EP2197906A1 - Polypeptide de fusion comprenant des unités de répétition de s-antigène - Google Patents

Polypeptide de fusion comprenant des unités de répétition de s-antigène

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Publication number
EP2197906A1
EP2197906A1 EP08802876A EP08802876A EP2197906A1 EP 2197906 A1 EP2197906 A1 EP 2197906A1 EP 08802876 A EP08802876 A EP 08802876A EP 08802876 A EP08802876 A EP 08802876A EP 2197906 A1 EP2197906 A1 EP 2197906A1
Authority
EP
European Patent Office
Prior art keywords
fusion polypeptide
antigen
disease
seq
fragment
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP08802876A
Other languages
German (de)
English (en)
Inventor
Kirsten Falk
Olaf RÖTZSCHKE
Richard Lucius
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Humboldt Universitaet zu Berlin
Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft
Original Assignee
Humboldt Universitaet zu Berlin
Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft
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Filing date
Publication date
Application filed by Humboldt Universitaet zu Berlin, Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft filed Critical Humboldt Universitaet zu Berlin
Priority to EP08802876A priority Critical patent/EP2197906A1/fr
Publication of EP2197906A1 publication Critical patent/EP2197906A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/445—Plasmodium
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/001—Preparations to induce tolerance to non-self, e.g. prior to transplantation
    • 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
    • A61K39/35—Allergens
    • 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
    • A61K39/385—Haptens or antigens, bound to carriers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a fusion polypeptide comprising a) at least one fragment of the Plasmodium falciparum S-antigen or a variant thereof, wherein the fragment comprises one or a plurality of Plasmodium falciparum S-antigen repeat unit(s) or variant(s) thereof and b) at least one antigen different from the S-antigen.
  • the present invention also relates to a pharmaceutical composition comprising that fusion polypeptide as well as to the use thereof for the treatment or prevention of an autoimmune disease, an allergic reaction, a transplantation-related complication, e.g. graft rejection or 'graft vs. host disease', and another inflammatory disease and a method for treatment or prevention of said conditions.
  • the immune system protects the body from infectious agents and diseases.
  • the immune system can be the cause of illness.
  • autoimmunity is the failure of an organism to recognize its own constituent parts as own, which results in an immune response against its own cells and tissues.
  • Autoimmune diseases such as multiple sclerosis, diabetes mellitus type 1, systemic lupus erythematosus or rheumatic arthritis, to name only a few prominent members, are caused by the attack of autoreactive immune cells leading to destruction of healthy own cells.
  • a second example where the immune system can cause illness is during tissue or organ transplantation. Except in the cases of genetically identical animals, such as monozygotic twins, tissue and organ transplants are rejected by the recipient's immune system as foreign. The immune reaction against transplants is even more pronounced in transplantation across species or xenotransplantation.
  • a third example where the immune system harms the host is during an allergic reaction where the immune system is activated by a generally innocuous antigen causing inflammation and in some cases tissue damage.
  • immunosuppressive drugs such as cyclosporin A, tacrolimus, IFN- ⁇ , Cop-1 and corticosteroids
  • cyclosporin A may cause decreased renal function, hypertension, toxicity and it must be administered for the life of the patient.
  • Corticosteroids may cause decreased resistance to infection, painful arthritis, osteoporosis and cataracts.
  • immune suppressants of this class trigger a general shut-down of the immune system.
  • opportunistic infections may result from the incapacitated immune system as one of the side-effects.
  • antibody therapies are often used in connection with immune suppression.
  • anti-T cell antibodies may cause fever, hypertension, diarrhea or sterile meningitis and are expensive, especially when a long term administration is necessary.
  • the US patent application 20070026057 describes a method of modulating an immune response by administration of a composition comprising antigen-containing vesicles or liposomes, an immunomodulatory factor (e.g. an interleukin) and a ligand for a dendritic cell (e.g. an antibody).
  • an immunomodulatory factor e.g. an interleukin
  • a ligand for a dendritic cell e.g. an antibody
  • the liposomes are directed and attached to den- dritic cells by the ligand.
  • Subsequent release of the antigen and the immunomodulatory factor can modify the immune response. That strategy may overcome the disadvantages described above for systemically administered broadly acting suppressive drugs.
  • a further approach is based on oligomers of a single T cell epitope as describe, for example, in the international application WO 98/05684.
  • oligomers of up to 32 repeats of one T cell epitope (HA306-318) linked by specific amino acid sequences were shown to modulate an immune response against the corresponding epitope.
  • the epitope oligomers according to WO 98/05684 have shown to target cell surface MHC molecules on certain antigen presenting cells (APC) and to suppress the clinical symptoms in experimental autoimmune model systems of multiple sclerosis (MS), autoimmune neuritis and type I diabetes.
  • APC antigen presenting cells
  • the technical problem underlying the present invention was to provide alternative immunosuppressors which overcome the disadvantages of systemically administered broadly acting suppressive drugs as well as the above described problems connected with epitope oligomers.
  • the solution to the technical problem described above is achieved by providing the embodiments characterized in the claims.
  • the present invention relates to a fusion polypeptide comprising a) at least one fragment of the Plasmodium falciparum S-antigen or a variant thereof, wherein the fragment comprises one or a plurality of Plasmodium falciparum S-antigen repeat unit(s) or variant(s) thereof and b) at least one antigen different from the S-antigen.
  • the fusion polypeptide according to the present invention is highly specific for particular antigens. Therefore, it is able to induce immunosuppression without causing the negative effects as described for broadly acting unspecific immunosuppressors, e.g. opportunistic infections or other systemic disorders. Suppression of the immune system using the fusion polypeptide according to the present invention is selective and evident only for the cell or tissue expressing the antigen.
  • a further advantage of the fusion polypeptide according to the present invention is the simple and inexpensive way of production.
  • the fusion polypeptide may be synthetically produced and purified according to standard biotechnological methods.
  • the fusion polypeptide may also be produced by recombinant techniques known in the art.
  • S-antigen S-Ag
  • body fusion polypeptides comprising fragments of the S-antigen
  • any antigen can be combined with the S-antigen fragment without causing solubility problems.
  • the antigen within the fusion polypeptide is not restricted to low molecular sizes due to a different mechanism underlying the action of the fusion polypeptide as compared to epitope oligomers. While the principle of epitope oligomers is the direct binding of the epitope to an MHC molecule, fusion polypeptides according to the present invention work in a different way.
  • the S-antigen sequence seems to target the fusion polypeptide to a tolerance-inducing antigen-presenting cell (APC). Therefore, the antigen attached to the S- antigen sequence does not have to consist of a short (peptide) sequence but rather can comprise of larger components covering multiple antigenic epitopes.
  • fusion polypeptides based on the S-antigen aim at a repair of the immune system by re-establishing the natural balance between the suppressor and effector cells.
  • fusion polypeptide refers to a polypeptide or protein having at least two peptides, polypeptides or proteins linked, either directly or via a linker such as an amino acid, peptide, non-peptide or chemical linker.
  • the linkage is preferably covalent.
  • the components forming the fusion polypeptide are typically linked C-terminus to N-terminus, although they can also be linked C-terminus to C-terminus or N-terminus to N- terminus.
  • the peptides, polypeptides or proteins of the fusion polypeptide can be in any order.
  • the fusion polypeptide may comprise from 20 to 10.000, preferably from 30 to 1 .000, more preferably from 50 to 500 and most preferably from 70 to 300 amino acids.
  • the fusion polypeptide comprises at least one fragment of the Plasmodium falciparum S- antigen or a variant thereof, wherein the fragment comprises one or a plurality of Plasmodium falciparum S-antigen repeat unit(s) or variant(s) thereof.
