WO2016131911A1 - Procédés de diagnostic et de traitement du diabète de type i - Google Patents

Procédés de diagnostic et de traitement du diabète de type i Download PDF

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Publication number
WO2016131911A1
WO2016131911A1 PCT/EP2016/053446 EP2016053446W WO2016131911A1 WO 2016131911 A1 WO2016131911 A1 WO 2016131911A1 EP 2016053446 W EP2016053446 W EP 2016053446W WO 2016131911 A1 WO2016131911 A1 WO 2016131911A1
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Prior art keywords
amino acid
acid residue
peptide
hla
seq
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PCT/EP2016/053446
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English (en)
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Christian Boitard
Sandrine LUCE
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Université Paris Descartes
Centre National De La Recherche Scientifique (Cnrs)
Assistance Publique-Hôpitaux De Paris (Aphp)
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Publication of WO2016131911A1 publication Critical patent/WO2016131911A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to methods for the treatment and diagnosis of Type 1
  • Type 1 Diabetes is a major health problem due to onset before the age of 18 in 50% of patients, an increasing incidence in all countries that have been scrutinized, the absence of treatment other than lifelong insulin replacement therapy, the high vascular burden relating with residual hyperglycaemia in insulin-treated patients and a major social impact.
  • T1D pathogenesis mostly based on studies in the NOD mouse 1 , efforts to apply immunotherapy 2 have been unsuccessful when translated into clinical trials.
  • major differences have been reported between the diabetes phenotype seen in animal models and the human disease 3 .
  • the present invention relates to methods for the treatment and diagnosis of Type 1 Diabetes.
  • the present invention is defined by the claims. DETAILED DESCRIPTION OF THE INVENTION:
  • the present invention relates to human preproinsulin peptide for use in a method for treating Type 1 Diabetes in a subject in need thereof.
  • the term "insulin-dependent diabetes mellitus,” “IDDM,” “type I diabetes mellitus,” and “T1DM,” refer to diseases characterized by the autoimmune destruction of the ⁇ cells in the pancreatic islets of Langerhans.
  • the subject was early diagnosed with T1DM.
  • the expression “early diagnosis of T1DM” or “early diagnosed” T1DM refers to the patient in whom T1DM has either been recently or newly diagnosed, e.g. wherein the patient has been diagnosed with T1DM within about 3 months of initial treatment, and/or wherein the patient's T1DM is in early stages or is not advanced, e.g.
  • a blood test such as C-peptide in which a detectable level of C-peptide (e.g. > 0.03 nMol/L either fasting or stimulated by a caloric load such as a mixed-meal tolerance test (MMTT) indicates the presence of functioning beta cells and a MMTT-stimulated C-peptide level >0.2 nMol/L indicates a clinically meaningful level of beta cell function.
  • a detectable level of C-peptide e.g. > 0.03 nMol/L either fasting or stimulated by a caloric load
  • MMTT mixed-meal tolerance test
  • treatment or “treating a subject” is defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease.
  • Treatment can slow, cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease.
  • treatment of a subject can slow, improve, or stop the ongoing autoimmunity, e.g., a reaction against pancreatic ⁇ -cells, in a subject before, during, or after the clinical onset of type 1 diabetes. Therefore the method of the invention can prevent diabetes mellitus, or prevent or delay loss of residual ⁇ -cell mass, providing a longer remission period reducing short term complications and/or delaying the onset of diabetes-related complications at a later stage of the life.
  • the onset of T1DM may be delayed by the method as described herein such that insulin is not needed by the subject for a longer length of time.
  • the present method may extend the "honeymoon phase" in an already diabetic subject.
  • the honeymoon phase is where insulin is secreted by the pancreas, causing high blood sugar levels to subside, and resulting in normal or near normal glucose levels due to responses to insulin injections and treatment.
  • the method of the present invention is also used to arrest the autoimmune destruction of tissue, e.g., pancreatic ⁇ -cells.
  • the method of the present invention is suitable to arrest the autoimmune destruction, even at a late stage at the time of clinical onset of type 1 diabetes mellitus or after clinical onset. For example, at the time of clinical onset of type 1 diabetes mellitus, significant number of insulin producing ⁇ -cells are destroyed.
  • the autoimmune process can be arrested even in this late stage or as far as residual secretion can be restored, these cells can be preserved.
  • the ⁇ -cells have some limited capacity to replicate and precursors may form new ⁇ -cells.
  • the phrase "delaying the progression" as used herein in the context of delaying the progression of diabetes mellitus means that the loss of functional residual ⁇ -cell mass, after the clinical onset of Type 1 diabetes is delayed.
  • the delayed progression of T1DM can be measured, for example, by measuring C-peptide production.
  • preproinsulin has its general meaning in the art.
  • the insulin mRNA is translated as a 110 amino acid single chain precursor called preproinsulin, and removal of its signal peptide during insertion into the endoplasmic reticulum generates proinsulin.
  • Proinsulin consists of three domains: an amino-terminal B chain, a carboxy-terminal A chain and a connecting peptide in the middle known as the C peptide.
  • proinsulin is exposed to several specific endopeptidases which excise the C peptide, thereby generating the mature form of insulin which consists of the A and B chain. Insulin and free C peptide are packaged in the Golgi into secretory granules which accumulate in the cytoplasm.
  • An exemplary amino acid sequence for the human preproinsulin polypeptide is SEQ ID NO: l .
  • SEQ ID NO: 1 Homo sapiens
  • the human preproinsulin peptide is a HLA-DQ8-restricted hPPI epitope selected from the group consisting of:
  • a peptide consisting of 8 consecutive amino acids selected in the sequence ranging from the amino acid residue at position 1 to the amino acid residue at position 15 in SEQ ID NO: l (“hPPIl-15")
  • the human preproinsulin peptide of the present invention comprises 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, or 35 amino acids.
  • the human preproinsulin peptide of the present invention is selected from the group consisting of
  • a peptide consisting of a sequence having at least 90% of identity with the sequence ranging from the amino acid residue at position 1 to the amino acid residue at position 15 in SEQ ID NO: l ("hPPIl-15")
  • a peptide consisting of a sequence having at least 90% of identity with the sequence ranging from the amino acid residue at position 16 to the amino acid residue at position 30 in SEQ ID NO: l("hPPI 16-30")
  • a peptide consisting of a sequence having at least 90%> of identity with the sequence ranging from the amino acid residue at position 20 to the amino acid residue at position 35 in SEQ ID NO: l("hPPI20-35")
  • a peptide consisting of a sequence having at least 90%> of identity with the sequence ranging from the amino acid residue at position 92 to the amino acid residue at position 110 in SEQ ID NO: l("hPPI92-l 10").
  • a first amino acid sequence having at least 90% of identity with a second amino acid sequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99; or 100% of identity with the second amino acid sequence.
  • Amino acid sequence identity is typically determined using a suitable sequence alignment algorithm and default parameters, such as BLAST P (Karlin and Altschul, 1990).
  • the human preproinsulin peptide of the present invention is selected from the group consisting of
  • the human preproinsulin peptide of the present invention consists of the sequence ranging from the amino acid residue at position 1 to the amino acid residue at position 30 in SEQ ID NO: 1.
  • the human preproinsulin peptide of the present invention is fused to a heterologous polypeptide to form a fusion protein.
  • a "fusion protein" comprises all or part (typically biologically active) of a human preproinsulin peptide of the present invention operably linked to a heterologous polypeptide (i.e., a polypeptide other than the human preproinsulin).
  • a heterologous polypeptide i.e., a polypeptide other than the human preproinsulin.
  • the term "operably linked” is intended to indicate that the human preproinsulin peptide of the present invention and the heterologous polypeptide are fused in-frame to each other.
  • the heterologous polypeptide can be fused to the N-terminus or C-terminus of the human preproinsulin peptide of the present invention.
  • the preproinsulin peptide of the present invention is fused either directly or via a linker to the heterologous polypeptide.
  • the term “directly” means that the (first or last) amino acid at the terminal end (N or C-terminal end) of the human preproinsulin polypeptide of the present invention is fused to the (first or last) amino acid at the terminal end (N or C-terminal end) of heterologous polypeptide.
  • the term "linker” refers to a sequence of at least one amino acid that links the human preproinsulin peptide of the present invention with the heterologous polypeptide. Linker are well known to one of ordinary skill in the art and typically comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids.
  • the heterologous polypeptide comprises at least one redox motif C— (X)2-[CST] or [CST]-(X)2-C.
  • the C— (X)2-[CST] or [CST]-(X)2- C motif is positioned N-terminally of the human preproinsulin peptide of the present invention.
  • the fusion protein of the invention contains the sequence motif C— X(2)- [CS] or [CS]— X(2)-C.
  • the fusion protein of the invention contain the sequence motif C—X(2)-S, S— X(2)-C or C— X(2)-C.
  • C— (X)2-[CST] or [CST]-(X)2-C motif As used herein, the symbol X is used for a position where any amino acid is accepted.
  • [CST] stands for an amino acid selected from Cys, Ser or Thr.
  • [CST] stands for an amino acid selected from Cys, Ser or Thr.
  • the different elements in a motif are separated from each other by a hyphen Repetition of an identical element within a motif can be indicated by placing behind that element a numerical value or a numerical range between parentheses.
