WO2013160865A1 - Protéines hybrides d'immuno-tolérisation pour le traitement de la sclérose en plaques - Google Patents

Protéines hybrides d'immuno-tolérisation pour le traitement de la sclérose en plaques Download PDF

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WO2013160865A1
WO2013160865A1 PCT/IB2013/053278 IB2013053278W WO2013160865A1 WO 2013160865 A1 WO2013160865 A1 WO 2013160865A1 IB 2013053278 W IB2013053278 W IB 2013053278W WO 2013160865 A1 WO2013160865 A1 WO 2013160865A1
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mog
plp
immunotolerizing
mbp
cells
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Nils Lycke
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Toleranzia Ab
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    • 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
    • A61K39/0007—Nervous system antigens; Prions
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]

Definitions

  • the present invention relates to new immunotolerizing fusion proteins comprising a mutant of the ADP-ribosylating Al-subunit of the cholera toxin (CTA1), one or more autoantigens associated with an autoimmune demyelinating disease, in particular multiple sclerosis, and a peptide capable of binding to a specific cellular receptor.
  • CTA1 ADP-ribosylating Al-subunit of the cholera toxin
  • Autoimmune disease is any disease caused by immune cells that become misdirected at healthy cells and/or tissues of the body. Autoimmune disease affects 3% of the U.S. population, and likely a similar percentage of the industrialized world population (Jacobson et al., Clin Immunol Immunopathol 84: 223-43, 1997). Autoimmune diseases are characterized by T and B lymphocytes that aberrantly target self -proteins, -polypeptides, - peptides, and/or other self-molecules, causing injury and or malfunction of an organ, tissue, or cell-type within the body (for example, pancreas, brain, thyroid or
  • Autoimmune diseases include diseases that affect specific tissues, as well as diseases that can affect multiple tissues. For some diseases, this may, in part, depend on whether the autoimmune responses are directed to an antigen confined to a particular tissue, or to an antigen that is widely distributed in the body.
  • the characteristic feature of tissue-specific autoimmunity is the selective targeting of a single tissue or individual cell type. Nevertheless, certain autoimmune diseases that target ubiquitous self-proteins can also affect specific tissues. For example, in polymyositis, the autoimmune response targets the ubiquitous protein histidyl-tRNA synthetase, yet the clinical manifestations primarily involve autoimmune destruction of muscle.
  • the immune system employs a highly complex mechanism designed to generate responses to protect mammals against a variety of foreign pathogens, while at the same time preventing responses against self-antigens.
  • the immune system In addition to deciding whether to respond (antigen specificity), the immune system must also choose appropriate effector functions to deal with each pathogen (effector specificity).
  • effector specificity A cell critical in mediating and regulating these effector functions is the CD4 + T cell.
  • CD4 + T cell A cell critical in mediating and regulating these effector functions.
  • cytokine production from long-term mouse CD4 + T cell clones was first published more than 20 years ago (Mosmann et al., J Immunol 136: 2348- 2357, 1986). In these studies, it was shown that CD4+ T cells produced two distinct patterns of cytokine production, which were designated T helper 1 (Thl) and T helper 2 (Th2). Thl cells were found to produce interleukin-2 (IL-2), interferon- ⁇ (IFN- ⁇ ) and lymphotoxin (LT), while Th2 clones predominantly produced IL-4, IL-5, IL-6, and IL-13 (Cherwinski et al., J Exp Med 169:1229-1244, 1987).
  • Thl interleukin-2
  • IFN- ⁇ interferon- ⁇
  • LT lymphotoxin
  • cytokines IL-9 and IL-10
  • Th2 clones Van Snick et al., J Exp Med 169:363-368, 1989
  • cytokines such as IL-3, granulocyte macrophage colony-stimulating factor (GM- CSF), and tumor necrosis factor-oc (TNF-00 were found to be secreted by both Thl and Th2 cells.
  • GM- CSF granulocyte macrophage colony-stimulating factor
  • TNF-00 tumor necrosis factor-oc
  • CD4+ T cells isolated from the inflamed joints of patients with Lyme disease contain a subset of IL-17-producing CD4+ T cells that are distinct from Thl and Th2 (Infante -Duarte et al., J. Immunol 165:6107-6115, 2000). These IL-17-producing CD4+ T cells are designated Thl7.
  • IL-17 a proinflammatory cytokine predominantly produced by activated T cells, enhances T cell priming and stimulates fibroblasts, endothelial cells, macrophages, and epithelial cells to produce multiple proinflammatory mediators, including IL-1, IL-6, TNF-a, NOS-2, metalloproteases, and chemokines, resulting in the induction of inflammation.
  • IL-17 expression is increased in patients with a variety of autoimmune diseases, such as multiple sclerosis, suggesting the contribution of IL-17 to the induction and/or development of such diseases.
  • Treg cells suppressor T cells
  • Treg cells suppressor T cells
  • Treg cells can be divided into two different subtypes, namely natural (or constitutive) and inducible (or adaptive) populations according to their origins (Mills, Nat Rev
  • Treg cell subsets have been identified according to their surface markers or cytokine products, such as CD4+ Treg cells (including natural
  • CD4+CD25+ Treg cells CD4+CD25+ Treg cells, IL-10-producting Trl cells, and TGF- ⁇ - producing Th3 cells
  • CD8+ Treg cells Veto CD8+ cells, ⁇ T cells, NKT (NK1.1+CD4-CD8-) cells, NK1.1- CD4-CD8- cells, etc.
  • Autoimmune disease encompasses a wide spectrum of diseases that can affect many different organs and tissues within the body (see, e.g., Paul, W.E. (1999), Fundamental Immunology, Fourth Edition, Lippincott-Raven, New York.)
  • Current therapies for human autoimmune disease include glucocorticoids, cytotoxic agents, and recently developed biological therapeutics.
  • the management of human systemic autoimmune disease is empirical and unsatisfactory.
  • broadly immunosuppressive drugs such as corticosteroids, are used in a wide variety of severe autoimmune disorders.
  • other immunosuppressive agents are used in management of the systemic autoimmune diseases.
  • Cyclophosphamide is an alkylating agent that causes profound depletion of both T- and B- lymphocytes and impairment of cell-mediated immunity.
  • Treatments for multiple sclerosis (MS) include interferon ⁇ and copolymer 1, which reduce relapse rate by 20-30% and only have a modest impact on disease progression.
  • MS is also treated with immunosuppressive agents including methylprednisolone, other steroids, methotrexate, cladribine and cyclophosphamide. These immunosuppressive agents have minimal efficacy in treating MS.
  • the introduction of the antibody Tysabri (natalizumab), an alpha 4-integrin antagonist, as treatment for MS has been overshadowed by incidences of progressive multifocal leucoencaphalopathy (PML) in patients receiving the therapy.