  • P. falciparum refers to a protozoan parasite that causes malaria in humans. It is transmitted by Anopheles mosquitoes. P. falciparum is the most dangerous of these infections as P. falciparum malaria has the highest rates of complications and mortality. In addition, it accounts for 80% of all human malarial infections and 90% of the deaths. It is more prevalent in sub-Saharan Africa than in other regions of the world. However, P. falciparum isolates could also be recovered from Asia as well as South America. Presently, 30 different isolates are known. Investigations have been undertaken on P. falciparum in view of a malaria vaccination. For examples, different P. falciparum antigens have been discovered which are suggested to be used in malaria vaccines.
  • P. falciparum S-antigen refers to the wild-type sequence of the S-antigen of P. falciparum.
  • Preferred S-antigen sequences or parts thereof are given in SEQ ID NOs: 7-12 below.
  • a "fragment” in the context of the invention is to be understood as a truncated (peptide, polypeptide or protein) sequence, i.e. a sequence which is N-terminally, C-terminally or intrasequentially truncated compared to the amino acid sequence of the original (wild-type) sequence.
  • a “fragment of the P. falciparum S-antigen” according to the invention is a part of the wild- type sequence of the S-antigen. However, a “fragment of the P. falciparum S-antigen” is not to be understood to be limited by the actual size of the wild-type sequence of the S-antigen.
  • a “fragment of the P. falciparum S-antigen” in the context of the invention can be extended by one or more additional Plasmodium falciparum S-antigen fragments, one or more additional repeat units or variants of a fragment of Plasmodium falciparum S-antigen fragments, or one or more additional amino acids. Therefore, the fragment according to the present invention may be larger than the natural occuring wild-type sequence of the Plasmodium falciparum S-antigen.
  • Plasmodium falciparum S-antigen repeat unit or variant thereof is to be understood as a characteristic protein or peptide sequence, which is repeated as identical or with minor variations and without any other protein or peptide sequences in the sequence of the S-antigen of Plasmodium falciparum.
  • a Plasmodium falcipa- rum S-antigen repeat unit or variant thereof according to the invention has a typically length of 6 to 18 amino acids, i.e. of 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 1 7, or 18 amino acids, preferably of 8 to 15 amino acids, i.e. of 8, 9, 10, 1 1 , 12, 13, 14, or 15 amino acids.
  • a study underlying the present invention encompassed an analysis of various genomes which revealed that tandem repeats of amino acid sequences are particularly frequent among parasites. Striking examples are the S-antigens of Plasmodium. Their repeats cover nearly two thirds of the protein.
  • Fig. 2 shows the sequence of the S-antigen of the Plasmodium falciparum isolate NF7 (SEQ ID NO: 10).
  • SEQ ID NO: 10 The protein is secreted in large amounts during the erythrocyte stage and function as a target for the immune system. Antibodies against these repeats are detectable in patients suffering from malaria and the region is highly polymorphic so that different variants of the S-antigen (S-Ag) exist.
  • Plasmodium S-antigens are characterized by a high content of repeat sequences. These repeat sequences consist, in general, of a stretch having several tandem repeats (repeat units) of 8 or 1 1 amino acids. However, also repeat units of 15 amino acids in length could be identified (e.g. isolate NF7). Table 1 illustrates some particular repeat units of Plasmodium falciparum isolates.
  • the fragment of the P. falciparum S-antigen or a variant thereof comprises one or a plurality of the P. falciparum S-antigen repeat unit(s).
  • the fusion polypeptide comprises at least 2, preferably at least 5, more preferably at least 8, even more preferably at least 16 and most preferably at least 24 repeat units or more.
  • the repeat units may or may not be identical, i.e. every repeat unit can be combined with another repeat unit.
  • one or two amino acids in the repeat units are interchangeable (e.g. NF7 isolate: R or L on position 2). Variants of the repeat units are also encompassed.
  • the fragment may further comprise sequences of the P. falciparum S- antigen which are not repetitive. For example, sequences positioned N- or C-terminal of the repeat units in the S-antigen may further be contained in the fragment.
  • Preferred repeat units according to the invention are those of Table 1 (SEQ ID NOs: 1 -6, and 13). However, any other repeat unit of a P. falciparum S-antigen may be used in accordance with the invention.
  • the fragment comprises at least 8 amino acids.
  • repeat units of a variety of the S-antigen of P. falciparum isolates are 8 amino acids in length. Therefore, according to this embodiment, the fragment corresponds to one repeat unit or a variant thereof.
  • the fragment comprises at least 16 amino acids or more. Accordingly, 16 amino acids correspond to two repeat units or variants thereof of 8 amino acids each which can either be identical or not.
  • the fragment comprises at least 19 or 23 amino acids or more. Accordingly, 19 und 23 amino acids, respectively, correspond to two repeat units or variants thereof which are not identical, i.e. one repeat unit of 8 amino acids and another repeat unit of 11 and 15 amino acids, respectively.
  • the fragment comprises at least 1 1 amino acids.
  • repeat units of the P. falciparum S-anigens can also be 1 1 amino acids in length.
  • the fragment of present embodiment corresponds to one repeat unit or a variant thereof.
  • the fragment comprises at least 22 amino acids or more, corresponding to two repeat units or variants thereof of 1 1 amino acids each which can ei- ther be identical or not.
  • the fragment comprises at least 26 amino acids or more. Accordingly 26 amino acids corresponds to at least two repeat units or variants thereof which are not identical, i.e. one repeat unit of 1 1 amino acids and another repeat unit of 15 amino acids.
  • the fragment comprises at least 15 amino acids.
  • falciparum S-antigens can also be 15 amino acids in length.
  • the fragment of present embodiment corresponds to one repeat unit or a variant thereof.
  • the fragment comprises at least 30 amino acids or more, corresponding to two repeat units or variants thereof of 15 amino acids each which can either be identical or not.
  • the fragment is derived from the Plasmodium falciparum isolate NF7 S-antigen (Fig. 2 and SEQ ID NO: 10) or a variant thereof.
  • the repeat unit has the sequence of SEQ ID NO: 1.
  • the repeat unit has the sequence of SEQ ID NO: 2.
  • the fragment comprises at least 2, preferably at least 5, more preferably at least 8, even more preferably at least 16 and most preferably at least 24 repeat units or more of the sequence of SEQ ID NO: 1 or of SEQ ID NO: 2.
  • the fusion polypeptide comprises the repeat units of SEQ
  • the fragment is derived from the Plasmodium falciparum isolate FC27 S-antigen (SEQ ID NO: 7) or a variant thereof.
  • the repeat unit has the sequence of SEQ ID NO: 3.
  • the repeat unit has the sequence of SEQ ID NO: 13.
  • the fragment comprises at least 2, preferably at least 5, more preferably at least 8, even more preferably at least 16 and most preferably at least 24 repeat units or more of the sequence of SEQ ID NO: 3 or of SEQ ID NO: 13.
  • the fusion polypeptide comprises the repeat units of SEQ ID NO: 7.
  • the fragment is derived from the Plasmodium falciparum isolate WELLCOME S-antigen (SEQ ID NO: 8) or a variant thereof.
  • the repeat unit has the sequence of SEQ ID NO: 4.
  • the fragment com- prises at least 2, preferably at least 5, more preferably at least 8, even more preferably at least 16 and most preferably at least 24 repeat units or more of the sequence of SEQ ID NO: 4.
  • the fusion polypeptide comprises the repeat units of SEQ ID NO: 8.
  • the fragment is derived from the Plasmodium falciparum isolate 3D7 S-antigen (SEQ ID NO: 9) or a variant thereof.
  • the repeat unit has the sequence of SEQ ID NO: 5.
