Yopm as delivery vehicle for cargo molecules and as biological therapeutic for immunomodulation of the inflammatory reactions

Abstract

The present invention relates to the use of Yersinia outer protein M (YopM), a YopM fragment, or a YopM variant, which is capable of autopenetrating the cell membrane and of integrating into the cell cytosol without the requirement of additional factors for delivering a cargo molecule across the membrane to the cytosol of a cell. The present invention also relates to a pharmaceutical composition comprising YopM, a YopM fragment, or a YopM variant being capable of autopenetrating the cell membrane and of integrating into the cell cytosol without the requirement of additional factors for the regulation of inflammatory reactions of the immune system and the treatment of diseases caused by autoimmunity of the host. The present invention further relates to a YopM fragment or variant, which is capable of autopenetrating the cell membrane and of integrating into the cell cytosol without the requirements of additional factors as well as such proteins or YopM linked to a cargo molecule.

1 . A pharmaceutical composition, comprising
 (i) an isolated YopM;   (ii) an isolated YopM fragment, comprising at least one alpha helix of YopM capable of mediating autopenetration; or   (iii) an isolated YopM variant, characterized in that said variant
 (a) consists of an amino acid sequence that is at least 80% identical to any of SEQ ID NOs: 1-8 or at least 80% identical to a fragment of any of SEQ ID NOs: 1-8; and 
 (b) comprises at least one alpha helix of YopM capable of mediating autopenetration; 
   in the absence of  Yersinia  bacteria;   wherein said YopM, YopM fragment or YopM variant is capable of autopenetrating the cell membrane and integrating into the cell cytosol of a eukaryotic cell without the assistance of a type III secretion system.   
     
     
         2 . The pharmaceutical composition of  claim 1 , wherein the YopM, YopM fragment or YopM variant comprises an immunomodulatory domain of YopM. 
     
     
         3 . The pharmaceutical composition of  claim 1 , wherein the YopM, YopM fragment or YopM variant is capable of downregulating cytokines and/or cytokine receptors and/or genes which respond to cytokines and/or cartilage-destroying molecules, and/or is capable of inhibiting osteoclastogenesis. 
     
     
         4 . A pharmaceutical composition, comprising an immunomodulatory domain of YopM wherein said immunomodulatory domain is (i) linked to a cell-penetrating peptide that is heterologous to YopM, (ii) has essentially no capability of autopenetrating the cell membrane and of integrating into the cell cytosol and (iii) is capable of downregulating cytokines, and/or cytokine receptors and/or genes which respond to cytokines and/or cartilage-destroying molecules and/or is capable of inhibiting osteoclastogenesis. 
     
     
         5 . The pharmaceutical composition of  claim 1 , further comprising at least one pharmaceutical carrier with which the YopM, YopM fragment or YopM variant is administered. 
     
     
         6 . The pharmaceutical composition of  claim 5 , wherein the carrier is selected from the group consisting of diluent, adjuvant, excipient, vehicle, vesicles, liposomes, microparticles, microcapsules, polymeric material, and sucrose solution. 
     
     
         7 . The pharmaceutical composition of  claim 1 , wherein the YopM, YopM fragment, YopM variant and/or immunomodulatory domain of YopM is present in a controlled release system, unit dosage form, lyophilized powder, water free concentrate, sealed container, ampoule, sachette, paste, ointment, lotion, cream, gel, transdermal patch, inhaler, nebulizer, composition for infusion, and/or composition for injection. 
     
     
         8 . The pharmaceutical composition of  claim 1 , wherein the YopM, YopM fragment YopM variant and/or immunomodulatory domain of YopM is present in a concentration of 0.01-100 wt % in the pharmaceutical composition. 
     
     
         9 . The pharmaceutical composition of  claim 3 , wherein said cytokine is selected from the group consisting of type 1 cytokines produced by Th1 T-helper cells, type 2 cytokines produced by Th2 T-helper cells, interleukins, chemokines, interferons, IL-1ra, CNTF, LIF, OSM, Epo, G-CSF, GH, PRL, IP10, I309, IFN-alpha, IFN-beta, IFN-gamma, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12 (p35+p40), IL13, IL14, IL15, IL16, IL17 A-F, IL18, IL19, IL20, IL21, IL22, IL23 (p19+p40), IL24, IL25, IL26, IL27 (p28−EBI3), IL28A, IL28B, IL29, IL30, IL31, IL32, IL33, IL35 (p35−EBI3), LT-alpha, LT-beta, LIGHT, TWEAK, APRIL, BAFF, TL1A, GITRL, OX40L, CD40L, FASL, CD27L, CD30L, 4-1BBL, TRAIL, RANK, GM-CSF, M-CSF, SCF, IL1-alpha, IL1-beta, aFGF, bFGF, int-2, KGF, EGF, TGF-alpha, TGF-beta, TNF-alpha, TNF-beta, betacellulin, SCDGF, amphiregulin, and HB-EGF. 
     