  • X(2) corresponds to X— X
  • X(2, 4) corresponds to X— X or X— X— X or X— X— X— X
  • A(3) corresponds to A- A-A.
  • C represents either cysteine or another amino acids with a thiol group such as mercaptovaline, homocysteine or other natural or non-natural amino acids with a thiol function.
  • the cysteines present in the redox motif should not occur as part of a cystine disulfide bridge. Nevertheless, the redox motif may comprise modified cysteines such as methylated cysteine, which is converted into cysteine with free thiol groups in vivo.
  • each of the amino acids X in the C— (X)2-[CST] or [CST]-(X)2- C motif can be any natural amino acid, including S, C, or T or can be a non-natural amino acid, whereby the two amino acids X are either the same or different.
  • X is an amino acid with a small side chain such as Gly, Ala, Ser or Thr.
  • X is not an amino acid with a bulky side chain such as Tyr.
  • at least one X in the [CST]-X(2)-[CST] motif is His or Pro.
  • the redox motif is placed either immediately adjacent to the human preproinsulin peptide sequence within the fusion protein, or is separated from the human preproinsulin peptide by a linker as defined herein.
  • the linker comprises an amino acid sequence of 7 amino acids or less.
  • the linker comprises 1, 2, 3, or 4 amino acids.
  • a linker may comprise 6, 8 or 10 amino acids. Typical aminoacids used in linkers are serine and threonine.
  • Example of peptides with linkers in accordance with the present invention are CXXC-G-human preproinsulin peptide, CXXC- GG- human preproinsulin peptide, CXXC— SSS-e human preproinsulin peptide, CXXC— SGSG- human preproinsulin peptide and the like.
  • the redox motif occurs several times (1, 2, 3, 4 or even more times) in the fusion protein, for example as repeats of the redox motif which can be spaced from each other by one or more amino acids (e.g. CXXC X CXXC X CXC), as repeats which are adjacent to each other (e.g.
  • one or more motifs are provided at both the N and the C terminus of the human preproinsulin peptide of the present invention.
  • Other variations envisaged for the fusion proteins of the present invention include fusion proteins containing repeats of a human preproinsulin peptide of the present invention wherein each peptide is preceded and/or followed by the redox motif (e.g. repeats of "motif- peptide” or repeats of "motif-peptide-moti ').
  • the redox motifs can all have the same sequence but this is not obligatory.
  • the fusion protein of the invention further comprises an amino acid sequence facilitating uptake of the peptide into (late) endosomes for processing and presentation.
  • the late endosome targeting is mediated by signals present in the cytoplasmic tail of proteins and correspond to well-identified peptide motifs such as the dileucine-based [DE]XXXL[LI] or DXXLL motif (e.g. DXXXLL), the tyrosine-based ⁇ 0 motif or the so- called acidic cluster motif.
  • the symbol 0 represents amino acid residues with a bulky hydrophobic side chains such as Phe, Tyr and Trp.
  • the late endosome targeting sequences allow for processing and efficient presentation of the human preproinsulin peptide of the present invention by antigen presenting cells (APC).
  • APC antigen presenting cells
  • Such endosomal targeting sequences are contained, for example, within the gp75 protein (Vijayasaradhi et al. (1995) J Cell Biol 130, 807-820), the human CD3 gamma protein, the HLA-DM ⁇ (Copier et al. (1996) J. Immunol. 157, 1017-1027), the cytoplasmic tail of the DEC205 receptor (Mahnke et al. (2000) J Cell Biol 151, 673-683).
  • the sequence can be that of a subdominant or minor T cell epitope from a protein, which facilitates uptake in late endosome without overcoming the T cell response towards the alloantigen-derived T cell epitope.
  • the fusion protein of the present is a fusion protein as described in WO2008035217. Accordingly, in some embodiments, the fusion protein of the present invention comprises:
  • an IgG binding moiety consisting of two or more IgG binding domains of streptococcal protein G placed in a tandem arrangement and optionally separated by a peptide linker, and
  • a cargo moiety comprising the human preproinsulin of the present invention.
  • the IgG binding moiety of a fusion protein of the invention comprises three IgG binding domains of streptococcal protein G. In some embodiments, it comprises four of five IgG binding domains of streptococcal protein G.
  • the IgG binding domains of streptococcal protein G may be identical or different. Any combination of these two or more domains among the Bl, B2, and B3 domains, in any order, can be used. Typical combinations include Bl-Bl, B1-B1-B1, Bl- B2, B1-B2-B1, B2-B1, B2-B1-B2, B2-B2 and B2-B2-B2.
  • the human preproinsulin peptide of the present invention is also fused to an ubiquitin domain to its N-terminal end, in order to enhance its degradation in the proteasome - dependent MHC class I antigen processing pathway.
  • an ubiquitin domain to its N-terminal end, in order to enhance its degradation in the proteasome - dependent MHC class I antigen processing pathway.
  • the presence of an N-terminal ubiquitin molecule may, upon protein translocation into the cytosol, enhance degradation of the polypeptide of interest by cellular proteasome complexes. This enhancing effect on degradation will not occur if one uses an ubiquitin variant wherein both the lysines at positions 29 and 48 are substituted by other amino-acid residues, e.g.
  • ubiquitin-antigen junctions obtained by replacing the C-terminal glycine of the ubiquitin sequence by another amino-acid residue (such as valine or cysteine), can be used if the objective is to induce more potent CTL responses, using the pathway of "ubiquitin fusion degradation".
  • the peptides and fusion proteins of the invention may be produced by any technique known per se in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination. Knowing the amino acid sequence of the desired sequence, one skilled in the art can readily produce said polypeptides, by standard techniques for production of polypeptides. For instance, they can be synthesized using well- known solid phase method, preferably using a commercially available peptide synthesis apparatus (such as that made by Applied Biosystems, Foster City, California) and following the manufacturer's instructions. Alternatively, the polypeptides and fusions proteins of the invention can be synthesized by recombinant DNA techniques as is now well-known in the art.
  • these fragments can be obtained as DNA expression products after incorporation of DNA sequences encoding the desired (poly) peptide into expression vectors and introduction of such vectors into suitable eukaryotic or prokaryotic hosts that will express the desired polypeptide, from which they can be later isolated using well-known techniques.
  • the human preproinsulin peptide of the present invention is fused or conjugated to an antibody for forming an "immunoconjugate".
  • antibody is thus used to refer to any antibody- like molecule that has an antigen binding region, and this term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, single domain antibodies (DABs or VHH), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody; kappa(lamda) bodies (scFv-CL fusions); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMIP ("small modular immunopharmaceutical” scFv-Fc dimer; DART (ds-stabilized diabody "Dual Affinity Re
  • the antibody is a chimeric antibody, a humanized antibody or a human antibody.
  • the techniques for preparing and using various antibody-based constructs and fragments are well known in the art. Significantly, as is well-known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, in general, Clark, W. R. (1986) The Experimental Foundations of Modern Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford).
  • the Fc' and Fc regions for example, are effectors of the complement cascade but are not involved in antigen binding.
  • an antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc' region designated an F(ab')2 fragment, retains both of the antigen binding sites of an intact antibody.
  • an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule.
  • Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd.
  • the Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.
  • the present invention also provides for F(ab') 2 Fab, Fv and Fd fragments.
  • Antibodies can be indeed fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments.
  • Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art. For example, each of Beckman et al., 2006; Holliger & Hudson, 2005; Le Gall et al., 2004; Reff & Heard, 2001 ; Reiter et al, 1996; and Young et al, 1995 further describe and enable the production of effective antibody fragments.
  • the various antibody molecules and fragments may derive from any of the commonly known immunoglobulin classes, including but not limited to IgA, secretory IgA, IgE, IgG and IgM.
  • IgG subclasses are also well known to those in the art and include but are not limited to human IgGl, IgG2, IgG3 and IgG4.
  • the present invention also includes so-called single chain antibodies.
  • the term "single domain antibody” (sdAb) or "VHH” refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®". According to the invention, sdAb can particularly be llama sdAb.
  • the antibody is directed against a surface antigen of an antigen presenting cell so that the human preproinsulin peptide of the present invention is targeted to said cell to elicit an immune response (e.g. tolerance).
  • an immune response e.g. tolerance
  • APC Antigen Presenting Cells
  • DCs Dendritic cells
  • These cells are characterized by their distinctive morphology, high levels of surface MHC-class II expression (Steinman, et al., Ann. Rev. Immunol. 9:271 (1991); incorporated herein by reference for its description of such cells). These cells can be isolated from a number of tissue sources, and conveniently, from peripheral blood, as described herein.
  • the antibody is selected from an antibody that specifically binds to dendritic cell immunoreceptor (DCIR), MHC class I, MHC class II, CD1, CD2, CD3, CD4, CD8, CD1 lb, CD 14, CD 15, CD 16, CD19, CD20, CD29, CD31, CD40, CD43, CD44, CD45, CD54, CD56, CD57, CD58, CD83, CD86, CMRF-44, CMRF-56, DCIR, DC-ASPGR, CLEC-6, CD40, BDCA-2, MARCO, DEC- 205, mannose receptor, Langerin, DECTIN-1, B7-1, B7-2, IFN- ⁇ receptor and IL-2 receptor, ICAM-1, Fey receptor, LOX-1, and ASPGR.
  • DCIR dendritic cell immunoreceptor
  • the antibody is specific for a cell surface marker of a professional antigen presenting cell.