  • the fragment comprises at least 2, preferably at least 5, more preferably at least 8, even more preferably at least 16 and most preferably at least 24 repeat units or more of the sequence of SEQ ID NO: 5.
  • the fusion polypeptide comprises the repeat units of SEQ ID NO: 9.
  • the fragment is derived from the Plasmodium falciparum isolate V1 S-antigen (SEQ ID NO: 12) or a variant thereof.
  • the repeat unit has the sequence of SEQ ID NO: 6.
  • the fragment comprises at least 2, preferably at least 5, more preferably at least 8, even more preferably at least 16 and most preferably at least 24 repeat units or more of the sequence of SEQ ID NO: 6.
  • the fusion polypeptide comprises the repeat units of SEQ ID NO: 12.
  • the fragment is derived from the Plasmodium falciparum isolate Palo Alto S-antigen (SEQ ID NO: 1 1 ) or a variant thereof.
  • a “variant” in the context of the invention refers to a (poly)peptide or protein having an amino acid sequence which differs from the original sequence in one or more mutation(s), such as one or more substituted, inserted and/or deleted amino acid(s), and having a sequence homology of at least 60%, preferably at least 70%, more preferably at least 80%, equally more preferably at least 85%, even more preferably at least 90% and most preferably at least 95% or more, to the wild-type sequence.
  • sequence homology encompasses sequence identity and sequence similarity.
  • sequences having a sequence homology are either identical or similar to each other to a certain extent, as defined below.
  • the sequences can be aligned in order to be subsequently compared to one another. Therefore, gaps can be inserted into the first sequence and the component at the corresponding position of the second sequence can be compared. If a position in the first sequence is occupied by the same component as is the case at a position in the second se- quence, the two sequences are identical at this position.
  • the percentage to which two sequences are identical is a function of the number of identical positions divided by the total number of positions.
  • the percentage to which two sequences are identical can be determined using a mathe- matical algorithm.
  • a preferred, but not limiting, example of a mathematical algorithm which can be used is the algorithm of Karlin et al. (1993), PNAS USA, 90:5873-5877 or Altschul et al. (1997), Nucleic Acids Res, 25:3389-3402. Such an algorithm is integrated in the BLAST program. Sequences which are identical to the sequences of the present invention to a certain extent can be identified by this program.
  • amino acids can be classified into amino acids having aliphatic side chains, polar side chains, e.g. positively or negatively charged side chains, hydrophobic side chains, aromatic groups in the side chains or amino acids, the side chains of which can enter into hydrogen bridges, e.g. side chains which have a hydroxyl function.
  • Amino acids belonging to one particular class are considered similar. For example serine and threonine, as well as leucine and isoleucine will be considered similar. Using a certain similarity matrix for the alignment which defines that similarity will not only identify identical but also similar sequences.
  • the fusion polypeptide further comprises at least one antigen different from the S-antigen.
  • antigen means a substance which leads to activation of the immune system and to a response by production of antibodies to the respective antigens (humoral immunity) and by production of immune competent cells (cellular immunity).
  • antigens are substances such as cells, proteins, peptides, nucleic acids, polysaccharides, lipids or other macromolecules or fragments thereof.
  • antigens are proteins and peptides or fragments thereof, such as epitopes of those proteins or peptides.
  • Epitopes also called "antigen determinants"
  • B-cell epitopes and T-cell epitopes are typically presented on MHC molecules, wherein e.g. MHC-I typically presents epitopes with a length of about 9 amino acids and MHC-II typically presents epitopes with a length of about 12-15 amino acids).
  • the aforementioned protein or peptide antigens may be modi- fied, i.e. in the form of derivatives of those proteins or peptides.
  • Derivatives of proteins or peptides are fragments and variants thereof as defined above.
  • derivatives of said protein antigens are functional derivatives, i.e. have immunological or immunogenic activity, in particular trigger an immune response in an organism.
  • fragments including an antigenic epitope are preferred.
  • antigens are exogenous or endogenous antigens.
  • Exogenous antigens are antigens that enter the cell or the body from outside, for example by inhalation, ingestion or injection, etc.
  • Antigens include e.g., pathogenic substances such as bacteria and viruses or parts thereof. These antigens are internalized by antigen-presenting cells ("APCs", such as dendritic cells or macrophages) and processed into fragments. APCs then present the fragments to T helper cells (e.g. CD4 + ) by the use of MHC Il molecules on their surface. Recognition of these antigen fragments by T cells leads to activation of the T cells and secretion of cytokines. Cytokines are substances that can activate proliferation of immune cells such as cytotoxic T cells, B cells or macrophages.
  • exogenous antigens are recognized and subsequently eliminated by the body's immune system.
  • the body does not just sense harmful substances such as pathogens, however, it sometimes develops an immune response against harmless sub- stances.
  • antigen causes an allergic reaction/allergy (hypersensitivity) which is characterized by the production of IgE antibodies.
  • IgE bind to Fc epsilon R1 (high affinity) receptors on the surface of mast cells and basophils, both involved in the acute inflammatory response.
  • the class switch in the plasma cell leading to IgE is tightly regulated by the immune system.
  • CD45 plays a critical regulatory role in receptor signaling through its protein tyrosine phosphatase and Janus kinase (JAK) phosphatase activities.
  • IL-4 is the primary interleukin which induces switch recombination.
  • Class switch recombination to IgE can also be triggered by the TH2 cytokine IL-13.
  • CD45 is able to function as JAK phosphatase in human B cells, and this activity is directly associated with negative regulation of the class switch recombination to IgE.
  • IgE-bearing epidermal dendritic cells have also been found.
  • IgE When IgE is first secreted, it binds to the Fc receptors on a mast cell or basophil, and such an IgE-coated cell is said to be sensitized to the allergen in question. A later exposure by the same allergen causes reactivation of these IgE, which then signals for the degranulation of the sensitized mast cell or basophil.
  • GPCRs such as chemokine receptors.
  • granules release histamine and other inflammatory chemical mediators (cytokines, interleukins, leukotrienes, and prostaglandins) into the surrounding tissue causing several systemic effects, such as vasodilation, mucous secretion, nerve stimulation and smooth muscle contraction.
  • cytokines cytokines, interleukins, leukotrienes, and prostaglandins
  • vasodilation cytokines, interleukins, leukotrienes, and prostaglandins
  • the fusion polypeptide comprises an allergen.
  • the allergen is an allergen in mammals, more preferably in humans.
  • an allergen can be seleceted from the group consisting of naturally occurring allergens or non-naturally occurring allergens such as those contained in allergy vac- cines including allergens from: mites such as, but not limited to, Dermatophagoides farinae, Dermatophagoides pteronyssinus, Acarus siro, Blomia tropicalis, Chortoglyphus arcuatas, Euroglyphus maynei, Lepidoglyphus destructor, Tyrophagus putrescentiae, and Glyphagus demesticus; venoms such as, but not limited to, Bombus spp., Vespa crabro, Apis mellifera, Dolichovespula spp., Polistes spp., Vespula spp., Dolichovespula maculata, and Dolichovespula arenaria; insects such as, but not limited to, Camponotus pennsylvanicus
  • weeds such as, but not limited to, Atriplex polycarpa, Baccharis halimifolia, Baccharis sarothroides, Hymenoclea salsola, Amaranthus hybridus, Xanthium strumarium (commune), Rumex crispus, Eupathium capillifolium, Solidago spp., Amaranthus tuberculatus (Acnida tamariscina), Allenrolfea occidentalis, Chenopodium botrys, Kochia scoparia, Cheno- podium album, Iva xanthifolia, Iva angustifolia, Chenopodium ambrosioides, Artemisia vulgaris, Artemisia ludoviciana, Urtica dioica, Am
  • botrytis Fagopyrum esculentum, Brassica oleracea var. capitata, Theobroma cacao, Cucumis melo, Daucus carota, Brassica oleracea var. botrytis, Apium graveolens var.
