially lacks a functional MHC class I interacting epitope.
  • IDDM includes IDDM, SPIDDM and gestational IDDM.
  • the regulatory T cells induced will depend on the form of antigen and its route of administration. For example, when an undegraded, conformationally-intact polypeptide or whole protein molecule is administered (e.g. insulin), CD8 T cells and, more particularly, CD8 ⁇ T cells are induced. Smaller peptides such as proinsulin peptides (e.g. proinsulin peptide 24-36) generally induce CD4 T cells and, more particularly, CD4 ⁇ T cells. Whole proteins may be degraded to peptides to generate predominantly CD4 regulatory T cells, particularly if administration is via the oral route.
  • an undegraded, conformationally-intact polypeptide or whole protein molecule e.g. insulin
  • CD8 T cells and, more particularly, CD8 ⁇ T cells are induced. Smaller peptides such as proinsulin peptides (e.g. proinsulin peptide 24-36) generally induce CD4 T cells and, more particularly, CD4 ⁇ T cells.
  • Whole proteins may be degraded to peptid
  • the absence of a functional MHC class I interacting epitope includes a single or multiple amino acid deletion encompassing all or part of the epitope region.
  • the epitope may be blocked by other means such as by an antibody or other molecular interaction.
  • a particularly preferred form of administration is intranasal administration via an aerosol spray, drip or vapour.
  • the preferred antigen associated with IDDM used for intranasal administration or other route of administration is preproinsulin or proinsulin as well as insulin and their immune response stimulatory derivatives thereof such as but not limited to peptide fragments of proinsulin, preproinsulin or insulin, provided that such antigens lack or substantially lack a functional MHC class I-associating region which is involved in inducing CTL immunity.
  • Immune response stimulation preferably includes regulatory T cell stimulation.
  • any islet antigen may be employed such as, but not limited to, glutamic acid decarboxylase (GAD) in its various isoforms (for example, GAD 65 and GAD 67) or derivatives thereof and tyrosine phosphatase IN-2 or derivatives thereof.
  • the antigens may be from human or any non-human species such as mouse.
  • the most preferred antigen is a proinsulin peptide modified to inactivate the MHC class I interacting region, defined by amino acids 24 to 33.
  • the interacting region may be modified by generating peptides lacking one or more key MHC class I anchor residues or comprising modified residues such that MHC class I binding is reduced.
  • the proinsulin peptide undergoes a C-terminal truncation to inactivate the MHC class I epitope.
  • CTL tolerance including CTL tolerance.
  • another aspect of the present invention contemplates a method of inducing, suppressing or otherwise ameliorating or preventing IDDM in a subject, said method comprising administering proinsulin peptide truncated at its C-terminal antigen end to disable the function of any MHC class I restricted epitope for a time and under conditions sufficient to prevent or reduce CTL immunity and otherwise induce immune tolerance, including CTL tolerance.
  • the methods of the present invention may also be used in combination with a strategy to block CTL induction and/or maturation.
  • a strategy to block CTL induction and/or maturation for example, the CD40-CD40 ligand (CD40L) interaction is blocked.
  • derivatives includes fragments, parts, portions, chemical equivalents, mutants, homologues and analogues of the antigens.
  • Analogues may be derived from natural synthetic or recombinant sources and include fusion proteins.
  • Chemical equivalents of an antigen can act as a functional analog of an antigen. Chemical equivalents may not necessarily be derived from an antigen but may share certain conformational similarities. Alternatively, chemical equivalents may be specifically designed to mimic certain physiochemical properties of an antigen. Chemical equivalents may be chemically synthesised or may be detected following, for example, natural product screenings.
  • a homologue of an antigen contemplated herein includes but is not necessarily limited to antigens derived from human or any non-human species such as mouse.
  • Derivatives include one or more insertions, deletions or substitutions of amino acids.
  • Amino acid insertional derivatives include amino and/or carboxylic terminal fusions as well as intrasequence insertions of single or multiple amino acids.
  • Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in said peptide although random insertion is also possible with suitable screening of the resulting product.
  • Deletional variants are characterised by the removal of one or more amino acids from the sequence.
  • Substitional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place.
  • Additions to amino acid sequences include fusions with other peptides or polypeptides. It is possible, for example, that the subject preferred peptides may be substituted by other peptides or functional homologues or analogues.
  • a hybrid peptide may comprise a combination of peptides.
  • aerosol is used in its most general sense to include any formulation capable of administration via nasal, pharyngeal, bronchial or oral passages. Aerosols generally comprise particles of liquid or solid suspended in a gas or vapour. Conveniently, the aerosol is a colloidal system such as a mist in which the dispersion medium is a gas.
  • the method of administering the aerosol formulation may be by any means and may be achieved using a hand pump, electric pump, pressurized dispenser, nasal drip or other convenient means. Furthermore, drop size may determine lung penetration and the size of the droplets may need to be manipulated to maximize efficacy of administration. It should be understood that the method of the present invention extends to direct application of said formulations to intranasal surfaces.