  • PML progressive multifocal leucoencaphalopathy
  • Soluble protein antigens have been administered systemically to inhibit the subsequent immune response to that antigen.
  • Such therapies include delivery of myelin basic protein, its dominant peptide, or a mixture of myelin proteins to animals with experimental autoimmune encephalomyelitis and humans with multiple sclerosis (Brocke et al., Nature 379: 343-6, 1996; Critchfield et al., Science 263: 1139-43, 1994; Weiner et al., Annu Rev Immunol 12: 809-37, 1994).
  • Another approach is the attempt to design rational therapeutic strategies for the systemic
  • TCR T cell receptor
  • Still another approach is the induction of oral tolerance by ingestion of peptide or protein antigens (see e.g. Weiner, Immmunol Today 18:335, 1997).
  • Treg cells that secrete anti-inflammatory cytokines, i.e. active tolerance (von Herrath, Res Immunol. 148:541-554, 1997).
  • Treg cells usually belong to the class of CD4 (helper) T cells. Instillation of intact protein antigen onto the nasopharyngeal mucosa also induces Treg cells that are protective. In this case, both CD4 and CD8 T cells may be induced.
  • Regulatory Treg cells induced after oral or intranasal antigen administration produce anti-inflammatory cytokines such as IL-4, IL-10 and TGF- ⁇ .
  • antigen can also be given in the form of aerosol.
  • regulatory T cells Repeated exposure to antigen in each case is able to induce regulatory T cells, but the nature of these cells differs, depending on the route and form of antigen.
  • regulatory cells induced by oral antigen are CD4 T cells and express T cell receptors (TCR) consisting of ⁇ heterodimers
  • TCR T cell receptors
  • the regulatory cells can also be CD8 T cells expressing a ⁇ heterodimer TCR (i.e. ⁇ T cells).
  • ⁇ T cells Some of these cells may also have a CD8 receptor that is an ⁇ homodimer instead of the conventional ⁇ -heterodimer TCR.
  • a majority of cells that carry the CD80C0C and ⁇ TCR reside in skin or mucosal tissues.
  • the hepatitis B virus vaccine contains recombinant hepatitis B virus surface antigen, a non-self antigen, formulated in aluminum hydroxide, which serves as an adjuvant. This vaccine induces an immune response against hepatitis B virus surface antigen to protect against infection.
  • An alternative approach involves delivery of an attenuated, replication deficient, and/or non-pathogenic form of a virus or bacterium, each a non-self antigen, to elicit a host protective immune response against the pathogen.
  • the oral polio vaccine is composed of a live attenuated virus, a non-self antigen, which infects cells and replicates in the vaccinated individual to induce effective immunity against polio virus, a foreign or non-self antigen, without causing clinical disease.
  • the inactivated polio vaccine contains an inactivated or 'killed' virus that is incapable of infecting or replicating and is administered subcutaneously to induce protective immunity against polio virus.
  • DNA therapies have been described for treatment of autoimmune diseases.
  • Such DNA therapies include DNA molecules encoding the antigen-binding regions of the T cell receptor to alter levels of autoreactive T cells driving the autoimmune response (Waisman et al., Nat Med 2:899-905, 1996; U.S. Patent 5,939,400).
  • DNA molecules encoding autoantigens were attached to particles and delivered by gene gun to the skin to prevent MS and collagen induced arthritis (WO 97/46253; Ramshaw et al.,
  • DNA molecules encoding adhesion molecules, cytokines (e.g., TNFa), chemokines (e.g., C-C chemokines), and other immune molecules (e.g., Fas-ligand) have been used for treatment of autoimmune diseases in animal models (Youssef et al., J Clin Invest 106:361- 371, 2000; Wildbaum et al., J Clin Invest 106:671-679, 2000; Wildbaum et al., J Immunol 165:5860- 5866, 2000).
  • Bacterial enterotoxins are used as immunostimulating adjuvants in vaccines for the prevention of infectious diseases.
  • Cholera toxin (CT) and the closely related E.coli heat-labile toxin (LT) are perhaps the most powerful and best studied mucosal adjuvants in experimental use today (Rappuoli et al., Immunol Today 20:493-500), but when exploited in the clinic, their potential toxicity and association with cases of Bell's palsy (paralysis of the facial nerve) have led to their withdrawal from the market (Gluck et al., J Infect Dis 181: 1129-1132, 2000; Gluck et al., Vaccine 20 (Suppl.l): S42-44, 2001; Mutsch et al., N Engl J Med.
  • CT and LT have proven to be effective immunoenhancers in experimental animals as well as in humans (Freytag et al., Curr Top Microbiol Immunol 236: 215-236, 1999). Structurally, these enterotoxins are AB 5 complexes, and consist of one ADP-ribosyltransf erase active Al subunit and an A2 subunit that links the Al to a pentamer of B subunits.
  • the holotoxins bind to most mammalian cells via the B subunit (CTB), which specifically interacts with the GMl-ganglioside receptor in the cell membrane.
  • CTB B subunit
  • CT and LT Although less toxic mutants of CT and LT have been engineered with substantial adjuvant function, such molecules still carry a significant risk of causing adverse reactions (Giuliani et al., J Exp Med 187: 1123-1132, 1998; Yamamoto et al., J Exp Med 185: 1203-1210, 1997), especially when considering that the adjuvanticity of CT and LT appears to be a combination of the ADP-ribosyltransferase activity of the A subunit and the ability to bind ganglioside receptors on the target cells (Soriani et al., Microbiology 148: 667-676, 2002). These observations and others preclude the use of CT or LT holotoxins in vaccines for humans.
  • CTA1-DD is nontoxic and has retained excellent immunoenhancing functions.
  • CTA1-DD provides comparable adjuvant effect to that of intact CT, greatly augmenting both cellular and humoral immunity against specific immunogens coadministered with the adjuvant. It also functions as a mucosal adjuvant and should be safe, as it is devoid of the B subunit that is a prerequisite of CT holotoxin toxicity.
  • CTA1-DD cannot bind to ganglioside receptors; rather, it targets B cells, limiting the CTA1-DD adjuvant to a restricted repertoire of cells that it can interact with.
  • CTA1(R7K)-DD and more specifically the immunomodulating complexes comprising CTA1(R7K)- DD linked to the shared immunodominant collagen II peptide comprising amino acids 260-273 (CII260-273) (Hasselberg et al, The Journal of Immunolgy, vol. 184, no. 6, p. 2776-2784, 2010).
  • CTB may not be suited for human use due to its GMl-ganglioside- binding properties and potential neurotoxic effects, as discussed above.