  • the antibody is specific for a cell surface marker of a dendritic cell, for example, CD83, CMRF-44 or CMRF- 56.
  • the antibody may be specific for a cell surface marker of another professional antigen presenting cell, such as a B cell or a macrophage.
  • CD40 is expressed on both dendritic cells, B cells, and other antigen presenting cells so that a larger number of antigen presenting cells would be recruited.
  • nucleic acid molecule is covalently attached to lysines or cysteines on the antibody, through N- hydroxysuccinimide ester or maleimide functionality respectively.
  • TDCs cysteine-based site-specific conjugation
  • ADCs cysteine-based site-specific conjugation
  • Conjugation to unnatural amino acids that have been incorporated into the antibody is also being explored for ADCs; however, the generality of this approach is yet to be established (Axup et al., 2012).
  • Fc-containing polypeptide engineered with an acyl donor glutamine-containing tag e.g., Gin-containing peptide tags or Q- tags
  • an endogenous glutamine that are made reactive by polypeptide engineering (e.g., via amino acid deletion, insertion, substitution, or mutation on the polypeptide).
  • a transglutaminase can covalently crosslink with an amine donor agent (e.g., a small molecule comprising or attached to a reactive amine) to form a stable and homogenous population of an engineered Fc-containing polypeptide conjugate with the amine donor agent being site- specifically conjugated to the Fc-containing polypeptide through the acyl donor glutamine- containing tag or the accessible/exposed/reactive endogenous glutamine (WO 2012059882).
  • an amine donor agent e.g., a small molecule comprising or attached to a reactive amine
  • the peptides, fusion proteins and immunoconjugates as described herein may be administered as part of one or more pharmaceutical compositions.
  • pharmaceutical composition refers to a composition described herein, or pharmaceutically acceptable salts thereof, with other agents such as carriers and/or excipients.
  • the pharmaceutical compositions as provided herewith typically include a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical-Sciences, Sixteenth Edition, E.
  • W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional carrier medium is incompatible with the peptides of the present invention, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention.
  • materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatine; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil, sesame oil; olive oil; corn oil and soybean oil; glycols; such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogenfree water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium
  • the peptides, fusion proteins and immunoconjugates as described herein are particularly suitable for preparing vaccine composition.
  • the term "vaccine composition” is intended to mean a composition which can be administered to humans or to animals in order to induce an immune system response; this immune system response can result in the activation of certain cells, in particular antigen- presenting cells, T lymphocytes and B lymphocytes.
  • the vaccine composition of the present invention is thus particularly suitable for the treatment of T1DM.
  • the vaccine composition of the present invention comprises an adjuvant.
  • adjuvant can be a compound that lacks significant activity administered alone but can potentiate the activity of another therapeutic agent.
  • an adjuvant is selected from the group consisting of buffers, anti-microbial preserving agents, surfactants, antioxidants, tonic regulators, antiseptics, thickeners and viscosity improvers.
  • the adjuvant is Incomplete Freund's adjuvant (IF A) or other oil based adjuvant is present between 30-70%, preferably between 40-60%>, more preferably between 45-55%> proportion weight by weight (w/w).
  • the active ingredient i.e. peptides, fusions proteins or immunoconjugates
  • IFA or other oil based adjuvant are present in about a 50/50 weight by weight ratio.
  • the pharmaceutical composition is free of contaminants, e.g., pyrogens.
  • the active ingredient (i.e. peptides, fusions proteins or immunoconjugates) is lyophilized.
  • the oil- based carrier is a composition that includes at least 10% by weight of a natural or synthetic oil suitable for administration to a human in conjunction with a therapeutic agent is one preferred embodiment.
  • the carrier includes at least 20, 30, 50, 70, 80, 90, 95, 98, or 99% oil by weight.
  • the oil-based carrier can include less than 70, 60, 50, 40, or 20% oil by weight.
  • the oil will be in the range of 10 to 95%), preferably 20 to 90%> or 30 to 70%> oil by weight.
  • the oil should be chosen such that it provides for sustained release of a substance dispersed within it when administered to a subject.
  • Suitable oils include mineral oil (e.g., Drakeol 6 VR light mineral oil), vegetable oil, squalene, or liquid paraffin.
  • the oil-based carrier can contain more than one type of oil.
  • the oil-based carrier can include an immune stimulator, e.g., an immunostimulating glucan, but it is much preferred that the oil-based carrier does not include an immune stimulator, e.g., an immunostimulating glucan, a bacterial component, e.g., a mycobacterial component.
  • the oil-based carrier does not include an alum component.
  • Oil based carriers optionally include an emulsifier or surfactant component.
  • the emulsifier or surfactant (and the amount of emulsifier or surfactant) is chosen such that it facilitates the mixture or dispersion of a substance, e.g., an antigen preparation, with the oil.
  • An oil-based carrier can include 0.1 to 50%>, preferably 1 to 30%>, more preferably 5 to 20%> by weight of a surfactant or emulsifier.
  • emulsifiers or surfactants include Arlacel A, mannide oleate (e.g., Montanide 80-mannide monooleate), anhydrous mannitol/oleic acid ester, polyoxyethylene or polyoxypropylene.
  • An oil-based carrier or adjuvant typically consists of two components: (1) an oil, and (2) an emulsifier or surfactant, mixed with water.
  • Suitable oils and emulsifiers are known in the art.
  • the oil can be mineral oil, vegetable oil, squalene or liquid paraffin.
  • the emulsifier or surfactant can be, e.g., Arlacel A, mannide oleate, anhydrous mannitol/oleic acid ester, polyoxyethylene or polyoxypropylene.
  • Exemplary oil- based adjuvants include conventional IFA, Montanide ISA adjuvants, or Hunter's TiterMax adjuvant.
  • the adjuvant includes 20 to 95%, preferably 30 to 90%, more preferably 40 to 70%> by weight of an oil phase, and 0.1 to 50%>, preferably 1 to 30%>, more preferably 5 to 20% by weight of a surfactant or emulsifier.
  • oil-based adjuvants are described, e.g., in U.S. Pat. No. 5,814,321, U.S. Pat. No. 6,299,884, U.S. Pat. No. 6,235,282, and U.S. Pat. No. 5,976,538.
  • Montanide ISA Adjuvants are a group of oil/surfactant based adjuvants in which different surfactants are combined with either a non-metabolizable mineral oil, a metabolizable oil, or a mixture of the two. They are prepared for use as an emulsion with aqueous Ag solution.
  • the surfactants of the Montanide group undergo strict quality control to guard against contamination by any substances that could cause excessive inflammation, as has been found for some lots of Arlacel A used in Freund's adjuvant.
  • the various Montanide ISA group of adjuvants are used as water-in-oil emulsions, oil-in-water emulsions, or water-in-oil-in-water emulsions.
  • the different adjuvants accommodate different aqueous phase/oil phase ratios, because of the variety of surfactant and oil combinations.
  • Hunter's TiterMax (CytRx Corp., Norcross, Ga.) is an oil/surfactant-based adjuvant prepared as a water-in-oil emulsion in a manner similar to that used for conventional Freund's adjuvants. However, it uses a metabolizable oil (squalene) and a nonionic surfactant that has good protein antigen-binding capacity as well as adjuvant activity.
  • the adjuvant activity may relate, in part, to the surfactant's ability to activate complement and bind complement components, as this helps target the Ag to follicular dendritic cells in the spleen and lymph nodes.
  • the surfactant used in the commercially available adjuvant is one of a number of synthetic nonionic block copolymers of polyoxyethylene and polyoxypropylene developed by Hunter (Hunter et al, 1991 Vaccine 9:250-256).
  • the utilization of copolymer-coated microparticles to stabilize the emulsion permits formation of stable emulsions with less than 20% oil, an important factor in minimizing total adjuvant injected.
  • the vaccine composition of the present invention comprise at least one Toll-Like Receptor (TLR) agonist which is selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, and TLR8 agonists.
  • TLR Toll-Like Receptor
  • compositions of the present invention can be delivered to a patient using a wide variety of routes or modes of administration. Suitable routes of administration include, but are not limited to, inhalation, transdermal, oral, rectal, transmucosal, intestinal and parenteral administration, including intramuscular, subcutaneous and intravenous injections.
  • the pharmaceutical compositions e.g. vaccine composition
  • Injection is a preferred method of administration for the compositions of the current invention.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • pharmaceutical composition herein may be administered parenterally, by injection subcutaneously, or intramuscularly.
  • a preferred mode of administration is intramuscularly.
  • the Type 1 diabetes autoantigen as described herein can be given as an intramuscular injection, preferably a deep intramuscular injection, in a small volume, e.g., 1 ml.
  • the autoantigen can be administered once, or more than once. It can be given, for example, before, or after the onset of Type 1 diabetes mellitus.
  • Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension may also contain suitable stabilizers or agents, which increase the solubility of the compounds to allow for the preparation of highly, concentrated solutions.
  • the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • the active ingredient of the present invention i.e. peptides, fusion proteins and immunoconjugates as described herein
  • a therapeutically effective amount is meant a sufficient amount of the active ingredient of the present invention to treat T1DM at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts.
  • the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
  • compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated.
  • a medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, in particular from 1 mg to about 100 mg of the active ingredient.