  • the aerosol is delivered at a rate of from about 1 to about 20 litres/min and preferably from about 2 to about 15 litres/min at a droplet size of from about 0.1 to about 10 ⁇ m and more preferably from about 0.1 to about 6 ⁇ m.
  • a stock solution of antigen is prepared at a concentration of from about 0.5 to about 20 mg/ml or more preferably from about 1.0 to about 10 mg/ml of carrier solution.
  • Commercially available insulin is particularly useful which is about 4 mg/ml.
  • a useful dose is from about 50 1 to 1000 ⁇ l and preferably 100 ⁇ l to 500 ⁇ l from the stock solution.
  • the antigen may be administered alone or by formulation in or with an adjuvant.
  • the adjuvant is selected from a range of adjuvants which enhance an immunoregulatory response including cholera toxin B, heat labile toxin of E. coli, saponin, Quill A extracts and other derivatives of saponin, D ⁇ A ⁇ -dextran, dextran sulphate, aluminium salts, and non-ionic block co-polymers.
  • the adjuvant may include other immunomodulators, such as cytokines (for example, IL-4 or IL-13), muramyl-dipeptide and derivatives, and cell wall components, for example, cell wall lipoprotein from Gram-ve bacteria such as E.coli, from species of Mycobacteria or Corynebacteria.
  • the adjuvant formulation may include a combination of two or more of the adjuvants listed. These lists are not to be taken as exhaustive.
  • the selection of adjuvant is in part dependent on the species being targeted and is based on the level and duration of the immune response required and on the lack of reactogenicity (i.e. tissue compatibility). The level of active component and adjuvant are chosen to achieve the desired level and duration of immune response.
  • the antigen is administered in a therapeutically effective amount.
  • a therapeutically effective amount means that amount necessary at least partly to attain the desired effect, or to delay the onset of, inhibit the progression of, or halt altogether, the onset or progression of the particular condition being treated. Such amounts will depend, of course, on the particular conditions being treated, the severity of the condition and individual patient parameters including age, physical conditions, size, weight and concurrent treatment. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgement. It will be understood by those of ordinary skill in the art, however, that a lower dose or tolerable dose may be administered for medical reasons, psychological reasons or for virtually any other reasons.
  • daily oral doses of antigen will be from about 0.01 mg/ per dose per subject per day to 1000 mg/per dose per subject per day.
  • Small doses (0.01-1 mg) may be administered initially, followed by increasing doses up to about 1000 mg/kg per day.
  • higher doses or effective higher doses by a different, more localized delivery route
  • a single dose may be administered or multiple doses may be required on an hourly, daily, weekly or monthly basis.
  • Effective amounts of antigen vary depending on the individual but may range from about 0.1 ⁇ g to about 100 mg, preferably from about 1 ⁇ g to about 10 mg and more preferably from about 5 ⁇ g to 20 mg per dose per subject.
  • lower doses may be contemplated for aerosol or intranasal administration, for example, ng- ⁇ g doses may be optimal.
  • the subject undergoing treatment may be any human or animal in need of therapeutic or prophylactic treatment.
  • the immune status generally, and specifically levels of regulatory T cells and cytokine profiles may be readily determined throughout any treatment regime using conventional methods known to those skilled in the art.
  • regulatory T cell levels may be monitored by cytometric analysis following labelling with commercially available antibodies specific to T-cell subsets.
  • Other examples of methods suitable for determining the status of the subject include purification of peripheral blood mononuclear cells by density centrifugation followed by stimulation by incubation with well known antigens such GAD, IA-2 family members, preproinsulin, proinsulin or insulin or peptide sequences from these antigens. Resulting proliferation may be quantified by assaying for inco oration of H 3 thymidine.
  • the cytokine profile can be determined approximately 24- 72 hours after stimulation by antigen.
  • Said cytokines can be detected using, for example, specific cytokine antibodies.
  • stimulated cells can be phenotypically characterized by, for example, flow cytometric analysis of activation marker expression (for example, CD69, CD44, CTLA4, CD25).
  • activation marker expression for example, CD69, CD44, CTLA4, CD25.
  • said cells may be further fixed and incubated with fluorochrome labelled antibodies to specific cytokines to determine intracellular cytokine levels.
  • cells may be further assessed by double labelling assays. The double labelled cells may be analysed utilizing flow cytometric analysis or fluorescence microscopy.
  • compositions comprising an antigen associated with an autoimmune disease in an aerosol formulation including one or more pharmaceutically acceptable carriers and/or diluents.
  • the autoimmune disease is IDDM.
  • the antigen is an islet antigen such as modified forms of insulin, or a precursor thereof such as preproinsulin, proinsulin or their derivatives (e.g. proinsulin peptide 24-36) or GAD or tyrosine phosphatases IA-2 or derivatives thereof wherein said antigens are modified to prevent an MHC class I epitope from functioning.
  • an islet antigen such as modified forms of insulin, or a precursor thereof such as preproinsulin, proinsulin or their derivatives (e.g. proinsulin peptide 24-36) or GAD or tyrosine phosphatases IA-2 or derivatives thereof wherein said antigens are modified to prevent an MHC class I epitope from functioning.