  • the present invention relates to new immunotolerizing fusion proteins and pharmaceutical compositions comprising them, as well as uses thereof for the prevention or treatment of autoimmune demyelinating diseases, in particular multiple sclerosis.
  • the immunotolerizing fusion proteins according to the present invention comprise a mutant ADP-ribosylating Al-subunit of the cholera toxin (CTA1), one or more autoantigens associated with said diseases, and a peptide capable of binding to a specific cellular receptor.
  • Administration of a therapeutically or prophylactically effective amount of the immunotolerizing fusion protein to a subject elicits suppression of an immune response against an antigen associated with the disease, thereby treating or preventing the disease.
  • the present invention provides an immunotolerizing fusion protein comprising:
  • a peptide capable of binding to a specific cellular receptor wherein the peptide specifically binds to a receptor expressed on an antigen presenting cell selected from the group consisting of lymphocytes, monocytes, macrophages, dendritic cells, and Langerhans cells.
  • autoimmune demyelinating diseases include multiple sclerosis and neuromyelitis optica (NMO, also known as Devic's disease or Devic's syndrome), optic neuritis, Guillain-Barre syndrome and Anti-MAG peripheral neuropathy.
  • NMO neuromyelitis optica
  • optic neuritis include Guillain-Barre syndrome and Anti-MAG peripheral neuropathy.
  • the one or more autoantigens or epitopes thereof are associated with multiple sclerosis (MS).
  • the one or more autoantigens are selected from the group of antigens consisting of myelin antigens, glial antigens, and neuronal antigens.
  • the one or more autoantigens are myelin antigens.
  • the myelin antigens are selected from the group of antigens consisting of antigens from myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), proteolipid protein (PLP), myelin-associated oligodendrocytic basic protein (MOBP), myelin-associated glycoprotein (MAG), oligodendrocyte-specific protein (OSP), myelin- associated neurite outgrowth inhibitor Nogo-A, and cyclic nucleotide phosphodiesterase (CNPase).
  • MBP myelin basic protein
  • MOG myelin oligodendrocyte glycoprotein
  • PBP proteolipid protein
  • MOBP myelin-associated oligodendrocytic basic protein
  • MAG myelin-associated glycoprotein
  • OSP oligodendrocyte-specific protein
  • CNPase cyclic nucleotide phosphodiesterase
  • the present invention further relates to an isolated nucleic acid encoding an immunotolerizing fusion protein in accordance with the present invention.
  • the nucleic acids of the invention can be DNA or RNA.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising one or more immunotolerizing fusion proteins or an isolated nucleic acid encoding such immunotolerizing fusion proteins in accordance with the present invention, and their use in the prevention or treatment of an autoimmune demyelinating disease, such as multiple sclerosis or neuromyelitis optica.
  • the pharmaceutical composition is made suitable for mucosal delivery, preferably intranasal delivery.
  • the present invention also relates to the use of an immunotolerizing fusion protein or a pharmaceutical composition in accordance with the present invention for the prevention or treatment of an autoimmune demyelinating disease, preferably multiple sclerosis.
  • a method for the prevention or treatment, of multiple sclerosis comprising administering to a subject an immunotolerizing fusion protein in accordance with the present invention comprising one or more autoantigens or epitopes thereof associated with MS.
  • the amino acid sequence of the ADP-ribosylating Al-subunit of the cholera toxin (CTA1) can be found e.g. in GenBank Accesion Nos. AAM22586.1, ADG44926.1, AAM74170.1, CAE11218.1, or AAA27514.1.
  • a subunit of the ADP-ribosylating Al-subunit of the cholera toxin (CTA1) refers to a polypeptide comprising at least a sequence corresponding to the sequence from amino acid 7, lysine, to amino acid 187, cysteine, of the sequence of the mature ADP-ribosylating Al-subunit of the cholera toxin (CTA1), such as a polypeptide comprising at least a sequence corresponding to the sequence from amino acid 1, aspargine, to amino acid 187, cysteine, of the sequence of the mature ADP-ribosylating Al-subunit of the cholera toxin (CTA1), or at least a sequence corresponding to the sequence from amino acid 1, aspargine, to amino acid 194, serine, of the sequence of the mature ADP- ribosylating Al-subunit of the cholera toxin (CTA1).
  • polynucleotide and nucleic acid refer to a polymer composed of a multiplicity of nucleotide units (ribonucleotide or deoxyribonucleotide or related structural variants) linked via phosphodiester bonds.
  • a polynucleotide or nucleic acid can be of substantially any length, typically from about six (6) nucleotides to about 10 9 nucleotides or larger.
  • Polynucleotides and nucleic acids include RNA, DNA, synthetic forms, and mixed polymers, both sense and antisense strands, double - or single-stranded, and can also be chemically or biochemically modified or can contain non-natural or derivatized nucleotide bases, as will be readily appreciated by the skilled artisan.
  • Antigen refers to any molecule that can be recognized by the immune system that is by B cells or T cells, or both.
  • Autoantigen refers to an endogenous molecule, typically a polysaccharide or a protein or fragment thereof, that elicits a pathogenic immune response. Autoantigen includes glycosylated proteins and peptides as well as proteins and peptides carrying other forms of post- translational modifications, including citrullinated peptides.
  • the autoantigen or epitope thereof When referring to the autoantigen or epitope thereof as "associated with an autoimmune demyelinating disease,” it is understood to mean that the autoantigen or epitope is involved in the pathophysiology of the disease either by inducing the pathophysiology (i.e., associated with the etiology of the disease), mediating or facilitating a pathophysiologic process; and/or by being the target of a pathophysiologic process.
  • the immune system aberrantly targets autoantigens, causing damage and dysfunction of cells and tissues in which the autoantigen is expressed and/or present.
  • autoantigens are ignored by the host immune system through the elimination, inactivation, or lack of activation of immune cells that have the capacity to recognize the autoantigen through a process designated "immune tolerance.”
  • epitope is understood to mean a portion of a polysaccharide or polypeptide having a particular shape or structure that is recognized by either B -cells or T-cells of the animal's immune system.
  • An epitope can include portions of both a polysaccharide and a polypeptide, e.g. a glycosylated peptide.
  • Autoantigenic epitope refers to an epitope of an autoantigen that elicits a pathogenic immune response.
  • polypeptide polypeptide
  • peptide protein
  • protein protein
  • amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.
  • Self-protein self-polypeptide
  • self-peptide any protein, polypeptide, or peptide, or fragment or derivative thereof that: is encoded within the genome of the animal; is produced or generated in the animal; may be modified posttranslationally at some time during the life of the animal; and, is present in the animal non-physiologically.
  • non- physiological or “non-physiologically” when used to describe the self-protein(s), -polypeptide(s), or - peptide(s) of this invention means a departure or deviation from the normal role or process in the animal for that self-protein, - polypeptide, or -peptide.
