EP2066347A1 - Mit virusartigen teilchen verbundene interleukin-1-muteine zur behandlung von erkrankungen im zusammenhang mit il-1 - Google Patents

Mit virusartigen teilchen verbundene interleukin-1-muteine zur behandlung von erkrankungen im zusammenhang mit il-1

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Publication number
EP2066347A1
EP2066347A1 EP07727479A EP07727479A EP2066347A1 EP 2066347 A1 EP2066347 A1 EP 2066347A1 EP 07727479 A EP07727479 A EP 07727479A EP 07727479 A EP07727479 A EP 07727479A EP 2066347 A1 EP2066347 A1 EP 2066347A1
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EP
European Patent Office
Prior art keywords
seq
amino acid
acid sequence
composition
mutein
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Withdrawn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
EP07727479A
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English (en)
French (fr)
Inventor
Martin Bachmann
Gunther Spohn
Alain Tissot
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Cytos Biotechnology AG
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Cytos Biotechnology AG
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Publication date
Priority claimed from PCT/EP2006/066866 external-priority patent/WO2007039552A1/en
Application filed by Cytos Biotechnology AG filed Critical Cytos Biotechnology AG
Priority to EP07727479A priority Critical patent/EP2066347A1/de
Publication of EP2066347A1 publication Critical patent/EP2066347A1/de
Withdrawn legal-status Critical Current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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/545—IL-1
    • 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
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • 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
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/18011—Details ssRNA Bacteriophages positive-sense
    • C12N2795/18111—Leviviridae
    • C12N2795/18123—Virus like particles [VLP]

Definitions

  • the present invention is in the fields of medicine, public health, immunology, molecular biology and virology.
  • the invention provides compositions comprising a virus-like particle (VLP) or a virus particle and at least one antigen, wherein said antigen is an Interleukin-1 (IL-I) protein, an IL-I fragment or peptide or an IL-I mutein covalently linked to the VLP or the virus particle, wherein most preferably the antigen is an IL-I mutein, preferably mutein of IL-I beta or IL-I alpha.
  • the invention also provides a process for producing the compositions.
  • the compositions of this invention are useful in the production of vaccines for the treatment of various human disorders, including rheumatoid arthritis osteoarthritis and others.
  • the compositions of the invention hereby induce efficient immune responses, in particular antibody responses.
  • IL-I is a potent proinflammatory cytokine produced by various cell types, including macrophages, dendritic cells, B-cells and T-cells (Dinarello C. A., 1991. Blood 77(8):1627- 1652). It consists of two molecular species, IL- l ⁇ and IL- l ⁇ , which share only limited sequence identity but exert similar biological activities through binding to IL-I receptor type I (IL-IRI) (Dinarello CA. et al, 1997, Cytokine & Growth Factor Rev. 8:253).
  • IL-IRI IL-I receptor type I
  • Both IL-I molecules also bind to a second IL-I receptor (IL-IRII), which lacks the intracellular signalling domain, and is believed to play a regulatory role as a decoy receptor (Dinarello CA. et al., 1997, Cytokine & Growth Factor Rev. 8:253).
  • IL-IRII the IL-I receptor antagonist
  • IL- Ira IL-I receptor antagonist
  • IL- Ira binds to both receptors without exerting any agonistic activity.
  • IL-lra together with IL-IRII and the shed forms of IL-IRI and IL-IRII counteract the activity of IL-I ⁇ and IL- l ⁇ and ensure a tight regulation of the inflammatory response.
  • a dysregulation of the IL-I -mediated inflammatory response is observed in many human disorders, including rheumatoid arthritis, inflammatory bowel disease, kidney diseases, osteoporosis and others. In each of these diseases either overproduction of IL-I and/or underproduction of IL-lra predisposes to the development of disease (Arend W.P., 2002, Cytokine & Growth Factor Reviews 13:323-340).
  • a recombinant version of IL-lra (anakinra, Kineret®) is efficacious in reducing inflammation and preventing tissue damage in several inflammatory disorders, but the need for high systemic concentrations and the short half life of the drug require frequent (daily) administrations of high doses (-100 mg), resulting in high cost of goods and potential patient compliance problems (Kineret ® prescribing information, Amgen; Granowitz E.V. et al. 1992, Cytokine 4:353). In addition, a large proportion of patients develop antibodies against Kineret®, which potentially neutralize the biological activity of the drug (Fleischmann R.M., et al., 2003, Arthritis Rheum 46:2287).
  • New therapeutic techniques therefore focus on active immunization strategies, which induce the production of IL-I -neutralizing antibodies by the immune system of the patient.
  • Svenson and co-workers 2000, J. Immunol. Methods 236:1-8 immunized mice with recombinant IL- l ⁇ chemically crosslinked to purified protein derivative of tuberculin (PPD), and observed the induction of antibodies which neutralized the biological activity of IL-I ⁇ .
  • PPD tuberculin
  • This strategy relies on the delivery of T-cell help to autoreactive B-cells by physical linkage of the self-antigen to a foreign antigen.
  • US patent 6,093,405 discloses a method of reducing the level of a circulating cytokine by immunization with an immunogenic composition containing the chemically or physically inactivated cytokine itself. Whereas in this method native cytokines are rendered immunogenic by physical or chemical treatment, the present invention discloses a method for making native cytokines immunogenic by presenting them in a highly repetitive fashion on the surface of VLPs.
  • WO2003/084979 furthermore describes the use of immunogenic compounds containing cytokine-derived peptides of 5-40 amino acids length for the treatment of diseases associated with an overproduction of cytokines.
  • inventive compositions and vaccines comprising at least one IL-I molecule, preferably a IL-I mutein, are not only capable of inducing immune responses against IL-I, and hereby in particular antibody responses, but are, furthermore, capable of neutralizing the pro-inflammatory activity of IL-I in vivo.
  • IL-I molecule when covalently linked to the VLP in accordance with the invention, can protect from inflammation and from clinical signs of arthritis in a mouse model of rheumatoid arthritis.
  • compositions of the invention protected mice better from the development of arthritis symptoms than the recombinant IL-I receptor antagonist Kineret ® , which is approved for the treatment of human rheumatoid arthritis (Example 7).
  • IL-I receptor antagonist Kineret ® which is approved for the treatment of human rheumatoid arthritis
  • compositions of the invention were able to inhibit the development of atherosclerotic symptoms, when injected into genetically susceptible mice (Example 4) and therefore are an efficient treatment for atherosclerosis.
  • IL-I ⁇ is involved in the pathogenesis of atherosclerosis.
  • muteins of IL-I beta and IL-I alpha showing reduced biological activity can be obtained (Examples 11 and 16), and that such muteins of IL-I beta are capable of inducing antibodies with neutralizing activity in vitro (Example 12 D).
  • structurally similar regions of IL-I beta and IL-I alpha have been identified where mutations, especially amino acid exchanges or deletions, result in muteins which are useful in the context of the invention.
  • the present invention provides a composition which comprises (a) a virus-like particle (VLP) with at least one first attachment site; and (b) at least one antigen with at least one second attachment site, wherein said at least one antigen an IL-I molecule, preferably selected from the group consisting of IL-I protein, IL-I mature fragment, IL-I peptide and IL-I mutein, wherein (a) and (b) are linked through said at least one first and said at least one second attachment site, preferably to form an ordered and repetitive antigen array.
  • VLP virus-like particle
  • antigen an IL-I molecule, preferably selected from the group consisting of IL-I protein, IL-I mature fragment, IL-I peptide and IL-I mutein
  • the virus-like particles suitable for use in the present invention comprises recombinant protein, preferably recombinant coat protein, mutants or fragments thereof, of a virus, preferably of an RNA bacteriophage.
  • the inventive composition comprises at least one IL-I mature fragment, preferably comprising the biological activity of IL-I.
  • the present invention uses the presentation of the self-antigen in a highly repetitive fashion on virus-like particles to stimulate autoreactive B-cells.
  • the invention provides a composition
  • a composition comprising (a) a virus-like particle (VLP) with at least one first attachment site; and (b) at least one, preferably one, antigen with at least one, preferably one, second attachment site; wherein said at least one antigen is an IL-I mutein, and wherein said IL-I mutein comprises at least one, preferably one, mutated amino acid sequence derived from a wild type amino acid sequence, wherein said wild type amino acid sequence is an IL-I beta amino acid sequence selected from the group consisting of: (1) position 3 to 11 of SEQ ID NO:64; (2) position 46 to 56 of SEQ ID NO:64; (3) position 88 to 109 of SEQ ID NO:64; and (4) position 143 to 153 of SEQ ID NO:64; or wherein said wild type amino acid sequence is an IL-I alpha amino acid sequence selected from the group consisting of: (5) position 9 to 20 of SEQ ID NO:63; (6) position 52 to 62 of S
  • said IL-I mutein is an IL-I beta mutein, wherein said IL-I beta mutein comprise or preferably consist of a polypeptide having the amino acid sequence of SEQ ID NO: 136.
  • said IL-I mutein is an IL-I alpha mutein, wherein said IL-I alpha mutein comprise or preferably consist of a polypeptide having the amino acid sequence of
  • the present invention provides a vaccine composition.
  • the present invention provides a method to administering the vaccine composition to a human or an animal, preferably a mammal.
  • the inventive vaccine composition is capable of inducing strong immune response, in particular antibody response, typically and preferably without the presence of at least one adjuvant.
  • the vaccine is devoid of an adjuvant. The avoidance of using adjuvant may reduce a possible occurrence of unwanted inflammatory T cell responses.
  • the VLP is a VLP of an RNA bacteriophage.
  • said RNA bacteriophage is an RNA bacteriophage selected from the group consisting of: Q ⁇ , fr, GA and AP203n a further preferred embodiment said
  • VLP of an RNA bacteriophage comprised by the composition and the vaccine composition, respectively is recombinantly produced in a host and the VLP of an RNA bacteriophage is essentially free of host RNA, preferably host nucleic acid. It is advantageous to reduce, or preferably to eliminate, the amount of host RNA to avoid unwanted T cell responses as well as other unwanted side effects, such as fever.
  • the present invention provides a method of treating a disease selected from the group consisting of: (a) vascular diseases; (b) inherited IL-I -dependent inflammatory diseases; (c) chronic autoimmune inflammatory diseases; (d) bone and cartilage degenerative diseases; (e) allergic diseases; and (f) neurological disease; in which diseases IL-
  • IL-I protein mediates, or contributes to the condition
  • the method comprises administering the inventive composition or the invention vaccine composition, respectively, to an animal, preferably human.
  • Diseases, in which IL-I protein mediates, or contributes to the condition are, for example, atherosclerosis, familial Mediterranean fever, rheumatoid arthritis, osteoarthritis, and allergy.
  • the present invention provides a pharmaceutical composition comprising the inventive composition and an acceptable pharmaceutical carrier.
  • the present invention provides for a method of producing the composition of the invention comprising (a) providing a VLP with at least one first attachment site; (b) providing at least one antigen, wherein said antigen is an IL-I molecule, an IL-I protein, an IL-I mature fragment, an IL-I peptide or an IL-I mutein, with at least one second attachment site; and (c) combining said VLP and said at least one antigen to produce said composition, wherein said at least one antigen and said VLP are linked through said at least one first and said at least one second attachment sites.
  • Proteins were analyzed on a 12 % SDS-polyacrylamide gel under reducing conditions. The gel was stained with Coomassie Brilliant Blue. Molecular weights of marker proteins are given in kDa on the left margin, identities of protein bands are indicated on the right margin. Lane 1: Pre stained protein marker (New England Bio labs). Lane 2: derivatized Q ⁇ capsid protein. Lane 3: free reduced mIL-l ⁇ n 9- 269 protein. Lane 4: Q ⁇ -mIL-l ⁇ ii 9- 269 coupling reaction.
  • Figure 2 Coupling of mIL-lccii 7- 2 7 o protein to Q ⁇ capsid protein
  • Proteins were analyzed on a 12 % SDS-polyacrylamide gel under reducing conditions. The gel was stained with Coomassie Brilliant Blue. Molecular weights of marker proteins are given in kDa on the left margin, identities of protein bands are indicated on the right margin. Lane 1: Prestained protein marker (New England Bio labs). Lane 2: derivatized Q ⁇ capsid protein. Lane 3: free reduced mlL-lcci 17.270 protein. Lane 4: Q ⁇ -mIL-lcci 17.270 coupling reaction.
  • Adjuvant refers to non-specific stimulators of the immune response or substances that allow generation of a depot in the host which when combined with the vaccine and pharmaceutical composition, respectively, of the present invention may provide for an even more enhanced immune response.
  • Preferred adjuvants are complete and incomplete Freund's adjuvant, aluminum containing adjuvant, preferably aluminium hydroxide, and modified muramyldipeptide.
  • Further preferred adjuvants are mineral gels such as aluminum hydroxide, surface active substances such as lyso lecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and human adjuvants such as BCG (bacille Calmette Guerin) and Corynebacterium parvum. Such adjuvants are also well known in the art.
  • compositions of the invention include, but are not limited to, Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18, CRL1005, Aluminum salts (Alum), MF-59, OM- 174, OM- 197, OM-294, and Virosomal adjuvant technology.
  • the adjuvants can also comprise a mixture of these substances.
  • VLP have been generally described as an adjuvant.
  • the term "adjuvant" refers to an adjuvant not being the VLP used for the inventive compositions, rather it relates to an additional, distinct component.
  • Antigen refers to a molecule capable of being bound by an antibody or a T-cell receptor (TCR) if presented by MHC molecules.
  • TCR T-cell receptor
  • An antigen is additionally capable of being recognized by the immune system and/or being capable of inducing a humoral immune response and/or cellular immune response leading to the activation of B- and/or T- lymphocytes. This may, however, require that, at least in certain cases, the antigen contains or is linked to a Th cell epitope and is given in adjuvant.
  • An antigen can have one or more epitopes (B- and T-epitopes).
  • antigen will preferably react, typically in a highly selective manner, with its corresponding antibody or TCR and not with the multitude of other antibodies or TCRs which may be evoked by other antigens.
  • Antigens as used herein may also be mixtures of several individual antigens.
  • the term "antigen” as used herein preferably refers to the IL-I molecule, the IL-I protein, IL-I mature fragment, the IL-I fragment, the IL-I peptide and the IL-I mutein, most preferably "antigen” refers to the IL-I mutein. If not indicated otherwise, the term "antigen” as used herein does not refer to the virus-like particle.
  • epitope refers to continuous or discontinuous portions of an antigen, preferably a polypeptide, wherein said portions can be specifically bound by an antibody or by a T-cell receptor within the context of an MHC molecule. With respect to antibodies, specific binding excludes non-specific binding but does not necessarily exclude cross-reactivity.
  • An epitope typically comprise 5-10 amino acids in a spatial conformation which is unique to the antigenic site.
  • Specific binding antibody / antigen: Within this application, antibodies are defined to be specifically binding if they bind to the antigen with a binding affinity (Ka) of 10 6 M “1 or greater, preferably 10 7 M “1 or greater, more preferably 10 s M “1 or greater, and most preferably 10 9 M “1 or greater.
  • Ka binding affinity
  • the affinity of an antibody can be readily determined by one of ordinary skill in the art (for example by Scatchard analysis, by ELISA or by Biacore analysis).
  • Specific binding IL-I / IL-I receptor: The interaction between a receptor and a receptor ligand can be characterized by biophysical methods generally known in the art, including, for example, ELISA or Biacore analysis.
  • An IL-I molecule is regarded as capable of specifically binding an IL-I receptor, when the binding affinity (Ka) of said IL-I to said IL-I receptor is at least 10 5 M "1 , preferably at least 10 6 M “1 , more preferably at least 10 7 M “1 , still more preferably at least 10 s M “1 , and most preferably at least 10 9 M “1 ; wherein preferably said IL-I receptor is an IL-I receptor from mouse or human, most preferably human.
  • said IL-I receptor comprises or more preferably consists of any one of the sequences SEQ ID NO: 166 to SEQ ID NO: 169, most preferably said IL-I receptor comprises or preferably consists of any one of the sequences SEQ ID NO: 166 and SEQ ID NO: 167.
  • association refer to all possible ways, preferably chemical interactions, by which two molecules are joined together. Chemical interactions include covalent and non-covalent interactions.
  • non-covalent interactions are ionic interactions, hydrophobic interactions or hydrogen bonds
  • covalent interactions are based, by way of example, on covalent bonds such as ester, ether, phosphoester, amide, peptide, carbon-phosphorus bonds, carbon-sulfur bonds such as thioether, or imide bonds.
  • first attachment site refers to an element which is naturally occurring with the VLP or which is artificially added to the VLP, and to which the second attachment site may be linked.
  • the first attachment site preferably is a protein, a polypeptide, an amino acid, a peptide, a sugar, a polynucleotide, a natural or synthetic polymer, a secondary metabolite or compound (biotin, fluorescein, retinol, digoxigenin, metal ions, phenylmethylsulfonylfluoride), or a chemically reactive group such as an amino group, a carboxyl group, a sulfhydryl group, a hydroxyl group, a guanidinyl group, histidinyl group, or a combination thereof.
  • a preferred embodiment of a chemically reactive group being the first attachment site is the amino group of an amino acid, preferably of lysine.
  • the first attachment site is located, typically on the surface, and preferably on the outer surface of the VLP. Multiple first attachment sites are present on the surface, preferably on the outer surface of virus-like particle, typically in a repetitive configuration.
  • the first attachment site is associated with the VLP, through at least one covalent bond, preferably through at least one peptide bond.
  • the first attachment site is naturally occurring with the VLP.
  • the first attachment site is artificially added to the VLP.
  • Attachment Site refers to an element which is naturally occurring with or which is artificially added to the IL-I molecule and to which the first attachment site may be linked.