  • the antigen and route of administration induce regulatory T cells, such as in relation to whole molecules such as insulin CD8 T cells and most preferably CD8 ⁇ T cells or, in relation to smaller molecules such as proinsulin peptide 24-36, CD4 T cells and most preferably CD4 ⁇ T cells.
  • regulatory T cells such as in relation to whole molecules such as insulin CD8 T cells and most preferably CD8 ⁇ T cells or, in relation to smaller molecules such as proinsulin peptide 24-36, CD4 T cells and most preferably CD4 ⁇ T cells.
  • a nucleic acid molecule encoding an IDDM-associated autoantigen is administered.
  • intranasal or other suitable administration of a nucleic acid molecule such as DNA encoding proinsulin or a modified form thereof induces a population of CD4 T cells which suppresses development of diabetes.
  • the nucleic acid molecule encodes a peptide lacking a functional MHC class I interacting molecule.
  • the nucleic acid molecule is preferably DNA such as cDNA or genomic DNA or is a DNA:RNA hybrid. It is particularly preferred to have the nucleic acid molecule in the form of a plasmid or vector.
  • the nucleic acid molecule may also contain additional or substitution analogues of nucleotide bases in order to enhance stability.
  • another aspect of the present invention contemplates a method of inducing immune tolerance, including CTL tolerance, while substantially avoiding CTL immunity in response to a mucosal antigen, said method comprising administering to a subject a nucleic acid molecule or analogue thereof encoding said mucosal antigen but wherein said antigen substantially lacks a functional MHC class I-restricted epitope for a time and under conditions sufficient to prevent or reduce induction of CTL immunity.
  • the present invention contemplates a method of preventing or suppressing IDDM while substantially avoiding CTL immunity, said method comprising administering to a subject a nucleic acid molecule or analogue thereof encoding an IDDM- associated autoantigen which substantially lacks a functional MHC class I-restricted epitope for a time and under conditions sufficient to prevent or reduce the effects of IDDM.
  • Still yet another aspect of the present invention contemplates other methods for dissociating CTL immunity from CTL tolerance.
  • the maturation of CTL to effector "killer" cells requires priming by antigen-presenting cells such as dendritic cells.
  • the dendritic cells present the antigenic peptide (i.e. epitope) as a complex with MHC class I molecules to the T-cell receptor of CD8 CTL.
  • the dendritic cell itself is primed to perform this function by prior interaction with a "helper" CD4 T cell through the interaction between CD40 ligand (CD40L) on the T cell and CD40 on the dendritic cell (see Figure 11).
  • CD8 T cells themselves have also been shown to express CD40L.
  • an antagonist of CD40L-CD40 interaction is a CD40L antibody, such as a monoclonal antibody.
  • the antagonist of CD40L-CD40 interaction may be administered before, simultaneously with or sequentially with the administration of the mucosal antigen. Sequential administration includes within seconds, minutes, hours, days or weeks. Simultaneous includes substantially simultaneously. This extra treatment may be in conjunction with the administration of a mucosal antigen or a nucleic acid molecule encoding a mucosal antigen.
  • Another aspect of the present invention contemplates a method of inducing tolerance to a mucosal antigen while substantially avoiding CTL immunity to said antigen, said method comprising administering said mucosal antigen or a nucleic acid encoding same for a time and under conditions sufficient to prevent or reduce CTL immunity, before, simultaneously or sequentially with the administration of an antagonist of CTL induction and/or maturation.
  • the present invention contemplates a method of inducing tolerance to a mucosal antigen while substantially avoiding CTL immunity to said antigen, said method comprising administering said mucosal antigen or a nucleic acid encoding same for a time and under conditions sufficient to prevent or reduce CTL immunity, before, simultaneously or sequentially with the administration of an antagonist of CD40L-CD4 interaction.
  • Yet another aspect of the present invention provides a method of inducing tolerance to a mucosal antigen while substantially avoiding CTL immunity to said antigen, said method comprising administering an agent for a time and under conditions sufficient to block or otherwise delay CTL induction and/or maturation.
  • the present invention provides a method of inducing tolerance to a mucosal antigen while substantially avoiding CTL immunity to said antigen, said method comprising administering an agent for a time and under conditions sufficient to block or otherwise delay CTL induction and/or maturation wherein said agent blocks or otherwise disrupts CD40-CD40L interaction.
  • Still another aspect of the present invention contemplates the use of an agent in the manufacture of a medicament for the treatment or prophylaxis of a disease condition, said agent capable of blocking CTL induction and/or maturation.
  • Retro- orbital venous blood was sampled at least every 28 days from 100 days of age and mice considered to be diabetic if their blood glucose, confirmed by a repeat test, was >11 mM.
  • Glucose was measured with BM-Test Glycemie (registered trademark) strips and a Reflolux (registered trademark) II meter (Boehringer-Mannheim), on a drop of blood aspirated via a glass capillary tube from the retro-orbital venous plexus of unanesthetized mice.