  • Prunus sp. Cinnamomum verum, Coffea arabic, Zea mays, Vaccin- ium macrocarpon, Cucumis sativus, Allium sativum, Zingiber officinale, Vitis sp., Citrus paradisi, Humulus lupulus, Citrus limon, Lactuca sativa, Agaricus campestris, Brassica sp., Myristica fragrans, Avena sativa, Olea europaea, Allium cepa var.
  • rapa Vanilla planifolia, Citrullus lanatus var. lanatus, and Triticun aestivum
  • fish and shellfish such as, but not limited to, Micropterus sp., lctalurus punctatus, Mercenaria mercenaria, Gadus morhua, Callinectes sapidus, Platichthys sp., Hippoglossus sp., Homarus americanus, Scomber scombrus, Crassostrea virginica, Se- bastes marinus, Salmo salar, Clupeiformes, Pecten magellanicus, Penaeus sp., Salvelinus sp., and Thunnus sp.; animal foods such as, but not limited to, Bos taurus, Ovis aries, and Sus scrofa; poultry products such as, but not limited to, chicken (Gallus gallus) products and turkey (Meleagris gallopavo) products
  • dust such as, but not limited to, barley grain dust, corn grain dust, house dust, mattress dust, oat grain dust, wheat grain dust, and upholstery dust, and fragments and variants thereof.
  • endogenous antigens are antigens which have been generated within the cell, e.g. as a result of normal cell metabolism or when the normal metabolism is disturbed (e.g. such as for tumor antigens). Fragments of these antigens are presented on MHC I molecules on the surface of APCs. These antigens are recognized by activated antigen-specific cyto- toxic CD8 + T cells. After recognition, those T cells react in secretion of different toxins that cause lysis or apoptosis of the antigen-presenting cell.
  • Endogenous antigens comprise antigens, e.g. proteins or peptides encoded by a foreign nucleic acid inside the cell as well as proteins or peptides encoded by the genetic information of the cell itself, or antigens from intracellularly occurring viruses.
  • An autoantigen is usually a normal protein or complex of proteins (and sometimes DNA or RNA) that is recognized by the immune system of patients suffering from a specific autoimmune disease. These antigens should under normal conditions not be the target of the immune system, but due to mainly genetic and environmental factors the normal immune tolerance for such a substance has been lost in these patients.
  • the fusion polypeptide may comprise an autoantigen.
  • the autoantigen is an autoantigen in mammals, more preferably in humans.
  • the autoantigen is selected from the group consisting of myelin basic protein (MBP), myelin oligodendrocyte protein (MOG), proteolipid protein (PLP), HLA (class II), HLA-DQ, HLA-B27, BIP, 21 -hydroxylase, ardiolipin, Lupus coagulant, CD151 , G6PI, mitochondrial malate dehydrogenase (mMDH), complex III, La/SS-B, Ro/SS- A, islet cell autoantigen (ICA1 , ICA69), p200, LRP2, HC gp-39, NAC, hnRNPA (2, 3), RA33, giantin, RCD8, limkain b1 , seminal vesicle autoantigen (SVA), PM-Scl-75, protein
  • MBP myelin basic protein
  • any known antigen, a fragment or a variant thereof can be used in the fusion polypeptide.
  • other unknown antigens may be used which can be identified, e.g., using sequence comparison algorithms, as described herein, or other methods known to those of skill in the art, e.g., hybridization assays and antibody binding assays.
  • the fusion polypeptide may comprise a transplantation antigen.
  • the transplantation antigen is an antigen in mammals, more preferably in humans.
  • the transplantation antigen is selected from the group consisting of genes of the Major Histocompatibility Complex (MHC) or other genes (minor H antigens) differing in their amino acid sequence between donor and recipi- ent or being expressed only by the donor.
  • MHC molecules are HLA-DR, -DP, - DQ and HLA-A, -B, -C.
  • Examples for minorH antigens encoded by the Y chromosome are SMCY, UTY, DFFRY, and DBY.
  • examples for other polymorphic minorH antigens are HA-1 , HA-2, HA-8, and HB-1.
  • the fusion polypeptide according to the invention may comprise one or more different or similar Plasmodium S-antigen fragment(s) and antigen(s). These components can be combined arbitrarily and can be in any order.
  • the fusion polypeptide according to the invention may comprise further components.
  • the fusion polypeptide may comprise one or more linkers, e.g. to join the Plasmodium S-antigen fragment(s) and/or repeat unit(s) and the antigen(s), as well as other non-S- antigen sequences.
  • Linkers which may be used in accordance with the invention are known to a person skilled in the art. They encompass, without being limited to, covalent linkers such as amino acid linkers, (poly)peptide linkers, or chemical linkers, e.g. disulfide bonds or ethylendiamine, glycol, PEG, glycerol and glycerol derivatives, diol or pyrrolidine linkers.
  • Amino acid linkers consist of one or a few, preferably 1 to 5, more preferably 2 to 4 identical, similar or differ- ent amino acids.
  • (Poly)peptide linkers encompass a stretch of at least 6 identical, similar or different amino acids.
  • the fusion polypeptide may further comprise one or more purification tag(s).
  • tags in- elude, without being limited to, (poly)peptide or protein tags such as polyhistidine (His) tag (such as hexahistidine tag), streptavidin (Strep) tag, SBP (streptavidin-binding) tag, GST (glutathione S-transferase) tag, FLAG tag, HA tag, Myc tag, Swa1 1 epitope tag, calmodulin tag.
  • MBP maltose-binding protein
  • the tag(s) may either be located on the C-terminus, the N-terminus or within the sequence of the fusion polypep- tide.
  • the fusion polypeptide may also comprise further peptide or non-peptide components such as, e.g., lipids, carbohydrates such as polysaccharides, and other biological and non- biological polymers (e.g. PEG).
  • further peptide or non-peptide components such as, e.g., lipids, carbohydrates such as polysaccharides, and other biological and non- biological polymers (e.g. PEG).
  • the fusion polypeptide according to the invention may be produced chemically or via recombinant methods for production of polypeptides. Those methods are well known in the art. A general description of recombinant methods is given, for example, in Maniatis et al. (2001 ), "Molecular Cloning-A Laboratory Manual”.
  • SPPS solid phase peptide synthesis
  • the production of the fusion polypeptide can either be performed for the fusion polypeptide as such or for the components of the fusion polypeptide (fragments, repeat units, antigens) separately which are linked subsequently to the fusion protein, e.g. by the linkers as described above.
  • the synthesized fusion polypeptide or its components may be purification using methods known in the art, e.g. chromatography, such as, for example, affinity chromatography (HPLC, FPLC, etc.), ion exchange chromatography, gel chromatography, size exclusion chromatography, gas chromatography, PAGE, or other biochemical or biophysical methods. Chromatography methods can employ tags as described above.
  • chromatography such as, for example, affinity chromatography (HPLC, FPLC, etc.), ion exchange chromatography, gel chromatography, size exclusion chromatography, gas chromatography, PAGE, or other biochemical or biophysical methods.
  • Chromatography methods can employ tags as described above.
  • Fusion polypeptides according to the invention are effective inducers of antigen-specific immune tolerance.
  • Induction of suppressor cells such as CD25+Foxp3+ Treg cells and reversal of suppression by antibodies blocking the IL-10 receptor clearly indicate that it is mediated by active immune tolerance, i.e. recruitment of antigen-specific suppressor cells.