  • An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
  • the peptides, fusions proteins and immunoconjugates as herein described are used in combination with, for example, any known therapeutic agent or method for treating T1DM.
  • known therapeutics for treating T1DM include insulin, insulin analogs, islet transplantation, stem cell therapy including PROCHYMAL®, non-insulin therapies such as IL-lbeta inhibitors including (Anakinra, Kineret®), Diamyd, alefacept (Ameviv®), anti-CD3 antibodies such as Otelixizumab, DiaPep277 (Hsp60 derived peptide), Alpha 1- Antitrypsin, Prednisone, azathioprine, Ciclosporin, El-INT (an injectable islet neogenesis therapy comprising an epidermal growth factor analog and a gastrin analog), statins including Zocor®, Simlup®, Simcard®, Simvacor®, Sitagliptin (dipeptidyl peptidas
  • the peptides, fusions proteins and immunoconjugates as herein described are used in combination with a GABA agonist.
  • GABA receptor agonists include, but are not limited to, certain barbiturates (e.g., thiopental, thiamylal, pentobarbital, secobarbital, hexobarbital, butobarbital, amobarbital, barbital, mephobarbital, phenobarbital, primidone, and the like), certain benzodiazepines (e.g., midazolam, triazolam, lometazepam, flutazolam, nitrazepam, fluritrazepam, nimetazepam, diazepam, medazepam, oxazolam, prazeam, tofisopam, rilmazafonoe, lorazepam, temazepam, oxazep
  • CTLA4 molecule is a molecule comprising a cytotoxic T-lymphocyte- associated antigen 4 (CTLA4) extracellular domain.
  • CTLA4 cytotoxic T-lymphocyte- associated antigen 4
  • the extracellular domain of CTLA4 comprises a portion of the CTLA4 protein that recognizes and binds to at least one B7 (CD80/86) antigen such as a B7 antigen expressed on B cells and on antigen presenting cells (APCs).
  • B7 CD80/86 antigen
  • APCs antigen presenting cells
  • the B-cells and APCs may be activated.
  • the extracellular domain may also include fragments or derivatives of CTLA4 that bind a B7 antigen.
  • the CTLA4 extracellular domain can also recognize and bind CD80 and/or CD86.
  • the extracellular domain may also include fragments or derivatives of CTLA4 that bind a binds CD80 and/or CD86.
  • the CTLA4 molecule may be a fusion protein, where a fusion protein is defined as one or more amino acid sequences joined together using methods well known in the art. The joined amino acid sequences thereby form one fusion protein.
  • the CTLA4 molecule contains at least a portion of an immunoglobulin, such as the Fc portion of an immunoglobulin.
  • the CTLA4 molecule is an isolated and purified CTLA4 molecule.
  • the CTLA4 molecule is a protein containing at least a portion of an immunoglobulin, such as the Fc portion of an immunoglobulin.
  • the CTLA4 molecule is an isolated and purified CTLA4 molecule.
  • the CTLA4 molecule is abatacept.
  • Abatacept is a soluble fusion protein that consists of the extracellular domain of human CTLA-4 linked to the modified Fc (hinge, CH2, and CH3 domains) portion of human immunoglobulin Gl (IgGl).
  • Abatacept is produced by recombinant DNA technology in a mammalian cell expression system. The apparent molecular weight of abatacept is 92 kilodaltons.
  • Abatacept was developed by Bristol-Myers Squibb and is disclosed, for example, in U.S. Pat. No. 5,851,795, U.S. Pat. No. 7,455,835, and U.S. Pat. Pub. 20011/311529.
  • a further object of the present invention also relates to a nucleic acid molecule encoding for a peptide or a fusion protein as herein described for use in a method for treating Type 1 Diabetes in a subject in need thereof.
  • said nucleic acid molecule is a DNA or RNA molecule, which may be included in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or a viral vector.
  • the acid nucleic molecule of the present invention can be formulated with cationic polymers including cationic liposomes.
  • Other liposomes also represent effective means to formulate and deliver self-acid nucleic molecule.
  • the DNA can be incorporated into a viral vector, viral particle, or bacterium for pharmacologic delivery.
  • Viral vectors can be infection competent, attenuated (with mutations that reduce capacity to induce disease), or replication-deficient. Methods utilizing DNA to prevent the deposition, accumulation, or activity of pathogenic self proteins may be enhanced by use of viral vectors or other delivery systems that increase humoral responses against the encoded autoantigen.
  • the DNA can be conjugated to solid supports including gold particles, polysaccharide-based supports, or other particles or beads that can be injected, inhaled, or delivered by particle bombardment (ballistic delivery).
  • solid supports including gold particles, polysaccharide-based supports, or other particles or beads that can be injected, inhaled, or delivered by particle bombardment (ballistic delivery).
  • particle bombardment ballistic delivery.
  • Methods for delivering nucleic acid preparations are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466.
  • a number of viral based systems have been developed for transfer into mammalian cells. For example, retroviral systems have been described (U.S. Patent No.
  • Adeno-associated virus (AAV) vector systems have also been developed for nucleic acid delivery.
  • AAV vectors can be readily constructed using techniques well known in the art. See, e.g. , U.S. Patent Nos. 5,173,414 and 5,139,941 ; International Publication Nos.
  • the nucleic acid molecule of the present invention is delivered without a viral vector.
  • the nucleic acid molecule can be packaged in liposomes prior to delivery to the subject. Lipid encapsulation is generally accomplished using liposomes which are able to stably bind or entrap and retain nucleic acid.
  • liposomes as carriers for delivery of nucleic acids, see, e.g. , Hug et al , Biochim. Biophys. Acta. 1097: 1-17 (1991); Straubinger et al. , in Methods of Enzymology, 101 : 512-527 (1983).
  • the nucleic acid molecule is delivered via electroporation (i.e. muscular delivery by electroporation).
  • the acid nucleic molecule is delivered by intramuscular ("IM") injection.
  • the acid nucleic molecule of the present invention is delivered intranasally, orally, subcutaneously, intradermally, intravenously, mucosally, impressed through the skin, or attached to particles delivered to or through the dermis.
  • nucleic acid molecules can be delivered into skin cells by topical application with or without liposomes or charged lipids.
  • the acid nucleic molecule is formulated in solutions containing higher quantities of Ca++, e.g., between 1 mM and 2M.
  • the acid nucleic molecule may be formulated with other cations such as zinc, aluminum, and others. Alternatively, or in addition, the acid nucleic molecule may be formulated either with a cationic polymer, cationic liposome-forming compounds, or in non- cationic liposomes. Examples of cationic liposomes for DNA delivery include liposomes generated using l,2-bis(oleoyloxy)-3-(trimethylammionio) propane (DOTAP) and other such molecules.
  • DOTAP l,2-bis(oleoyloxy)-3-(trimethylammionio) propane
  • the human preproinsulin peptide of the present invention is loaded in MHC class II multimers. Typically MHC class II multimers are well known in the art (Fierabracci A. The potential of multimer technologies in type 1 diabetes prediction strategies.
  • the HLA class II multimers according to the invention are preferably HLA-DQ8 multimers loaded with the human preproinsulin peptide of the present invention.
  • MHC class II multimers include (i) the ectopic expression of a membrane -bound MHC class II heterodimer on the surface of eukaryotic cells by use of a lipid tether (GPI anchor), see Wettstein et al, 1991, J. of Exp.
  • a recombinant MHC class II molecule which comprises (i) all or part of the extracellular portion of an MHC class II a chain; (ii) all or part of the extracellular portion of an MHC class II ⁇ chain; wherein (i) and (ii) provide a functional peptide binding domain and wherein (i) and (ii) are linked by a disulphide bond between cysteine residues located in the a2 domain of said a chain and the ⁇ 2 domain of said ⁇ chain, wherein said cysteine residues are not present in native MHC class II a2 and ⁇ 2 domains.
  • the native MHC class II molecules comprise an a- and a ⁇ -chain, both of which have transmembrane regions and belong to the immunoglobulin (Ig) superfamily.
  • the extracellular portion of each chain is composed of two domains, each consisting of approximately 90 amino acid residues, of which the two membrane distal domains, the al and ⁇ domains, form an inter-latticed ⁇ / ⁇ structure essential for the peptide binding property of the T cell epitopes.
  • the molecules of US20130171668 comprise all or part of the extracellular portion of an MHC class II a chain and all or part of the extracellular portion of an MHC class II ⁇ chain.
  • the extracellular portion of an MHC class II a chain comprises a signal sequence, a membrane distal l domain and a membrane proximal a2 domain (which forms a discrete Ig domain). There is also a spacer region between the transmembrane domain and the a2 domain.
  • the extracellular portion of an MHC class II ⁇ chain comprises a signal sequence, a membrane distal ⁇ domain, a membrane proximal ⁇ 2 domain (which forms a discrete Ig domain) and a spacer region.
  • Other examples include dextramer reagents commercially available from Immudex (Copenhagen, Denmark http://www.immudex.com/about-products/dextramer-descrip.aspx).
  • multimers thus comprise two or three or four or five or more recombinant MHC class II molecules associated with one another. Such association can be carried out using methods known and described in the art but is generally mediated via another linkage such as a linker molecule.
  • Suitable linker molecules are well known and described in the art and particularly appropriate linker molecules will have multiple binding sites to which the recombinant molecules can be attached.