  • the self-protein, -polypeptide or -peptide may be modified in form or structure and thus be unable to perform its physiological role or process or may be involved in the pathophysiology of the condition or disease either by inducing the pathophysiology; mediating or facilitating a pathophysiologic process; and/or by being the target of a pathophysiologic process.
  • autoimmune disease the immune system aberrantly attacks self -proteins causing damage and dysfunction of cells and tissues in which the self-protein is expressed and/or present.
  • the self -protein, - polypeptide or -peptide can itself be expressed at non-physiological levels and/or function non- physiologically.
  • posttranslational modifications of self-protein(s), - polypeptide(s) or - peptide(s) are glycosylation, addition of lipid groups, reversible phosphorylation, addition of dimethylarginine residues, citrullination, and proteolysis, and more specifically citrullination of MBP.
  • immune protein(s), polypeptide(s) or peptide(s) are proteins comprising the T-cell receptor, immunoglobulins, cytokines, including the type I interleukins, and the type II cytokines, including the interferons and IL-10, TNF, lymphotoxin, and the chemokines, such as macrophage inflammatory protein -1 alpha and beta, monocyte -chemotactic protein and RANTES, and other molecules directly involved in immune function, such as Fas-ligand.
  • immune protein(s), polypeptide(s) or peptide(s) that are included in the self -protein, -polypeptide or -peptide of the invention and they are: class I MHC membrane glycoproteins, class II MHC glycoproteins and osteopontin.
  • Modulation of refers to any alteration of an existing or potential immune responses against an autoimmune or allergy provoking epitope, including, e.g., nucleic acids, lipids, phospholipids, carbohydrates, self-polypeptides, protein complexes, or ribonucleoprotein complexes, that occurs as a result of administration of an autoimmune or allergy provoking epitope, including, e.g., nucleic acids, lipids, phospholipids, carbohydrates, self-polypeptides, protein complexes, or ribonucleoprotein complexes, that occurs as a result of administration of an autoimmune or allergy provoking epitope, including, e.g., nucleic acids, lipids, phospholipids, carbohydrates, self-polypeptides, protein complexes, or ribonucleoprotein complexes, that occurs as a result of administration of an autoimmune or allergy provoking epitope, including, e.g., nucleic acids, lipids, phospho
  • Immune cells include B cells, T cells, NK cells, NK T cells, professional antigen-presenting cells, non-professional antigen-presenting cells, inflammatory cells, or any other cell capable of being involved in or influencing an immune response.
  • Modulation includes any change imparted on an existing immune response, a developing immune response, a potential immune response, or the capacity to induce, regulate, influence, or respond to an immune response. Modulation includes any alteration in the expression and/or function of genes, proteins and/or other molecules in immune cells as part of an immune response.
  • Modulation of an immune response includes, for example, the following: elimination, deletion, or sequestration of immune cells; induction or generation of immune cells that can modulate the functional capacity of other cells such as autoreactive lymphocytes, antigen presenting cells, or inflammatory cells; induction of an unresponsive state in immune cells (i.e., anergy); increasing, decreasing, or changing the activity or function of immune cells or the capacity to do so, including, but not limited to, altering the pattern of proteins expressed by these cells. Examples include altered production and/or secretion of certain classes of molecules such as cytokines, chemokines, growth factors, transcription factors, kinases, costimulatory molecules, or other cell surface receptors; or any combination of these modulatory events.
  • administration of an immunomodulating complex or a polynucleotide encoding an immunomodulating complex can modulate an immune response by eliminating, sequestering, or inactivating immune cells mediating or capable of mediating an undesired immune response; inducing, generating, or turning on immune cells that mediate or are capable of mediating a protective immune response; changing the physical or functional properties of immune cells; or a combination of these effects.
  • measurements of the modulation of an immune response include, but are not limited to, examination of the presence or absence of immune cell populations (using flow cytometry, immunohistochemistry, histology, electron microscopy, polymerase chain reaction (PCR));
  • a signal such as using T cell proliferation assays and pepscan analysis based on 3 H-thymidine incorporation following stimulation with anti-CD3 antibody, anti-T cell receptor antibody, anti-CD28 antibody, calcium ionophores, PMA, antigen presenting cells loaded with a peptide or protein antigen; B cell proliferation assays); measurement of the ability to kill or lyse other cells (such as cytotoxic T cell assays); measurements of the cytokines, chemokines, cell surface molecules, antibodies and other products of the cells (e.g., by flow cytometry, enzyme-linked immunosorbent assays, Western blot analysis, protein microarray analysis, immunoprecipitation analysis); measurement of biochemical markers of activation of immune cells or signaling pathways within immune cells (e.g., Western blot and immunoprecipitation analysis of tyrosine, serine or threonine phosphorylation, poly
  • Immunotolerizing shall mean “immunomodulating” in which pro-inflammatory immune responses - that are elicited by autoantigens and that are targeting autoantigens - are downregulated. This downregulation may be accompanied by upregulation of regulatory T cells. The downregulation would suppress adaptive immunity (T and B effector cells) and innate immunity (macrophages, dendritic cells, neutrophils).
  • Subjects shall mean any animal, such as, for example, a human, non-human primate, horse, cow, dog, cat, mouse, rat, guinea pig or rabbit.
  • Treating", “treatment”, or “therapy” of a disease or disorder shall mean slowing, stopping or reversing the disease's progression, as evidenced by decreasing, cessation or elimination of either clinical or diagnostic symptoms, by administration of an immunotolerizing fusion protein or a polynucleotide encoding an immunotolerizing fusion protein, either alone or in combination with another compound as described herein.
  • Treating, “treatment”, or “therapy” also means a decrease in the severity of symptoms in an acute or chronic disease or disorder or a decrease in the relapse rate as, for example, in the case of a relapsing or remitting autoimmune disease course or a decrease in inflammation in the case of an inflammatory aspect of an autoimmune disease.
  • treating a disease means reversing or stopping or mitigating the disease's progression, ideally to the point of eliminating the disease itself.
  • ameliorating a disease and treating a disease are equivalent.
  • Preventing, "prophylaxis”, or “prevention” of a disease or disorder as used in the context of this invention refers to the administration of an immunotolerizing fusion protein or a polynucleotide encoding an immunotolerizing fusion protein, either alone or in combination with another compound as described herein, to prevent the occurrence or onset of a disease or disorder or some or all of the symptoms of a disease or disorder or to lessen the likelihood of the onset of a disease or disorder.
  • a “therapeutically or prophylactically effective amount" of an immunotolerizing fusion protein refers to an amount of the immunotolerizing fusion protein that is sufficient to treat or prevent the disease as, for example, by ameliorating or eliminating symptoms and/or the cause of the disease.