  • a fusion polypeptide was constructed comprising a fragment of the myelin oligodendrocyte protein (MOG, residues 35-55) attached to repeat units (SEQ ID NO: 1 ) of the Plasmodium falciparum isolate NF7 (24 units) (S-AgMOG35-55/tandem repeat-MOG peptide).
  • MOG myelin oligodendrocyte protein
  • SEQ ID NO: 1 the Plasmodium falciparum isolate NF7 (24 units)
  • Myelin is a collection of lipids and proteins that sheaths around neural axons.
  • the myelin In multiple sclerosis, the myelin is destructively removed from around the axon which slows down nerve impulses in a process known as demyelination.
  • axons are demy- elinated in inflammatory patches called lesions. As the disease progresses, oligodendrocytes and, ultimately, the axons themselves are destroyed. The destruction is caused by the body's own immune system.
  • EAE experi- mental autoimmune encephalomyelitis
  • SEQ ID NO:1 , 24 units were linked to a fragment of the ⁇ -chain of human insulin protein (INSB 9-23).
  • INLB 9-23 a fragment of the ⁇ -chain of human insulin protein
  • Insulin is an autoantigen, suspected to be a major target antigen in the autoimmune attack leading to the destruction of insulin-producing islet cells. The elimination of these cells leads to the manifestation of type 1 diabetes and is characterized by the inability of the patient to produce insulin to reduce blood sugar levels.
  • the repeat units (SEQ ID NO: 1 , 24 units) as described above were also fused to a fragment of ovalbumin (OVA, residues 323-339).
  • OVA ovalbumin
  • the resulting fusion polypeptide S- AgOVA323-339 (S-AgOVA/OVA-peptid) was tested in the delayed-type hypersensitivity (DTH) model.
  • DTH delayed-type hypersensitivity
  • the administration of the construct was able to reduce the amount of ovalbumin-specific IgGI antibodies (Fig. 14).
  • suppressor cells were generated in mice in reaction to that fusion polypeptide after administration.
  • these by-stander mechanisms can be based on cell/cell contact or the release of suppressive cytokines such as TGF-b or IL-10. In any case, these by-stander mechanisms result in an effective shut-down of proinflammatory effector cells located in the proximal vicinity to the suppressor T cell.
  • Suppressor T cells can be generated de novo in the thymus as well as by "polarization" of na ⁇ ve CD4+ T cells in the periphery. The latter pathway is of particular importance for the induction of peripheral tolerance in therapeutic settings (Fig.1 B). Polarization was initially described for effector CD4+ T cells.
  • na ⁇ ve T cells can differen- tiate either into TH-1 cells producing cytokines that address macrophages effector or to TH- 2 cells inducing antibody production by affecting B cells.
  • APC antigen presenting cell
  • na ⁇ ve T cells can differen- tiate either into TH-1 cells producing cytokines that address macrophages effector or to TH- 2 cells inducing antibody production by affecting B cells. Recent studies have shown, however, that similar processes can also lead to the generation of suppressor T cells. This applies both for IL-10 secreting Tr1 cells as well as for regulatory T cells (Treg), characterized by the expression of the marker CD25
  • immune tolerance can be induced against short peptides representing T cell epitopes.
  • larger polypeptides and even antigenic proteins can be linked to the repeat sequence.
  • the fact, that immune tolerance induction against the epitope requires the physical linkage to the repeat structure fur- ther indicates that the repeat may act as a targeting sequence hitting a receptor on tolerance inducing APC.
  • Short repetitive amino acid sequences can apparently target tolerance inducing antigen presenting cells.
  • the fusion polypeptide accord- ing to the invention may be used in medicine, in particular for the treatment or prevention of an autoimmune disease, an allergic reaction, a transplantation-related complication, e.g. graft rejection or 'graft vs. host disease', or another inflammatory disease.
  • the fusion polypeptide according to the invention may also be used for the preparation of a pharmaceutical composition for the treatment or prevention of an autoimmune disease, an allergic reaction, a transplantation-related complication, e.g. graft rejection or 'graft vs. host disease', or another inflam- matory disease.
  • a method for the treatment or prevention of an autoimmune disease, an allergic reaction, a transplantation-related complication, e.g. graft rejection or 'graft vs. host disease', or another inflammatory disease by administering a fusion polypeptide according to the invention is also encompassed.
  • Autoimmune diseases to be prevented or treated in accordance with the invention are selected from the group consisting of Acute disseminated encephalomyelitis (ADEM), Addi- son's disease, Ankylosing spondylitis, Antiphospholipid antibody syndrome (APS), Aplastic anemia, Autoimmune encephalomyelitis, Autoimmune hepatitis, Autoimmune neuritis, Autoimmune oophoritis, Coeliac disease, Crohn's disease, Diabetes mellitus type 1 , Gestational pemphigoid, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome (GBS), Hashimoto's disease, Idiopathic thrombocytopenic purpura, Kawasaki's Disease, Lupus erythematosus, Multiple sclerosis, Myasthenia gravis, Opsoclonus myoclonus syn- drome (OMS), Optic neuritis, Ord's thyroiditis,
  • Allergic diseases to be prevented or treated in accordance with the invention are selected from the group consisting of eczema, hives, hay fever, asthma, food allergies, and reactions to the venom of stinging insects such as wasps and bees.
  • a preferably allergic disease to be prevented or treated is asthma.
  • Still another subject matter of the present invention is a composition, preferably a pharmaceutical composition comprising the fusion polypeptide as described above.
  • the (pharmaceutical) composition may optionally a (pharmaceutically) acceptable carrier and/or further auxiliary substances and additives.
  • the (pharmaceutical) composition employed according to the present invention typically comprises a safe and effective amount of the fusion polypeptide.
  • safety and effective amount means an amount of the fusion polypeptide according to the invention such as is sufficient to induce a significantly positive change of a state to be treated, e.g. allergic reactions, autoimmune diseases. At the same time, however, a "safe and effective amount” is low enough to avoid serious side effects in the therapy of these diseases that is to say to render possible a reasonable ratio of advantage and risk. Determination of these limits typically lies within the range of reasonable medical judgement.
  • concentration of the fusion polypeptide according to the invention in such (pharmaceu- tical) compositions may therefore vary, for example, without being limited to, within a wide range of from e.g. 0.1 ng to 1,000 mg/ml.
  • concentration of the fusion polypeptide according to the invention can vary in connection with the particular state to be treated and the age and the physical state of the patient to be treated, the severity of the state, the duration of the treatment, the nature of the concomitant therapy, of the particular (pharmaceutically) acceptable carrier used and similar factors within the knowledge and experience of the treating doctor.
  • the pharmaceutical composition described here can be employed for human and also for veterinary medicine purposes.
  • the (pharmaceutical) composition according to the invention described here can optionally comprise a (pharmaceutically) acceptable carrier.
  • the term "(pharmaceutically) acceptable carrier” as used herein preferably includes one or more compatible solid or liquid fillers, or diluents or encapsulating compounds which are suitable for administration to a person.
  • the term "compatible” as used here means that the constituents of the composition are capable of being mixed together with the fusion polypeptide according to the invention and the aux- iliary substance optionally contained in the composition, as such and with one another in a manner such that no interaction occurs which would substantially reduce the (pharmaceutical) effectiveness of the composition under usual condition of use, such as e.g. would reduce the (pharmaceutical) activity of the fusion polypeptide.
  • (Pharmaceutically) acceptable carrier must of course have a sufficiently high purity and a sufficiently low toxicity to render them suitable for administration to a person to be treated.