  • multiple attachment molecules such as avidin and streptavidin (or any other molecule which binds biotin in a multivalent manner) may be used which each have multiple binding sites for biotin.
  • avidin and streptavidin or any other molecule which binds biotin in a multivalent manner
  • biotin incorporation of biotin into the recombinant MHC molecules by methods known in the art (e.g. using AviTag or other BirA substrates to enable enzymatic biotinylation) will allow the formation of multimers, e.g. tetramers of MHC molecules.
  • Labels can also conveniently be incorporated into multimeric forms, e.g.
  • labels include fluorochromes such as, for example, fluorescein isothiocyanate (FITC), phycoerythrin, phycocyanin or allophycocyanin.
  • the peptides and multimers as described herein are also suitable for preparing polyclonal T cells and T cell lines or clones recognizing a human preproinsulin peptide of the invention.
  • the human preproinsulin peptides of the present invention are suitable for preparing Treg cells that can be used in adoptive T cell transfer.
  • "regulatory T cells” or “Tregs” has its general meaning in the art and refers to the subset of T cells which actively suppress or tolerize activation of the immune system (e.g. reactive immune cells), maintain immune system homeostasis and/or prevent pathological self- reactivity.
  • Regulatory T cells of the present invention include cells that express e.g.
  • T regulatory cells also encompass other T cells that have suppressive function.
  • the regulatory T cells of the present invention encompass both "naturally-occurring" Tregs as well as Tregs generated in vitro.
  • Tregs of the present invention are typically capable of suppressing a variety of cells, such as other T cells (e.g. effector T cells), B cells (e.g. effector B cells) and/or antigen presenting cells (e.g. effector APCs such as monocytes and dendritic cells).
  • Tregs exert their function via TCR/MHC class II interaction following cell to cell contact, however, Tregs may also suppress effector cells by secretion of immunosuppressive cytokines (e.g. TGF-beta and IL-10).
  • immunosuppressive cytokines e.g. TGF-beta and IL-10.
  • the T cells are prepared according to the method described in WO 2010119307.
  • the a method for producing T cells displaying specific immunological properties from a subject comprising the steps consisting of culturing said blood or PBMC sample in an appropriate culture medium which comprises an amount of Granulocyte/Macrophage Colony-Stimulating Factor (GM-CSF) and/or IL-4 and/or FMS-like tyrosine kinase 3 (Flt-3) ligand and/or IL-lbeta and an amount of a least human preproinsulin peptide of the present invention and isolating at least one T cell displaying a specificity for the human preproinsulin peptide.
  • Said specific immunological properties include, but are not limited to, recognition by the isolated T cells of the peptide.
  • T cells producing IL- 10 can be used as regulatory T cells for the treatment T1DM by adoptive T cell transfer.
  • the person skilled in the art is also familiar with methods suitable to isolate said Ag-specific T cells in a viable state based on different immunological properties.
  • selection of IL-10- producing T cells may be obtained by Miltenyi cytokine capture assays.
  • said T cells can be isolated by means of MHC Class I or Class II multimers [Mallone et al, Diabetes 53:971, 2004; Mallone et al, Blood 106:2798, 2005; Skowera et al, J.Clin.Invest.
  • the peptides as described herein are also suitable for detecting autoreactive T cells specific for a human preproinsulin polypeptide of the invention. Therefore the human preproinsulin polypeptides of the present invention are thus particularly suitable for diagnosing T1DM in a subject.
  • the diagnostic method of the present invention is performed as described in WO 2010119307.
  • the method comprises the steps consisting of culturing a blood or PBMC sample obtained from the subject in an appropriate culture medium which comprises an amount of Granulocyte/Macrophage Colony- Stimulating Factor (GM-CSF) and/or IL-4 and/or FMS-like tyrosine kinase 3 (Flt-3) ligand and/or IL-lbeta and an amount of a least human preproinsulin peptide of the present invention and detecing at least one T cell displaying a specificity for the human preproinsulin peptide.
  • GM-CSF Granulocyte/Macrophage Colony- Stimulating Factor
  • Flt-3 FMS-like tyrosine kinase 3
  • the diagnostic method of the present invention contemplated use of a human preproinsulin peptide of the present invention that is loaded on multimers as described above, so that the isolated CD8+ T cells from the subject are bringing into contact with said tetramers. There is no requirement for in vitro T cell activation or expansion. Following binding, and washing of the T cells to remove unbound or non-specifically bound multimer, the number of CD8+ cells binding specifically to the HLA-peptide tetramer may be quantified by standard flow cytometry methods, such as, for example, using a FACSCalibur Flow cytometer (Becton Dickinson).
  • the multimers can also be attached to paramagnetic particles or magnetic beads to facilitate removal of non-specifically bound reporter and cell sorting.
  • Such particles are readily available from commercial sources (eg. Beckman Coulter, Inc., San Diego, Calif , USA). Multimer staining does not kill the labeled cells; therefore cell integrity is maintained for further analysis.
  • FIGURES are a diagrammatic representation of FIGURES.
  • FIG. 1 Ex vivo proliferative responses to hPPI in YES-RIP-hB7.1 mice. Full- length hPPI and a series of hPPI peptides covering the whole molecule were tested, hPPIi-15, hPPIi6-3o, hPPl2o-35, hPPl25-4o and I1PPI33-47.
  • Splenocytes from diabetic YES-RIP-hB7.1 ( ⁇ ), non-diabetic YES-RIP-hB7.1 ( ⁇ ) and YES mice (O) were re-stimulated in vitro for 3 days and proliferation was evaluated by measuring BrdU incorporation, ns, non-significant, * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001 ; Mann- Whitney test.
  • mice were obtained by crossing the hINS mouse that lacks the expression of the two mINS genes, expresses the human insulin gene (hINS) as a transgene under the control of 353 bp of the human insulin gene promoter on a DBA/CBA (H-2 k , IA k ) mix genetic background (kindly given by J Jami and R Joshi, Institut Cochin, Paris) 15 and a double transgenic HLA- A*02:01/HLA-DQ8 mouse with C57BL/6J as main genetic background.
  • hINS human insulin gene
  • the double transgenic HLA-A*02:01/HLA-DQ8 mouse was obtained (F Lemonnier) by crossing a H-2D b , mouse B2- microglobuline mfilni) double KO mice lacking the expression of mouse H-2 class I molecules, expressing under the control of HLA-A*02:01 gene promoter a unique species of MHC class I molecules (a chimeric HLA-A*02:01 HHD monochain containing the HLA-A*02:01 al and a2 domains and the H2-D b a3 domain linked to the human ⁇ 2 ⁇ by a 15 -residue linker peptide) 16 with a mouse lacking the expression of the mouse IA b and IE b genes 11 , but expressing the HLA-DQ8 DQA1 *03:01 a and DQB1 *03:02 ⁇ chains under the control of their natural MHC class II promoter 18 .
  • YES-RIP-hB7.1 mice were obtained by lentiviral transgenesis at Institut Pasteur, Paris.
  • the RIP-hB7.1 transgene was inserted in a recombinant lentiviral vector (LV- RIP-hB7.1; 4,640 bp) derived from Human Immunodefiency Virus as previously reported 19 .
  • Retroviral pseudotypes were injected in fertilized eggs obtained from superovulated YES female mice mated with YES-male mice. Fertile and transduced eggs reimplanted into pseudopregnant C57BL/6-CBA Fl mice. All mice were maintained under specific pathogen- free conditions at the Hospital Cochin-St Vincent de Paul animal facility, and experimental studies were performed in accordance with the Institutional Animal Care and Use Guidelines and accredited by the ethic committee under the number CEEA34.CB.024.11.
  • Genotyping Real time quantitative PCR based on 3 -point dilutions using QuantiTect SybrGreen PCR Kit (Qiagen) on LC480 (Roche) were performed to select mice carrying a single allele of mlNSl and/or mINS2 genes for further crosses.
  • Roche® Probes were optimised after performing a preliminary Comparative Genomic Hybridization (CGH) experiment, ⁇ g of each genomic DNA was used for sample library preparation (Illumina TruSeq DNA Sample Preparation Kit, ROCHE) and was amplified using Ligated-Mediated PCR (LM-PCR) in preparation for hybridization to the SeqCap EZ Developer Library. Captured multiplex DNA samples were recovered, amplified and captured-sequences reads on a Roche 454 GS-FLX System at ICM, Paris. All reads were analysed by IGV software with C57BL/6NJ genome as reference. Captured sequences were filtered by complete alignment with the primary sequence LV-RIP-hB7.1 sequence in BLASTN.
  • CGH Comparative Genomic Hybridization
  • RNA extractions were performed on liver, pancreas, thymus, spleen, kidney and inguinal lymph node with RNeasy Mini Prep and Dnasel treatment (Qiagen).
  • RNeasy Mini Prep and Dnasel treatment Qiagen.
  • One-step real time RT-PCR were performed with RT-PCR one-step QuantiTect SYBRGeen RT-PCR Kit (Qiagen) and passed on Light Cycler LC480 (Roche).