  • the second attachment site of the IL-I molecule preferably is a protein, a polypeptide, a peptide, an amino acid, a sugar, a polynucleotide, a natural or synthetic polymer, a secondary metabolite or compound (biotin, fluorescein, retinol, digoxigenin, metal ions, phenylmethylsulfonylfluoride), or a chemically reactive group such as an amino group, a carboxyl group, a sulfhydryl group, a hydroxyl group, a guanidinyl group, histidinyl group, or a combination thereof.
  • a preferred embodiment of a chemically reactive group being the second attachment site is the sulfhydryl group, preferably of an amino acid cysteine.
  • the term "IL-I molecule with at least one second attachment site” refers, therefore, to a construct comprising the IL-I molecule and at least one second attachment site.
  • a construct typically and preferably further comprises a "linker”.
  • the second attachment site is associated with the IL-I molecule through at least one covalent bond, preferably through at least one peptide bond.
  • the second attachment site is naturally occurring within the IL-I molecule.
  • the second attachment site is artificially added to the IL-I molecule through a linker, wherein said linker comprises or alternatively consists of a cysteine.
  • the linker is fused to the IL-I molecule by a peptide bond.
  • Coat protein refers to a viral protein, preferably a subunit of a natural capsid of a virus, preferably of a RNA-phage, which is capable of being incorporated into a virus capsid or a VLP.
  • IL-I molecule refers to any polypeptide having an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably at least 95 %, even more preferably at least 99 % and most preferably 100 % sequence identity with any one of the sequences selected from the group consisting of SEQ ID NO:36 to SEQ ID NO: 116, SEQ ID NO: 130 to SEQ ID NO: 140 and SEQ ID NO: 163 to SEQ ID NO: 165.
  • IL-I -molecule preferably refers to any IL-I protein, IL-I fragment, IL-I mature fragment, IL-I peptide or IL-I mutein comprising or alternatively consisting of a polypeptide having an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably at least 95 %, even more preferably at least 99 % and most preferably 100 % sequence identity with any one of the sequences selected from the group consisting of SEQ ID NO:36 to SEQ ID NO:116, SEQ ID NO: 130 to SEQ ID NO: 140 and SEQ ID NO: 163 to SEQ ID NO: 165.
  • IL-I molecule also typically and preferably refers to orthologs of IL-I proteins of any animal species.
  • An IL-I molecule is preferably, but not necessarily, capable of binding to the IL-I receptor and further preferably comprises biological activity.
  • IL-I alpha molecule refers to an IL-I alpha protein, IL-I alpha fragment, IL-I alpha mature fragment, IL-I alpha peptide or IL-I alpha mutein comprising or alternatively consisting of an polypeptide having an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably at least 95 %, even more preferably at least 99 % and most preferably 100 % sequence identity with any one of the sequences selected from the group consisting of SEQ ID NO:36 to 48, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67 to SEQ ID BO:88, and SEQ ID NO: 163.
  • a specifically preferred embodiment of IL-I alpha is human IL-I alpha 119-271 (SEQ ID NO:63).
  • IL-I beta molecule refers to an IL-I beta protein, IL-I beta fragment, IL-I beta mature fragment, IL-I beta peptide or IL-I beta mutein comprising or alternatively consisting of an polypeptide having an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably at least 95 %, even more preferably at least 99 % and most preferably 100 % sequence identity with any one of the sequences selected from the group consisting of SEQ ID NO:49 to SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:89 to SEQ ID NO: 116, SEQ ID NO:130 to SEQ ID NO:140, SEQ ID NO:164, and SEQ ID NO:165.
  • IL-I beta is human IL-I beta 117-269 (SEQ ID NO:64).
  • IL-I protein refers to a naturally occurring protein, wherein said naturally occurring protein has an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably at least 95 %, even more preferably at least 99 % and most preferably 100 % sequence identity with any one of SEQ ID NO:36 to SEQ ID NO:62; or wherein said naturally occurring protein.is capable of binding the IL-I receptor and preferably comprises biological activity.
  • IL-I protein preferably refers to a naturally occurring protein, wherein said naturally occurring protein has an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably at least 95 %, even more preferably at least 99 % and most preferably 100 % sequence identity with any one of SEQ ID NO:36 to SEQ ID NO:62; and wherein said naturally occurring protein.is capable of binding the IL-I receptor and preferably comprises biological activity.
  • IL-I protein refers to at least one naturally occurring protein, wherein said protein is capable of binding the IL-I receptor and comprises biological activity, and wherein further said protein comprises or alternatively consists of a polypeptide having an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably at least 95 %, even more preferably at least 99 % and most preferably 100 % sequence identity with any one of SEQ ID NO:36 to SEQ ID NO:62.
  • IL-I alpha protein relates to an IL-I protein comprising or alternatively consisting of a polypeptide having an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably at least 95 %, even more preferably at least 99 % and most preferably 100 % sequence identity with any one of SEQ ID NO:36 to SEQ ID NO:48
  • IL-I beta protein relates to an IL-I protein comprising or alternatively consisting of a polypeptide having an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably at least 95 %, even more preferably at least 99 % and most preferably 100 % sequence identity with any one of SEQ ID NO:49 to SEQ ID NO:62.
  • IL-I fragment refers to a polypeptide comprising a consecutive stretch of an IL-I protein, wherein said polypeptide is at least 50, preferably at least 100, most preferably at least 150 amino acids in length. Typically and preferably said IL-I fragment is at most 300, more preferably at most 250, and most preferably at most 200 amino acids in length. Typically and preferably, IL-I fragments are capable of binding the IL-I receptor and further preferably comprises biological activity. Accordingly, the terms "IL-I alpha fragment” and "IL-I beta fragment” relate to an IL-I fragment as defined, wherein said IL-I protein is an IL-I alpha protein or an IL-I beta protein, respectively.
  • IL-I mature fragment The term "IL-I mature fragment”, as used herein, relates to a IL-I fragment, wherein said IL-I fragment is a naturally occurring maturation product of an IL-I protein. Accordingly, the terms "IL-I alpha mature fragment” and "IL-I beta mature fragment”, as used herein relate to IL-I mature fragments as defined, wherein said IL-I protein is an IL-I alpha protein or an IL-I beta protein, respectively.
  • Preferred embodiments of IL-I alpha mature fragments are SEQ ID NO:63, SEQ ID NO:65 and SEQ ID NO: 163.
  • Preferred embodiments of IL-I beta mature fragments are SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO: 130, SEQ ID NO: 164, and SEQ ID NO: 165.
  • Preferred IL-I alpha mature fragments comprise or preferably consist of an amino acid sequence selected from the group consisting of: (a) human IL-I alpha 119-271 (SEQ ID NO:63); (b) mouse IL-I alpha 117-270 (SEQ ID NO:65); (c) mouse IL-I alpha 117-27Os (SEQ ID NO: 163); and (e) an amino acid sequence which is at least 80 %, or preferably at least 90 %, more preferably at least 95 %, or most preferably at least 99 % identical with any one of SEQ ID NO:63, SEQ ID NO:65 and SEQ ID NO:163.
  • Preferred IL-I beta mature fragments comprise or preferably consist of an amino acid sequence selected from the group consisting of: (a) human IL-I beta 117-269 (SEQ ID NO:64); (b) human IL-I beta 116-269 (SEQ ID NO:165); (c) mouse IL-I beta 119-269 (SEQ ID NO:66); (d) mouse IL-I beta 119-269s (SEQ ID NO: 164); and (e) an amino acid sequence which is at least 80 %, or preferably at least 90 %, more preferably at least 95 %, or most preferably at least 99 % identical with any one of SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO: 164 and SEQ ID NO: 165.
  • IL-I peptide relates to a polypeptide comprising a consecutive stretch of a naturally occurring protein, wherein said protein is capable of binding the IL-I receptor and preferably comprises biological activity, wherein said polypeptide is 4 to 49, preferably 6 to 35, most preferably 10 to 25 amino acids in length.
  • the IL-I peptide may be, but typically is not, capable of binding the IL-I receptor and typically has no biological activity.
  • IL-I alpha peptide and "IL-I beta peptide”, as used herein relate to IL-I peptides as defined, wherein said naturally occurring protein is an IL-I alpha protein or an IL-I beta protein, respectively.
  • Preferred IL-I peptides are SEQ ID NO: 82 to SEQ ID NO: 116.
  • IL-I mutein comprise or preferably consist of any polypeptide derived from an IL-I molecule, preferably from an IL-I alpha or an IL-I beta protein, an IL-I alpha or an IL-I beta fragment, an IL-I alpha or an IL-I beta mature fragment or an IL-I alpha or an IL-I beta peptide, wherein preferably said polypeptide exhibits reduced biological activity as compared to the IL-I molecule it is derived from.
  • IL-I alpha muteins and IL-I beta muteins are IL-I muteins as defined, wherein said polypeptide is derived from an IL-I alpha molecule or an IL-I beta molecule, respectively.
  • Very preferred IL-I beta muteins are IL-I beta muteins derived from IL-I beta mature fragments, preferably from human IL- l ⁇ i 17.20 9 (SEQ ID NO:64).
  • Very preferred IL-I alpha muteins are derived from IL-I alpha mature fragments, preferably from human IL-I ⁇ ii 9- 27i (SEQ ID NO:63).
  • said biological activity is less than 80 %, more preferably less than 60 %, still more preferably less than 40 %, still more preferably less than 20 % of the biological activity of the IL-I molecule it is derived from, wherein further preferably said biological activity is determined by the capacity of said IL-I mutein to induce IL-6 in human PBMCs, wherein most preferably said biological activity is determined essentially as described in Example 11 B.
  • said biological activity is less than 80 %, more preferably less than 60 %, still more preferably less than 40 %, still more preferably less than 20 % of the biological activity of the IL-I beta molecule it is derived from, wherein preferably said IL-I beta molecule is an IL-I beta mature fragment, preferably human IL- l ⁇ i 17-26 9 (SEQ ID NO:64), and wherein further preferably said biological activity is determined by the capacity of said IL-I beta mutein to induce IL-6 in human PBMCs, wherein most preferably said biological activity is determined essentially as described in Example 11 B.
  • said biological activity is less than 80 %, more preferably less than 60 %, still more preferably less than 40 %, still more preferably less than 20 % of the biological activity of the IL-I alpha molecule it is derived from, wherein preferably said IL-I alpha molecule is an IL-I alpha mature fragment, preferably human human IL-I (X 1 ⁇ -271 (SEQ ID NO:63), and wherein further preferably said biological activity is determined by the capacity of said IL-I alpha mutein to induce IL-6 in human PBMCs, wherein most preferably said biological activity is determined essentially as described in Example 16.
  • Further preferred IL-I muteins are derived from an IL-I mature fragment, wherein the biological activity of said IL-I mutein is less than 80 %, more preferably less than 60 %, still more preferably less than 40 %, still more preferably less than 20 % of the biological activity of the IL-I mature fragment said IL-I mutein is derived from. Very preferred IL-I muteins do not exhibit biological activity.
  • IL-I muteins are capable of specifically binding an IL-I receptor.
  • compositions of the invention comprising a preferred IL-I mutein as the sole antigen induce a titer of antibodies capable of specifically binding the IL-I molecule said IL-I mutein is derived from, wherein said titer is at least 20 %, preferably at least 40 %, still more preferably at least 60 %, still more preferably at least 80 % and most preferably at least 100 % of the titer obtained with a composition comprising the IL- 1 molecule said IL-I mutein is derived from as the sole antigen, wherein preferably said titer is determined essentially as described in Example 12 D.
  • compositions of the invention comprising a preferred IL-I beta mutein as the sole antigen induce a titer of antibodies capable of specifically binding the IL-I beta molecule said IL-I beta mutein is derived from, wherein preferably said IL-I beta molecule is an IL-I beta mature fragment, most preferably human IL- l ⁇ i 17.26 9 (SEQ ID NO:64), wherein said titer is at least 20 %, preferably at least 40 %, still more preferably at least 60 %, still more preferably at least 80 % and most preferably at least 100 % of the titer obtained with a composition comprising the IL-I beta molecule said IL-I beta mutein is derived from, preferably said IL-I beta mature fragment, most preferably said human IL-l ⁇ n7.26 9 (SEQ ID NO:64) as the sole antigen, wherein further preferably said titer is determined essentially as described in Example 12
  • compositions of the invention comprising a preferred IL-I alpha mutein as the sole antigen induce a titer of antibodies capable of specifically binding the IL-I alpha molecule said IL-I alpha mutein is derived from, wherein preferably said IL-I alpha molecule is an IL-I alpha mature fragment, most preferably human IL-I (X 1 1 9 -271 (SEQ ID NO:63), wherein said titer is at least 20 %, preferably at least 40 %, still more preferably at least 60 %, still more preferably at least 80 % and most preferably at least 100 % of the titer obtained with a composition comprising the IL-I alpha molecule said IL-I alpha mutein is derived from, preferably said IL-I alpha mature fragment, most preferably said human IL-I (X 1 ⁇ -271 (SEQ ID NO:63)as the sole antigen, wherein further preferably
  • IL-I mutein is an IL-I mutein, wherein said biological activity is less than 80 %, more preferably less than 60 %, still more preferably less than 40 %, still more preferably less than 20 % of the biological activity of the IL-I molecule it is derived from, wherein further preferably said biological activity is determined by the capacity of said IL-I mutein to induce IL-6 in human PBMCs, wherein most preferably said biological activity is determined essentially as described in Example 11 B, and wherein additionally compositions of the invention comprising said very preferred IL-I mutein as the sole antigen induce a titer of antibodies capable of specifically binding the IL-I molecule said very preferred IL-I mutein is derived from, wherein said titer is at least 20 %, preferably at least 40 %, still more preferably at least 60 %, still more preferably at least 80 % and most preferably at least 100 % of the titer obtained with
  • IL-I muteins derived from (i) an IL-I protein, preferably from SEQ ID NO:36 to SEQ ID NO:62; or (ii) more preferably of an IL-I mature fragment, preferably from any one of SEQ ID NO:63 to SEQ ID NO:66, SEQ ID NO: 130, and SEQ ID NO:163 to SEQ ID NO:165.
  • IL-I muteins useful in the context have been described in Kamogashira et al. (1988) J. Biochem. 104:837-840; Gehrke et al. (1990) The Journal of Biological Chemistry 265(11):5922-5925; Conca et al. (1991) The Journal of Biological Chemistry 266(25): 16265- 16268;Ju et al. (1991) PNAS 88:2658-2662; Auron et al. (1992) Biochemistry 31:6632-6638; Guinet et al. (1993) Eur. J.
  • Preferred IL-I muteins comprise or preferably consist of a polypeptide having an amino acid sequence which differs from the amino acid sequence of an IL-I protein, an IL-I fragment, an IL-I mature fragment or an IL-I peptide in 1 to 10, preferably 1 to 6, more preferably 1 to 5, still more preferably 1 to 4, still more preferably 1 to 3, still more preferably 1 to 2, and most preferably in exactly 1 amino acid residue(s), wherein preferably said amino acid residue(s) are (i) deleted from said polypeptide, (ii) inserted into said polypeptide, (iii) exchanged by another amino acid residue, or (iv) any combination of (i) to (iii).
  • said amino acid residues are in one consecutive stretch.
  • Further preferred IL-I muteins comprise or preferably consist of a polypeptide having an amino acid sequence which differs from the amino acid sequence of an IL-I protein, an IL-I fragment, or an IL-I mature fragment, preferably of an IL-I mature fragment, in 1 to 10, preferably 1 to 6, more preferably 1 to 5, still more preferably 1 to 4, still more preferably 1 to 3, still more preferably 1 to 2, and most preferably in exactly 1 amino acid residue(s), wherein preferably said amino acid residue(s) are (i) deleted from said polypeptide, (ii) inserted into said polypeptide, (iii) exchanged by another amino acid residue, or (iv) any combination of (i) to (iii).
  • Further preferred IL-I muteins comprise or more preferably consist of a polypeptide having an amino acid sequence which differs from the amino acid sequence of any one of SEQ ID NO:36 to SEQ ID NO:48 or SEQ ID NO:49 to SEQ ID NO:62 in 1 to 10, preferably 1 to 6, more preferably 1 to 5, still more preferably 1 to 4, still more preferably 1 to 3, still more preferably 1 to 2, and most preferably in exactly 1 amino acid residue(s), wherein preferably said amino acid residue(s) are (i) deleted from said polypeptide, (ii) inserted into said polypeptide, (iii) exchanged by another amino acid residue, or (iv) any combination of (i) to (iii).
  • IL-I muteins comprise or preferably consist of a polypeptide having an amino acid sequence which differs from the amino acid sequence selected from the group consisting of (i) any one of SEQ ID NO: 63, SEQ ID NO: 65, and SEQ ID NO: 163, most preferably SEQ ID NO:63; or (ii) of any one selected from the group consisting of SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:130, SEQ ID NO:164, and SEQ ID NO:165, most preferably SEQ ID NO:64 in 1 to 10, preferably 1 to 6, more preferably 1 to 5, still more preferably 1 to 4, still more preferably 1 to 3, still more preferably 1 to 2, and most preferably in exactly 1 amino acid residue(s), wherein preferably said amino acid residue(s) are (i) deleted from said polypeptide, (ii) inserted into said polypeptide, (iii) exchanged by another amino acid residue, or (iv) any combination of (i) to (iii).
  • IL-I alpha muteins are IL-I alpha muteins, wherein said IL-I alpha muteins comprise or more preferably consist of a polypeptide having an amino acid sequence which differs from the amino acid sequence of any one of SEQ ID NO:36 to SEQ ID NO:48 in 1 to 6, preferably 1 to 5, more preferably 1 to 4, still more preferably 1 to 3, still more preferably 1 to 2, and most preferably in exactly 1 amino acid residue(s), wherein preferably said amino acid residue(s) are (i) deleted from said polypeptide, (ii) inserted into said polypeptide, (iii) exchanged by another amino acid residue, or (iv) any combination of (i) to (iii).