  • Carriers are chosen dependent on the way of administration; they may be in solid or liquid form. Accordingly, the choice of a (pharmaceutically) acceptable carrier as described above is determined in particular by the mode in which the (pharmaceutical) composition according to the invention is administered.
  • the (pharmaceutical) composition according to the invention can be administered, for example, systemically.
  • Administration routes include e.g. intra- or transdermal, oral, parenteral, including subcutaneous, intramuscular, i.a. or intra- venous injections, topical and/or intranasal routes.
  • the suitable amount of the (pharmaceutical) composition according to the invention which is to be used can be determined by routine experiments using animal models. Such models include, but without being limited thereto, models of the rabbit, sheep, mouse, rat, dog and non-human primate models.
  • the carrier may be selected from pyrogen- free water; isotonic saline solution and buffered solutions, e.g. phosphate buffered solutions.
  • Preferred unit dose forms for injection include sterile solutions of water, physiological saline solution or mixtures thereof, e.g. Ringer-Lactat solution. The pH of such solutions should be adjusted to about 7. 0 to about 7.6, preferably about 7.4.
  • the (pharmaceutical) composition according to the invention can comprise, for example, an aqueous injection buffer or water which contains, with respect to the total (pharmaceutical) composition, if this is in liquid form, a sodium salt, preferably at least 50 mM sodium salt, a calcium salt, preferably at least 0.01 mM calcium and/or magnesium salt, and optionally a potassium salt, preferably at least 3 mM potassium salt.
  • a sodium salt preferably at least 50 mM sodium salt
  • a calcium salt preferably at least 0.01 mM calcium and/or magnesium salt
  • optionally a potassium salt preferably at least 3 mM potassium salt.
  • the sodium salts, calcium and/or magnesium salts and optionally potassium salts contained in such an injection buffer are in the form of halides, e.g. chlorides, iodides or bromides, or in the form of their hydroxides, carbonates, bicarbonates or sulfates.
  • the injection buffer can also contain organic anions of the abovementioned cations.
  • such an injection buffer contains as salts sodium chloride (NaCI), calcium chloride (CaCI 2 ) and optionally potassium chloride (KCl), it is also being possible for other anions to be present in addition to the chlorides.
  • the injection buffer can be in the form of both hypertonic and isotonic or hypotonic injection buffers.
  • the injection buffer is hypertonic, isotonic or hypotonic in each case with respect to the particular reference medium, i.e.
  • the injection buffer has either a higher, the same or a lower salt content compared with the particular reference medium, such concentrations of the abovementioned salts which do not lead to damage to the cells caused by osmosis or other concentration effects preferably being employed.
  • Reference media are, for example, liquids which occur in “in vivd' methods, such as, for example, blood, lymph fluid, cytosol fluids or other fluids which occur in the body, or liquids or buffers conventionally employed in “in vitro” methods. Such liquids and buffers are known to a person skilled in the art.
  • the injection buffer optionally contained in the (pharmaceutical) composition according to the invention can also contain further components, for example sugars (mono-, di-, tri- or polysaccharides), in particular glucose or mannitol. In a preferred embodiment, however, no sugars are present in the injection buffer used. It is also preferable for the injection buffer precisely to contain no non-charged components, such as, for example, sugars.
  • the injection buffer typically contains exclusively metal cations, in particular from the group consisting of the alkali or alkaline earth metals, and anions, in particular the anions described above.
  • the pH of the injection buffer used is preferably between 1 and 8.5, preferably between 3 and 5, more preferably between 5.5 and 7.5, in particular between 5.5 and 6.5.
  • the injection buffer can also contain a buffer system which fixes the injection buffer at a buffered pH. This can be, for example, a phosphate buffer system, HEPES or Na 2 HPO 4 ZNaH 2 PO 4 .
  • the injection buffer used very particularly preferably contains none of the abovementioned buffer systems or contains no buffer system at all.
  • the injection buffer optionally contained in the (pharmaceutical) composition according to the invention can contain, in addition to or as an alternative to the monovalent and divalent cations described, divalent cations, in particular from the group consisting of alkaline earth metals, such as, for example, magnesium (Mg 2+ ), or also iron (Fe 2+ ), and monovalent cations, in particular from the groups consisting of alkali metals, such as, for example, lithium (Li + ).
  • These monovalent cations are preferably in the form of their salts, e.g. in the form of halides, e.g. chlorides, iodides or bromides, or in the form of their hydroxides, carbonates, bicarbonates or sulfates.
  • lithium salt LiCI, LiI, LiBr, Li 2 CO 3 , LiHCO 3 , Li 2 SO 4 for the magnesium salt MgCI 2 , MgI 2 , MgBr 2 , MgCO 3 , MgSO 4 , and Mg(OH) 2
  • iron salt FeCI 2 , FeBr 2 , FeI 2 , FeF 2 , Fe 2 O 3 , FeCO 3 , FeSO 4 , Fe(OH) 2 All the combinations of di- and/or monovalent cations, as described above, are likewise included.
  • Such injection buffers which contain only divalent, only monovalent or di- and monovalent cations can thus be used in the (pharmaceutical) composition according to the invention.
  • Such injection buffers which contain only one type of di- or monovalent cations, particularly preferably e.g. only Ca 2+ cations, or a salt thereof, e.g. CaCI 2 , can likewise be used.
  • the molarities given above for Ca 2+ (as a divalent cation) and Na 1+ (as a monovalent cation) (that is to say typically concentrations of at least 50 mM Na + , at least 0.01 mM Ca 2+ and optionally at least 3 mM K + ) in the injection buffer can also be taken into consideration if another di- or monovalent cation, in particular other cations from the group consisting of the alkaline earth metals and alkali metals, are employed instead of some or all the Ca 2+ or, respectively, Na 1+ in the injection buffer used according to the in- vention for the preparation of the injection solution.
  • All the Ca 2+ or Na 1+ can indeed be replaced by in each case other di- or, respectively, monovalent cations in the injection buffer used, for example also by a combination of other divalent cations (instead of Ca 2+ ) and/or a combination of other monovalent cations (instead of Na 1+ ) (in particular a combination of other divalent cations from the group consisting of the alka- line earth metals or, respectively, of other monovalent cations from the group consisting of the alkali metals), but it is preferable to replace at most some of the Ca 2+ or Na 1+ , i.e.
  • the injection buffer optionally contained in the pharmaceutical composition according to the invention contains exclusively Ca 2+ as a divalent cation and Na 1+ as a monovalent cation, that is to say, with respect to the total pharmaceutical composition, Ca 2+ represents 100 % of the total molarity of divalent cations, just as Na 1+ represents 100 % of the total molarity of monovalent cations.
  • the aqueous solution of the injection buffer can contain, with respect to the total pharmaceutical composition, up to 30 mol% of the salts contained in the solution, preferably up to 25 mol%, preferably up to 20 mol%, furthermore preferably up to 15 mol%, more preferably up to 10 mol%, even more preferably up to 5 mol%, likewise more preferably up to 2 mol% of insoluble or sparingly soluble salts.
  • Salts which are sparingly soluble in the context of the present invention are those of which the solubility product is ⁇ 10 "4 .
  • Salts which are readily soluble are those of which the solubility product is > 10 ⁇ 4 .
  • the injection buffer optionally contained in the pharmaceutical composition according to the invention is from 50 mM to 800 mM, preferably from 60 mM to 500 mM, more preferably from 70 mM to 250 mM, particularly preferably 60 mM to 1 10 mM in sodium chloride (NaCl), from 0.01 mM to 100 mM, preferably from 0.5 mM to 80 mM, more preferably from 1 .5 mM to 40 mM in calcium chloride (CaCl 2 ) and optionally from 3 mM to 500 mM, preferably from 4 mM to 300 mM, more preferably from 5 mM to 200 mM in potassium chloride (KCI).