  • therapeutically effective amounts fall within broad range(s) and are determined through clinical trials, and for a particular patient is determined based upon factors known to the skilled clinician, including, e.g., the severity of the disease, weight of the patient, age, and other factors.
  • Autoimmune demyelinating disease refers to any autoimmune condition that results in damage to the myelin sheath surrounding nerve fibers, whether in relation to the central nervous system or in relation to the peripheral nervous system.
  • autoimmune demyelinating diseases of the central nervous system would include, but are not limited to Multiple sclerosis, Neuromyelitis Optica (NMO, also known as Devic's disease or Devic's syndrome) and Optic neuritis.
  • NMO Neuromyelitis Optica
  • Figure 4 DNA construct encoding the immunotolerizing fusion protein K-CTAl(R7K/C187A)-human MOG(10-60)-DD
  • Figure 7 The therapeutic effect of K-CTAl(R7K/C187A)-MOG(35-55)-DD is peptide specific in the mouse EAE model.
  • Figure 8 The therapeutic effect of K-CTAl(R7K/C187A)-MOG(35-55)-DD on the incidence of disease is peptide specific in the mouse EAE model.
  • the present invention relates to new immunotolerizing fusion proteins and pharmaceutical compositions comprising them, as well as uses thereof for the prevention or treatment of autoimmune demyelinating diseases, in particular multiple sclerosis.
  • the immunotolerizing fusion proteins according to the present invention comprise a mutant ADP-ribosylating Al-subunit of the cholera toxin (CTA1), one or more autoantigens or epitopes thereof associated with said diseases, and a peptide capable of binding to a specific cellular receptor.
  • Administration of a therapeutically or prophylactically effective amount of the immunotolerizing fusion protein to a subject elicits suppression of an immune response against an antigen associated with the disease, thereby treating or preventing the disease.
  • the present invention provides an immunotolerizing fusion protein comprising:
  • a peptide capable of binding to a specific cellular receptor wherein the peptide specifically binds to a receptor expressed on an antigen presenting cell selected from the group consisting of lymphocytes, monocytes, macrophages, dendritic cells, and Langerhans cells.
  • mutant subunit of SEQ ID NO: l the amino acid lysine (K) has been added to the N-terminus of CTA1(1-194), amino acid 7, arginine (R), has been replaced by lysine (K), and amino acid 187 cysteine (C), has been replaced by alanine (A), and in the context of the present invention this sequence is abbreviated as K-CTA1(R7K/C187A).
  • the fusion proteins according to the present invention comprising K-CTA1(R7K/C187A) provide surprising and advantageous effects as compared to CTAl(R7K)-containing fusion proteins according to the prior art WO 2009/078796.
  • the therapeutic effect of K-CTA1(R7K C187A)- MOG(35-55)-DD has surprisingly been found to be significantly better than the therapeutic effect of CTAl(R7K)-MOG(35-55)-DD, as shown in the Examples below.
  • K-CTAl(R7K C187A)-MOG(35-55)-DD can effectively and significantly reduce disease scores in an EAE model induced by another epitope, PLP(178-191), indicating that the MOG(35-55) epitope is central to the EAE disease and may thus be central to MS in humans suggesting that K-CTAl(R7K C187A)-MOG(35-55)-DD may be effective in therapeutically treating MS in human subjects.
  • autoimmune demyelinating diseases include multiple sclerosis and neuromyelitis optica (NMO, also known as Devic's disease or Devic's syndrome), optic neuritis, Guillain-Barre syndrome and Anti-MAG peripheral neuropathy.
  • NMO neuromyelitis optica
  • optic neuritis include Guillain-Barre syndrome and Anti-MAG peripheral neuropathy.
  • the one or more autoantigens or epitopes thereof are associated with multiple sclerosis (MS).
  • MS multiple sclerosis
  • MS Multiple sclerosis
  • Diagnosis of MS is based upon a history including at least two distinct attacks of neurologic dysfunction that are separated in time, produce objective clinical evidence of neurologic dysfunction, and involve separate areas of the CNS white matter.
  • Laboratory studies providing additional objective evidence supporting the diagnosis of MS include magnetic resonance imaging (MRI) of CNS white matter lesions, cerebral spinal fluid (CSF) oligoclonal banding of IgG, and abnormal evoked responses.
  • MRI magnetic resonance imaging
  • CSF cerebral spinal fluid
  • IgG cerebral spinal fluid
  • the autoantigen targets of the autoimmune response in autoimmune demyelinating diseases may comprise epitopes from proteolipid protein (PLP); myelin basic protein (MBP); myelin oligodendrocyte glycoprotein (MOG); cyclic nucleotide phosphodiesterase (CNPase); myelin-associated glycoprotein (MAG) and myelin-associated oligodendrocytic basic protein (MBOP); alpha-B-crystalin (a heat shock protein); viral and bacterial mimicry peptides, e.g.
  • the integral membrane protein PLP is a dominant autoantigen of myelin. Determinants of PLP antigenicity have been identified in several mouse strains, and include residues 139-151, 103-116, 215-232, 43-64 and 178-191. At least 26 MBP epitopes have been reported (Meinl et al., J Clin Invest 92, 2633-43, 1993). Notable are residues 1-11, 59-76 and 87- 99. Immunodominant MOG epitopes that have been identified in several mouse strains include residues 1-22, 35-55, 64-96.
  • EAE Experimental autoimmune encephalomyelitis
  • MS multiple sclerosis
  • CNS central nervous system
  • T-cells specific for MOG34-56 have been implicated in the disease progression to clinically evident EAE (Jagessar et al., J Neuropathol Exp Neurol 69:372-385, 2010; Kap et al., J Immunol 180: 1326-1337, 2008).
  • T-cells have an effector memory phenotype and cytotoxic function triggered by MOG40-48 epitope presented by Caja-E molecules (Jagessar et al., Eur J Immunol 42:217-227, 2012).
  • MOG40-48 epitope presented by Caja-E molecules
  • myelin proteins and epitopes were identified as targets of the autoimmune T and B cell response.
  • Antibody eluted from MS brain plaques recognized myelin basic protein (MBP) peptide 83-97 (Wucherpfennig et al., J Clin Invest 100: 1114-1122, 1997).
  • MBP myelin basic protein
  • T and B cell (brain lesion-eluted Ab) response focused on MBP 87-99 (Oksenberg et al., Nature 362: 68-70, 1993).
  • MBP 87-99 the amino acid motif HFFK is a dominant target of both the T and B cell response (Wucherpfennig et al., J Clin Invest 100: 1114-22, 1997).
  • Another study observed lymphocyte reactivity against myelin-associated oligodendrocytic basic protein (MOBP), including residues MOBP 21-39 and MOBP 37-60 (Holz et al., J Immunol 164: 1103-9, 2000).