  • IL-I alpha muteins comprise or preferably consist of a polypeptide having an amino acid sequence which differs from the amino acid sequence selected from the group consisting of (i) any one of SEQ ID NO:63, SEQ ID NO:65, and SEQ ID NO:163, most preferably SEQ ID NO:63, in 1 to 6, preferably 1 to 5, more preferably 1 to 4, still more preferably 1 to 3, still more preferably 1 to 2, and most preferably in exactly 1 amino acid residue(s), wherein preferably said amino acid residue(s) are (i) deleted from said polypeptide, (ii) inserted into said polypeptide, (iii) exchanged by another amino acid residue, or (iv) any combination of (i) to (iii).
  • Very preferred IL-I alpha muteins comprise or preferably consist of a polypeptide having an amino acid sequence which differs from the amino acid sequence of SEQ ID NO:63 in 1 to 10, preferably 1 to 6, more preferably 1 to 5, still more preferably 1 to 4, still more preferably 1 to 3, still more preferably 1 to 2, and most preferably in exactly 1 amino acid residue(s), wherein preferably said amino acid residue(s) are (i) deleted from said polypeptide, (ii) inserted into said polypeptide, (iii) exchanged by another amino acid residue, or (iv) any combination of (i) to (iii).
  • Still more preferred IL-I alpha muteins comprise or preferably consist of a polypeptide having an amino acid sequence selected from any one of the group consisting of SEQ ID NO:210 to SEQ ID NO:218.
  • Further preferred IL-I muteins are IL-I beta muteins, wherein said IL-I beta muteins comprise or more preferably consist of a polypeptide having an amino acid sequence which differs from the amino acid sequence of any one of SEQ ID NO:49 to SEQ ID NO:62 in 1 to 6, preferably 1 to 5, more preferably 1 to 4, still more preferably 1 to 3, still more preferably 1 to 2, and most preferably in exactly 1 amino acid residue(s), wherein preferably said amino acid residue(s) are (i) deleted from said polypeptide, (ii) inserted into said polypeptide, (iii) exchanged by another amino acid residue, or (iv) any combination of (i) to (iii).
  • IL-I beta muteins comprise or preferably consist of a polypeptide having an amino acid sequence which differs from the amino acid sequence selected from the group consisting of SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO: 130, SEQ ID NO: 164, and SEQ ID NO: 165, most preferably SEQ ID NO:64, in 1 to 6, preferably 1 to 5, more preferably 1 to 4, still more preferably 1 to 3, still more preferably 1 to 2, and most preferably in exactly 1 amino acid residue(s), wherein preferably said amino acid residue(s) are (i) deleted from said polypeptide, (ii) inserted into said polypeptide, (iii) exchanged by another amino acid residue, or (iv) any combination of (i) to (iii).
  • Very preferred IL-I beta muteins comprise or preferably consist of a polypeptide having an amino acid sequence which differs from the amino acid sequence of SEQ ID NO:64 in 1 to 10, preferably 1 to 6, more preferably 1 to 5, still more preferably 1 to 4, still more preferably 1 to 3, still more preferably 1 to 2, and most preferably in exactly 1 amino acid residue(s), wherein preferably said amino acid residue(s) are (i) deleted from said polypeptide, (ii) inserted into said polypeptide, (iii) exchanged by another amino acid residue, or (iv) any combination of (i) to (iii).
  • Still more preferred IL-I beta muteins comprise or preferably consist of a polypeptide having an amino acid sequence selected from any one of the group consisting of SEQ ID NO: 131 to SEQ ID NO: 140 and SEQ ID NO:205 to SEQ ID NO:209.
  • amino acid sequence which is derived from another amino acid sequence means that said amino acid sequence is essentially identical with the amino acid sequence it is derived from, with the exception of certain mutations, wherein said mutations are selected from the group consisting of (i) amino acid exchanges, (ii) deletions, (iii) insertions, and (iv) any combination of (i) to (iii), wherein preferably said mutations are selected from (i) amino acid exchanges and (ii) deletions.
  • a mutated amino acid sequence derived from a wild type amino acid sequence preferably differs from said wild type amino acid sequence in 1 to 10, preferably 1 to 6, more preferably 1 to 5, still more preferably 1 to 4, still more preferably 1 to 3, still more preferably 1 to 2, and most preferably in exactly 1 amino acid residue(s), wherein preferably said amino acid residue(s) are (i) exchanged by another amino acid, (ii) deleted from said wild type amino acid, (iii) inserted into said wild type sequence, and (iv) any combination of (i) to (iii), wherein most preferably said amino acid residue(s) are (i) exchanged by another amino acid, or (ii) deleted from said wild type amino acid.
  • Deletions of more than one amino acid residue preferably occur as a deletion of a consecutive stretch of amino acid residues of said wild type amino acid sequence.
  • a mutated amino acid sequence which is derived from a wild type amino acid sequence preferably has at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, and most preferably at least 99 % sequence identity with said wild type amino acid sequence.
  • mutein derived from a IL-I molecule refers to a mutein, wherein said mutein comprises or preferably consists of a polypeptide having an amino acid sequence which is essentially identical to that of the IL-I molecule it is derived from, with the exception of certain mutations, wherein said mutations are selected from the group consisting of (i) amino acid exchanges, (ii) deletions, (iii) insertions, and (iv) any combination of (i) to (iii), wherein preferably said mutations are selected from (i) amino acid exchanges and (ii) deletions.
  • an IL-I mutein derived from an IL-I molecule differs from said IL-I molecule in 1 to 10, preferably 1 to 6, more preferably 1 to 5, still more preferably 1 to 4, still more preferably 1 to 3, still more preferably 1 to 2, and most preferably in exactly 1 amino acid residue(s), wherein preferably said amino acid residue(s) are (i) exchanged by another amino acid, (ii) deleted from said wild type amino acid, (iii) inserted into said wild type sequence, and (iv) any combination of (i) to (iii), wherein most preferably said amino acid residue(s) are (i) exchanged by another amino acid, or (ii) deleted from said wild type amino acid.
  • Deletions of more than one amino acid residue preferably occur as a deletion of a consecutive stretch of amino acid residues of the IL-I molecule said IL-I mutein is derived from.
  • a mutein derived from a wild type amino acid sequence preferably has at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, and most preferably at least 99 % sequence identity with the IL-I molecule said IL-I mutein is derived from.
  • Amino acid exchange refers to the exchange of an amino acid residue in a certain position of an amino acid sequence by any other amino acid residue.
  • Agonistic effect/biological activity of the IL-I refers to the ability of the IL-I molecule to induce the production of IL-6 after systemical administration into animals, preferably as outlined in Example 2 E. and in Example 3 E.
  • biological activity of the IL-I molecule is also meant the ability to induce the proliferation of thymocytes (Epps et al., Cytokine 9(3): 149-156 (1997), D10.G4.1 T helper cells (Orencole and Dinarello, Cytokine 1(1): 14-22 (1989), or the ability to induce the production of IL-6 from MG64 or HaCaT cells (Boraschi et al., J. Immunol. 155:4719-4725 (1995) or fibroblasts (Dinarello et al., Current Protocols in Immunology 6.2.1-6-2-7 (2000)), or the production of IL-2 from EL-4 thymoma cells (Simon et al., J.
  • the term biological activity of an IL-I molecule or an IL-I mutein refers to the capacity of a composition of the invention comprising said IL-I molecule or said IL-I mutein to induce IL-6 in human PBMCs, wherein preferably said IL-I molecule or said IL-I mutein is the sole antigen in said composition, and wherein most preferably said biological activity is determined essentially as described in Example 11 B.
  • Linked refers to all possible ways, preferably chemical interactions, by which the at least one first attachment site and the at least one second attachment site are joined together. Chemical interactions include covalent and non-covalent interactions. Typical examples for non-covalent interactions are ionic interactions, hydrophobic interactions or hydrogen bonds, whereas covalent interactions are based, by way of example, on covalent bonds such as ester, ether, phosphoester, amide, peptide, carbon-phosphorus bonds, carbon-sulfur bonds such as thioether, or imide bonds.
  • first attachment site and the second attachment site are linked through at least one covalent bond, preferably through at least one non-peptide bond, and even more preferably through exclusively non-peptide bond(s).
  • first attachment site and the second attachment site are linked through at least one covalent bond, preferably through at least one peptide bond, and even more preferably through exclusively peptide bond(s).
  • first attachment site and the second attachment site are linked exclusively by peptide bounds, preferably by genetic fusion, either directly, or, preferably, via an amino acid linker.
  • the second attachment site is linked to the C-terminus of said first attachment site exclusively by peptide bounds, preferably by genetic fusion.
  • Linker A "linker”, as used herein, either associates the second attachment site with the IL-I molecule or already comprises, essentially consists of, or consists of the second attachment site.
  • a “linker”, as used herein already comprises the second attachment site, typically and preferably - but not necessarily - as one amino acid residue, preferably as a cysteine residue.
  • a “linker” as used herein is also termed “amino acid linker", in particular when a linker according to the invention contains at least one amino acid residue.
  • linker and “amino acid linker” are interchangeably used herein.
  • linker consists exclusively of amino acid residues, even if a linker consisting of amino acid residues is a preferred embodiment of the present invention.
  • the amino acid residues of the linker are, preferably, composed of naturally occurring amino acids or unnatural amino acids known in the art, all-L or all-D or mixtures thereof.
  • Further preferred embodiments of a linker in accordance with this invention are molecules comprising a sulfhydryl group or a cysteine residue and such molecules are, therefore, also encompassed within this invention.
  • linkers useful for the present invention are molecules comprising a C1-C6 alkyl-, a cycloalkyl such as a cyclopentyl or cyclohexyl, a cycloalkenyl, aryl or heteroaryl moiety.
  • linkers comprising preferably a C1-C6 alkyl-, cycloalkyl- (C5, C6), aryl- or heteroaryl- moiety and additional amino acid(s) can also be used as linkers for the present invention and shall be encompassed within the scope of the invention.
  • Association of the linker with the IL-I molecule is preferably by way of at least one covalent bond, more preferably by way of at least one peptide bond.
  • a linker may be absent or preferably is an amino acid linker, more preferably an amino acid linker consisting exclusively of amino acid residues.
  • Very preferred linkers for genetic fusion are flexible amino acid linkers. In the context of linkage by genetic fusion linkers preferred consist of 1 to 20, more preferably of 2 to 15, still more preferably of 2 to 10, still more preferably of 2 to 5, and most preferably of 3 amino acids.
  • Very preferred linkers for genetic fusion comprise or preferably consist of GSG (SEQ ID NO: 189).
  • Ordered and repetitive antigen array generally refers to a repeating pattern of antigen or, characterized by a typically and preferably high order of uniformity in spacial arrangement of the antigens with respect to virus-like particle, respectively.
  • the repeating pattern may be a geometric pattern.
  • Certain embodiments of the invention are typical and preferred examples of suitable ordered and repetitive antigen arrays which, moreover, possess strictly repetitive paracrystalline orders of antigens, preferably with spacing of 1 to 30 nanometers, preferably 2 to 15 nanometers, even more preferably 2 to 10 nanometers, even again more preferably 2 to 8 nanometers, and further more preferably 1.6 to 7 nanometers.
  • the term "packaged” as used herein refers to the state of a polyanionic macro molecule or immunostimulatory substances in relation to the VLP.
  • the term "packaged” as used herein includes binding that may be covalent, e.g., by chemically coupling, or non-covalent, e.g., ionic interactions, hydrophobic interactions, hydrogen bonds, etc.
  • the term also includes the enclosement, or partial enclosement, of a polyanionic macromolecule.
  • the polyanionic macromolecule or immunostimulatory substances can be enclosed by the VLP without the existence of an actual binding, in particular of a covalent binding.
  • the at least one polyanionic macromolecule or immunostimulatory substances is packaged inside the VLP, most preferably in a non-covalent manner.
  • said immunostimulatory substances is nucleic acid, preferably a DNA
  • the term packaged implies that said nucleic acid is not accessible to nucleases hydrolysis, preferably not accessible to DNAse hydrolysis (e.g. DNaseI or Benzonase), wherein preferably said accessibility is assayed as described in Examples 11-17 of [0060]
  • Polypeptide The term "polypeptide” as used herein refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds).
  • Recombinant VLP refers to a VLP that is obtained by a process which comprises at least one step of recombinant DNA technology.
  • VLP recombinantly produced refers to a VLP that is obtained by a process which comprises at least one step of recombinant DNA technology.
  • recombinant VLP and “VLP recombinantly produced” are interchangeably used herein and should have the identical meaning.
  • virus particle refers to the morphological form of a virus. In some virus types it comprises a genome surrounded by a protein capsid; others have additional structures (e.g., envelopes, tails, etc.).
  • Virus-like particle refers to a non-replicative or noninfectious, preferably a non-replicative and non-infectious virus particle, or refers to a non- replicative or non-infectious, preferably a non-replicative and non- infectious structure resembling a virus particle, preferably a capsid of a virus.
  • non-replicative refers to being incapable of replicating the genome comprised by the VLP.
  • non- infectious refers to being incapable of entering the host cell.
  • a virus-like particle in accordance with the invention is non-replicative and/or non-infectious since it lacks all or part of the viral genome or genome function.
  • a virus- like particle is a virus particle, in which the viral genome has been physically or chemically inactivated.
  • a virus-like particle lacks all or part of the replicative and infectious components of the viral genome.
  • a virus-like particle in accordance with the invention may contain nucleic acid distinct from their genome.
  • a typical and preferred embodiment of a virus-like particle in accordance with the present invention is a viral capsid such as the viral capsid of the corresponding virus, bacteriophage, preferably RNA bacteriophage.
  • viral capsid refers to a macromolecular assembly composed of viral protein subunits. Typically, there are 60, 120, 180, 240, 300, 360 and more than 360 viral protein subunits. Typically and preferably, the interactions of these subunits lead to the formation of viral capsid or viral-capsid like structure with an inherent repetitive organization, wherein said structure is, typically, spherical or tubular.
  • the capsids of RNA bacteriophages or HBcAgs have a spherical form of icosahedral symmetry.
  • RNA bacteriophage refers to a virus-like particle comprising, or preferably consisting essentially of or consisting of coat proteins, mutants or fragments thereof, of an RNA bacteriophage.
  • virus-like particle of an RNA bacteriophage resembling the structure of an RNA bacteriophage, being non replicative and/or non-infectious, and lacking at least the gene or genes encoding for the replication machinery of the RNA bacteriophage, and typically also lacking the gene or genes encoding the protein or proteins responsible for viral attachment to or entry into the host.
  • This definition should, however, also encompass virus-like particles of RNA bacteriophages, in which the aforementioned gene or genes are still present but inactive, and, therefore, also leading to non-replicative and/or non-infectious virus-like particles of an RNA bacteriophage.
  • VLPs derived from RNA bacteriophages exhibit icosahedral symmetry and consist of 180 subunits (monomers).
  • Preferred methods to render a virus-like particle of an RNA bacteriophage non replicative and/or non-infectious is by physical, chemical inactivation, such as UV irradiation, formaldehyde treatment, typically and preferably by genetic manipulation.
  • the amino acid sequence identity of polypeptides can be determined conventionally using known computer programs such as the Bestfit program.
  • Bestfit or any other sequence alignment program preferably using Bestfit, to determine whether a particular sequence is, for instance, 95 % identical to a reference amino acid sequence, the parameters are set such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.
  • This aforementioned method in determining the percentage of identity between polypeptides is applicable to all proteins, polypeptides or a fragment thereof disclosed in this invention.
  • compositions of the invention comprise: (a) a core particle with at least one first attachment site, wherein said core particle is a virus-like particle (VLP) or a virus particle; and (b) at least one antigen with at least one second attachment site, wherein the at least one antigen is an IL-I molecule, preferably selected from the group consisting of IL-I protein, IL-I mature fragment, IL-I peptide and IL-I mutein, wherein (a) and (b) are covalently linked through the at least one first and the at least one second attachment site.
  • VLP virus-like particle
  • antigen is an IL-I molecule, preferably selected from the group consisting of IL-I protein, IL-I mature fragment, IL-I peptide and IL-I mutein, wherein (a) and (b) are covalently linked through the at least one first and the at least one second attachment site.
  • said IL-I molecule is linked to the core particle, so as to form an ordered and repetitive antigen- VLP array.
  • at least 20, preferably at least 30, more preferably at least 60, again more preferably at least 120 and further more preferably at least 180 IL-I molecules are linked to the core particle.
  • Any virus known in the art having an ordered and repetitive structure may be selected as a VLP or a virus particle of the invention.
  • Illustrative DNA or RNA viruses, the coat or capsid protein of which can be used for the preparation of VLPs have been disclosed in WO 2004/009124 on page 25, line 10-21, on page 26, line 11-28, and on page 28, line 4 to page 31, line 4.
  • Virus or virus-like particle can be produced and purified from virus-infected cell cultures.
  • the resulting virus or virus-like particle for vaccine purpose should be preferably non-replicative or non- infectious, more preferably non-replicative and non- infectious.
  • UV irradiation, chemical treatment, such as with formaldehyde or chloroform, are the general methods known to skilled person in the art to inactivate virus.
  • the core particle is a virus particle, and wherein preferably said virus particle is a bacteriophage, and wherein further preferably said bacteriophage is an RNA bacteriophage, and wherein even further preferably said RNA bacteriophage is an RNA bacteriophage selected from Q ⁇ , fr, GA or AP205.
  • the core particle is a VLP.