  • Spleens, thymus, inguinal lymph nodes, lymphocytes and bone marrow cells were re- suspended at 10 6 cells/ml in RPMI 1640 / FCS 10 and stained with the following antibodies: anti-mouse-CD38-PE, anti-mouse-CD38-APC, anti-mouse-CD8a-PerCP, anti-mouse-CD4- efluor450, anti-mouse-CD44-FITC, anti-mouse-CD 19-APC, anti-mouse-CDl lc-APC, anti- mouse-CDl lb-efluor450, anti-mouse-CDl lb-APC, anti-human- 2microglobulin-FITC (clone TLI99, BD-Biosciences), anti-human-HLA-DQ-Biot (Hybridoma SVPL3), SAV-FITC, anti- H2-D k , anti-IA k , anti -mous
  • Immunizations were performed by injecting YES mice with either of Keyhole Limpet Hemocyanin (KLH), 50 ⁇ g in complete Freund's adjuvant (CFA), or 100 ⁇ g denaturised (3 min at 94°C) hINS, or 100 ⁇ g HLA-A*02:01 -restricted Influenza matrix protein 2 peptide GILGFVFTL (MatA2 5 8-6e) in CFA, or 100 ⁇ g of hPPI 6 -i4, hPPIis-24 or hPPI 3 3-42 peptides with 140 ⁇ g HLA-DQ8-restricted-helper Nef peptide Cyst 6 6-97 in incomplete Freund's adjuvant (IF A); or 100 ⁇ g hPPI or 50 ⁇ g HLA-DQ8-restricted hPPIi_ 3 i, hPPIi-is, hPPIi 6 -3o, hPPIis-3o, hPPl2o-35, hPPI 2
  • DNA immunizations were performed by pre -treating YES mice with 10 ⁇ cardiotoxin (Carloxan Lab) intramuscularly, and with inject i.m 5 days later 100 ⁇ g of pCMV-BlO-hPPI DNA vector or pCMV-BlO-CTL DNA vector as control followed by a boost two weeks after. Mice were anesthetized during each injection. Control immunizations were realized injecting PBS IX under the same conditions.
  • IFNy-ELIspot assays were performed as previously reported 20 . Spots were counted using Bioreader 5000 Pro-SF (Bio-Sys GmbH). Data are mean of triplicate wells and expressed as spot-forming cells (SFC) per 10 6 cells evaluating the background IFNy responses from triplicates in the absence of peptide. Positive controls were triplicates of cells stimulated by 1 ⁇ g/ml ConA (Sigma- Aldrich). In some experiments, the response was inhibited using 50 ⁇ g/ml anti-HLA-A*02:01 antibody (BB7.2) for 20 min.
  • SFC spot-forming cells
  • Immunostainings were performed on formalin-fixed paraffin pancreas sections that were deparaffinised in Xylene and dehydrated by Ethanol. After washing, antigen retrieval was realized by hot incubation, followed by permeabilization (20 min in PBS lX/0,4% TritonXl 00) and saturation (20 min PBS1X/1% Horse Serum) before immunostaining with biotynilized rat anti-human CD3s (ABDSerotec) and polyclonal rabbit anti-glucagon (DAKO) antibodies overnight.
  • Tetramers (TMrs) associating the chimeric HLA-A*02:01-HHD to hPPI peptides were obtained from the NIH Tetramer Core Facility. Stainings were performed as previously reported 21 . Incubation of cells with TMRs was performed for 30 min at RT. The following antibodies were used: anti-CD3s-AlexaFluo700, anti-CD8a-APC, anti-CD 19-PerCP-Cy5 and anti- CD 14-PercP-Cy5 antibodies (BD/Pharmingen and eBioscience). Cells were analysed with a BD LSR Fortessa flow cytometer and the Flow Jo software (Tree Star Inc).
  • CD8a-APC-labeled, anti-CD 19-PerCP-Cy5.5-labeled and anti-CD 14-PercP- Cy5.5-labeled antibodies were then added for 15 min at 4°C.
  • Lymphocytes were gated according to forward/side scatter profiles.
  • CD8 + /TMr + T-cells were sorted with a FACSAria II at 1 cell/well into 96-well PCR plates containing 5 ⁇ 1 PBS IX treated with diethyl pyrocarbonate (DEPC) (Sigma) and immediately frozen at -80°C.
  • DEPC diethyl pyrocarbonate
  • Islets of Langerhans were isolated as described previously 22 . Once handpicked, islets were dissociated with Cell Dissociation Solution Non-Enzymatic (SIGMA) 30 min at RT. Cells were filtered and stained with anti-mouse-HLA-DQ-FITC (BD Biosciences), anti-human-B2m- PE (BD Biosciences), guinea pig-anti-bovine -insulin (DAKO), anti-guinea pig-Biot and SAV- PE-Cy7, anti-mouse CD4-AlexaFluo700, anti-mouse-CD8-APC anti-mouse-CD 19- PercpCy5.5 and anti-mouse-TCRB-efluo450 antibodies (e-biosciences). Acquisition was done with a BD FACSAria flow cytometer and analyses using Flow Jo software (Tree Star Inc). Infiltrate staining
  • Handpicked islets were pooled in 24-well-plate well for 24h in RPMI supplemented with 10% FCS, 1% penicillin-streptomycin and 5.10 ⁇ 5 M B-mercapto ethanol allowing a spontaneous extrusion of infiltrating lymphocytes.
  • Infiltrating lymphocytes were recovered and stained with anti-mouse-CD38-AlexaFluo700, anti-mouse-CD 19-PerCP-Cy5, anti-mouse- CD1 lb-efluo450 and anti-mouse-CD 1 lc-APC antibodies, anti-mouse-CD 103-FITC, anti- HLA-DQ-Biot (SVPL3 Hybridoma), SAV-BrillantViolet650 (Biolegend), anti-mouse-CD4- PE, anti-mouse-CD8a-PE-Cy7 antibodies from e-biosciences. Acquisition was done with a BD FACSAria flow cytometer and analyses using Flow Jo software (Tree Star). Results
  • HLA-A*02:01/HHD was further confirmed by flow cytometry on immune cell subsets in spleen, blood, and iLN.
  • the expression of HLA- A*02:01 was comparable in YES mice and in parental HLA-A*02:01/HLA-DQ8 double transgenic mice (data not shown).
  • HLA-A*02:01 In the basal state, a lower percentage of spleen B and DCs cells and of blood CD4 + T cells expressed HLA-A*02:01 in YES mice as compared to the corresponding cells subsets that expressed H2-D b in C57BL/6 mice. In addition, an increased percentage of iLN macrophages expressed HLA-A*02:01 in YES mice.
  • the expression of HLA-DQ8 was comparable in YES and in parental HLA-A*02:01/HLA-DQ8 double transgenic mice (data not shown). As for HLA-A*02:01, several differences were seen when comparing the expression of HLA-DQ8 on immune cells in YES mice to that of H-2IA b in C57BL/6 mice. In the basal state, a lower percentage of spleen and blood DCs and of spleen macrophages expressed HLA-DQ8 as compared to the corresponding subsets that expressed H- 2IA
  • the dominant CD3 +low T-cell subset which is mainly constituted of CD3 W CD4 + CD8 + double positive cells was comparable in YES and C57BL/6 mice (92% and 88%, respectively).
  • Respective numbers of spleen B and T-cells were 34,4xl0 6 ⁇ 8,1 and 6,9xl0 6 ⁇ 1,6, respectively, in YES mice, as compared to 45xl0 6 ⁇ 6,9 and 21,7xl0 6 ⁇ 3,2, respectively, in C57BL/6 mice.
  • the total number of T-cells was 16,2xl0 6 ⁇ 2,5 in YES mice, as compared to 29,4xl0 6 ⁇ 4 in C57BL/6 mice.
  • CD4 + CD25 + Foxp3 + regulatory T-cells which were not significantly different in YES and in C57BL/6 mice.
  • NC Native cells
  • DN3 CD44 " CD25 +
  • DN2 CD44 + CD25 + , 5.2%) pro-T cells
  • DN4 CD44 + CD25 " , 16.3% pre-T cells as compared to 3.4%, 32.2%, 3.1% and 61.2%, respectively in hINS transgenic mice.
  • DN cells were CD62L " , suggesting that they correspond to immature T-cells blocked at an immature stage and devoid of migratory capacity to the peripheral lymph node.
  • mice showed differences in the composition of their peripheral immune system, we evaluated immune responses to conventional antigens.
  • spleen cells from immunized mice were shown cytotoxic against MatA258-66-pulsed target cells. Cytotoxicity was inhibited by pre -incubating target cells with an anti-HLA-A*02:01 antibody (BB7.2).
  • YES mice were immunized against a series of hPPI peptides covering a large part of the hPPI sequence: hPPIi- 15, hPPIie-30, hPPIis-30, hPPI 2 o-35, hPPI 2 5-4o, hPPl33-47, hPPIs5-7o, hPPI 8 o-9vand I1PPI92-110.
  • Proliferative responses were obtained against B-chain peptides I1PPI25-40 and I1PPI33-47 and against the leader sequence-B-chain junction I1PPI20-35.
  • hINS binding to the mouse insulin receptor is in the normal range 24
  • the secretion of hINS was expected to allow normoglycemia in YES mice.
  • YES mice maintained a normal weight in the long-term range when compared to hINS transgenic and HLA-A*02:01/HLA- DQ8 double transgenic parental strains.
  • the overall morphology of the islets of Langerhans was comparable in YES and HLA-A*02:01/HLA-DQ8 parental mice, as were insulin, glucagon and PDX1 stainings.