  • KCI potassium chloride
  • Organic anions can also occur as further anions in addition to the abovementioned inorganic anions, for example halides, sulfates or carbonates.
  • succinate, lactobionate, lactate, malate, maleate etc. which can also be present in combination.
  • An injection buffer optionally contained in the (pharmaceutical) composition according to the invention preferably contains lactate. If it contains an organic anion, such an injection buffer particularly preferably contains exclusively lactate as the organic anion. Lactate in the context of the invention can be any desired lactate, for example L-lactate and D-lactate.
  • Lactate salts which occur in connection with the present invention are typically sodium lactate and/or calcium lactate, especially if the injection buffer contains only Na + as a monovalent cation and Ca 2+ as a divalent cation.
  • composition according to the invention and as described above preferably contains, with respect to the total pharmaceutical composition, from 15 mM to 500 mM, more preferably from 15 mM to 200 mM, and even more most preferably from 15 mM to 100 mM lactate.
  • the pharmaceutical com- position of the invention may be contain compounds which can serve as acceptable carriers or constituents thereof, e.g. sugars, such as, for example, lactose, glucose and sucrose; starches, such as, for example, corn starch or potato starch; cellulose and its derivatives, such as, for example, sodium carboxymethylcellulose, ethylcellulose, cellulose acetate; pulverized tragacanth; malt; gelatine; tallow; solid lubricants, such as, for example, stearic acid, magnesium stearate; calcium sulfate; plant oils, such as, for example, groundnut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil from Theobroma; polyols, such as, for example, polypropylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid.
  • sugars such as, for example, lactose, glucose and sucrose
  • emulsifiers such as, for example, Tween ®
  • wetting agents such as, for example, sodium lauryl sulfate
  • colouring agents such as, for example, sodium lauryl sulfate
  • flavouring agents such as, for example, medicament carriers; tablet- forming agents; stabilizers; antioxidants; preservatives.
  • acceptable carriers for injection include hydrogels, devices for controlled or delayed release, polylactic acid and collagen matrices.
  • Acceptable carriers which can be used here include those which are suitable for use in lotions, creams, gels and the like. If the compound is to be administered perorally, tablets, capsules and the like are the preferred unit dose form.
  • the acceptable carriers for the preparation of unit dose forms which can be used for oral administration are well-known in the prior art. Their choice will depend on secondary considerations, such as flavour, cost and storage stability, which are not critical for the purposes of the present invention and can be implemented without difficulties by a person skilled in the art.
  • Fig.1 is a schematic representation of 'active suppression' by suppressor T cells:
  • APC antigen presenting cell
  • Fig. 2 shows the repeat sequences (underlined) within the S-antigen of P. falciparum isolate NF7 (SEQ ID NO: 10).
  • Fig. 3 represents a theoretical plot of the secondary structure of the S-antigen of Fig. 2.
  • Fig. 4 illustrates the design of fusion polypeptides with repeat sequences of the S-antigen (S- Ag) of P. falciparum.
  • the repeat sequence consisting of 24 repeats (24mer) of an 8 amino acid unit (A(LVR)KSDEAE; i.e. SEQ ID NO: 1 ) derived from the NF7 isolate was linked to the N-terminus of CD4+ T cell epitope Ova323-339 (-> S-AgOVA323-339). Since the natural sequence contained either the amino acid L or R in their repeat unit (see Fig. 2) building blocks were used in which the two amino acids alternate in tandem repeats.
  • Fig. 5 demonstrates the expression of fusion polypeptides in E. coli by an SDS-Page analysis of crude products isolated by Ni-NTA affinity chromatography prior to RP-HLPC purification.
  • 8mer, 16mer and 24mer refers to the number of repeat units (of SEQ ID NO: 1 ) attached to the epitope.
  • Fig. 6 represents the results of an in vivo delayed-type hypersensitivity (DTH) model using S- AgOVA323-339 (SAgOVA).
  • DTH delayed-type hypersensitivity
  • SAgOVA S- AgOVA323-339
  • the measured values of footpad swelling are shown for mice which were either treated with S-AgOVA323-339 (left column) or did not receive any treatment with S-AgOVA323-339 (middle column).
  • Right column is a control showing mice after treatment with S-AgOVA323-339 which was not primed before with the peptide (Ova323-339).
  • Fig. 7 shows the in vivo induction of Foxp3+CD25+ suppressor Treg cells.
  • FACS analysis is shown for mice treated with 50 ⁇ g (left) or 10 ⁇ g of S-AgOVA323-339 (middle) or not treated (right).
  • the population shows transferred Ova-specific T cells, gated on CFSE- fluorescence and on a clonotypic antibody specific for the Ova-specific T cell receptor (KJ26.1 ). Double-staining was carried out with antibodies specific for the Treg markers CD25 and Foxp3. Treg cells are located in the upper right quadrant; percentage of cells of each quadrant is indicated.
  • Fig. 8 is a diagram of the treatment of EAE with S-AgMOG35-55.
  • EAE was induced in C57/BI6 mice by priming with the MOG35-55 peptide.
  • Mice were treated either with the S- Ag repeat sequence (tandem repeat only, i.e. SEQ ID NO: 1 ) only, S-Ag repeat sequence (SEQ ID NO: 1 ) plus free MOG35-55 (tandem repeat & free MOGpeptide) or with the S- AgMOG35-55 fusion polypeptide (tandem repeat-MOG peptide) by a single i.v. injection of 50 ⁇ g 7 days prior to priming.
  • Fig. 9 is a diagram of the treatment of EAE with S-AgPLPI 39-151 .
  • EAE was induced in SJL mice by priming with the PLP139-151 peptide.
  • Mice were treated with S-AgPLPI 39-151 fusion polypeptide by a single i.v. injection of 50 ⁇ g 7 days after induction.
  • Fig. 10 shows the reversal of S-AgMOG35-55 (repeat-MOG) induced suppression by ⁇ - ILI OR antibodies.
  • EAE was induced in C57/BI6 mice by priming with the MOG35-55 peptide.
  • Mice were treated with the S-AgMOG35-55 fusion polypeptide by a single i.v. injection of 50 ⁇ g on day 7.
  • Some animals received 1 mg of ⁇ -IL10R antibodies or of a nonspecific IgG (control) on days 7, 9, 1 1 and 13.
  • Fig. 1 1 shows the inhibition of EAE by a synthetic variant of the S-antigen fusion protein.
  • Mice were vaccinated 7 days prior to the disease induction with the recombinant S- AgPLPI 39-151 containing 24 units of SEQ ID NO: 1 (S-Ag-PLP) or with fully synthetic version containing 5 repeat units of SEQ ID NO: 1 (synth-5-PLP). The experiment was carried out as described in Fig. 9.
  • Fig. 12 shows the inhibition of EAE by recombinant S-Ag fusion protein containing the repeats of the plasmodium falciparum isolate FC 27 (24 units of the repeat unit SEQ ID NO: 13). The suppression is shown for the construct fused to the MOG fragment (FC27-MOG) used in Fig. 8.
  • Fig. 13 shows the inhibition of experimental type 1 diabetes in NOD mice.
  • NOD mice de- velop spontaneously diabetes. The effect is shown here for a fusion construct in which the
  • S-Ag repeats (SEQ ID NO: 1 ; 24 units) are linked to a fragment of the ⁇ -chain of human insulin 9-23 (S-Ag-INS). While the blood sugar level of healthy mice is around 100 mg/dl
  • mice with a level above 250 mg/dl are considered diabetogenic (middle panel).
  • the lower panel shows the effect of 1 .5 nmol S-Ag-INS injected i.v. within one week after the blood sugar levels were crossing the threshold concentration.