  • the VLP is a recombinant VLP. Almost all commonly known viruses have been sequenced and are readily available to the public. The gene encoding the coat protein can be easily identified by a skilled artisan.
  • the virus-like particle comprises, or alternatively consists of, recombinant proteins, mutants or fragments thereof, of a virus selected form the group consisting of: a) RNA bacteriophages; b) bacteriophages; c) Hepatitis B virus, preferably its capsid protein (Ulrich, et al, Virus Res.
  • VLP comprising more than one different recombinant proteins is generally referred, in this application, as mosaic VLP.
  • the VLP is a mosaic VLP, wherein said mosaic VLP comprises, or consists of, more than one recombinant protein, preferably of two recombinant proteins, most preferably of two recombinant capsid proteins, mutants or fragments thereof.
  • fragment of a recombinant protein or the term “fragment of a coat protein”, as used herein, is defined as a polypeptide, which is of at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% the length of the wild-type recombinant protein, or coat protein, respectively and which preferably retains the capability of forming VLP.
  • the fragment is obtained by at least one internal deletion, at least one truncation or at least one combination thereof. Further preferably, the fragment is obtained by at most 5, 4, 3 or 2 internal deletions, by at most 2 truncations or by exactly one combination thereof.
  • fragment of a recombinant protein or "fragment of a coat protein” shall further refer to a polypeptide, which has at least 80%, preferably 90%, even more preferably 95% amino acid sequence identity with the "fragment of a recombinant protein” or “fragment of a coat protein", respectively, as defined above and which is preferably capable of assembling into a virus-like particle.
  • mutant coat protein refers to a polypeptide having an amino acid sequence derived from the wild type recombinant protein, or coat protein, respectively, wherein the amino acid sequence is at least 80 %, preferably at least 85 %, 90 %, 95 %, 97 %, or 99 % identical to the wild type sequence and preferably retains the ability to assemble into a VLP.
  • the virus-like particle of the invention is of Hepatitis B virus. The preparation of Hepatitis B virus-like particles has been disclosed, inter alia, in WO 00/32227, WO 01/85208 and in WO 01/056905. AU three documents are explicitly incorporated herein by way of reference.
  • HBcAg suitable for use in the practice of the present invention
  • page 34-39 of WO 01/056905 Other variants of HBcAg suitable for use in the practice of the present invention have been disclosed in page 34-39 of WO 01/056905.
  • a lysine residue is introduced into the HBcAg polypeptide, to mediate the linking of IL-I molecule to the VLP of HBcAg.
  • VLPs and compositions of the invention are prepared using a HBcAg comprising, or alternatively consisting of, amino acids 1-144, or 1-149, 1-185 of SEQ ID NO:1, which is modified so that the amino acids at positions 79 and 80 are replaced with a peptide having the amino acid sequence of Gly-Gly-Lys-Gly-Gly (SEQ ID NO: 170).
  • This modification changes the SEQ ID NO:1 to SEQ ID NO:2.
  • the cysteine residues at positions 48 and 110 of SEQ ID NO:2, or its corresponding fragments, preferably 1-144 or 1-149 are mutated to serine.
  • the invention further includes compositions comprising Hepatitis B core protein mutants having above noted corresponding amino acid alterations.
  • the invention further includes compositions and vaccines, respectively, comprising HBcAg polypeptides which comprise, or alternatively consist of, amino acid sequences which are at least 80%, 85%, 90%, 95%, 97% or 99% identical to SEQ ID NO:2.
  • the virus-like particle of the invention comprises, consists essentially of, or alternatively consists of, recombinant coat proteins, mutants or fragments thereof, of an RNA bacteriophage.
  • the RNA bacteriophage is selected from the group consisting of a) bacteriophage Q ⁇ ; b) bacteriophage Rl 7; c) bacteriophage fr; d) bacteriophage GA; e) bacteriophage SP; f) bacteriophage MS2; g) bacteriophage Mi l; h) bacteriophage MXl; i) bacteriophage NL95; k) bacteriophage f2; 1) bacteriophage PP7 and m) bacteriophage AP205.
  • the composition comprises coat protein, mutants or fragments thereof, of RNA bacteriophages, wherein the coat protein has amino acid sequence selected from the group consisting of: (a) SEQ ID NO: 3 referring to ⁇ ) CP; (b) a mixture of SEQ ID NO:3 and SEQ ID NO:4 (Q ⁇ Al protein); (c) SEQ ID NO:5 (Rl 7 capsid protein); (d) SEQ ID NO:6 (fr capsid protein); (e) SEQ ID NO:7 (GA capsid protein); (f) SEQ ID NO:8 (SP capsid protein); (g) a mixture of SEQ ID NO:8 and SEQ ID NO:9; (h) SEQ ID NO: 10 (MS2 capsid protein); (i) SEQ ID NO: 11 (Mi l capsid protein); (j) SEQ ID NO: 12 (MXl capsid protein); (k) SEQ ID NO: 13 (NL95 capsid protein); (1)
  • the VLP is a mosaic VLP comprising or alternatively consisting of more than one amino acid sequence, preferably two amino acid sequences, of coat proteins, mutants or fragments thereof, of an RNA bacteriophage.
  • the VLP comprises or alternatively consists of two different coat proteins of an RNA bacteriophage, said two coat proteins have an amino acid sequence of CP Q ⁇ (SEQ ID NO: 3) and CP Q ⁇ Al (SEQ ID NO:4), or of CP SP (SEQ ID NO:8) and CP SP Al (SEQ ID NO:9).
  • the virus-like particle of the invention comprises, or alternatively consists essentially of, or alternatively consists of recombinant coat proteins, mutants or fragments thereof, of the RNA-bacteriophage Q ⁇ , fr, AP205 or GA.
  • the VLP of the invention is a VLP of RNA bacteriophage Q ⁇ .
  • the capsid contains 180 copies of the coat protein, which are linked in covalent pentamers and hexamers by disulfide bridges (Golmohammadi, R. et al., Structure 4:543-5554 (1996)), leading to a remarkable stability of the Q ⁇ capsid.
  • Capsids or VLPs made from recombinant Q ⁇ coat protein may contain, however, subunits not linked via disulfide bonds to other subunits within the capsid, or incompletely linked.
  • the capsid or VLP of Q ⁇ shows unusual resistance to organic solvents and denaturing agents. Surprisingly, we have observed that DMSO and acetonitrile concentrations as high as 30 %, and guanidinium concentrations as high as 1 M do not affect the stability of the capsid.
  • the high stability of the capsid or VLP of Q ⁇ is an advantageous feature, in particular, for its use in immunization and vaccination of mammals and humans in accordance of the present invention.
  • RNA bacteriophages in particular of Q ⁇ and fr in accordance of this invention are disclosed in WO 02/056905, the disclosure of which is herewith incorporated by reference in its entirety.
  • Particular example 18 of WO 02/056905 gave detailed description of preparation of VLP particles from Q ⁇ .
  • the VLP of the invention is a VLP of RNA bacteriophage AP205.
  • Assembly-competent mutant forms of AP205 VLPs, including AP205 coat protein with the substitution of proline at amino acid 5 to threonine, may also be used in the practice of the invention and leads to other preferred embodiments of the invention.
  • WO 2004/007538 describes, in particular in Example 1 and Example 2, how to obtain VLP comprising AP205 coat proteins, and hereby in particular the expression and the purification thereto.
  • WO 2004/007538 is incorporated herein by way of reference.
  • the VLP of the invention comprises or consists of a mutant coat protein of a virus, preferably an RNA bacteriophage, wherein the mutant coat protein has been modified by removal of at least one lysine residue by way of substitution and/or by way of deletion.
  • the VLP of the invention comprises or consists of a mutant coat protein of a virus, preferably an RNA bacteriophage, wherein the mutant coat protein has been modified by addition of at least one lysine residue by way of substitution and/or by way of insertion.
  • the deletion, substitution or addition of at least one lysine residue allows varying the degree of coupling, i.e. the amount of IL-I molecule per subunits of the VLP of a virus, preferably of an RNA bacteriophages, in particular, to match and tailor the requirements of the vaccine.
  • the compositions and vaccines of the invention have an antigen density being from 0.5 to 4.0.
  • antigen density refers to the average number of IL-I molecules which is linked per subunit, preferably per coat protein, of the VLP, and hereby preferably of the VLP of an RNA bacteriophage. Thus, this value is calculated as an average over all the subunits of the VLP, preferably of the VLP of the RNA bacteriophage, in the composition or vaccines of the invention.
  • VLPs or capsids of Q ⁇ coat protein display a defined number of lysine residues on their surface, with a defined topology with three lysine residues pointing towards the interior of the capsid and interacting with the RNA, and four other lysine residues exposed to the exterior of the capsid.
  • the at least one first attachment site is a lysine residue, pointing to or being on the exterior of the VLP.
  • the virus-like particle comprises, consists essentially of or alternatively consists of mutant Q ⁇ coat proteins.
  • these mutant coat proteins comprise or alternatively consist of an amino acid sequence selected from the group of a) Q ⁇ -240 (SEQ ID NO: 16, Lysl3-Arg of SEQ ID NO: 3) b) Q ⁇ -243 (SEQ ID NO:17, AsnlO-Lys of SEQ ID NO:3); c) Q ⁇ -250 (SEQ ID NO:18, Lys2-Arg of SEQ ID NO:3) d) Q ⁇ -251 (SEQ ID NO:19, Lysl6-Arg of SEQ ID NO:3); and e) Q ⁇ -259 (SEQ ID NO:20, Lys2-Arg, Lysl6-Arg of SEQ ID NO:3).
  • Q ⁇ -240 SEQ ID NO: 16, Lysl3-Arg of SEQ ID NO: 3
  • Q ⁇ -243 SEQ ID NO:17, AsnlO-Lys of SEQ ID NO:3
  • Q ⁇ -250 SEQ ID NO:18, Lys2-Arg of SEQ ID NO:3
  • Q ⁇ -251 SEQ ID
  • the virus-like particle comprises, or alternatively consists essentially of, or alternatively consists of mutant coat protein of Q ⁇ , or mutants or fragments thereof, and the corresponding Al protein.
  • the virus-like particle comprises, or alternatively consists essentially of, or alternatively consists of mutant coat protein with amino acid sequence SEQ ID NO: 16, 17, 18, 19, or 20 and the corresponding Al protein.
  • RNA bacteriophage coat proteins have also been shown to self-assemble upon expression in a bacterial host (Kastelein, RA. et al., Gene 23:245-254 (1983), Kozlovskaya, TM. et al., Dokl. Akad. Nauk SSSR 287:452-455 (1986), Adhin, MR. et al., Virology 170:238-242 (1989), Priano, C. et al., J. MoI. Biol. 249:283-297 (1995)).
  • GA Biochemical properties of GA (Ni, CZ., et al., Protein Sci.
  • RNA bacteriophages have been determined.
  • the crystal structure of several RNA bacteriophages has been determined (Golmohammadi, R. et al., Structure 4:543-554 (1996)). Using such information, surface exposed residues can be identified and, thus, RNA bacteriophage coat proteins can be modified such that one or more reactive amino acid residues can be inserted by way of insertion or substitution.
  • Another advantage of the VLPs derived from RNA bacteriophages is their high expression yield in bacteria that allows production of large quantities of material at affordable cost.
  • the composition of the invention comprises at least one antigen, preferably one to four, more preferably one to three, still more preferably one to two and most preferably exactly one antigen, wherein said antigen is an IL-I molecule, preferably an IL-I protein, an IL-I fragment, an IL-I mature fragment, an IL-I peptide or an IL-I mutein, wherein said IL-I molecule preferably comprises or even more preferably consists of a polypeptide having an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably at least 95 %, even more preferably at least 99 % and most preferably 100 % sequence identity with any one of SEQ ID NO:36 to SEQ ID NO: 116, SEQ ID NO: 130 to SEQ ID NO: 140 and SEQ ID NO: 163 to SEQ ID NO: 165.
  • said antigen is an IL-I molecule, preferably an IL-I protein, an IL-I fragment, an IL-I mature
  • said antigen is an IL-I molecule derived from an organism selected from the group consisting of: (a) humans; (b) primates; (c) rodents; (d) horses; (e) sheep; (f) cat; (g) cattle; (h) pig; (i) rabbit; (j) dog; (k) mouse; and (1) rat.
  • said IL-I molecule is derived from humans, preferably comprising or even more preferably consisting of a polypeptide having at least 80 %, preferably at least 90 %, more preferably at least 95 %, even more preferably at least 99 % and most preferably 100 % sequence identity with any one of the sequences selected from the group consisting of SEQ ID NO:36, SEQ ID NO:49, SEQ ID NO:63, SEQ ID NO:64, any one of SEQ ID NO:67 to 110, and one of SEQ ID NO: 130-140, and SEQ ID NO: 165.
  • IL-I molecule is an IL-I alpha molecule, preferably an IL-I alpha protein, an IL-I alpha fragment, an IL-I alpha mature fragment, an IL-I alpha peptide or an IL-I alpha mutein, wherein said IL-I alpha molecule preferably comprises or even more preferably consists of a polypeptide having an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably at least 95 %, even more preferably at least 99 % and most preferably 100 % sequence identity with any one of the sequences selected from the group consisting of SEQ ID NO:36 to 48, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67 to 88, and SEQ ID NO: 165.
  • IL-I alpha molecules are human IL-I alpha molecules, preferably human IL-I alpha proteins, human IL-I alpha fragments or human IL-I alpha mature fragments, wherein said IL-I alpha molecules preferably comprise or even more preferably consist of a polypeptide having an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably at least 95 %, even more preferably at least 99 % and most preferably 100 % sequence identity with any one of SEQ ID NO:36, SEQ ID NO:63, and SEQ ID NO:163, most preferably SEQ ID NO:63.
  • said IL-I molecule is an IL-I beta molecule, preferably an IL-I beta protein, an IL-I beta fragment, an IL-I beta mature fragment, an IL-I beta peptide or an IL-I beta mutein, wherein said IL-I beta molecule preferably comprises or even more preferably consists of a polypeptide having an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably at least 95 %, even more preferably at least 99 % and most preferably 100 % sequence identity with any one of the sequences selected from the group consisting of SEQ ID NO:49 to 62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:89 to 116, SEQ ID NO:130 to SEQ ID NO:140, SEQ ID NO:164, and SEQ ID NO:165.
  • IL-I beta molecules are human IL-I beta molecules, preferably human IL-I beta proteins, human IL-I beta fragments or human IL-I beta mature fragments, wherein said IL-I beta molecules preferably comprises or even more preferably consists of a polypeptide having an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably at least 95 %, even more preferably at least 99 % and most preferably 100 % sequence identity with any one of SEQ ID NO:49, SEQ ID NO:64, SEQ ID NO: 130 to SEQ ID NO: 140 and SEQ ID NO: 165, most preferably SEQ ID NO:64.
  • said IL-I molecule is an IL-I protein, an IL-I fragment or, preferably, an IL-I mature fragment, wherein said IL-I protein, IL-I fragment or IL-I mature fragment preferably are capable of binding to the IL-I receptor and, still more preferably, additionally also comprise biological activity.
  • said IL-I molecule is an IL-I protein, wherein said IL-I protein preferably comprises or even more preferably consists of a polypeptide having an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably at least 95 %, even more preferably at least 99 % and most preferably 100 % sequence identity with any one of SEQ ID NO:36 to SEQ ID NO:62.
  • said IL-I protein is an IL-I alpha protein, wherein said IL-I alpha protein preferably comprises or even more preferably consists of a polypeptide having an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably at least 95 %, even more preferably at least 99 % and most preferably 100 % sequence identity with any one of the sequences selected from the group consisting of SEQ ID NO:36 to SEQ ID NO:48.
  • said IL-I alpha protein is a human IL-I alpha protein, wherein said human IL-I alpha protein preferably comprises or even more preferably consists of a polypeptide having least 80 %, preferably at least 90 %, more preferably at least 95 %, even more preferably at least 99 % and most preferably 100 % sequence identity with SEQ ID NO:36.
  • said said IL-I protein is an is an IL-I beta protein, wherein said IL-I beta protein preferably comprises or even more preferably consists of a polypeptide having an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably at least 95 %, even more preferably at least 99 % and most preferably 100 % sequence identity with any one of the sequences selected from the group consisting of SEQ ID NO:49 to SEQ ID NO:62.
  • said IL-I beta protein is a human IL-I beta protein, wherein said human IL-I beta protein preferably comprises or even more preferably consists of a polypeptide having least 80 %, preferably at least 90 %, more preferably at least 95 %, even more preferably at least 99 % and most preferably 100 % sequence identity with SEQ ID NO:49.
  • said IL-I molecule is an IL-I fragment, preferably an IL-I mature fragment, and wherein said IL-I fragment or said IL-I mature fragment preferably is derived from mouse or human, most preferably human.
  • said IL-I fragment or said IL-I mature fragment comprises or even more preferably consists of a polypeptide having an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably at least 95 %, even more preferably at least 99 % and most preferably 100 % sequence identity with any one of SEQ ID NO:63 to SEQ ID NO:66, SEQ ID NO: 130, and SEQ ID NO: 163 to SEQ ID NO: 165.
  • said IL-I mature fragment is an IL-I alpha mature fragment, wherein said IL-I alpha mature fragment preferably comprises biological activity and wherein further said IL-I alpha mature fragment preferably comprises or even more preferably consists of a polypeptide having an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably at least 95 %, even more preferably at least 99 % and most preferably 100 % sequence identity with any one of SEQ ID NO:63 or SEQ ID NO:65, most preferably SEQ ID NO:63.