  • MOBP myelin-associated oligodendrocytic basic protein
  • the shared immunodominant epitope may be selected from any suitable autoantigen known to be associated with an autoimmune demyelinating disease.
  • the epitope may, for instance, be selected from any of the autoantigens associated with the disease.
  • epitopes with a high content of cysteine may counteract the advantageous effect provided by the replacement of amino acid 187, cysteine, by an alanine in K-CTA1(R7K/C187A) of the immunotolerizing fusion protein according to the present invention as compared to CTA1(R7K). Therefore, it is preferable that epitopes according to the present invention are choosen in such a way as to avoid high contents of cysteine.
  • autoantigens and epitopes thereof associated with an autoimmune demyelinating disease in particular multiple sclerosis, can be found using antigen microarrays as described by Quintana et al. in Proc Natl Acad Sci USA 105: 18889-18894, 2008.
  • the one or more autoantigens are selected from the group of antigens consisting of myelin antigens (MBP, PLP, MOG, MOBP, MAG, OSP, Nogo-A, CNPasw), glial antigens (GFAP, S100 , ⁇ -crystallin), and neuronal antigens (Neurofilament-L, Neurofilament- M, ⁇ -Synuclein, Contactin-2, Neurofascin), as described by Krishnamoorthy and Wekerle in Eur J Immunol 39: 1991-2058, 2009.
  • the one or more autoantigens are myelin antigens.
  • Autoantigens or epitopes thereof of different species including mice, rats, primates, and humans can be used in accordance with the present invention.
  • human autoantigens or epitopes thereof are used in accordance with the present invention.
  • a description of suitable, including human, autoantigens/epitopes can be found, for example, in the articles by Krishnamoorthy and Wekerle in Eur J Immunol 39: 1991-2058, 2009, and Kaushansky et al. in PLoS ONE 6(11): e27860 (November 2011).
  • the myelin antigens are selected from the group of antigens consisting of antigens from myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), proteolipid protein (PLP), myelin-associated oligodendrocytic basic protein (MOBP), myelin-associated glycoprotein (MAG), oligodendrocyte-specific protein (OSP), myelin- associated neurite outgrowth inhibitor Nogo-A, and cyclic nucleotide phosphodiesterase (CNPase).
  • MBP myelin basic protein
  • MOG myelin oligodendrocyte glycoprotein
  • PBP proteolipid protein
  • MOBP myelin-associated oligodendrocytic basic protein
  • MAG myelin-associated glycoprotein
  • OSP oligodendrocyte-specific protein
  • CNPase cyclic nucleotide phosphodiesterase
  • the myelin antigens are selected from the group of antigens consisting of antigens from myelin basic protein (MBP), myelin oligodendrocyte
  • MOG glycoprotein
  • PBP proteolipid protein
  • MOBP myelin-associated oligodendrocytic basic protein
  • MAG myelin-associated glycoprotein
  • OSP oligodendrocyte-specific protein
  • the myelin antigens are selected from the group of autoantigenic epitopes consisting of PLP(41-60), PLP(43-64), PLP(45-53), PLP(56- 70), PLP(103-116), PLP(104-117), PLP(139-151), PLP(175-194), PLP(178-191), PLP(215-232), PLP(215-235), PLP(258-276), MBP(l-ll), MBP(ll-30), MBP(21-35), MBP(29-84), MBP(59-76),
  • epitopes and their amino acid sequences are known in the art and have been described, e.g., by Krishnamoorthy and Wekerle in Eur J Immunol 39: 1991-2058, 2009, and Kaushansky et al. in PLoS ONE 6(11): e27860 (November 2011), and in references cited therein.
  • the epitope amino acid sequences vary in length and sometimes overlap. In any case, these amino acid sequences contain one or more autoantigenic epitopes, of which the exact amino acid sequence may not always be known.
  • peptides comprising one or more of the above epitope amino acid sequences can be used in accordance with the present invention.
  • the length of the peptides does not matter as long as it contains one or more autoantigenic epitope amino acid sequences.
  • the person skilled in the art of MS-related autoantigens is able to select one or more suitable, preferably human, epitopes for incorporation into one or more immunotolerizing fusion proteins to be used in accordance with the present invention.
  • the myelin antigens are selected from the group of autoantigenic epitopes consisting of hPLP(45-53), hPLP(139-151), hPLP(175-194), hMBP(84-102), hMBP(85-99), hMBP(89-104), hMOG( 10-60), hMOG(34-56), hMOBP(15-36), hMOBP(55-77), hOSP(55-74), and hOSP(55-80).
  • the immunotolerizing fusion protein in accordance with the present invention is selected from the group consisting of K-CTAl-R7K/C187A-MOG(35-55)-DD (SEQ ID NO:2), K- CTAl-R7K/C187A-MOG(35-55)-PLP(178-191)-DD (SEQ ID NO:3), K-CTA1-R7K/C187A- PLP(178-191)-DD (SEQ ID NO:4) and K-CTAl(R7K/C187A)-human MOG(10-60)-DD.
  • the fusion protein comprises a peptide that specifically binds to a receptor expressed on a cell capable of antigen presentation, especially cells expressing MHC class I or MHC class II antigen.
  • the antigen presenting cell is selected from the group consisting of lymphocytes, such as B-lymphocytes, monocytes, macrophages, dendritic cells, and Langerhans cells.
  • the peptide is a peptide that binds to receptors of the above cells, preferably to an Ig or Fc receptor expressed by said antigen presenting cell and most preferably to receptors of B-lymphocytes and dendritic cells.
  • said peptide is constituted by protein A or a fragment thereof in single or multiple copies, such as one or more D subunits thereof.
  • said peptide is constituted by an antibody fragment, such as a single chain antibody fragment, that specifically binds to a receptor expressed on a cell capable of antigen presentation.
  • peptides capable of binding to receptors such as:
  • DEC205 an endocytic receptor for antigen uptake and processing expressed at high levels on a subset of dendritic cells
  • CDllc a cell surface receptor for numerous soluble factors and proteins (LPS, fibrinogen, iC3b) found primarily on myeloid cells,
  • LPS soluble factors and proteins
  • iC3b soluble factor and proteins
  • the mannose receptor present on dendritic cells, macrophages and other antigen presenting cells
  • CD103 an integrin alpha chain expressed by a subset of dendritic cells
  • the peptide is preferably such that the resulting fusion protein is in possession of water solubility and capability of targeting the fusion protein to a specific cell receptor different from receptors binding to the native toxin, thereby mediating intracellular uptake of at least said subunit.
  • the present invention further relates to an isolated nucleic acid encoding an immunotolerizing fusion protein in accordance with the present invention.
  • the nucleic acids of the invention can be DNA or RNA.