  • Fig. 14 shows the reduction of ovalbumin-specific IgGI antibodies by S-AgOVA323-339 (OVA-peptid) in an experimental asthma model.
  • S-AgOVA323-339 OVA-peptid
  • Balb/c mice were sensitized against ovalbumin by 6 consecutive intraperitoneal immunizations with 10 ⁇ g ovalbunmin.
  • the mice received 50 mg of S-AgOVA323-339 either 1 week before (i.p.) or on day 24 (i.n.) and were challenged on day 26 and day 28 with 50 ⁇ g ovalbumin i.n..
  • the mice were killed on day 30 and the ovalbumin-specific IgGI titer was determined by ELISA.
  • Fig. 15 shows the tolerization of female recipients against male spleen cell grafts.
  • the injec- tion of 50 mg of the S-antigen repeat (SEQ ID NO: 1 ; 24 units) fused to a male minor histocompatibility antigen encoded by the Y Chromosome (HY) is sufficient to prevent the rejection of the graft.
  • Female mice accept spleen cells from other female mice of the same strain but eliminate cells from male donors.
  • the figure shows the FACS staining of mice that received male spleen cells labeled with bright CSFE fluorescence and female cells with lower fluorescence. Staining is shown for day 3 and day 25 after transplantation. Mice received either only the HY peptide (upper panel) or the S-Ag repeats (middle panel) or the S-Ag-HY fusion protein (lower panel).
  • gedevsngre dkvsnggede vsngredkvs ngredkvsng gedevsngre dkvsnggede 361 vsngredkvs ngredkvsng redevsngre dkvsnggede vsngredkvs ngredkvsngredkvsngng
  • SEQ ID NO: 12 P. falciparum isolate V1 :
  • recombinant fusion polypeptides were generated in which 24 units of the repeat unit SEQ ID NO: 1 were fused to defined epitopes of CD4+ T cells.
  • the fusion polypeptides were tested in vivo using experimental mouse models of allergy (delayed-type hypersensitivity; DTH), asthma, type 1 diabetes and multiple sclerosis (experimental autoimmune encephalomyelitis; EAE).
  • DTH delayed-type hypersensitivity
  • EAE experimental autoimmune encephalomyelitis
  • the generation and production was carried out by recombinant techniques in £ coli as de- scribed in Rotzschke et al., Proc Natl Acad Sci USA 1997, 94: 14642-7.
  • the polypeptides were isolated using a His-tag located at the C-terminal site of the polypeptide and purified by RP-HPLC to remove endotoxins (Fig. 5).
  • the fully synthetic version of the fusion protein synth-5-PLP, consisting of 5 S-Ag repeats of SEQ ID NO: 1 linked to the PLP fragment was generated by standard peptide synthesis (Fig. 1 1).
  • polypeptides containing a CD4+ T cell epitope of ovalbumin were initially tested in a DTH model.
  • T cells iso- lated from a mouse strain transgenic for an Ova-specific T cell receptor (DO1 1.10) were transferred into a Balb/c recipient.
  • Mice were primed with the Ova323-339 antigen the next day and challenged by an injection of the peptide into the foot pad 7 days later.
  • 50 ⁇ g of S- AgOVA323-339 was injected i.v. on day 8 and the swelling of the footpad in response to priming was measured on day 9.
  • no swelling was detected in treated mice, indicating that proinflammatory response has been effectively prevented (Fig. 6).
  • mice received Ova-specific T cells from DO11 .10 which were labeled with the green fluorescent dye CFSE (5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester).
  • CFSE green fluorescent dye
  • CFSE fluorescent dye
  • S-AgOVA323-339 was injected 1 day later, and the lymph node cells were isolated and analyzed by flow cytometry (FACS) on day 4.
  • FACS flow cytometry
  • polypeptides were generated in which the S-Ag repeat sequence (SEQ ID NO: 1 ; 24 units) was linked to an epitope derived from myelin oligodendrocyte glycoprotein (MOG).
  • MOG35-55 is an encephalitogenic CD4+ T cell epitope known to induce EAE in C57/BI6 mice.
  • a fusion polypeptide was generated in which the Ova-epitope was replaced by MOG35-55.
  • S-AgMOG35-55 efficiently suppressed clinical symptoms of the disease.
  • the effect occurred only by administration of the fusion polypeptide and not by administration of a mixture of S-Ag repeat sequence (SEQ ID NO: 1 ) and free MOG35-55 peptide. That indicates that a physical link between the T cell epitope and the repeat sequence is required (Fig. 8).
  • Example 4 Type 1 diabetes model
  • a construct was generated in which the S-Ag repeats (SEQ ID NO: 1 ; 24 units) were fused to a fragment of the insulin ⁇ -chain (INS 9-23).
  • NOD mice spontaneously developed diabetes, indicated by increased blood glucose levels.
  • blood glucose levels of a cohort of NOD mice were determined twice weekly from blood samples using a commercially available blood glucose meter (Fig. 13). 1 .5 nmol of the fusion protein were injected when the blood glucose level was above 250 mg/dl in two consecutive readings.
  • treated mice had a significantly more stable blood glucose level, demonstrating effectiveness of the fusion protein in this disease model.
  • mice In Balb/c mice experimental asthma can be induced after sensitizing the mice with ovalbu- min protein. For this, the mice received 6 repeated i.p. injections of 10 ⁇ g on days (1 , 3, 5,
  • the allergic response was triggered on days 26 and 27 by i.n. application of 50 ⁇ g ovalbumin.
  • the strength of the response was then determined on day 30 by the amount of antibodies generated against the allergen (Fig. 14).
  • the antibody titer In this example the antibody titer
  • HY antigens Due to the lack of tolerance against proteins encoded by the male-specific Y-chromosome female mice reject grafts from male mice even when they are from the same strain.
  • the transplantation antigens encoded by the Y-chromosome are called HY antigens.
  • S-Ag fusion protein was gener- ated in which the repeat region (SEQ ID NO: 1 ; 24 units) was linked to a mouse HY antigen (S-Ag-HY).
  • S-Ag-HY mouse HY antigen
  • mice received i.v. injections of 1.5 nmol HY peptide (upper panels), S-Ag repeats only (middle panel) or the S-Ag-HY fusion rotein (lower panel).
  • Blood samples of the mice were taken on days 3 (left panels) and 25 (right panels) and analysed by FACS. While on day 3 both male and female cells were still present in either of the mice, on day 25 the male cells had disappeared from the mice that received only the HY peptide only or the S-Ag without the HY antigen. In contrast both male and female cells were still present in mice that were vaccinated with the S-Ag-HY fusion protein.
  • S-Ag-HY was able to tol- erize the mice to accept the 'foreign' graft.

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Abstract

La présente invention concerne un polypeptide de fusion comprenant a) au moins un fragment du S-antigène Plasmodium falciparum ou un variant de celui-ci, le fragment comprenant une ou plusieurs unités de répétition de S-antigène de Plasmodium falciparum ou un ou plusieurs variants de celles-ci et b) au moins un antigène différent du S-antigène. La présente invention concerne également une composition pharmaceutique comprenant ce peptide de fusion ainsi que l'utilisation de celle-ci pour le traitement ou la prévention d'une maladie auto-immune, d'une réaction allergique, d'une complication liée à une transplantation, par exemple un rejet de greffe ou la maladie du greffon contre hôte, et une autre maladie inflammatoire, et sur un procédé pour le traitement ou la prévention desdits états.
EP08802876A 2007-10-11 2008-10-13 Polypeptide de fusion comprenant des unités de répétition de s-antigène Withdrawn EP2197906A1 (fr)

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