     
         10 . The pharmaceutical composition of  claim 1 , wherein said YopM, YopM fragment, YopM variant and/or immunomodulatory domain of YopM is linked to a cargo molecule, wherein optionally
 a) said cargo molecule comprises at least one compound selected from the group consisting of nucleic acids, polypeptides, organic molecules, small organic molecules, metals, nano-particles, viruses, modified viruses, viral vectors, plasmids, therapeutic proteins, suicide proteins, tumor suppressor proteins, transcription factors, kinase inhibitors, kinases, regulatory proteins, apoptotic proteins, anti-apoptotic proteins, microbial antigens, viral antigens, bacterial antigens, parasitic antigens, cellular antigens, differentiation factors, immortalisation factors, toxines, enzymes, antisense constructs, diagnostic imaging or contrast agents, isotopes, dyes, antibacterial agents, antifungal agents, antiviral agents, antiproliferative agents, cytostatics, immunosuppressive agents, histamine receptor antagonists, vitamins, analgesic agents, anti-neoplastic agents, hormones, antiinflammatory agents, adhesion-molecules, receptor-molecules, therapeutic organic molecules, organic inhibitors, peptide inhibitors, and antiaging agents; and/or   b) said cargo molecule displays a therapeutic and/or diagnostic activity.   
     
     
         11 . The pharmaceutical composition of  claim 1 , wherein said YopM, YopM fragment, YopM variant and/or immunomodulatory domain of YopM is linked to a cell-specific targeting agent. 
     
     
         12 . The pharmaceutical composition of  claim 1 , wherein said YopM, YopM fragment, YopM variant and/or immunomodulatory domain of YopM is modified, optionally by
 a) a modification that increases the solubility, stability or half-life, decreases immunogenicity and/or modulates the hydrophilic or hydrophobic character of said YopM, YopM fragment, YopM variant and/or immunomodulatory domain of YopM; or   b) chemical derivatization of said YopM, YopM fragment, YopM variant and/or immunomodulatory domain of YopM, such as derivatization with polymers, saccharides and/or lipids.   
     
     
         13 . The pharmaceutical composition of  claim 1 , wherein said YopM, YopM fragment, YopM variant and/or immunomodulatory domain of YopM comprises at least one Leucin-rich repeat (LRR) of YopM, preferably one, two, three, four, five, six, seven or eight LRRs of YopM. 
     
     
         14 . The pharmaceutical composition of  claim 1 , wherein said YopM fragment or YopM variant comprises one of the alpha helices of YopM, two of the alpha helices of YopM, one of the alpha helices of YopM and 1 to 3 YopM leucine-rich repeats or two of the alpha helices of YopM and 1 to 3 YopM leucine-rich repeats. 
     
     
         15 . The pharmaceutical composition of  claim 13 , wherein said leucine-rich repeats are leucine-rich repeats 1 to 3 of YopM, preferably amino acids 74 to 133 of SEQ ID NO: 4. 
     
     
         16 . The pharmaceutical composition of  claim 1 , wherein said YopM fragment or YopM variant comprises a nuclear localization sequence. 
     
     
         17 . The pharmaceutical composition of  claim 1 , wherein
 a) said YopM, YopM fragment, YopM variant and/or immunomodulatory domain of YopM is selected from YopM of a  Yersinia  strain naturally comprising a YopM encoding virulence plasmid;   b) said YopM, YopM fragment, YopM variant and/or immunomodulatory domain of YopM is selected from YopM of the species  Yersinia enterocolitica, Yersinia pseudotuberculosis  or  Yersinia pestis;      c) said YopM comprises the amino acid sequence of any sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8;   d) said pharmaceutical composition comprises said YopM, YopM fragment, YopM variant and/or immunomodulatory domain of YopM in a therapeutically effective amount.   
     