  • mice were killed by CO 2 inhalation and the pancreas and salivary glands immediately removed into Bouin's fixative and embedded in paraffin.
  • the insulitis score a measure of the severity of islet infiltration, was determined blindly by two independent investigators by grading and then averaging a minimum of 15 separate islets in serial 6 ⁇ m pancreas sections stained with haematoxylin and eosin.
  • the grading scale was: 0, no filtration, islet intact; 1, ⁇ 10 peri-islet lymphoid cells, islet intact; 2, 10-20 peri-and intra-islet lymphoid cells, islet intact; 3, >20 peri- and intra-islet lymphoid cells, ⁇ 50% of islet replaced or destroyed; 4, massive lymphoid infiltrate with >50% of islet replaced or destroyed.
  • Infiltration of the salivary glands was graded by the number of lymphoid cells in clusters: 0, no cells; 1, ⁇ 10 cells; 2, 10-50 cells; 3, >50 cells.
  • Spleen cells from individual normoglycemic mice were treated with a red cell lysis buffer, resuspended and incubated in quadruplicate at 2 x 10 5 /200 ⁇ l of serum-free HL-1 medium (Hycor, Irvine, CA) containing 50 ⁇ m 2-mercaptoethanol, in round-bottom wells with the indicated concentrations of antigen.
  • GAD65 was the recombinant human form expressed with a C-terminal hexahistidine in a baculovirus system and purified by Ni 2+ chelation affinity chromatography. It was resolved as a single band in SDS-PAGE and was endotoxin-free by the quantitative Limulus lysate assay (BioWhittaker, Walkersville, MD).
  • IL-2, -4, -10 and IFN- ⁇ were measured by ELISAs with monoclonal antibody pairs (Pharmingen); the lower limits of detection were 62, 16, 16 and 55 pg/ml, respectively.
  • TGF-/31 was measured with an ELISA kit (Promega) with a lower limit of detection of 16 pg/ml.
  • 125 I-labelled human insulin (approximately 100,000 cpm: specific activity 120 ⁇ Ci/ ⁇ g) was incubated with or without excess unlabelled insulin (10 ⁇ g/ml) in phosphate-buffered saline containing a mixture of protease inhibitors and serial log dilutions of mouse serum, for 5 days at 4°C. Complexes were then precipitated with rabbit anti-mouse globulin anti-serum, washed and counted in a gamma counter. Positive control sera (guinea pig anti-porcine insulin serum, human IDDM sera) maximally precipitated 37-54% of the total radioactivity. Non-specific binding, in the presence of excess unlabelled insulin, was ⁇ .3%. EXAMPLE 4 Adoptive transfer of diabetes
  • mice aged 6-9 weeks (16/group) were irradiated (800R) from a Cobalt source and 3-6 hours later received 2 x 10 7 pooled splenocytes from recently-diabetic 14-19 week- old female NOD mice, together with 2 x 10 7 splenocytes (or cells fractionated from this number) from either aerosol insulin- or ovalbumin-treated mice, in 200 ⁇ l via the tail vein.
  • the onset of diabetes was then monitored by measuring blood glucose starting two weeks after transfer.
  • Spleen cells were treated with red cell lysis buffer and resuspended in mouse tonicity phosphate buffered saline. Total T cells were purified by non-adherence to nylon wool.
  • CD4 and CD8 cells were positively selected/depleted magnetically with monoclonal antibodies directly bound to MACS MicroBeads (Milteny Biotec, GmbH, Germany) according to the manufacturer's protocols, and counted as viable cells (trypan blue stain negative). Flow cytometry revealed 95% depletion of CD4 or CD8 cells, with recoveries -80% and -50% respectively.
  • ⁇ T cells were positively selected/depleted by incubating T cells from aerosol-treated mice first with biotinylated GL3-1A antibody (Pharmingen, San Diego, CA) and then with streptavidin-MACS MicroBeads, followed by magnetic separation. By flow cytometry, ⁇ cells comprised 1-2% of NOD splenocytes and were totally depleted with GL3-1A antibody.
  • CD8 T cells were first magnetically selected from total T cells with anti-CD8-FITC conjugate and anti-FITC MicroBeads. The MicroBeads were then released according to the Miltenyi Biotec protocol, and the CD8 cells magnetically separated into ⁇ positive and depleted fractions. Double staining and FACS analysis demonstrated total depletion of ⁇ cells and their recovery as a GL3-1 A high and low expressing CD8 population.
  • Aerosol human insulin or ovalbumin were administered in different schedules to female NOD mice from 28 days of age, the earliest time at which insulitis is detectable in the colony of mice, and their incidence of diabetes and severity of insulitis subsequently measured.
  • mice (32/group) were given either aerosol insulin or ovalbumin for 10 consecutive days and then weekly from 28 days of age. At 105 days of age, five non-diabetic mice from each group were killed for pancreas histology. The insulitis score is expressed as mean ⁇ SD.
  • ovalbumin mice had significantly higher responses than insulin mice to human glutamic acid decarboxylase 65 (GAD65), previously reported to stimulate splenic T cells in NOD mice (25).