  • said IL-I mature fragment is an IL-I beta mature fragment, wherein said IL-I beta mature fragment preferably comprises biological activity and wherein further said IL-I beta mature fragment preferably comprises or even more preferably consists of a polypeptide having an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably at least 95 %, even more preferably at least 99 % and most preferably 100 % sequence identity with any one of SEQ ID NO:64, SEQ ID NO:66, and SEQ ID NO: 130, most preferably SEQ ID NO:64.
  • said IL-I molecule is an IL-I peptide, wherein said IL-I peptide is derived from mouse, rat or human, most preferably human.
  • said IL-I peptide comprises or even more preferably consists of a polypeptide having an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably at least 95 %, even more preferably at least 99 % and most preferably 100 % sequence identity with any one of SEQ ID NO:67 to SEQ ID NO:116.
  • said IL-I molecule is an IL-I mutein, wherein preferably said IL-I mutein comprises reduced or more preferably no biological activity, and wherein further said IL-I mutein is capable of binding the IL-I receptor.
  • said IL-I mutein comprises or preferably consists of a polypeptide having an amino acid sequence which differs from the amino acid sequence of an IL-I mature fragment in 1 to 3, more preferably in 1 to 2, and most preferably in exactly 1 amino acid residue.
  • said IL-I mutein comprises at least one, preferably one, mutated amino acid sequence derived from a wild type amino acid sequence, wherein said wild type amino acid sequence is an IL-I beta amino acid sequence selected from the group consisting of: (1) position 3 to 11 of SEQ ID NO:64; (2) position 46 to 56 of SEQ ID NO:64; (3) position 88 to 109 of SEQ ID NO:64; and (4) position 143 to 153 of SEQ ID NO:64; or wherein said wild type amino acid sequence is an IL-I alpha amino acid sequence selected from the group consisting of: (5) position 9 to 20 of SEQ ID NO:63; (6) position 52 to 62 of SEQ ID NO:63; (7) position 94 to 113 of SEQ ID NO:63; and (8) position 143 to 153 of SEQ ID NO:63; and wherein said at least one mutated amino acid sequence is characterized by an amino acid exchange in one to four positions, preferably in one, two or three positions, more
  • said IL-I mutein comprises at most one mutated amino acid sequence derived from each of said L-I beta amino acid sequences (1) to (4); or wherein said IL-I mutein comprises at most one mutated amino acid sequence derived from each of said IL-I alpha amino acid sequences (5) to (8).
  • said IL-I mutein comprises exactly one of said at least one mutated amino acid sequence, wherein preferably said exactly one mutated amino acid sequence is derived from a wild type amino acid sequence, wherein said wild type amino acid sequence is position 143 to 153 of SEQ ID NO:64 or position 143 to 153 of SEQ ID NO:63.
  • said at least one mutated amino acid sequence is characterized by a deletion of one to three, preferably of one to two, consecutive amino acids of said wild type amino acid sequence it is derived from.
  • said at least one mutated amino acid sequence is characterized by a deletion of exactly one amino acid of said wild type amino acid sequence it is derived from.
  • said at least one mutated amino acid sequence is derived from a wild type amino acid sequence, wherein said wild type amino acid sequence is position 143 to 153 of SEQ ID NO:64 or position 143 to 153 of SEQ ID NO:63. Most preferably said at least one mutated amino acid sequence is derived from position 143 to 153 of SEQ ID NO:64.
  • said at least one mutated amino acid sequence is derived from a wild type amino acid sequence, wherein said wild type amino acid sequence is position 46 to 56 of SEQ ID NO:64 or position 52 to 62 of SEQ ID NO:63, wherein preferably said at least one mutated amino acid sequence is characterized by a deletion of one to four, preferably of two to three, consecutive amino acids of said wild type amino acid sequence it is derived from.
  • said IL-I mutein comprises or preferably consists of a polypeptide having the amino acid sequence of SEQ ID NO: 137 or SEQ ID NO:138.
  • said at least one mutated amino acid sequence is derived from a wild type amino acid sequence, wherein said wild type amino acid sequence is position 88 to 109 of SEQ ID NO:64 or position 94-113 of SEQ ID NO:63, wherein said at least one mutated amino acid sequence is characterized by the deletion of one to four, preferably of one to three, more preferably of one to two consecutive amino acids of said wild type amino acid sequence it is derived from.
  • said at least one mutated amino acid sequence is characterized by an amino acid exchange in one or two positions, preferably in exactly one position, as compared to said wild type amino acid sequence it is derived from.
  • said wild type amino acid sequence is position 143 to 153 of SEQ ID NO: 64 or position 143 to 153 of SEQ ID NO: 63 and said at least one mutated amino acid sequence is characterized by an amino acid exchange in one or two positions, preferably in exactly one position, as compared to said wild type amino acid sequence, wherein further preferably said exactly one position is position 145 of SEQ ID NO: 64 or position 145 of SEQ ID NO: 63, wherein still further preferably said amino acid exchange is an exchange of aspartic acid (D) to an amino acid selected from the group consisting of lysine (K), tyrosine (Y), phenylalanine (F), asparagine (N) and arginine (R).
  • D aspartic acid
  • said wild type amino acid sequence is position 143 to 153 of SEQ ID NO: 64 or position 143 to 153 of SEQ ID NO: 63 and said at least one mutated amino acid sequence is characterized by an amino acid exchange in exactly one position as compared to said wild type amino acid sequence, wherein further preferably said exactly one position is position 146 of SEQ ID NO:64 or position 146 of SEQ ID NO:63, wherein still further preferably said amino acid exchange is an exchange of phenylalanine (F) to an amino acid selected from the group consisting of asparagine (N), glutamine (Q), and serine (S).
  • F phenylalanine
  • said IL-I mutein is an IL-I beta mutein, preferably a human IL-I beta mutein, most preferably a human IL-I beta mutein selected from SEQ ID NO: 131 to SEQ ID NO: 140.
  • said IL-I mutein is an IL-I beta mutein, wherein preferably said IL-I beta mutein comprises or preferably consists of a polypeptide having an amino acid sequence, wherein said amino acid sequence differs from the amino acid sequence of SEQ ID NO:64 in 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 amino acid residues. Most preferably said amino acid sequence differs from the amino acid sequence of SEQ ID NO:64 in exactly 1 amino acid residue.
  • said IL-I beta mutein comprises or preferably consists of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 131 to SEQ ID NO: 140 and SEQ ID NO:205 to SEQ ID NO:209, wherein most preferably said IL-I beta mutein comprises or preferably consists of a polypeptide having the amino acid sequence of SEQ ID NO: 136.
  • said IL-I mutein is an IL-I alpha mutein, wherein preferably said IL-I alpha mutein comprises or preferably consists of a polypeptide having an amino acid sequence, wherein said amino acid sequence differs from the amino acid sequence of SEQ ID NO:63 in 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 amino acid residues. Most preferably said amino acid sequence differs from the amino acid sequence of SEQ ID NO:63 in exactly 1 amino acid residue.
  • said IL-I alpha mutein comprise or preferably consist of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:210 to SEQ ID NO:218, wherein most preferably said IL-I alpha mutein comprises or preferably consists of a polypeptide having the amino acid sequence of SEQ ID NO:210.
  • the present invention provides for a method of producing the composition of the invention comprising (a) providing a VLP with at least one first attachment site; (b) providing at least one antigen, wherein said antigen is an IL-I molecule, an IL-I protein, an IL-I fragment, preferably an IL-I mature fragment, an IL-I peptide or an IL-I mutein, with at least one second attachment site; and (c) combining said VLP and said at least one antigen to produce said composition, wherein said at least one antigen and said VLP are linked through the first and the second attachment sites.
  • the provision of the at least one antigen i.e.
  • IL-I molecule an IL-I protein, an IL-I fragment, preferably an IL-I mature fragment, an IL-I peptide or an IL-I mutein, with the at least one second attachment site is by way of expression, preferably by way of expression in a bacterial system, preferably in E. coli.
  • a purification tag such as His tag, Myc tag, Fc tag or HA tag is added to facilitate the purification process.
  • the IL-I peptides or IL-I muteins with no longer than 50 amino acids are chemically synthesized.
  • the VLP with at least one first attachment site is linked to the IL-I molecule with at least one second attachment site via at least one peptide bond.
  • a gene encoding an IL-I molecule, preferably an IL-I mature fragment is in-frame ligated, either internally or preferably to the N- or the C-terminus to the gene encoding the coat protein of the VLP. Fusion may also be effected by inserting sequences of the IL-I into a mutant coat protein where part of the coat protein sequence has been deleted, that are further referred to as truncation mutants. Truncation mutants may have N- or C-terminal, or internal deletions of part of the sequence of the coat protein.
  • amino acids 79-80 are replaced with a foreign epitope.
  • the fusion protein shall preferably retain the ability of assembly into a VLP upon expression which can be examined by electromicroscopy.
  • Flanking amino acid residues may be added to increase the distance between the coat protein and foreign epitope. Glycine and serine residues are particularly favored amino acids to be used in the flanking sequences. Such a flanking sequence confers additional flexibility, which may diminish the potential destabilizing effect of fusing a foreign sequence into the sequence of a VLP subunit and diminish the interference with the assembly by the presence of the foreign epitope.
  • the at least one IL-I molecule preferably the IL-I mature fragment can be fused to a number of other viral coat protein, as way of examples, to the C- terminus of a truncated form of the Al protein of Q ⁇ (Kozlovska, T. M., et al., Intervirology 39:9-15 (1996)), or being inserted between position 72 and 73 of the CP extension.
  • the IL-I can be inserted between amino acid 2 and 3 of the fr CP, leading to a IL-l-fr CP fusion protein (Pushko P. et al., Prot. Eng. 6:883-891 (1993)).
  • IL-I can be fused to the N-terminal protuberant ⁇ -hairpin of the coat protein of RNA bacteriophage MS-2 (WO 92/13081).
  • the IL-I can be fused to a capsid protein of papillomavirus, preferably to the major capsid protein Ll of bovine papillomavirus type 1 (BPV-I) (Chackerian, B. et al., Proc. Natl. Acad. Sci.USA 96:2373-2378 (1999), WO 00/23955).
  • BPV-I bovine papillomavirus type 1
  • US 5,698,424 describes a modified coat protein of bacteriophage MS-2 capable of forming a capsid, wherein the coat protein is modified by an insertion of a cysteine residue into the N-terminal hairpin region, and by replacement of each of the cysteine residues located external to the N-terminal hairpin region by a non-cysteine amino acid residue.
  • the inserted cysteine may then be linked directly to a desired molecular species to be presented such as an epitope or an antigenic protein.
  • capsids may lead to oligomerization of capsids by way of disulfide bridge formation.
  • attachment between capsids and antigenic proteins by way of disulfide bonds are labile, in particular, to sulfhydryl-moiety containing molecules, and are, furthermore, less stable in serum than, for example, thioether attachments (Martin FJ. and Papahadjopoulos D. (1982) Irreversible Coupling of Immunoglobulin Fragments to Preformed Vesicles. J. Biol. Chem. 257: 286-288).
  • the association or linkage of the VLP and the at least one antigen, i.e. IL-I molecule does not comprise a disulfide bond.
  • the at least one second attachment comprise, or preferably is, a sulfhydryl group.
  • the association or linkage of the VLP and the at least one IL-I molecule does not comprise a sulphur-sulphur bond.
  • the at least one second attachment comprise, or preferably is, a sulfhydryl group.
  • said at least one first attachment site is not or does not comprise a sulfhydryl group. In again a further very preferred embodiment, said at least one first attachment site is not or does not comprise a sulfhydryl group of a cysteine. [00129] In a further preferred embodiment said at least one first attachment comprises an amino group and said second attachment comprises a sulfhydryl group.
  • only one of said second attachment sites associates with said first attachment site through at least one non-peptide covalent bond leading to a single and uniform type of binding of said IL-I molecule to said core particle, wherein said only one second attachment site that associates with said first attachment site is a sulfhydryl group, and wherein said IL-I molecule and said core particle interact through said association to form an ordered and repetitive antigen array.
  • an IL-I molecule preferably an IL-I protein, more preferably an IL-I mature fragment, still more preferably an IL-I mature fragment comprising or consisting of amino acid sequenced SEQ ID NO:63 to SEQ ID NO:66, most preferably SEQ ID NO: 63 or SEQ ID NO: 64, is fused to either the N- or the C-terminus, preferably the C-terminus, of a coat protein, mutants or fragments thereof, of RNA bacteriophage AP205.
  • VLPs comprising fusion proteins of coat protein of bacteriophage AP205 with an antigen are generally disclosed in WO2006/032674A1 which is incorporated herein by reference.
  • the fusion protein further comprises a linker, wherein said linker is fused to the coat protein, fragments or mutants thereof, of AP205 and the IL-I molecule.
  • said IL-I molecule is fused to the C-terminus of said coat protein, fragments or mutants thereof, of AP205 via said linker.
  • IL-I molecules in particular IL-I proteins and IL-I fragments comprising at least 100 and up to 300 amino acids, typically and preferably about 140 to 160 amino acids, and most preferably about 155 amino acids, can be fused to coat protein of bacteriophages, preferably to coat protein of AP205, while maintaining the ability of the coat protein to self assemble into a VLP.
  • AP205-IL-1 As the level of incorporation of AP205-IL-1 fusion protein into the mosaic VLP is depending on the level of suppression, AP205-IL-1 is expressed in E.coli cells already containing a plasmid overexpressing a suppressor t-RNA.
  • plasmid pISM3001 (Smiley, B.K., Minion, F. C. (1993) Enhanced readthrough of opal (UGA) stop codons and production of Mycoplasma pneumoniae Pl epitopes in Escherichia coli. Gene 134, 33-40), which encodes a suppressor t-RNA recognizing the opal stop codon and introducing Trp is used.
  • Plasmid pISM579 was generated by excising the trpT176 gene from pISM3001 with restriction endonuclease EcoRI and replacing it by an EcoRI fragment from plasmid pMY579 (gift of Michael Yarus) containing an amber t-RNA suppressor gene.
  • This t-RNA suppressor gene is a mutant of trpT175 (Raftery LA. Et al. (1984) J. Bacteriol.
  • E.coli JM 109 may generate a proportion of AP205-IL-1 fusion proteins with a GIn instead of Trp introduced at the amber stop codon, in addition to AP205-IL-1 fusion proteins with a Trp introduced at the amber stop codon.
  • the identity of the amino acid translated at the stop codon may therefore depend on the combination of suppressor t-RNA overexpressed, and strain phenotype. As described by Miller JH et al. ((1983) J.
  • the efficiency of suppression is context dependent.
  • the codon 3' of the stop codon and the first base 3 ' from the stop codon are particularly important.
  • stop codons followed by a purine base are in general well suppressed.
  • said VLP is a mosaic VLP, wherein said mosaic VLP comprises or preferably consists of at least one, preferably one, first polypeptide and of at least one, preferably one, second polypeptide, wherein said first polypeptide is a recombinant capsid protein, mutant or fragments thereof; and wherein said second polypeptide is a genetic fusion product of a recombinant capsid protein, mutant or fragments thereof, preferably of said first polypeptide, with an IL-I molecule.
  • said first polypeptide is a recombinant capsid protein of bacteriophage AP205 or a mutant or fragment thereof.
  • said first polypeptide is selected from SEQ ID N0:21, SEQ ID NO:22, SEQ ID NO:23. In a very preferred embodiment said first polypeptide is SEQ ID NO:21.
  • Mosaic VLPs of bacteriophage AP205 comprising an antigen are generally disclosed in WO2006/032674A1, in particular in paragraph 107 of said publication.
  • said second polypeptide is a genetic fusion product of a recombinant capsid protein, mutant or fragments thereof, preferably of said first polypeptide, with an IL-I molecule, wherein said IL-I molecule is fused to the C-terminus of said recombinant capsid protein, mutant or fragments thereof, preferably via an amino acid linker.
  • said IL-I molecule comprises or preferably consists of 100 to 300 amino acids, typically and preferably about 140 to 160 amino acids, and most preferably about 155 amino acids.
  • the molar ratio of said first polypeptide and said second polypeptide in said mosaic VLP is 10:1 to 5:1, preferably 8:1 to 6:1, most preferably about 7:1.
  • the composition comprises or alternatively consists essentially of a virus-like particle with at least one first attachment site linked to at least one antigen, i.e. an IL-I molecule, with at least one second attachment site via at least one covalent bond, wherein preferably the covalent bond is a non-peptide bond.
  • the first attachment site comprises, or preferably is, an amino group, preferably the amino group of a lysine residue.
  • the second attachment site comprises, or preferably is, a sulfhydryl group, preferably a sulfhydryl group of a cysteine.
  • the at least one first attachment site is an amino group, preferably an amino group of a lysine residue and the at least one second attachment site is a sulfhydryl group, preferably a sulfhydryl group of a cysteine.
  • the IL-I molecule is linked to the VLP by way of chemical cross-linking, typically and preferably by using a heterobifunctional cross-linker.
  • the hetero-bifunctional cross-linker contains a functional group which can react with the preferred first attachment sites, preferably with the amino group, more preferably with the amino groups of lysine residue(s) of the VLP, and a further functional group which can react with the preferred second attachment site, i.e. a sulfhydryl group, preferably of cysteine(s) residue inherent of, or artificially added to the IL-I molecule, and optionally also made available for reaction by reduction.
  • a functional group which can react with the preferred first attachment sites preferably with the amino group, more preferably with the amino groups of lysine residue(s) of the VLP
  • a further functional group which can react with the preferred second attachment site, i.e. a sulfhydryl group, preferably of cysteine(s) residue inherent of, or artificially added to the IL-I molecule, and optionally also made available for reaction by reduction.
  • cross-linkers include the preferred cross-linkers SMPH (Pierce), Sulfo-MBS, Sulfo-EMCS, Sulfo-GMBS, Sulfo-SIAB, Sulfo-SMPB, Sulfo- SMCC, SVSB, SIA and other cross-linkers available for example from the Pierce Chemical Company, and having one functional group reactive towards amino groups and one functional group reactive towards sulfhydryl groups.