  • the ⁇ -cell mass was comparable in YES mice and parental hINS and HLA-A*02:01/HLA-DQ8 mice at 15 weeks of age.
  • mice remained normoglycemic up to more than one year of age and showed no evidence of insulitis within the same age range, indicating that they were basically immune tolerant to the hINS transgene.
  • TID human insulin gene promoter
  • infiltrating cells were composed of 37.8 to 84.7% CD3 + T-cells, of which 65.7-84.2% were CD8 + T-cells and 9.9-24.0% were CD4 + T-cells.
  • Other cells included 4.1-7.3% CD4 CD8 hi CDl lc + CDl lb-, 3.4-11.0% CD4 CD8 l0 CDl lc + CDl lb + DCs and 0.8-2.9% CD19 + cells.
  • mice carrying the 1 lqB5 transgene insertion in association with either the 19qA or 14qE5 insertions, 7/9 and 17/35, respectively.
  • mice carrying the 1 lqB5 transgene insertion in association with either the 19qA or 14qE5 insertions, 7/9 and 17/35, respectively.
  • mice carrying the l lqB5, 14qE5 and 19qA insertions developed diabetes, but corresponding mice were obtained early along the F-36 lineage and were thus unlikely to carry homozygous LV-RIP- hB7.
  • the age at onset of hyperglycaemia was variable from one mouse to another, spanning from 6 weeks to 51 weeks independently of the lineage expansion and diabetes was definitive in all mice observed.
  • hPPI-SPECIFIC T-LYMPHOCYTES ARE DETECTED IN YES-RIP-hB7.1 MICE
  • IFNy-ELIspot assay we observed that most selected hPPI peptides were recognized by T-cells obtained from diabetic YES-RIP-hB7.1 mice. A significant response was detected against hPPl6-i4, hPPIi5-24, and I1PPI34-42.
  • mice When considering responses that were over the mean ⁇ 3SD of responses seen in YES mice as controls, 56.2% diabetic YES-RIP-hB7.1 mice responded to hPPl6-i4, hPPIi5-24 and I1PPI34-42. In addition, individual responses were observed to hPPb-n (43.7%), hPPl3o-39 (6.2%), hPPI 3 3-42 (18.7%), hPPI 42 -5i (37.5%) and hPPIioi-109 (12.5%) although they did not reach statistical significance in the whole mouse population analysed (Table 2).
  • YES-RIP-hB7.1 diabetic mice showed a positive response, defined as over mean ⁇ 3SD of responses seen in YES mice, against at least one hPPI peptide (Table 2).
  • insulitis was detected in non-diabetic YES- RIP-hB7.1 mice, although to a lower extent than in diabetic YES-RIP-hB7.1 mice.
  • mice Individual non-diabetic YES-RIP-hB7.1 mice showed a positive IFNy-ELIspot response, defined as previously, to either hPPh-n (1/6), hPPIe-i4 (2/6), hPPIis-24 (0/6), hPPbo-39 (2/6), hPPI 3 3-42 (1/6), hPPI 34 -42 (1/6), hPPl42-5i (1/6) and hPPIioi-109 (1/6), thus 50% non-diabetic YES-RIP-hB7.1 mice showed a response to at least one hPPI peptide (Table 2).
  • CD8 + T-cells were seen in 70.6% YES-RIP-hB7.1 mice against hPPl6-i4, hPPIis-24 or I1PPI33-42, which does not preclude that expansions driven by other hPPI peptides may have occurred.
  • Single hPPI-specific CD8 + T-cells showed gene expression profiles that were characteristic of memory T-cells.
  • hPPI-specific CD8 + T-cells (59.38%) from diabetic YES-RIP-hB7.1 mice expressed GzmA, among which 100% co-expressed Ccr7, while only 4.33% hPPI-specific CD8 + T-cells from YES-RIP-hB7.1 non-diabetic mice or YES control mice expressed GzmA, of which ⁇ . ⁇ % co-expressed Ccr7, suggesting a na ' ive T-cells profile of most hPPI-specific CD8 + T-cells in non-diabetic mice.
  • HLA-DQ8-restricted T cell responses to hPPI in YES-RIP-hB7.1 mice.
  • a proliferation assay Using a proliferation assay, a significant response to full-length hPPI was detected in diabetic and non-diabetic YES-RIP-hB7.1 mice, as compared to YES mice.
  • hPPI regions were located in the leader sequence, at the leader sequence-B chain junction and within the B chain ( Figure 1) as indicated by significant T-cell proliferations detected in the presence of hPPIi-15, I1PPI16-30, hPPI 20-35 , I1PPI25-40 and hPPl33-47 peptides in YES-RIP-hB7.1 diabetic mice as compared to YES controls and similar proliferative responses, although not significant, observed in non-diabetic YES-RIP-hB7.1 mice.
  • CD8 + T-cell responses individual responses were observed against peptides covering the whole hPPI sequence.
  • antigen-specific immunotherapy has failed in preserving ⁇ -cells from further destruction in recent-onset T 1 D patients or in preventing T 1 D in autoantibody-positive subj ects at risk for its development.
  • the autoantigen dose, the delivery route, the stage of the disease process at which immunotherapy is applied and the use of peptides rather than full-length autoantigens remain difficult issues in the human.
  • preclinical models that would allow direct translation of experimentally-defined peptides in human are presently lacking and the NOD mouse shows a unique phenotype that is unlikely to summarize T1D heterogeneity.
  • mice We developed a new model in which we introgressed genes encoding for HLA- A*02:01, HLA-DQ8 and the human insulin genes into mice that were deficient for murine class I, class II and insulin genes.
  • Mice carrying the same hINS transgene have been reported as presenting with normal insulin levels and glucose homeostasis 25 .
  • YES mice indeed remain normoglycemic up to one year of follow up, maintain normoglycemia to an intraperitoneal glucose challenge in the long-term range, have a normal ⁇ -cell mass and show a glucose response to insulin that is in the range of control mice up to 6 months of age.
  • mice express two insulin genes located on chromosome 19 and chromosome 2, respectively.
  • Diabetes has been observed at several crosses in males expressing a single mINS 1 allele, as reported on the NOD mouse background 26 . Indeed, we observed acute diabetes in male mice at some crosses in which mice expressed a single mINSI allele. However, diabetes was not observed at further crosses and in mice with complete lack of murine insulin genes that express two copies of the hINS transgene. YES mice are able to mount normal immune responses to conventional antigens despite showing an inverted CD4 + /CD8 + T-cell ratio, presumably due to a lower efficiency of class II HLA-DQ8 than class I HLA-A*02:01 to select in the thymus and maintain in periphery the HLA-restricted T-cells.
  • the YES immune phenotype is basically identical to that of the parental HLA-A*02:01/HLA-DQ8 mice strain.
  • YES mice remain tolerant to hPPI and to islets, including the human insulin transgene, and remain insulitis-free in the long-term range.
  • induction of hB7.1 expression on ⁇ -cells in YES mice has been obtained by injecting a HIV-derived recombinant lentiviral vector in which a RIP-hB7.1 transgene has been inserted.
  • Diabetic YES-RIP-hB7.1 mice showed extensive insulitis that was composed of both CD8 + and CD4 + infiltrating T-cells, and of CDl lc + cells among which over 38% CD4 CD8 hi CDl lc + CDl lb " lymphoid DCs (data not shown). Insulitis was also detected in non-diabetic YES-RIP-hB7.1 mice, although to a lower extent than in diabetic mice, with a minority of mice showing extensive insulitis, presumably at an autoimmune stage that was close to diabetes. The T-cell infiltrate was predominantly composed of CD8 + T-cells in non-diabetic YES-RIP-hB7.1 mice.
  • a CD8 + T-cell response was observed against a wide array of hPPI epitopes using an IFNy- ELIspot assay, with a predominant response to two leader sequence peptides, hPPl6 i4 and hPPIi5-24, and to the B chain peptide I1PPI34-42. But overall, YES mice showed responses to a diverse peptide repertoire and individual mice showed responses to up to 3 peptides. Responses were observed against epitopes spanning the whole hPPI sequence. CD8 + T-cell responses were also observed in a minority of non-diabetic YES-PJP-hB7.1 mice.
  • HLA-A*02:01 NOD transgenic mouse responses have been reported against peptides that were largely overlapping with the HLA-A*02:01 -restricted peptides that we have identified, in particular hPPIs-i i and hPPI 5 -i4 28 .
  • HLA-A*02:01 -restricted TMrs we detected expansions of CD8 + T-cells against the three dominants HLA-A*02:01 -restricted hPPI peptides.
  • CD8 + T-cells Fifty five to 95% CD8 + T-cells were shown to express ccr7, suggesting that CD8 + T-cells were mostly central memory cells in diabetic YES-RIP-hB7.1 mice, while CD8 + T-cells mostly showed a naive phenotype in non-diabetic YES-RIP-hB7.1 mice, as confirmed by increased expression of Foxol.
  • HLA-DQ8-restricted CD4 + T-cell responses were also detected against full-length hPPI. A majority of responses were seen against a region overlapping the B-chain and B-chain- C-peptide junction.
  • HLA-A*02:01/HLA-DQ8 double transgenic mice diabetes was largely CD4 + T-cell-independent as in transgenic mice that lacked the expression of class II molecules and CD4 + T-cells 34 .