  • GAD65 human glutamic acid decarboxylase 65
  • proliferative responses to non-antigen-specific stimulation by concanavalin A or T-cell receptor CD3 monoclonal antibody, 145-2C11 were similar (Table 2) and no different to untreated mice, indicating that aerosol treatment did not cause general immunosuppression.
  • IL-2, IFN- ⁇ and TGF- ⁇ l secretion in response to insulin B chain 9-23 were not significantly different between insulin- and ovalbumin-treated mice; however, the levels of IL-4 and particularly IL-10 were higher from cells of insulin-treated mice (Table 3).
  • This increase in the "level" of insulin antibodies after aerosol insulin, together with the suppression of T cell proliferation and the increase in IL-4 and IL-10 responses to insulin B-chain peptide is consistent with the phenomenon of immune deviation, as described after oral MBP in Lewis rats (1) and intranasal GAD peptides in NOD mice (28).
  • ⁇ -cell destruction within the DTH lesion of IDDM is an example of Thl -mediated process (10,11), whose inhibition by aerosol insulin might be expected to shift the Thl/Th2 balance towards Th2 in response to key islet antigens.
  • Defective suppressor T-cell function has been postulated to shift the balance towards Thl in IDDM (11). It seems unlikely that the reduced T cell proliferative response to GAD could reflect "bystander" suppression due to the secretion of the Th2 cytokines IL4 and IL- 10 (1) by insulin aerosol-induced regulatory cells because, apart from an absence of added insulin in the cultures with GAD, responses to conA and anti-CD3 were not impaired.
  • spleen cells from diabetic NOD female mice transferred intravenously to young, irradiated non-diabetic syngeneic male or female recipients cause clinical diabetes in the majority within 4 weeks.
  • 2 x 10 7 spleen cells were co-injected from older, diabetic mice with an equal number of spleen cells from aerosol ovalbumin mice, the majority of young recipients developed diabetes within 4-5 weeks; in contrast, after co-injection with spleen cells from aerosol insulin mice, only a minority developed diabetes (Figure 4A). Diabetes incidence was suppressed by >75% in six separate experiments with either splenocytes or nylon wool-non-adherent splenocytes (enriched for T cells) from aerosol insulin mice.
  • T cells bearing ⁇ receptors have been shown to have an immunoregulatory role (31-36). Interestingly, it has been reported that total peripheral blood ⁇ cells decrease concomitantly with loss of ⁇ -cell function in humans with sub-clinical IDDM (37).
  • the inventors fractionated spleen cells with the anti- ⁇ T-cell monoclonal antibody, GL3- 1A (38). Depletion of ⁇ T cells, like that of CD8 cells, completely abrogated the ability of nylon wool non-adherent spleen cells from insulin aerosol-treated mice to suppress adoptive transfer of diabetes (Figure 5A). Conversely, relatively small numbers of ⁇ T cells from insulin aerosol-treated mice could suppress transfer.
  • Aerosol inhalation as a mode of insulin delivery to the mucosa was as effective as oral insulin (22,23) in reducing diabetes incidence in the NOD mouse.
  • the fact that it was therapeutic after the onset of insulitis is especially relevant to the prevention of IDDM in at-risk humans with sub-clinical disease in whom the presence of circulating islet-antigen reactive antibodies and T cells is taken to reflect underlying insulitis.
  • NOD mice compared to humans with recently-diagnosed IDDM, NOD mice have more intense insulitis and the majority of females to progress to diabetes (10,11,24). Aerosol insulin had no obvious metabolic effect but induced a population of regulatory CD8 ⁇ T cells, small numbers of which suppressed the ability of pathogenic effector T cells to adoptively transfer diabetes.
  • These antigen-induced "suppressor" T cells protective against cell-mediated autoimmune pathology have not been previously described.
  • Oral tolerance has been associated with a decrease in cellular and sometimes an increase in humoral antigen-specific immunity, and with either CD8 or CD4 T cells that secrete, respectively, TGF- ⁇ or IL-4, IL-10 and TGF- ⁇ l (8).
  • CD8 or CD4 T cells that secrete, respectively, TGF- ⁇ or IL-4, IL-10 and TGF- ⁇ l (8).
  • these regulatory cells have not been identified as bearing ⁇ receptors.
  • oral tolerance to insulin was attributed to regulatory CD4 T cells (21).
  • CD8 ⁇ T cells account for the regulatory cells induced by aerosol insulin.
  • insulin at 4 mg/ml, or proinsulin or proinsulin peptide 24-36 at 1-4 mg/ml in either insulin carrier solution or mouse tonicity-phosphate buffered saline, was applied in a volume of 10-20 ⁇ l to the nostrils of unanaesthetized, restrained NOD female mice at either 28 or 56 days of age. Note that by 56 days of age all mice exhibit underlying islet inflammation (insulitis). -*Single doses of insulin, proinsulin or proinsulin peptide 24-36 at either 28 or 56 days of age each significantly delayed the onset of diabetes in NOD female mice, compared to control proteins ovalbumin or hen egg lysozyme.