  • the above mentioned cross-linkers all lead to formation of an amide bond after reaction with the amino group and a thioether linkage with the sulfhydryl groups.
  • cross-linkers suitable in the practice of the invention is characterized by the introduction of a disulfide linkage between the IL-I molecule and the VLP upon coupling.
  • Preferred cross-linkers belonging to this class include, for example, SPDP and Sulfo-LC-SPDP (Pierce).
  • the composition of the invention further comprises a linker.
  • Engineering of a second attachment site onto the IL-I molecule is achieved by the association of a linker, preferably containing at least one amino acid suitable as second attachment site according to the disclosures of this invention. Therefore, in a preferred embodiment of the present invention, a linker is associated to the IL-I molecule by way of at least one covalent bond, preferably, by at least one, preferably one peptide bond.
  • the linker comprises, or alternatively consists of, the second attachment site.
  • the linker comprises a sulfhydryl group, preferably of a cysteine residue.
  • the amino acid linker is a cysteine residue.
  • the selection of a linker will be dependent on the nature of the IL-I molecule, on its biochemical properties, such as pi, charge distribution and glycosylation. In general, flexible amino acid linkers are favored.
  • the linker consists of amino acids, wherein further preferably the linker consists of at least one and at most 25, preferably at most 20, more preferably at most 15 amino acids. In an again preferred embodiment of the invention, the amino acid linker contains 1 to 10 amino acids.
  • Preferred linkers according to this invention are glycine linkers (G)n further containing a cysteine residue as second attachment site, such as N-terminal glycine linker (GCGGGG, SEQ ID NO: 174) and C-terminal glycine linker (GGGGCG, SEQ ID NO: 182).
  • GGKKGC C-terminal glycine- lysine linker
  • CGKKGG N-terminal glycine- lysine linker
  • GGCG SEQ ID NO: 188
  • GGC SEQ ID NO: 178
  • GGC-NH2 SEQ ID NO: 179, "NH2" stands for amidation
  • linkers at the C-terminus of the peptide or CGG (SEQ ID NO: 171) at its N- terminus.
  • glycine residues will be inserted between bulky amino acids and the cysteine to be used as second attachment site, to avoid potential steric hindrance of the bulkier amino acid in the coupling reaction.
  • Other methods of linking the IL-I molecule to the VLP include methods wherein the IL-I molecule is cross-linked to the VLP, using the carbodiimide EDC, and NHS.
  • the IL-I molecule may also be first thiolated through reaction, for example with SATA, SATP or iminothiolane. The IL-I molecule, after deprotection if required, may then be coupled to the VLP as follows.
  • the IL-I molecule is reacted with the VLP, previously activated with a hetero- bifunctional cross-linker comprising a cysteine reactive moiety, and therefore displaying at least one or several functional groups reactive towards cysteine residues, to which the thiolated IL-I molecule can react, such as described above.
  • a hetero- bifunctional cross-linker comprising a cysteine reactive moiety, and therefore displaying at least one or several functional groups reactive towards cysteine residues, to which the thiolated IL-I molecule can react, such as described above.
  • low amounts of a reducing agent are included in the reaction mixture.
  • the IL-I molecule is attached to the VLP, using a homo-bifunctional cross-linker such as glutaraldehyde, DSG, BM[PEO]4, BS3, (Pierce) or other known homo-bifunctional cross-linkers with functional groups reactive towards amine groups or carboxyl groups of the VLP.
  • a homo-bifunctional cross-linker such as glutaraldehyde, DSG, BM[PEO]4, BS3, (Pierce) or other known homo-bifunctional cross-linkers with functional groups reactive towards amine groups or carboxyl groups of the VLP.
  • the composition comprises or alternatively consists essentially of a virus-like particle linked to IL-I molecule via chemical interactions, wherein at least one of these interactions is not a covalent bond.
  • Linking of the VLP to the IL-I molecule can be effected by biotinylating the VLP and expressing the IL-I molecule as a streptavidin- fusion protein.
  • One or several antigen molecules can be attached to one subunit of the VLP, preferably of RNA bacteriophage coat proteins, preferably through the exposed lysine residues of the coat proteins of RNA bacteriophage VLP, if sterically allowable.
  • VLPs of RNA bacteriophage and in particular of the Q ⁇ coat protein VLP is thus the possibility to couple several antigens per subunit. This allows for the generation of a dense antigen array.
  • the IL-I molecule is linked via a cysteine residue, having been added to either the N-terminus or the C-terminus of, or a natural cysteine residue within an IL-I molecule, to lysine residues of coat proteins of the VLPs of RNA bacteriophage, and in particular to the coat protein of Q ⁇ .
  • lysine residues are exposed on the surface of the VLP of Q ⁇ coat protein. Typically and preferably these residues are derivatized upon reaction with a cross-linker molecule. In the instance where not all of the exposed lysine residues can be coupled to an antigen, the lysine residues which have reacted with the cross-linker are left with a cross-linker molecule attached to the ⁇ -amino group after the derivatization step. This leads to disappearance of one or several positive charges, which may be detrimental to the solubility and stability of the VLP.
  • Q ⁇ -240 (Lysl3-Arg; SEQ ID NO: 16), Q ⁇ -250 (Lys 2-Arg, Lysl3-Arg; SEQ ID NO: 18), Q ⁇ -259 (Lys 2-Arg, Lysl6-Arg; SEQ ID NO:20) and Q ⁇ -251; (Lysl6-Arg, SEQ ID NO: 19).
  • Q ⁇ mutant coat protein with one additional lysine residue Q ⁇ -243 (Asn 10-Lys; SEQ ID NO: 17), suitable for obtaining even higher density arrays of antigens.
  • the VLP of an RNA bacteriophage is recombinantly produced by a host and wherein said VLP is essentially free of host RNA, preferably host nucleic acids.
  • the composition further comprises at least one polyanionic macromolecule bound to, preferably packaged in or enclosed in, the VLP.
  • the polyanionic macromolecule is poly glutamic acid and/or polyaspartic acid.
  • the composition further comprises at least one immunostimulatory substance bound to, preferably packaged in or enclosed in, the VLP.
  • the immunostimulatory substance is a nucleic acid, preferably DNA, most preferably an unmethylated CpG containing oligonucleotide.
  • Essentially free of host RNA, preferably host nucleic acids refers to the amount of host RNA, preferably host nucleic acids, comprised by the VLP, which amount typically and preferably is less than 30 ⁇ g, preferably less than 20 ⁇ g, more preferably less than 10 ⁇ g, even more preferably less than 8 ⁇ g, even more preferably less than 6 ⁇ g, even more preferably less than 4 ⁇ g, most preferably less than 2 ⁇ g, per mg of the VLP.
  • Host refers to the host in which the VLP is recombinantly produced.
  • RNA preferably nucleic acids
  • the typical and preferred method to determine the amount of RNA, preferably nucleic acids, in accordance with the present invention is described in Example 17 of WO2006/037787A2.
  • Identical, similar or analogous conditions are, typically and preferably, used for the determination of the amount of RNA, preferably nucleic acids, for inventive compositions comprising VLPs other than Q ⁇ .
  • the modifications of the conditions eventually needed are within the knowledge of the skilled person in the art.
  • the numeric value of the amounts determined should typically and preferably be understood as comprising values having a deviation of ⁇ 10%, preferably having a deviation of ⁇ 5%, of the indicated numeric value.
  • Polyanionic macromolecule refers to a molecule of high relative molecular mass which comprises repetitive groups of negative charge, the structure of which essentially comprises the multiple repetition of units derived, actually or conceptually, from molecules of low relative molecular mass.
  • a polyanionic macromolecule should have a molecular weight of at least 2000 Dalton, more preferably of at least 3000 Dalton and even more preferably of at least 5000 Dalton.
  • polyanionic macromolecule typically and preferably refers to a molecule that is not capable of activating toll-like receptors.
  • polyanionic macromolecule typically and preferably excludes Toll-like receptors ligands, and even more preferably furthermore excludes immunostimulatory substances such as Toll-like receptors ligands, immunostimulatory nucleic acids, and lipopolysacchrides (LPS). More preferably the term “polyanionic macromolecule” as used herein, refers to a molecule that is not capable of inducing cytokine production. Even more preferably the term “polyanionic macromolecule” excludes immunostimulatory substances.
  • immunostimulatory substance refers to a molecule that is capable of inducing and/or enhancing immune response specifically against the antigen comprised in the present invention.
  • Host RNA preferably host nucleic acids:
  • the RNA, preferably nucleic acids may, however, undergo chemical and/or physical changes during the procedure of reducing or eliminating the amount of RNA, preferably nucleic acids, typically and preferably by way of the inventive methods, for example, the size of the RNA, preferably nucleic acids, may be shortened or the secondary structure thereof may be altered. However, even such resulting RNA or nucleic acids is still considered as host RNA, or host nucleic acids.
  • RNA and to reduce the amount of RNA comprised by the VLP have disclosed in US provisional application filed by the same assignee on October 5, 2004 and thus the entire application is incorporated herein by way of reference.
  • Reducing or eliminating the amount of host RNA, preferably host nucleic minimizes or reduces unwanted T cell responses, such as inflammatory T cell response and cytotoxic T cell response, and other unwanted side effects, such as fever, while maintaining strong antibody response specifically against IL-I.
  • this invention provides a method of preparing the inventive compositions and VLP of an RNA-bacteriophage the invention, wherein said VLP is recombinantly produced by a host and wherein said VLP is essentially free of host RNA, preferably host nucleic acids, comprising the steps of: a) recombinantly producing a virus-like particle (VLP) with at least one first attachment site by a host, wherein said VLP comprises coat proteins, variants or fragments thereof, of a RNA-bacteriophage; b) disassembling said virus-like particle to said coat proteins, variants or fragments thereof, of said RNA- bacteriophage; c) purifying said coat proteins, variants or fragments thereof; d) reassembling said purified coat proteins, variants or fragments thereof, of said RNA-bacteriophage to a virus-like particle, wherein said virus-like particle is essentially free of host RNA, preferably host nucleic acids; and e)
  • the invention provides a vaccine comprising the composition of the invention.
  • the IL-I molecule which is linked to the VLP in the vaccine composition may be of animal, preferably mammal or human origin.
  • the IL-I of the invention is of human, bovine, dog, cat, mouse, rat, pig or horse origin.
  • the vaccine composition further comprises at least one adjuvant.
  • the administration of the at least one adjuvant may hereby occur prior to, contemporaneously or after the administration of the inventive composition.
  • adjuvant refers to non-specific stimulators of the immune response or substances that allow generation of a depot in the host which when combined with the vaccine and pharmaceutical composition, respectively, of the present invention may provide for an even more enhanced immune response.
  • the vaccine composition is devoid of adjuvant.
  • An advantageous feature of the present invention is the high immunogenicity of the composition, even in the absence of adjuvants. The absence of an adjuvant, furthermore, minimizes the occurrence of unwanted inflammatory T-cell responses representing a safety concern in the vaccination against self antigens.
  • the administration of the vaccine of the invention to a patient will preferably occur without administering at least one adjuvant to the same patient prior to, contemporaneously or after the administration of the vaccine.
  • the invention further discloses a method of immunization comprising administering the vaccine of the present invention to an animal or a human.
  • the animal is preferably a mammal, such as cat, sheep, pig, horse, bovine, dog, rat, mouse and particularly human.
  • the vaccine may be administered to an animal or a human by various methods known in the art, but will normally be administered by injection, infusion, inhalation, oral administration, or other suitable physical methods.
  • the conjugates may alternatively be administered intramuscularly, intravenously, transmucosally, transdermally, intranasally, intraperitoneally or subcutaneously.
  • Components of conjugates for administration include sterile aqueous (e.g., physiological saline) or non-aqueous solutions and suspensions.
  • Vaccines of the invention are said to be "pharmacologically acceptable” if their administration can be tolerated by a recipient individual. Further, the vaccines of the invention will be administered in a "therapeutically effective amount" (i.e., an amount that produces a desired physiological effect). The nature or type of immune response is not a limiting factor of this disclosure. Without the intention to limit the present invention by the following mechanistic explanation, the inventive vaccine might induce antibodies which bind to IL-I and thus reducing its concentration and/or interfering with its physiological or pathological function.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the composition as taught in the present invention and an acceptable pharmaceutical carrier.
  • vaccine of the invention When administered to an individual, it may be in a form which contains salts, buffers, adjuvants, or other substances which are desirable for improving the efficacy of the conjugate.
  • examples of materials suitable for use in preparation of pharmaceutical compositions are provided in numerous sources including Remington's Pharmaceutical Sciences (Osol, A, ed., Mack Publishing Co., (1990)).
  • the invention teaches a process for producing the composition of the invention comprising the steps of: (a) providing a VLP with at least one first attachment site; (b) providing a IL-I molecule with at least one second attachment site, and (c) combining said VLP and said IL-I molecule to produce a composition, wherein said IL-I molecule and said VLP are linked through the first and the second attachment sites.
  • the step of providing a VLP with at least one first attachment site comprises further steps: (a) disassembling said virus-like particle to said coat proteins, mutants or fragments thereof, of said RNA-bacteriophage; (b) purifying said coat proteins, mutants or fragments thereof; (c) reassembling said purified coat proteins, mutants or fragments thereof, of said RNA-bacteriophage to a virus-like particle, wherein said virus-like particle is essentially free of host RNA, preferably host nucleic acids.
  • the reassembling of said purified coat proteins is effected in the presence of at least one polyanionic macromolecule.
  • the invention provides a method of using the compositions of the invention for treating and/or attenuating diseases or conditions in which IL-I exerts an important pathological function in an animal or in human.
  • the invention further provides for use of the compositions of the invention or the vaccine of the invention or the pharmaceutical composition of the invention for the manufacture of a medicament for treatment of a disease in an animal, preferably dog, cat horse or human, most preferably human, wherein said disease is preferably selected from the group consisting of: (a) vascular diseases, preferably coronary artery disease, atherosclerosis and vasculitis, most preferably atherosclerosis; (b) inherited IL-I -dependent inflammatory diseases, preferably Familial Mediterranean Fever (FMF), Familial Cold Autoinflammatory Syndrome (FCAS) Neonatal Onset Multisystem Inflammatory Disease (NOMID) and Muckle Wells Syndrome, most preferably Familial Mediterranean Fever (FMF); (c) chronic autoimmune inflammatory diseases, preferably rheumatoid arthritis, systemic onset juvenile idiopathic arthritis, adult onset Still's disease, psoriasis, Crohn's disease and ulcerative colitis, most preferably rheumatoid at
  • the invention further provides for use of the compositions of the invention or the vaccine of the invention or the pharmaceutical composition of the invention for the manufacture of a medicament for treatment of a disease in an animal, preferably dog, cat horse or human, most preferably human, wherein said disease is a vascular disease, preferably coronary artery disease, atherosclerosis and vasculitis, most preferably atherosclerosis, and wherein said at least one antigen comprised by said composition, said vaccine or said pharmaceutical composition is an IL-I alpha molecule of the invention, preferably an IL-I alpha mature fragment, most preferably SEQ ID NO: 63 or a mutein thereof.
  • the invention further provides for use of the compositions of the invention or the vaccine of the invention or the pharmaceutical composition of the invention for the manufacture of a medicament for treatment of a disease in an animal, preferably dog, cat horse or human, most preferably human, wherein said disease is selected from the group consisting of: (a) inherited IL-I -dependent inflammatory diseases, preferably Familial Mediterranean Fever (FMF), Familial Cold Autoinfiammatory Syndrome (FCAS) Neonatal Onset Multisystem Inflammatory Disease (NOMID) and Muckle Wells Syndrome, most preferably Familial Mediterranean Fever (FMF); (b) chronic autoimmune inflammatory diseases, preferably rheumatoid arthritis, systemic onset juvenile idiopathic arthritis, adult onset Still's disease, psoriasis, Crohn's disease and ulcerative colitis, most preferably rheumatoid athritis; (c) bone and cartilage degenerative diseases, preferably gout, osteoporosis and osteoarthriti
  • the invention further provides for use of the compositions of the invention or the vaccine of the invention or the pharmaceutical composition of the invention for the manufacture of a medicament for treatment of a disease in an animal, preferably dog, cat horse or human, most preferably human, wherein said disease is an inherited IL-I -dependent inflammatory diseases, preferably Familial Mediterranean Fever (FMF); and wherein said at least one antigen comprised by said composition, said vaccine or said pharmaceutical composition is an IL-I beta molecule, preferably an IL-I beta mature fragment, most preferably SEQ ID NO: 64 or a mutein thereof.
  • FMF Familial Mediterranean Fever
  • the invention further provides for use of the compositions of the invention or the vaccine of the invention or the pharmaceutical composition of the invention for the manufacture of a medicament for treatment of a disease in an animal, preferably human, wherein said disease is a vascular disease, preferably atherosclerosis.
  • the invention further provides for use of the compositions of the invention or the vaccine of the invention or the pharmaceutical composition of the invention for the manufacture of a medicament for treatment of a disease in an animal, preferably human, wherein said disease is an inherited IL-I -dependent inflammatory diseases, preferably familial mediterranean fever (FMF).
  • FMF familial mediterranean fever
  • the invention further provides for use of the compositions of the invention or the vaccine of the invention or the pharmaceutical composition of the invention for the manufacture of a medicament for treatment of a disease in an animal, preferably human, wherein said disease is a chronic autoimmune inflammatory diseases, preferably rheumatoid arthritis.
  • the invention further provides for use of the compositions of the invention or the vaccine of the invention or the pharmaceutical composition of the invention for the manufacture of a medicament for treatment of a disease in an animal, preferably human, wherein said disease is a bone and cartilage degenerative diseases, preferably osteoarthritis.