  • the hPPI epitopes that were identified in non-diabetic and diabetic YES-RIP-hB7.1 mice are likely to be involved in the human disease.
  • CD8 + T-cells were detected against a wide array of epitopes covering the hPPI sequence. The dominant responses were observed against the same epitopes as those that we previously identified in the human. In the case of the two leader sequence epitopes, i.e.
  • hPPl6 i4 and hPPIis-24 we and others have previously provided evidence that they are naturally processed as hPPl6 i4, I1PPI15-24, and hPPl34-42-specific CD8 + T-cells are cytotoxic either to hPPI-transfected HLA-A*02:01-P815 target cells 23 or to human HLA-A*02:01 islets 35 .
  • the repertoire of hPPI epitopes that were recognized by CD8 + T- cells was very diverse. As we previously reported in the human, there was no correlation between the affinity of peptides for soluble HLA-A*02:01 and the prevalence of CD8 + T-cell responses in diabetic mice 19 ' 20 ' 23 .
  • HLA-DQ8-restricted hPPI epitopes have been ill defined in the human.
  • the dominant responses that were characterized by immunization of YES mice against hPPI peptides were comparable to the most frequent responses detected in diabetic YES-RIP-hB7.1 mice and corresponded to hPPI 20-35 , 11PPI25-40 and I1PPI33-47.
  • I-A g7 -restricted and HLA-DQ8-restricted T-cell responses were observed in the NOD mouse 10 ' 36 and in the human 10 against the B-chain peptide B9-23, that is identical to I1PPI33-47.
  • Epitopes located in the C-peptide region have also been characterized as recognized by T cell clones obtained from patients with T1D 37 .
  • the proliferative responses to I1PPI20-35 and I1PPI25-40 that overlap the hPPI leader sequence peptide and B-chain have not been previously reported.
  • mice showed responses to hPPIi-15, hPPIi6-3o, hPPI 5 5-7o and I1PPI92-110 spanning all over the hPPI sequence.
  • CD4 + T-cell responses have been detected against hPPIi-24 and I1PPI44-63 but not against I1PPI33-47 in conventional HLA-DQ8 transgenic mice 38 .
  • Such responses were not observed with YES and YES-RIP-hB7.1 mice.
  • Such marked differences might be related to the fact that by contrast with YES and YES-RIP-hB7.1 mice, conventional HLA-DQ8 transgenic mice expressed murine insulin genes.
  • the I1PPI33-47 sequence only differs from mINSI by a S/P substitution at position 33 and is identical to the mINS2 sequence.
  • I1PPI20-35 and I1PPI25-40 differ by 2 positions at hPPl27 and I1PPI33 from mINSI and by one position at I1PPI27 from mINS2, respectively.
  • differences between YES or YES-RIP-hB7.1 mice and parental HLA-DQ8 transgenic mice which have not been reported to respond to I1PPI33-47, possibly result from differences at the selection level in the thymus.
  • I1PPI25-40 and I1PPI33-47 both carry a glutamic acid at position 37 and 45, respectively, which are frequent anchor residues with the HLA-DQ8 P9 binding pocket 39 ⁇ 40 .
  • the YES-RIP-hB7.1 mouse is a new TID model that will allow evaluating hPPI-based immunotherapy that may directly apply to the human.
  • the YES mouse does not develop spontaneous diabetes, but we have preliminary evidence that TID is possibly induced in YES mice following immunization against recombinant hPPI in combination with the injection of TLR agonists.
  • the NOD mouse the YES mouse does not develop spontaneous diabetes, probably due to the absence of selection of TID susceptibility genes other than the HLA-DQ8 gene. Deciphering mechanisms of induction of TID in the YES model may thus prove informative as referred to the search for environmental factors that may combine to allow breaking of immune tolerance to the islets of Langerhans in the human.
  • Identifying peptides derived from autoantigens and that are presented to T lymphocytes, especially CD4+ T-cells, is a key issue in type 1 diabetes. It will open new avenues in setting up assays that will be used as tests in diabetes diagnosis and in developing strategies to induce immune tolerance to prevent type 1 diabetes in prediabetic individuals or dampen the autoimmune process in diabetic patients. Characterization of peptides derived from human insulin or its precursors (human preproinsulin, hPPI) is especially important considering the direct (Thebaut-Baumont et al. J Clin Invest 2003) and indirect evidence that it is the key autoantigen in the disease process. The YES mouse that we now report will be instrumental in both avenues.
  • HLA*0201 It has allowed us to define peptides that are recognized by CD8 + T cells upon their presentation by the class I allele HLA*0201, that is the most common in Caucasians and shows a genetic association with type 1 diabetes (see Table 3 as above mentioned).
  • HLA* 0201 -restricted PPI peptides that we identified in YES mice that develop autoimmune diabetes upon expression of the T-cell coactivator hB7.1 under the control of the rat insulin promotor (RIP), in particular two peptides located in the leader sequence and a peptide located in the insulin B chain, all were confirmed as relevant to the human disease whatever the detection techniques that we used in the human: either ⁇ -interferon elispot assays or peptide -HLA* 0201 tetramers that both detected an increased number of peptide-specific CD8 + T cells in the human (Luce et al.
  • HLA-DQ8 transgenic mice are highly susceptible to collagen-induced arthritis: a novel model for human polyarthritis. Journal of Experimental Medicine 183, 27-37 (1996).
  • Costimulator B7-1 confers antigen- presenting-cell function to parenchymal tissue and in conjunction with tumor necrosis factor alpha leads to autoimmunity in transgenic mice. Proc Natl Acad Sci U S A 91, 5138-5142 (1994).
  • CTLs are targeted to kill beta cells in patients with type 1 diabetes through recognition of a glucose-regulated preproinsulin epitope. / Clin Invest 118, 3390-3402 (2008).

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Abstract

La présente invention concerne des procédés de diagnostic et de traitement du diabète de type I. La présente invention concerne en particulier un peptide préproinsulinique humain choisi dans le groupe comprenant : un peptide constitué de huit acides aminés consécutifs choisis dans la séquence allant du résidu d'acide aminé en position 1 au résidu d'acide aminé en position 15 dans la SEQ ID NO : 1 (« hPPI1-15 ») ; un peptide constitué de huit acides aminés consécutifs choisis dans la séquence allant du résidu d'acide aminé en position 16 au résidu d'acide aminé en position 30 dans la SEQ ID NO : 1 (« hPPI16-30 ») ; un peptide constitué de huit acides aminés consécutifs choisis dans la séquence allant du résidu d'acide aminé en position 20 au résidu d'acide aminé en position 35 dans la SEQ ID NO : 1 (« hPPI20-35 ») ; un peptide constitué de huit acides aminés consécutifs choisis dans la séquence allant du résidu d'acide aminé en position 25 au résidu d'acide aminé en position 40 dans la SEQ ID NO : 1 (« hPPI25-40 ») ; un peptide constitué de huit acides aminés consécutifs choisis dans la séquence allant du résidu d'acide aminé en position 80 au résidu d'acide aminé en position 97 dans la SEQ ID NO : 1 (« hPPI80-97 ») ; un peptide constitué de huit acides aminés consécutifs choisis dans la séquence allant du résidu d'acide aminé en position 92 au résidu d'acide aminé en position 110 dans la SEQ ID NO : 1 (« hPPI92-110 »).
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019246408A3 (fr) * 2018-06-22 2020-02-06 Baylor College Of Medicine Immunothérapie à base de peptide lié à un anticorps pour le diabète de type 1
WO2020056037A1 (fr) * 2018-09-13 2020-03-19 Board Of Regents Of The University Of Nebraska Marqueurs biologiques destinés au diabète de type 1
WO2022264088A1 (fr) * 2021-06-17 2022-12-22 Phaim Pharma Ltd. Thérapie cellulaire individualisée à l'aide de lymphocytes t régulateurs spécifiques d'antigènes issus de patients

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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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DATABASE Geneseq [online] 18 July 2013 (2013-07-18), "Human insulin A-chain (residues 90-110), SEQ ID 6.", XP002742581, retrieved from EBI accession no. GSP:BAP08311 Database accession no. BAP08311 *
DATABASE Geneseq [online] 27 May 2010 (2010-05-27), "Human insulin B chain peptide, SEQ ID 8.", XP002742580, retrieved from EBI accession no. GSP:AXX42529 Database accession no. AXX42529 *
RAJU RAGHAVANPILLAI ET AL: "T cell recognition of human pre-proinsulin peptides depends on the polymorphism at HLA DQ locus: A study using HLA DQ8 and DQ6 transgenic mice", HUMAN IMMUNOLOGY, NEW YORK, NY, US, vol. 58, no. 1, 1 November 1997 (1997-11-01), pages 21 - 29, XP002677789, ISSN: 0198-8859 *
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019246408A3 (fr) * 2018-06-22 2020-02-06 Baylor College Of Medicine Immunothérapie à base de peptide lié à un anticorps pour le diabète de type 1
WO2020056037A1 (fr) * 2018-09-13 2020-03-19 Board Of Regents Of The University Of Nebraska Marqueurs biologiques destinés au diabète de type 1
WO2022264088A1 (fr) * 2021-06-17 2022-12-22 Phaim Pharma Ltd. Thérapie cellulaire individualisée à l'aide de lymphocytes t régulateurs spécifiques d'antigènes issus de patients

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