  • proinsulin and proinsulin peptide 24-36 were more effective than insulin. These effects are greater with repeated doses of these proteins or peptide.
  • female mice pretreated with a single intranasal dose (40 ⁇ g) of proinsulin 24-36 whole splenocytes, and whole splenocytes depleted of CD8 but not CD4 T cells, significantly suppressed the adoptive transfer of diabetes by splenocytes from diabetic mice (Figure 7).
  • Female mice were treated at 28 days of age, and then killed and their splenocytes taken for adoptive co-transfers at 56 days of age.
  • the intranasal insulin trial involves administration of intranasal insulin to at-risk but otherwise healthy first-degree relatives with immune markers of IDDM, including circulating antibodies and T cells reactive with islet autoantigens.
  • Our subjects have at least two antibodies, to insulin, GAD or tyrosine phosphatase IA-2, and peripheral blood T cell responses to insulin or proinsulin peptide 24-36, and sometimes to GAD and IA-2 peptides.
  • the rationale is to induce mucosa-mediated immune tolerance to insulin, based on the success of this approach in the NOD mouse, and to demonstrate safety.
  • Commercially-available human recombinant insulin is used, which is normally given routinely by subcutaneous or intravenous injection to people with IDDM.
  • the INIT trial examines the effect of intranasal insulin on the surrogate immune markers of IDDM.
  • the design is randomized, double-blind and placebo-controlled, with a crossover at six months.
  • the placebo is the carrier solution normally used for insulin.
  • the aim is to demonstrate significant effects on the levels of antibodies and T cells to insulin and other beta cell antigens.
  • first phase insulin release (FPIR) in response to an intravenous injection of glucose, a measure of beta cell function, is monitored at the start, six months and 12 months.
  • FPIR first phase insulin release
  • the crossover design gives all subjects the opportunity of treatment (an important issue for at-risk relatives), measures if any treatment effects are sustained and allows within- and between-group analyses. Treatment is administered initially daily for 10 consecutive days, then for two consecutive days weekly. After six months, treatment is crossed over (from insulin to placebo, or vice versa).
  • the administration dose of insulin per nostril is approximately 200 ⁇ l (800 ⁇ g) of the commercial 4 mg/ml solution.
  • the placebo is the carrier solution in which the insulin is normally dissolved.
  • proinsulin peptide 24-36 Following administration of the proinsulin peptide 24-36, although CD4 regulatory T cells were induced which almost completely blocked the adoptive transfer of diabetes (when isolated and transferred with effector "diabetogenic" T cell into young irradiated NOD mice), and the onset of spontaneous diabetes was delayed but not prevented.
  • the inventors observed that the proinsulin peptide contained predicted epitopes for MHC class I (H2- K d )-restricted CTLs, namely aa26-34 and aa25-34. Accordingly, administration of proinsulin peptide 24-36 could result in concomitant induction of CTL immunity and tolerance.
  • CTL cytotoxic T-lymphocyte
  • TCR for the MHC class I-restricted OVA 257 .
  • 264 peptide and OT-II mice bearing a transgenic CD4 TCR for the MHC class Il-restricted OVA 23 . 3 9 peptide were used between 6 and 12 weeks as donors of OVA-reactive T cells for adoptive transfer into Ly5.1/CD45.2 congenic C57B1/6 mice (OT-I cells) and into RIP-OVA transgenic mice (OT-I and OTII cells).
  • Oral tolerance was induced with two protocols corresponding to reported high and low dose OVA.
  • OVA (Grade V, Sigma, St. Louis, MO) was administered to female C57B1/6 mice either at 20 mg on three alternate days (high dose) or at 0.5 mg on five alternate days (low dose) via intragastric intubation under light methoxyflurane (Penthrane (trademark)) anaesthesia.
  • CD40L signaling was blocked by administration of the hamster IgGl anti-mouse CD40L mAb MR-1 (ATCC, Rockville, MD); the control was the hamster mAb 6C8 specific for human Bcl-2. Both mAbs were purified from hybridoma cell culture medium by affinity chromatography on protein G-Sepharose (Pharmacia, Uppsala, Sweden) and injected intraperitoneally (i.p.). in a dose of 250 ⁇ g as indicated.
  • CTL Cytotoxic T lymphocyte
  • mice were primed i.v. with 20 x 10 6 OVA-coated H-2K bm l spleen cells (dependant on CD4 T-cell help) or subcutaneously (s.c.) in the base of tail with 200 ⁇ g of OVA peptide 257-264 in CFA in 100 ⁇ l (independent of CD4 T-cell help).
  • mice were primed 2 or 3 weeks after receiving mAb and oral OVA.
  • Mice were killed 7 days after priming, and their spleen cells stimulated in vitro for another 6 days before being used as effectors in a 51 Cr release assay. Lytic units were calculated by dividing the total number of effectors generated from each spleen by the total number of effectors required for 30% OVA-specific lysis.