  • the present invention provides recombinant plasmids, vectors and expression systems comprising a nucleic acid according to the invention.
  • the recombinant expression systems can be adapted for eukaryotic or bacterial expression.
  • the invention further provides transformed cells containing a plasmid, vector or an expression system according to the invention.
  • the transformed cells can be transformed eukaryotic or bacterial cells.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising one or more immunotolerizing fusion proteins or an isolated nucleic acid encoding such immunotolerizing fusion proteins in accordance with the present invention, and their use in the prevention or treatment of an autoimmune demyelinating disease, in particular multiple sclerosis.
  • the pharmaceutical composition is made suitable for mucosal delivery, preferably intranasal delivery.
  • the immunotolerizing fusion protein is delivered orally, sublingually, subcutaneously, transcutaneously, intradermally, intravenously, or intramuscularly.
  • the present invention also relates to the use of an immunotolerizing fusion protein or a pharmaceutical composition in accordance with the present invention for the prevention or treatment of an autoimmune demyelinating disease, in particular multiple sclerosis.
  • a method is provided for the prevention or treatment of an autoimmune demyelinating disease, preferably multiple sclerosis (MS), comprising administering to a subject one or more immunotolerizing fusion proteins in accordance with the present invention comprising one or more autoantigens or epitopes thereof associated with MS.
  • Multiple immunotolerizing fusion proteins comprising different autoantigens or autoantigenic epitopes may be administered as a mixture or cocktail of fusion proteins, and each individual immunotolerizing fusion protein may comprise multiple autoantigens or autoantigenic epitopes.
  • the methods and compositions for the treatment, prophylaxis and/or prevention of an autoimmune disease further comprise the administration of the immunotolerizing fusion protein according to the invention in combination with other substances which may enhance tissue penetration, uptake into dendritic cells or stimulate tolerance response, such as, for example, polynucleotides comprising an immune modulatory sequence, immune stimulating complexes (ISCOMS), other pharmacological agents, adjuvants, cytokines, or vectors encoding cytokines.
  • other substances which may enhance tissue penetration, uptake into dendritic cells or stimulate tolerance response such as, for example, polynucleotides comprising an immune modulatory sequence, immune stimulating complexes (ISCOMS), other pharmacological agents, adjuvants, cytokines, or vectors encoding cytokines.
  • ISCOMS immune stimulating complexes
  • the immunotolerizing fusion proteins according to the invention can be produced by conventional recombinant DNA technology.
  • Plasmids, vectors and expression systems of the invention employs standard ligation and restriction techniques that are well-known in the art (see generally, e.g., Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience, 1989; Sambrook and Russell, Molecular Cloning, A Laboratory Manual 3rd ed. 2001). Isolated plasmids, DNA sequences, or synthesized oligonucleotides are cleaved, tailored, and religated in the form desired. Sequences of DNA constructs can be confirmed using, e.g., standard methods for DNA sequence analysis (see, e.g., Sanger et al. (1977), Proc. Natl. Acad. Sci., 74, 5463-5467).
  • RNA is isolated from, for example, cells, tissues, or whole organisms by techniques known to one skilled in the art.
  • cDNA Complementary DNA
  • poly-dT or random hexamer primers primers, deoxynucleotides, and a suitable reverse transcriptase enzyme.
  • the desired polynucleotide can then be amplified from the generated cDNA by PCR.
  • the polynucleotide of interest can be directly amplified from an appropriate cDNA library.
  • Primers that hybridize with both the 5' and 3' ends of the polynucleotide sequence of interest are synthesized and used for the PCR.
  • the primers may also contain specific restriction enzyme sites at the 5' end for easy digestion and ligation of amplified sequence into a similarly restriction digested plasmid vector.
  • Therapeutically and prophylactically effective amounts of the one or more immunotolerizing fusion proteins in accordance with the present invention are in the range of about 1 ⁇ g to about 10 mg. In one embodiment of the present invention, the therapeutic or prophylactically effective amount of an immunotolerizing fusion protein is in the range of about 5 ⁇ g to about 1 mg. In certain embodiments, the immunotolerizing fusion protein of the present invention is administered weekly or monthly, for example monthly for 6-12 months, and then every 3-12 months as a maintenance dose.
  • Alternative treatment regimens may be developed and may range from daily, to weekly, to every other month, to yearly, to a one-time administration, depending upon the severity of the disease, the age of the patient, the immunotolerizing fusion protein being administered, and such other factors as would be considered by the ordinary treating physician.
  • Example 1 Immunotolerizing fusion protein K-CTAl(R7K/C187A)-MOG(35-55)-DD (Figure 1)
  • amino acid sequences for the peptides A-B were used in BLAST searches on the NCBI homepage to confirm each nucleotide sequence in Mus Musculus.
  • the cloning was performed in competent DH5a cells. That is, annealed and phosphorylated oligonucleotides were ligated into BamHI cleaved and SAP (Shrimp Alkaline Phosphatase) treated vector and then transformed into competent DH5a cells. Correct nucleotide sequences were confirmed before each vector was transformed, into either competent DH5 bacteria for the K- CTAl(R7K C187A)-DD-based vectors or competent BL21 bacteria for the CTAl(R7K)-DD-based vector, for corresponding protein expression and production. "Bgll-MOG-B amffl-FP"
  • plasmids were then either transformed into E.coli, BL21 (for the CTAl(R7K)-DD-based vector) or DH5 (for the K-CTAl(R7K C187A)-DD-based vectors).
  • the transformation mixture was transferred onto agar/LB plates with antibiotics, (ampicillin or Kanamycin depending on the vector) and after overnight incubation five colonies from each construct were collected and cultivated in LB media, containing antibiotics, for eight hours.
  • antibiotics ampicillin or Kanamycin depending on the vector
  • the cultures were frozen and stored at -80°C as glycerol stock solutions.
  • the frozen glycerol stock solutions were thawed and cultivated in 2xYT media, containing antibiotics.
  • the protein expression levels were analyzed four hours after induction (IPTG).
  • the pellets and respective supernatants of the five different clones were lysed, separated by SDS- PAGE electrophoresis and Coomassie stained.
  • Western blot was performed on the selected clones to confirm the identity of the primarily expressed protein from the respective expression vector and the fusion protein was purified from the pellet using Q Sepharose FF and SEC Superdex 200 pg on the Akta FPLC system.
  • Antibody utilized in the Western blot Polyclonal Chicken Anti CTA1-DD, alkaline phosphatase (AP)- labelled antibody (Agrisera/MDAB).
  • the total protein concentration was measured using BCA assay (Pierce).
  • Enzymatic activity was determined in an ADP-ribosylation assay where the fusionproteins were compared to CT.
  • the Ig-binding capacity of the fusionproteins was determined using ELISA assay.