     
         18 . The pharmaceutical composition of  claim 1  for use in
 a) the treatment, prevention and/or amelioration of a disease; 
 b) human and/or and veterinary immunomodulatory therapy; 
 c) the regulation of inflammatory reactions of the immune system, the treatment of diseases caused by autoimmunity of the host, the treatment of inflammation, chronic inflammation, gastroenteritis, chronic gastritis, inflammatory bowel diseases (IBD), Colitis ulcerosa, psoriasis, allergic reactions, Morbus Crohn, rheumatoid arthritis, for treating bone diseases characterized by changes in bone resorption and/or for suppressing the immune system; 
 d) the treatment of acute disseminated encephalomyelitis (ADEM), Addison's disease, ankylosing spondylitis, antiphospholipid antibody syndrome (APS), aplastic anemia, autoimmune hepatitis, autoimmune Oophoritis, celiac disease, Crohn's disease (Morbus Crohn), diabetes mellitus type 1, gestational pemphigoid, good pasture's syndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's disease, idiopathic thrombocytopenic purpura, Kawasaki's disease, lupus erythematosus, Mixed Connective Tissue Disease, multiple sclerosis, myasthenia gravis, opsoclonus myoclonus syndrome (OMS), optic neuritis, Ord's thyroiditis, pemphigus, pernicious anaemia, primary biliary cirrhosis, rheumatoid arthritis, Reiter's syndrome, Sjögren's syndrome, Takayasu's arteritis, temporal arteritis, Warm autoimmune hemolytic anemia and Wegener's granulomatosis; 
 e) the treatment of osteoporosis, osteomyelitis, osteopenia, hypercalcemia, bone loss due to arthritis metastasis, immobilization or periodontal disease, Paget's disease, osteopetrosis, prosthetic loosening and the like; 
 f) the treatment of inflammatory disorders like asthma, chronic prostatitis, glomerulonephritis, hypersensitivities, inflammatory bowel diseases, pelvic inflammatory disease, reperfusion injury, arthritis, osteoarthritis, (juvenile) chronic arthritis, rheumatoid arthritis, psoriatic arthritis,  A. mutilans , septic arthritis, infectious arthritis and/or reactive arthritis, transplant rejection or vasculitis, allergic reactions, inflammatory myopathies, atherosclerosis, ischaemic heart disease, gastroenteritis, chronic gastritis, colitis ulcerosa, psoriasis, psoriasis arthritis; 
 g) in antirheumatic therapy against inflammation and structural damage; and/or 
 h) inhibition of osteoclastogenesis. 
 
     
     
         19 . A method of preventing, ameliorating and/or treating a disease, comprising administering to a subject a composition, comprising
 (i) an isolated YopM;   (ii) an isolated YopM fragment, comprising at least one alpha helix of YopM capable of mediating autopenetration;   or   (iii) an isolated YopM variant, characterized in that said variant
 (a) consists of an amino acid sequence that is at least 80% identical to any of SEQ ID NOs: 1-8 or at least 80% identical to a fragment of any of SEQ ID NOs: 1-8; and 
 (b) comprises at least one alpha helix of YopM capable of mediating autopenetration in the absence of  Yersinia  bacteria; 
   wherein said YopM, YopM fragment or YopM variant is capable of autopenetrating the cell membrane and integrating into the cell cytosol of a eukaryotic cell without the assistance of a type III secretion system.   
     
     
         20 . A kit or pharmaceutical package for use in the treatment, prevention and/or amelioration of a disease, comprising
 (i) an isolated YopM;   (ii) an isolated YopM fragment, comprising at least one alpha helix of YopM capable of mediating autopenetration;   or   (iii) an isolated YopM variant, characterized in that said variant
 (a) consists of an amino acid sequence that is at least 80% identical to any of SEQ ID NOs: 1-8 or at least 80% identical to a fragment of any of SEQ ID NOs: 1-8; and 
 (b) comprises at least one alpha helix of YopM capable of mediating autopenetration in the absence of  Yersinia  bacteria; 
   wherein said YopM, YopM fragment or YopM variant is capable of autopenetrating the cell membrane and of integrating into the cell cytosol without the assistance of a type III secretion system, and of downregulating cytokines and/or cytokine receptors and/or genes which respond to cytokines and/or cartilage-destroying molecules and/or of inhibiting osteoclastogenesis,   and wherein the kit or pharmaceutical package further comprises a documentation indicating the treatment regimen, use and/or employment of the YopM, YopM fragment or YopM variant for administration.   
     
     
         21 . A kit or pharmaceutical package for use in the treatment, prevention and/or amelioration of a disease, comprising
 an immunomodulatory domain of YopM, wherein said immunomodulatory domain is (i) linked to a cell-penetrating peptide that is heterologous to YopM, (ii) has essentially no capability of autopenetrating the cell membrane and of integrating into the cell cytosol and (iii) is capable of downregulating cytokines, and/or cytokine receptors and/or genes which respond to cytokine and/or cartilage-destroying molecules and/or of inhibiting osteoclastogenesis.