  • RIP- OVA mice which express OVA on their pancreatic ⁇ cells were adoptively transferred with 0.3 x 10 5 OT-1 cells and 0.2 x 10 6 OT-1 cells and given MR-1 or control 6C8 mAb on the day of transfer (day 0). Mice were then treated with oral OVA, 0.5 mg on five alternate days, starting from day 1. Blood glucose was measured on a drop of retro-orbital venous blood with a glucometer, on days 14 and 21 and values above 14 mmol/1 were considered to be diagnostic for diabetes.
  • Cell suspensions were prepared from spleens and nodes by mechanical disruption through a stainless steel mesh, washed, counted and resuspended in RPMI-1640 medium containing 2 mM glutamine, 5 x 10 "5 2-mercaptoethanol and 5% v/v fetal calf serum for assay of proliferative and cytokine responses to OVA.
  • IgG subclass antibodies to OVA were measured by ELISA using peroxidase-conjugated anti-mouse IgG 1, 2a, 2b or 3 antibodies (Southern Biotechnology Associates) as previously described .
  • Proliferative responses to OVA of splenocytes (1 x 10°) or inguinal lymph node cells (5 x 10 5 ) in 200 ⁇ l medium were measured in replicates of eight in round-bottom wells of 96- well Linbro plates (Flow Labs, McLean, VA), after incubation with or without 0.1 mg/ml OVA at 37°C in 5% CO 2 / air for 96 hours.
  • 3 H-thymidine (1 ⁇ Ci) was added to each well for the last 10-16 hours, the cells harvested and washed, and counted on a TopCount scintillation counter.
  • Splenocyte or inguinal lymph node cell IFN- ⁇ and IL-4 responses to OVA were measured by ELISPOT assay.
  • Membrane-bound cytokine was reacted with 4 ⁇ g/ml biotin-conjugated monoclonal rat anti-mouse IFN- ⁇ (clone XMG1.2) or IL-4 (clone BVD6-24G2) overnight at 4°C. After washing, colour was developed with streptavidin-peroxidase followed by 3-amino-9- ethylcarbazole (NEC; Dako, Carpinteria, CN). All monoclonal antibodies were from Pharmingen, San Diego, CA. Statistics
  • CD40L blockade impairs CTL induction by oral OVA
  • OVA-specific transgenic CTL (OT-I cells) into naive Ly5.1 congenic recipients and fed them OVA.
  • the role of CD40L in the response of OT-I cells to oral OVA was examined by pre-treating recipient mice with either control mAb 6C8 or anti-CD40L mAb MRl.
  • the inventors examined OT-I cells from the spleen 14 days after the last dose of oral OVA. This site and time corresponded to other protocols used, e.g. to measure OVA- induced CTL.
  • OT-I cells in the spleen expanded greatly (Figure 13B) and increased CD44 ( Figures 13 A, C) and decreased CD62L ( Figures 13 A, D) expression, indicating that many had acquired an activated/memory phenotype.
  • MRl the ability of oral OVA to induce expansion ( Figure 13B) and activation ( Figures 13 A, C, D) of OT-I cells was markedly impaired.
  • Anti-CD40L treatment prevents induction of diabetes by oral OVA in RIP-OVA ° mice
  • Anti-CD40L treatment does not prevent induction of oral tolerance
  • CD40L signalling is required for induction of oral tolerance (39).
  • this mutation affects the development of Peyer's patches (39) and germinal centres (40). Therefore, it was important to determine if oral tolerance could be induced in genetically unmanipulated mice treated short-term with anti-CD40L mAb.
  • oral OVA while inducing CTL immunity, paradoxically suppressed the further priming of strong CTL immunity by systemic OVA (41).
  • anti-CD40L treatment influenced this tolerogenic effect of oral OVA on CTL, C57B1/6 mice were given control mAb 6C8 or anti-CD40L mAb MRl 250 ⁇ g i.p.
  • Mucosal administration of antigen can tolerize subsequent immune responses to the antigen and, in the case of autoantigens, suppress development of autoimmune disease.
  • Mucosal administration, however, of the model protein antigen ovalbumin (OVA) also induces cytotoxic T-cell (CTL) immunity and this may cause disease.
  • OVA ovalbumin
  • CTL cytotoxic T-cell
  • the inventors show that oral OVA-induced tolerance and CTL immunity can be dissociated by targeting the interaction between CD40L and CD40.
  • Monoclonal antibody blockade of CD40L strengthened tolerance by preventing the simultaneous induction of CTL. This was reflected by inhibition of the activation and expansion of adoptively-transferred OVA- specific CTL (OT-1-CD8 cells) in response to oral OVA.
  • CD40L blockade significantly inhibited the development of CTL (OT-1 cell)-mediated autoimmune diabetes that followed oral administration of OVA.
  • Mouse proinsulin II cDNA or ovalbumin genomic DNA was subcloned into a plasmid vector derived from the mammalian expression vector, pCI, under the control of the CMV early promoter.
  • the vector was modified and is designated as CIGH.
  • Plasmids were prepared from E.coli and purified by PEG precipitation and Triton XI 14 phase partition, diluted to 2 mg DNA per ml in PBS and frozen at -20°C.