  • the cloning was performed in a manner analogous to the method described in Example 1.
  • the cloning was performed in a manner analogous to the method described in Example 1.
  • C57B1/6 mice were obtained from Harlan and were 7-9 weeks of age at the start of the experiment.
  • One hundred microliter induction mixture containing 100 microgram MOG(35-55) was mixed 1 : 1 with Complete Freund's Adjuvant containing 10 mg/ml Mycobacterium tuberculosis and the induction mixture was given by subcutaneous injection at the base of the tail at Day 0.
  • Two hundred nanogram Pertussis toxin was given intraperitoneally on Day 0 and on Day 2. Treatment was performed by intranasal immunization with the fusion proteins at indicated concentrations in a volume of 20 microliter per nostril on Days -2, 0, 2, 10, 12, 14.
  • Dexamethasone at a dose of 1 mg/kg was given intraperitoneally 5 Days per week (not weekends), starting on Day 0. Each animal was evaluated from Day 5 until Day 28 for EAE severity using the disability scoring system; 0, no disease; 0.5, tail paresis or partial paralysis; 1, complete tail paralysis or limb weakness; 1.5, limb weakness with partial tail paralysis; 2, paraparesis: limb weakness and tail paralysis; 2.5, partial limb paralysis; 3, complete hind- or front limb paralysis; 3.5, paraplegia; 4, quadriplegia, moribund; 5, death due to EAE. The highest clinical score of each mouse during the complete experimental period was determined. The experiment was terminated on Day 28.
  • K-CTA 1 (R7K/C 187 A)-MOG-DD 5 ⁇ g/dose intranasally, d -2, 0, 2, 10, 12, 14
  • MMS Mean Maximum Score
  • AMS Accumulated Mean Score
  • MOG(35-55)-DD 5 ⁇ g/dose intranasally, d -2, 0, 2, 10, 12, 14
  • MMS Mean Maximum Score
  • AMS Accumulated Mean Score
  • Example 7 The therapeutic effect of K-CTAl(R7K/C187A)-MOG(35-55)-DD is peptide specific in the mouse EAE model. ( Figure 7) EAE was induced in C57B1/6 mice in the same way as described in example 5 above.
  • K-CTA1(R7K/C187A)-DD 5 ⁇ g/dose intranasally, d -2, 0, 2, 10, 12, 14
  • MMS Mean Maximum Score
  • K-CTAlR7K(C187A)-MOG(35-55)-DD The therapeutic effect of K-CTAlR7K(C187A)-MOG(35-55)-DD was significantly better than treatment with the empty vector, K-CTA1R7K(C187A)-DD, which had no therapeutic effect indicating that the disease specific epitope, in this case MOG(35-55) is needed to achieve any therapeutic effect.
  • Example 8 The therapeutic effect of K-CTAl(R7K/C187A)-MOG(35-55)-DD on the incidence of disease is peptide specific in the mouse EAE model. ( Figure 8)
  • EAE was induced in C57B1/6 mice in the same way as described in example 5 above.
  • K-CTA1R7K(C187A)- MOG(35-55)-DD significantly fewer animals showed sign of disease following treatment with K-CTA1R7K(C187A)- MOG(35-55)-DD compared to control animals treated with PBS.
  • Example 9 The therapeutic effects of K-CTAl(R7K/C187A)-MOG(35-55)-DD and K- CTA1(R7K/C187A)-PLP(178-191)-DD in the mouse EAE model. ( Figure 9)
  • EAE was induced in C57B1/6 mice in the same way as described in example 5 above with the exception that 100 microgram PLP( 178-191) was used to induce EAE instead of 100 microgram MOG(35-55).
  • PBS intranasally, d -2, 0, 2, 10, 12, 14
  • K-CTAlR7K(C187A)-MOG(35-55)-DD 5 ⁇ g/dose intranasally, d -2, 0, 2, 10, 12, 14
  • K-CTA1R7K(C187A)-PLP(178-191)-DD 5 ⁇ g/dose intranasally, d -2, 0, 2, 10, 12, 14 Mean Maximum Score (MMS) was calculated:
  • AMS Accumulated Mean Score
  • K-CTAlR7K(C187A)-MOG(35-55)-DD The therapeutic effect of K-CTAlR7K(C187A)-MOG(35-55)-DD as well as of K-CTA1R7K(C187A)- PLP(178-191)-DD was significant on EAE disease as seen in the decrease of the disease score as compared with the control group (PBS).
  • K-CTAlR7K(C187A)-MOG(35-55)-DD had a significant therapeutic effect on EAE disease induced by PLP(178-191) indicating that this MOG-epitope is central to the EAE disease and thus potentially also in the human MS disease.
  • Example 10 The therapeutic effects of K-CTAl(R7K/C187A)-MOG(35-55)-DD, K-
  • EAE was induced in C57B1/6 mice in the same way as described in example 5 above with the exception that 100 microgram PLP(178-191) together with 100 microgram MOG(35-55) was used to induce EAE instead of only 100 microgram MOG(35-55) alone.
  • MMS Mean Maximum Score
  • K-CTAlR7K(C187A)-MOG(35-55)-DD, CTA1R7K(C187A)-PLP(178- 191)-DD as well as of K-CTAlR7K(C187A)-MOG(35-55)-PLP(178-191)-DD was significant on EAE disease as seen in the decrease of the disease score as compared with the control group (PBS). Further the fusion protein containing the combination of two epitopes, the K-CTA1R7K(C187A)-M0G(35- 55)-PLP(178-191)-DD had a significant better therapeutic effect compared to either K-

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Abstract

La présente invention concerne de nouvelles protéines hybrides d'immuno-tolérisation et des compositions pharmaceutiques les comportant, ainsi que leurs utilisations pour la prévention ou le traitement de maladies auto-immunes démyélinisantes, en particulier la sclérose en plaques. Les protéines hybrides d'immuno-tolérisation selon la présente invention comporte une sous-unité A1 mutante de l'ADP ribosylation de la toxine de choléra (CTA1), un ou des auto-antigène(s) ou épitope(s) de celui-ci associé(s) aux dites maladies, et un peptide capable de liaison à un récepteur cellulaire spécifique. L'administration d'une quantité à efficacité thérapeutique ou prophylactique de la protéine hybride d'immuno-tolérisation à un sujet provoque une suppression de la réponse immunitaire contre l'antigène/les antigènes présent(s) dans la protéine hybride ou contre un autre/d'autres antigène(s) associé(s) à la maladie, permettant ainsi le traitement ou la prévention de la maladie.
PCT/IB2013/053278 2012-04-26 2013-04-25 Protéines hybrides d'immuno-tolérisation pour le traitement de la sclérose en plaques WO2013160865A1 (fr)

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