  • the invention further provides for use of the compositions of the invention or the vaccine of the invention or the pharmaceutical composition of the invention for the manufacture of a medicament for treatment of a disease in an animal, preferably human, wherein said disease is a neurological disease, preferably multiple sclerosis.
  • the invention further provides a method of treating a disease, the method comprising administering the composition of the invention, the vaccine of the invention or the pharmaceutical composition of the invention to an animal, preferably dog, cat horse or human, most preferably human, wherein said disease is preferably selected from the group consisting of: (a) vascular diseases, preferably coronary artery disease, atherosclerosis and vasculitis, most preferably atherosclerosis; (b) inherited IL-I -dependent inflammatory diseases, preferably Familial Mediterranean Fever (FMF), Familial Cold autoinflammatory Syndrome (FCAS) Neonatal Onset Multisystem Inflammatory Disease (NOMID) and Muckle Wells Syndrome, most preferably Familial Mediterranean Fever (FMF); (c) chronic autoimmune inflammatory diseases, preferably rheumatoid arthritis, systemic onset juvenile idiopathic arthritis, adult onset Still's disease, psoriasis, Crohn's disease and ulcerative colitis, most preferably rheumatoid athriti
  • the invention further provides a method of treating a disease, the method comprising administering the composition of the invention, the vaccine of the invention or the pharmaceutical composition of the invention to an animal, preferably dog, cat horse or human, most preferably human, wherein said disease is a vascular diseases, preferably coronary artery disease, atherosclerosis and vasculitis, most preferably atherosclerosis, and wherein said at least one antigen comprised by said composition, said vaccine or said pharmaceutical composition is an IL-I alpha molecule, preferably an IL-I alpha mature fragment, most preferably SEQ ID NO:63 or a mutein thereof.
  • the invention further provides a method of treating a disease, the method comprising administering the composition of the invention, the vaccine of the invention or the pharmaceutical composition of the invention to an animal, preferably dog, cat horse or human, most preferably human, wherein said disease is preferably selected from the group consisting of: (a) inherited IL-I -dependent inflammatory diseases, preferably Familial Mediterranean Fever (FMF), Familial Cold Autoinfiammatory Syndrome (FCAS) Neonatal Onset Multisystem Inflammatory Disease (NOMID) and Muckle Wells Syndrome, most preferably Familial Mediterranean Fever (FMF); (b) chronic autoimmune inflammatory diseases, preferably rheumatoid arthritis, systemic onset juvenile idiopathic arthritis, adult onset Still's disease, psoriasis, Crohn's disease and ulcerative colitis, most preferably rheumatoid athritis; (c) bone and cartilage degenerative diseases, preferably gout, osteoporosis and osteoarthriti
  • the invention further provides a method of treating a disease, the method comprising administering the composition of the invention, the vaccine of the invention or the pharmaceutical composition of the invention to an animal, preferably dog, cat horse or human, most preferably human, wherein said disease is an inherited IL-I -dependent inflammatory diseases, preferably Familial Mediterranean Fever (FMF); and wherein said at least one antigen comprised by said composition, said vaccine or said pharmaceutical composition is an IL-I beta molecule, preferably an IL-I beta mature fragment, most preferably SEQ ID NO:64 or a mutein thereof.
  • FMF Familial Mediterranean Fever
  • the invention further provides a method of treating a disease, the method comprising administering the composition of the invention, the vaccine of the invention or the pharmaceutical composition of the invention to an animal, preferably human, wherein said disease is a vascular disease, preferably atherosclerosis.
  • the invention further provides a method of treating a disease, the method comprising administering the composition of the invention, the vaccine of the invention or the pharmaceutical composition of the invention to an animal, preferably human, wherein said disease is an inherited IL-I -dependent inflammatory diseases, preferably familial mediterranean fever (FMF).
  • FMF familial mediterranean fever
  • the invention further provides a method of treating a disease, the method comprising administering the composition of the invention, the vaccine of the invention or the pharmaceutical composition of the invention to an animal, preferably human, wherein said disease is a chronic autoimmune inflammatory diseases, preferably rheumatoid arthritis.
  • the invention further provides a method of treating a disease, the method comprising administering the composition of the invention, the vaccine of the invention or the pharmaceutical composition of the invention to an animal, preferably human, wherein said disease is a bone and cartilage degenerative diseases, preferably osteoarthritis. All references cited herein are incorporated entirely by reference.
  • the nucleotide sequence encoding amino acids 119-269 of the murine IL- l ⁇ precursor was amplified with oligonucleotides ILl ⁇ l (5 '-ATATATGCTAGCCCCCATTAGACAGCTGCACTACAGG-S '; SEQ ID NO:26) and ILl ⁇ 2 (5 '-ATATATCTCGAGGGAAGACACAGATTCCATGGTGAAG-S ';
  • the vector pModECl (SEQ ID NO:29) is a derivative of pET22b(+) (Novagen Inc.), and was constructed in two steps. In a first step the multiple cloning site of pET22b(+) was changed by replacing the original sequence between the Ndel and Xhol sites with the annealed oligos primerMCS-lF (5 '-TATGGATCCGGCTAGCGCTCGAGGGTTTA AACGGCGGCCGCAT-3'; SEQ ID NO:30) and primerMCS-lR (5'-TCGAATGCGGCCG CCGTTTAAACCCTCGAGCGCTAGCCGGATCCA-S'; SEQ ID NO:31) (annealing in 15 mM TrisHCl pH 8 buffer).
  • primerMCS-lF 5 '-TATGGATCCGGCTAGCGCTCGAGGGTTTA AACGGCGGCCGCAT-3'
  • primerMCS-lR 5'-TCGAATGCGGCCG CCGTTTAAACC
  • the resulting plasmid was termed pModOO, and had Ndel, BamHI, Nhel, Xhol, Pmel and Notl restriction sites in its multiple cloning site.
  • oligolR-C-glycine-linker (5'-GGCCGCGTTTAAACTTATTA ACCGCAACCACCACCACCACCACCACCC-3'; SEQ ID NO:35) were ligated together into the BamHI -Notl digested pModOO plasmid to obtain pModECl, which encodes an N-terminal hexahistidine tag, an enterokinase cleavage site and a C-terminal glycine linker containing one cysteine residue.
  • Escherichia coli BL21 cells harbouring either plasmid were grown at 37°C to an OD at 600 nm of 1.0 and then induced by addition of isopropyl- ⁇ -D-thiogalactopyranoside at a concentration of 1 mM.
  • Bacteria were grown for 4 more hours at 37°C, harvested by centrifugation and resuspended in 80 ml lysis buffer (10 mM Na 2 HPO 4 , 30 mM NaCl, pH 7.0). Cells were then disrupted by sonication and cellular DNA and RNA were digested by 30 min incubation at room temperature with 64 ⁇ l 2 M MgCl 2 and lO ⁇ l Benzonase.
  • a solution containing 1.3 mg/ml of the purified murine IL-l ⁇ n 9-2 6 9 protein from EXAMPLE 1 (SEQ ID NO: 66) in PBS pH 7.2 was incubated for 60 min at room temperature with an equimolar amount of TCEP for reduction of the C-terminal cysteine residue.
  • a solution of 6 ml of 2 mg/ml Q ⁇ capsid protein in PBS pH 7.2 was then reacted for 60 min at room temperature with 131 ⁇ l of a SMPH solution (65 mM in DMSO). The reaction solution was dialysed at 4 0 C against three 3 1 changes of 20 mM HEPES, 150 mM NaCl pH 7.2 over 24 hours.
  • Coupled products were analyzed on a 12 % SDS-polyacrylamide gel under reducing conditions.
  • the Coomassie stained gel is shown in Fig. 1. Several bands of increased molecular weight with respect to the Q ⁇ capsid monomer are visible, clearly demonstrating the successful cross-linking of the mouse IL-l ⁇ n 9-2 6 9 protein to the Q ⁇ capsid.
  • mice Five female balb/c mice were immunized with Q ⁇ -mIL-l ⁇ ii 9 .269 (SEQ ID NO:66). Fifty ⁇ g of total protein were diluted in PBS to 200 ⁇ l and injected subcutaneously (100 ⁇ l on two ventral sides) on day 0 and day 21. Mice were bled retroorbitally on day 0, 21, and 35, and sera were analyzed using mouse IL-l ⁇ n 9 .26 9 -specific ELISA.
  • ELISA plates were coated with mouse IL- l ⁇ i 1 9 .20 9 protein at a concentration of 1 ⁇ g/ml. The plates were blocked and then incubated with serially diluted mouse sera from day 0, 21, and 35. Bound antibodies were detected with enzymatically labeled anti-mouse IgG antibody. Antibody titers of mouse sera were calculated as the average of those dilutions which led to half maximal optical density at 450 nm. The average anti-mouse IL- l ⁇ i 1 9 .20 9 titer was 1:22262 at day 21 and 1:309276 at day 35. This demonstrates that immunization with Q ⁇ coupled to the mouse IL- l ⁇ i 1 9 .20 9 protein could overcome immunological tolerance and produce high titer antibodies which recognize specifically IL- l ⁇ i 1 9 .26 9
  • Sera of mice immunized with Q ⁇ -mIL-l ⁇ 119-269 were then tested for their ability to inhibit the binding of mouse IL- l ⁇ protein to its receptor.
  • ELISA plates were therefore coated with a recombinant mlL-lreceptorl-hFc fusion protein at a concentration of 1 ⁇ g/ml, and co-incubated with serial dilutions of sera from mice which had been immunized either with mouse IL- l ⁇ i 1 9 .20 9 coupled to Q ⁇ capsid or ⁇ fc ⁇ mouse IL- lain.
  • mice immunized against murine IL- l ⁇ i 19.269 inhibited completely the binding of mouse IL- l ⁇ i 19.269 to its receptor at concentrations of >0.4 %, whereas sera from mice immunized against mouse IL- l ⁇ i 17-270 did not show any inhibitory effect even at the highest concentration used (3.3 %).
  • immunization with mouse IL- l ⁇ i 1 9 .20 9 coupled to Q ⁇ capsid can yield antibodies which are able to neutralize the interaction of mouse IL- l ⁇ i 1 9 .26 9 and its receptor.
  • As a control on day 28 all mice were injected with 1 ⁇ g mIL-l ⁇ .
  • the clinical score was assigned over 3 consecutive weeks to each limb according to the following definitions: 0 normal, 1 mild erythema and/or swelling of digits/paw, 2 erythema and swelling extending over whole paw/joint, 3 strong swelling, deformation of paw/joint, stiffness.
  • Cumulative clinical scores of individual mice were calculated as the sum of clinical scores of all four limbs, resulting in a possible maximal cumulative score per mouse of 12.
  • EXAMPLE 3 A. Coupling of mouse IL-lccii 7- 2 7 o to Q ⁇ virus-like particles
  • a solution containing 1.8 mg/ml of the purified IL-Ia 117-270 protein from EXAMPLE 1 (SEQ ID NO: 65) in PBS pH 7.2 was incubated for 60 min at room temperature with an equimolar amount of TCEP for reduction of the C-terminal cysteine residue.
  • a solution of 6 ml of 2 mg/ml Q ⁇ capsid protein in PBS pH 7.2 was then reacted for 60 minutes at room temperature with 131 ⁇ l of a SMPH solution (65 mM in DMSO). The reaction solution was dialyzed at 4 0 C against three 3 1 changes of 20 mM HEPES, 150 mM NaCl pH 7.2 over 24 hours.
  • Coupled products were analyzed on a 12 % SDS-polyacrylamide gel under reducing conditions.
  • the Coomassie stained gel is shown in Fig. 2.
  • Several bands of increased molecular weight with respect to the Q ⁇ capsid monomer are visible, clearly demonstrating the successful cross-linking of the mouse IL-Ia 117-270 protein to the Q ⁇ capsid.
  • mice Five female balb/c mice were immunized with Q ⁇ -mIL-l ⁇ i I 7-270 Fifty ⁇ g of total protein were diluted in PBS to 200 ⁇ l and injected subcutaneously (100 ⁇ l on two ventral sides) on day O and day 21. Mice were bled retroorbitally on day 0, 21, and 35, and sera were analyzed using mouse IL-l ⁇ ii7-27o-specific ELISA.
  • ELISA plates were coated with mouse IL-Ia 117-270 protein at a concentration of 1 ⁇ g/ml. The plates were blocked and then incubated with serially diluted mouse sera from day 0, 21, and 35. Bound antibodies were detected with enzymatically labeled anti-mouse IgG antibody. Antibody titers of mouse sera were calculated as the average of those dilutions which led to half maximal optical density at 450 nm. The average anti-mouse IL-Ia 117-270 titer was 1:9252 at day 21 and 1:736912 at day 35. This demonstrates that immunization with Q ⁇ coupled to the mouse IL-Ia 117-270 protein could overcome immunological tolerance and produce high titer antibodies which recognize specifically IL-Ia 117-270
  • mice immunized with QP-InIL-IaIi 7-27 O were then tested for their ability to inhibit the binding of mouse IL-l ⁇ protein to its receptor.
  • ELISA plates were therefore coated with a recombinant mIL-lreceptorl-hFc fusion protein at a concentration of 1 ⁇ g/ml, and co- incubated with serial dilutions of sera from mice which had been immunized either with mouse IL-Ia 117-270 coupled to Q ⁇ capsid or with mousEL-l ⁇ i 1 9 .20 9 coupled to Q ⁇ capsid and 5 ng/ml of mouse IL-Ia 117-270 Binding of IL- IaH 7-270 to the immobilized mIL-lreceptorl-hFc fusion protein was detected with a biotinylated anti-mouse IL-l ⁇ antibody and horse radish peroxidase conjugated strep tavidin.
  • mice immunized against murine IL-IaH 7-270 inhibited completely the binding of mouse IL-IaH 7-270 to its receptor at concentrations of > 0.4%, whereas sera from mice immunized against mouse IL- l ⁇ i 1 9 .20 9 did not show a significant inhibitory effect even at the highest concentration used (3.3 %).
  • As a control on day 28 all mice were injected with 1 ⁇ g mIL-l ⁇ .
  • mice After a booster injection of 200 ⁇ g bovine type II collagen mixed with incomplete Freund's adjuvant at day 63 mice were examined on a daily basis for the development of arthritis symptoms. A clinical score as defined in EXAMPLE 2F was assigned to each limb according to the degree of reddening and swelling observed, and ankle thickness of all hind limbs was measured. Two weeks after the second collagen injection Q ⁇ - immunized mice showed an average cumulative clinical score of 4.44, while Q ⁇ -mIL- Ia 117- 27 o-immunized mice showed an average score of only 2.31.
  • the mice were fed initially with a normal chow diet, which was replaced on day 21 by a western diet (20 % fat, 0.15 % cholesterol, Provimi Kliba AG, Switzerland). Mice were bled at regular intervals throughout the experiment and the antibody response against IL-I alpha was measured in the sera.
  • Sacrifice was on day 159, and the aorta were isolated and prepared essentially as described (Tangirala R.K. et al. (1995) J. Lipid. Res. 36: 2320-2328).
  • hearts were removed and snap-frozen in liquid nitrogen for subsequent histologic preparation essentially as described by Paigen B. et al. (Atherosclerosis 1987;68:231-240) and Zhou X. et al. (Arterioscler Thromb Vase Biol 2001;21: 108-114). The animals were bled by cardiac puncture and perfused with cold PBS.
  • the aorta was then exposed, as much as possible of the adventitia removed in situ, and the aorta finally sectioned 2 mm from the heart.
  • the heart was sectioned in the middle, and the upper part was immediately frozen in Hank's balanced salt solution in a plastic tube in liquid nitrogen.
  • Serial sections (7 ⁇ m thickness) were cut in a cryostat through the origin of the aorta and harvested upon appearance of at least two valve cusps, until disappearance of the last valve cusps. Sections were fixed in formalin, stained with oil red O, and plaque load was evaluated in 4-7 sections (3 sections in one animal of the Q ⁇ group) per mouse by quantitative image analysis. An average plaque area was computed for each animal from the plaque area of each section used for the evaluation. An average group plaque area was computed for the Q ⁇ -mIL-lcci 17.270 and Q ⁇ group respectively. Statistical analysis was performed with a Student t-test. P ⁇ 0.05 was considered statistically significant.
  • the antibody response was measured in a classical ELISA, with recombinant IL- 1 alpha coated on the ELISA plate. Binding of specific antibodies was detected using a goat anti-mouse HRP conjugate. The titers against IL-I alpha were calculated as the reciprocal of the serum dilution giving half-maximal binding in the assay. Specificity of the response was assessed by measuring pre-immune serum. The pre-immune titer was below the lowest serum dilution used in the assay, and was assigned this lowest-serum dilution value.
  • mice Eight weeks old male SJL mice (5 per group) are injected subcutaneously three times at two week intervals with either 50 ⁇ g of Q ⁇ -mIL-l ⁇ i 17-270 or 50 ⁇ g Q ⁇ -mIL-l ⁇ ii 9- 269, or a mixture of 50 ⁇ g each of Q ⁇ -mIL-lcci 17.270 and Q ⁇ -mIL-l ⁇ i 19.269.
  • mice Two weeks after the last immunization, all mice are slightly anesthetized with Isofluran, and 1 mg of trinitrobenzesulfonic acid (TNBS) in 100 ⁇ l 50 % ethanol is administered intrarectally via a polyethylene catheter at a distance of 4 cm of the anus. Body weight is recorded daily as readout of disease progression, and 7 days after TNBS administration all mice are sacrificed. The colon of each mouse is removed, a specimen of colon located 2 cm proximal to the anus is fixed in PBS-buffered formalin, and the degree of inflammation is graded semi-quantitatively on hematoxylin- and eosin-stained colonic cross-sections according to Neurath M.F. et al.