  • NOD mice were bred and maintained in The Walter and Eliza Hall Institute of Medical Research. At 3 and 5 weeks of age, 25 ⁇ l of PBS containing 50 ⁇ g DNA was given intranasally in repeat 5 ⁇ l portions to non-anaesthetized female mice. In other experiments mice were given 25 ⁇ g DNA intranasally for four consecutive weeks beginning at 3 weeks of age.
  • Blood glucose was measured using the Advantage monitor (Boehringer Mannheim) on a drop of blood obtained via a fine glass capillary tube from the retro-orbital venous plexus. Mice were considered to be diabetic if their blood glucose was >11 mM on consecutive days. Diabetic donor mice used in adoptive transfer studies has an elevated blood glucose for ⁇ 1 week. RESULTS
  • spleen cells from intranasal DNA-treated mice at 10 weeks of age were enriched for T cells (hereafter referred to as splenic T cells) by passage through nylon wool, and than either co-transferred i.v. with spleen cells from recently-diabetic NOD mice into irradiated 6 week-old male NOD mice or transferred i.v. into cyclophosphamide- treated NOD females Cyclophosphamide treatment accelerates the onset of diabetes on NOD mice. In both experimental models, a significant reduction in diabetes incidence was observed in recipient mice that received cells from proinsulin DNA-treated donors.
  • the inventors next sought to identify the phenotype of the T cell responsible for protection by transferring fractional splenic T-cell population.
  • Splenic T cells were incubated with either anti-mouse CD4 or anti -mouse CD8 monoclonal antibodies conjugated to magnetic MACS MicroBeads and purified on positive selection columns (Miltenyi).
  • the purity of CD4 and CD8 T cells by FACS analyses was >95% and >85%, respectively.
  • Either 4 x 10 6 CD4 T cells or 1 x 10 6 CD8 T cells were then co-transferred with 2 x 10 7 diabetic spleen cells into 6 week-old irradiated males.
  • Diabetes incidence 4 weeks after transfer was 36% in recipients of CD4 T cells from proinsulin DNA-treated donors compared to 71% in recipients of CD4 T cells from ovalbumin DNA-treated controls (p 0.02) (Table 4). In contrast, there was no difference in diabetes incidence (94 v 83%) in recipients of co-transferred CD8 cells from either proinsulin DNA-or ovalbumin DNA-treated mice (Table 4). Similar results were obtained when either 4 x 10° CD4 T cells or 2.5 x 10 6 CD4- depleted (CD8 T cell-enriched) splenic T cells were injected into cyclophosphamide- treated 10 week-old female mice.

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Abstract

La présente invention porte sur un procédé de prophylaxie et de traitement de maladies auto-immunes et sur des agents utilisés pour traiter ces maladies. Selon une réalisation, l'invention porte sur l'utilisation d'antigènes des muqueuses et/ou d'agents capables de bloque ou du moins retarder l'induction et/ou la maturation des lymphocytes T cytotoxiques (CTL) pour empêcher ou du moins réduire le risque d'une maladie auto-immune induite par les CTL, Plus spécifiquement, cette invention porte sur la tolérance induite par les muqueuses pour protéger des symptômes associés à une pathologie auto-immune. Et, plus particulièrement, l'invention porte sur un procédé de prévention contre le diabète sucré insulinodépendant clinique, ou de prévention, ou réduction ou amélioration des effets du diabète sucré insulinodépendant clinique en administrant par aérosol des auto-antigènes associés au diabète sucré insulinodépendant sur les surfaces des muqueuses, ou en administrant des agents bloquant l'induction et/ou la maturation des CTL.
PCT/AU2000/001299 1999-10-22 2000-10-20 Procede de prophylaxie et de traitement WO2001030378A1 (fr)

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CA002387652A CA2387652A1 (fr) 1999-10-22 2000-10-20 Procede de prophylaxie et de traitement
JP2001532795A JP2003512435A (ja) 1999-10-22 2000-10-20 予防及び治療方法
AU11153/01A AU767688B2 (en) 1999-10-22 2000-10-20 A method of prophylaxis and treatment
EP00972430A EP1225912A4 (fr) 1999-10-22 2000-10-20 Procede de prophylaxie et de traitement

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AUPQ3627A AUPQ362799A0 (en) 1999-10-22 1999-10-22 A method of prophylaxis and treatment
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AUPQ4347A AUPQ434799A0 (en) 1999-11-26 1999-11-26 A method of propylaxis and treatment - II
AUPQ4347 1999-11-26
AUPQ7621A AUPQ762100A0 (en) 2000-05-19 2000-05-19 A method of prophylaxis and treatment - III
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WO2015006833A1 (fr) * 2013-07-19 2015-01-22 St. Vincent's Institute Of Medical Research Procédé de traitement du diabète de type 1

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WO2015006833A1 (fr) * 2013-07-19 2015-01-22 St. Vincent's Institute Of Medical Research Procédé de traitement du diabète de type 1

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JP2003512435A (ja) 2003-04-02

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