  • TNBS trinitrobenzesulfonic acid
  • Familial Mediterranean Fever is a recessively inherited inflammatory disorder characterized by recurrent fever as well as peritonitis, serositis, arthritis and skin rashes. Affected individuals carry a missense mutation in the MEFV gene, leading to expression of a truncated pyrin protein. Mice carrying a similar mutation in the MEFV gene show an increased caspase-1 activity, leading to overproduction of mature IL- l ⁇ and increased hypothermia and lethality after LPS administration.
  • mice Eight weeks old homozygote pyrin- truncation mice (5 per group) are immunized three times at two weeks intervals with 50 ⁇ g of Q ⁇ -mIL-l ⁇ i 1 9 .26 9 or 50 ⁇ g of Q ⁇ VLPs alone. Two weeks after the last immunizatn all mice are injected intraperitoneally with a mixture 20 mg D-Galactosamine and 0.01 ⁇ g/g LPS. Q ⁇ - mIL-l ⁇ n 9 .26 9 -immunized mice show a markedly reduced hypothermia and a reduced lethality in response to LPS administration, when compared to Q ⁇ -immunized controls.
  • Kineret® (Anakinra, Amgen) is a recombinant version of the human IL-I receptor antagonist, which is approved for the treatment of human rheumatoid arthritis. In order to reach a clinical benefit, relatively high amounts (100 mg) have to be applied via subcutaneous injection on a daily basis. The collagen- induced arthritis model was used to compare the efficacy of Q ⁇ -mIL-lcci 17.270 and Q ⁇ -mIL-l ⁇ i 19.209 immunization with daily applications of different doses of Kineret®.
  • a daily injection of 37.5 ⁇ g Kineret® per mouse corresponds roughly to a dose of 1.5 mg/kg, which is in the range of the recommended efficacious amount for humans (100 mg). All mice were boosted on day 63 by intradermal injection of 200 ⁇ g bovine type II collagen mixed with incomplete Freund's adjuvant, and examined on a daily basis for the development of arthritis symptoms.
  • EXAMPLE 8 Cloning, expression, and purification of virus-like particles consisting of AP205 coat protein genetically fused to mouse IL-lcc ⁇ 7- 2 7 o (AP2O5_mIL-lcc ⁇ 7- 2 7 o) [00214] Given the large size of interleukin-1 alpha and for steric reasons, an expression system producing so called mosaic particles, comprising AP205 coat proteins fused to interleukin-1 alpha as well as wt coat protein subunits was constructed. In this system, suppression of the stop codon yields the AP205-interleukin-l alpha coat protein fusion, while proper termination yields the wt AP205 coat protein.
  • Both proteins are produced simultaneously in the cell and assemble into a mosaic virus-like particle.
  • Two intermediary plasmids, pAP590 and pAP592, encoding the AP205 coat protein gene terminated by the suppressor codons TAG (amber, pAP590) or TGA (opal, pAP592) were made.
  • a linker sequence encoding the tripeptide Gly-Ser-Gly (SEQ ID NO: 189) was added downstream and in frame of the coat protein gene.
  • Kpn2I and HindIII sites were added for cloning sequences encoding foreign amino acid sequences at the C-terminus of the Gly-Ser-Gly amino acid linker, C-terminal to the AP205 coat protein.
  • AP590 SEQ ID NO: 117
  • AP592 SEQ ID NO: 118
  • telomere sequence obtained with oligonucleotides pi.44 (GGCAAATAAGCCAATGCAACCG-3'; SEQ ID NO: 119) and pINC-36 (5'- GTAAGCTTAGATGCATTATCCGGA TCCCTAAGC AGTAGTATCAGACGATACG-3 ' ; SEQ ID NO:120) was digested with Ncol and HindIII, and cloned into vector pQbl85, which had been digested with the same restriction enzymes.
  • pQbl85 is a vector derived from pGEM vector.
  • plasmid pAP592 was constructed by cloning a NcoI/Hindlll-digested PCR fragment obtained with oligonucleotides p 1.44 and pINC-40 (5 '-GTAAGCTTAGATGCATTATCCGGATCCTCAAGCAGTAGTA TCAGACGATACG-S'; SEQ ID NO: 121) into the same vector.
  • the obtained DNA fragment was digested with Kpn2I and HindIII and cloned into both vector pAP590, creating plasmid pAP594 (amber suppression), and into vector pAP592, creating plasmid pAP596 (opal suppression), respectively.
  • E.coli JM 109 cells containing plasmid pISM 579 or pISM 3001 were transformed with plasmid pAP594 or pAP596, respectively.
  • Plasmid pISM579 was generated by excising the trpT176 gene from pISM3001 with restriction endonuclease EcoRI and replacing it by an EcoRI fragment from plasmid pMY579 (gift of Michael Yarus) containing an amber t-RNA suppressor gene.
  • This t-RNA suppressor gene is a mutant of trpT175 (Raftery LA. Et al. (1984) J. Bacteriol.
  • trpT differs from trpT at three positions: G33, A24 and T35.
  • Five milliliters of LB liquid medium containing 20 ⁇ g/ml ampicillin and 10 ⁇ g/ml kanamycin were inoculated with a single colony, and incubated at 37 0 C for 16-24 h without shaking.
  • the prepared inoculum was diluted 5O x with M9 medium containing 20 ⁇ g/ml ampicillin and 10 ⁇ g/ml Kanamycin and incubated at 37 0 C overnight on a shaker. Cells were harvested by centrifugation.
  • Cells (I g, transformed with plasmid pAP594 and containing pISM579) were lysed by ultrasonication in lysis buffer (20 mM Tris-HCl, 5mM EDTA, 150 mM NaCl, pH 7.8, 0.1 % Tween 20). The lysate was cleared by centrifugation, and the cell debris were washed with lysis buffer. Pooled supernatant were loaded on a Sepharose CL-4B column eluted in TEN buffer (20 mM Tris-HCl, 5mM EDTA, 150 mM NaCl, pH 7.8).
  • capsids in the cleared lysate and wash supernatant was confirmed by agarose gel electrophoresis (1 % TAE, ethidium bromide stained gel and UV detection). Two peaks eluted from the column as determined by SDS-PAGE or UV-spectrometric analysis of light scattering at 310 nm. Fractions of the second peak, containing the capsids, were pooled and loaded on a Sepharose CL-6B column. Peak fractions from the CL-6B columne were pooled and concentrated using a centrifugal filter unit (Amicon Ultra 15 MWCO 30000, Millipore).
  • the protein was purified further by one additional round of gel filtration on a CL-4B column, and the resulting peak fractions were pooled and concentrated on a centrifugal filter unit as above.
  • the buffer was exchanged to 10 mM Hepes, pH 7.5, and glycerol was added to a final concentration of 50 %.
  • Purification of AP205_mIL-l ⁇ Ci 17.270 from plasmid pAP596 was performed essentially as described for pAP594 above, with the inclusion of an additional sucrose gradient purification step after the last CL-4B column.
  • the protein was layered on a gradient prepared with the following sucrose solutions: 9 ml 36 %, 3 ml 30 %, 6 ml 25 %, 8 ml 20 %, 6 ml 15 %, 6 ml 10 % and 3 ml 5% sucrose. Fractions were identified by UV spectroscopy, and pooled fractions containing the capsids were concentrated on a centrifugal filter unit as above, and the buffer exchanged to 10 mM Hepes, pH 7.5. Glycerol was finally added to a final concentration of 50 %.
  • mice Four female balb/c mice were immunized with AP205_-mIL-lcci 17.270 Twentyfive ⁇ g of total protein were diluted in PBS to 200 ⁇ l and injected subcutaneously (100 ⁇ l on two ventral sides) on day 0, day 14, and day 28. Mice were bled retroorbitally on days 0, 14, 28 and 35, and sera were analyzed using mouse ELISA.
  • ELISA plates were coated with mouse IL-Ia 117-270 protein at a concentration of 1 ⁇ g/ml. The plates were blocked and then incubated with serially diluted mouse sera from days 14, 28 and 35. Bound antibodies were detected with enzymatically labeled anti-mouse IgG antibody. Antibody titers of mouse sera were calculated as the average of those dilutions which led to half maximal optical density at 450 nm. The average anti-mouse IL-Ia 117-270 titer was 1:4412 at day 14, 1:27955 on day 28 and 1:34824 on day 35. This demonstrates that immunization with AP205_mIL-l ⁇ i 17-270 could overcome immunological tolerance and produce high titer antibodies which recognize specifically IL-Ia 117-270 .
  • mice immunized with AP205_mIL-l ⁇ i 17-270 were tested for their ability to inhibit the binding of mouse IL-l ⁇ protein to its receptor.
  • ELISA plates were therefore coated with a recombinant mlL-lreceptorl-hFc fusion protein at a concentration of 1 ⁇ g/ml, and co- incubated with serial dilutions of sera from mice which had been immunized either with AP205_mIL-l ⁇ i 17-270 or with AP205 alone and 100 ng/ml of mouse IL-Ia 117-270
  • Binding of mIL-lai 17-270 to the immobilized mIL-lreceptorl-hFc fusion protein was detected with a biotinylated anti-mouse IL- l ⁇ antibody and horse radish peroxidase conjugated streptavidin.
  • mice immunized AP205_mIL-l ⁇ i 17.270 inhibited completely the binding of mouse IL-Ia 117-270 to its receptor at concentrations of > 3.3 %, whereas sera from mice immunized with AP205 did not show a significant inhibitory effect at any concentration used.
  • mice immunized with AP205_mIL-l ⁇ i 17.270 showed an average increase of only 0.06 ⁇ 0.05 ng/ml (p ⁇ 0.01).
  • CIA murine collagen-induced arthritis model
  • Q ⁇ - immunized mice showed an average cumulative clinical score of 5.81, while AP205_mIL- l ⁇ n7-270-immunized mice showed an average score of only 2.06.
  • the average increase in hind ankle thickness was 19 % for AP205-immunized mice and only 9 % for mice which had been immunized with AP205_mIL-lcci 17.270 Taken together, these data show that immunization with AP205_mIL-lcci 17.270 strongly protects mice from inflammation and clinical signs of arthritis in the CIA model.
  • virus- like particles consisting of AP205 coat protein genetically fused to mouse IL-l ⁇ n 9- 269 (AP205_mIL-l ⁇ i 19-209)
  • virus- like particles consisting of AP205 coat protein genetically fused to human IL-l ⁇ 116-269 (AP205_hIL-l ⁇ i 16-20 9 )
  • human interleukin 1 beta was amplified from plasmid pET42T-hIL- l ⁇ i 16-26 9 coding for human interleukin 1 beta using primers pINC-74 (5'- GA TCC GGA GGT GGT GCC CCT GTA CGA TCA CTG AAC TG -3', SEQ ID NO: 194) and pINC-76 (5'- GTATGCATTAGGAAGACACAAATTGCATGGTGAAGTC-3. SEQ ID NO: 195), introducing a 5' Kpn2I and 3' MphllO3I site, respectively.
  • the obtained human-IL-l ⁇ fragment was digested with Kpn2I and MphllO3I and cloned in the same restriction sites into vector pAP590 (amber suppression) creating plasmid pAP649.
  • 5 ml of LB liquid medium with 20 ⁇ g/ml ampicillin and 10 ⁇ g/ml canamicin were inoculated with a single colony, and incubated at 37 0 C for 16-24 h without shaking.
  • the prepared inoculum was diluted 5Ox with M9 medium containing 20 ⁇ g/ml ampicillin and 10 ⁇ g/ml kanamycin and incubated at 37 0 C overnight on a shaker. Cells were harvested by centrifugation.
  • mice Four female C3H/HeJ mice were immunized with AP205_mIL- l ⁇ i 19.269 Twentyfive ⁇ g of total protein were diluted in PBS to 200 ⁇ l and injected subcutaneously (100 ⁇ l on two ventral sides) on day 0, day 14, and day 28. Mice were bled retroorbitally on days 0, 14, 28 and 35, and sera were analyzed using mIL-l ⁇ n 9 .26 9 -specific ELISA.
  • ELISA plates were coated with mouse IL- l ⁇ i 1 9 .20 9 protein at a concentration of 1 ⁇ g/ml. The plates were blocked and then incubated with serially diluted mouse sera from days 0, 14, 28, and 35. Bound antibodies were detected with enzymatically labeled anti-mouse IgG antibody. Antibody titers of mouse sera were calculated as the average of those dilutions which lead to half maximal optical density at 450 nm. The average anti-mouse IL- l ⁇ i 1 9 .26 9 titer was 1:19000 on day 14, 1:58200 on day 28 and 1:104700 on day 35. This demonstrates that immunization with AP205_mIL- l ⁇ i 1 9 .26 9 could overcome immunological tolerance and produce high titer antibodies which recognize specifically mouse IL- l ⁇ i 1 9 .26 9
  • mice immunized with AP205_mIL- l ⁇ i 1 9 .26 9 are then tested for their ability to inhibit the binding of mouse IL- l ⁇ protein to its receptor.
  • ELISA plates are therefore coated with a recombinant mIL-lreceptorl-hFc fusion protein at a concentration of 1 ⁇ g/ml, and co- incubated with serial dilutions of sera from mice immunized either with AP205_mIL- l ⁇ i 1 9 .26 9 or with AP205 alone, and 100 ng/ml of mouse IL- l ⁇ i 19.269 Binding of IL- l ⁇ i 19.269 to the immobilized mIL-lreceptorl-hFc fusion protein is detected with a biotinylated anti-mouse IL- l ⁇ antibody and horse radish peroxidase conjugated streptavidin.
  • mice immunized with AP205_mIL- l ⁇ i 1 9 .26 9 strongly inhibit the binding of mouse IL- l ⁇ i 1 9 .26 9 to its receptor, whereas sera from mice immunized with AP205 alone do not show any inhibitory effect.
  • AP205_mIL-l ⁇ i 1 9 .20 9 can yield antibodies which are able to neutralize the interaction of mouse IL- l ⁇ i 1 9 .20 9 and its receptor.
  • mice As readout of the inflammatory activity of the injected mIL-l ⁇ i 1 9 .20 9 , serum samples were withdrawn before and 3 h after injection and analysed for the relative increase in the concentration of the pro-inflammatory cytokine IL-6. AP205-immunized mice showed an increase of 0.28 ng/ml in serum IL-6 concentrations, whereas mice immunized with AP205_mIL-l ⁇ i 1 9 .26 9 showed no increase at all. These data indicate that the antibodies produced by immunization with AP205_mIL-l ⁇ i 1 9 .26 9 were able to neutralize specifically and efficiently the pro-inflammatory activity of IL-l ⁇ .
  • CIA murine collagen-induced arthritis model
  • a clinical score as defined in EXAMPLE 2F was assigned to each limb according to the degree of reddening and swelling observed, and ankle thickness of all hind limbs was measured. Twenty days after the second collagen injection AP205-immunized mice showed an average cumulative clinical score of 2.69, while AP205_mIL-l ⁇ n 9 .26 9 -immunized mice showed an average score of only 1.0. Moreover, the average increase in hind ankle thickness was 8.8% for AP205-immunized mice and only 0.6% for mice which had been immunized with AP205_mIL-l ⁇ i 1 9 .26 9 .
  • mice Four female C3H/HeJ mice were immunized with AP205_hIL-l ⁇ n6-269. Twentyfive ⁇ g of total protein were diluted in PBS to 200 ⁇ l and injected subcutaneously (100 ⁇ l on two ventral sides) on days 0, 14, and 28. Mice were bled retroorbitally on days 0, 14, 28 and 35, and sera were analyzed using human IL-l ⁇ n6-26 9 -specific ELISA.
  • ELISA plates were coated with human IL-l ⁇ n6-26 9 protein at a concentration of 1 ⁇ g/ml. The plates were blocked and then incubated with serially diluted mouse sera from days 0, 14, 28, and 35. Bound antibodies were detected with enzymatically labeled anti-mouse IgG antibody. Antibody titers of mouse sera were calculated as the average of those dilutions which lead to half maximal optical density at 450 nm. The average anti- human IL-l ⁇ n6-26 9 titer was 1:39600 on day 14, 1:58300 on day 28 and 1:65600 on day 35. This demonstrates that AP205_hIL-l ⁇ i 16-269 induces high titers of hIL-l ⁇ n6-269-specific antibodies in mice.
  • the nucleotide sequence encoding amino acids 116-269 of human IL- l ⁇ was amplified by PCR from a cDNA library of human liver tissue using oligonucleotides HIL-I (5'- ATATATGATATCCCTGTACGATCACTGAACTGCACG-S'; SEQ ID NO:124) and HIL-2 (5 '-ATATATCTCGAGGGAAGACA CAAATTGCATGGTGAAG-3'; SEQ ID NO: 125), digested with Xhol and EcoRV and cloned into the expression vector pET42T(+).
  • Plasmid pET-42T(+) was constructed by replacing the whole region between the T7 promoter and the T7 terminator of pET-42a(+) (Novagen) in two steps by new linker sequences, which facilitate the expression of a protein of interest as a fusion with a C-terminal tag (SEQ ID NO: 190) comprising a His-tag and a cysteine containing linker.
  • plasmid pET-42a(+) was digested with the restriction enzymes Ndel and Avrll, liberating a 958 bp fragment between the T7 promoter and T7 terminator composed of a GST-tag, S-tag, two His-tags and the multiple cloning site.
  • the residual 4972 bp fragment containing the vector backbone of pET-42a(+) was isolated and ligated to the annealed complementary oligonucleotides 42-1 (5 -TATGGATATCGAATTCAAGCTTCTGCAGCTGCTCGAGTAA TTGATT AC-3'; SEQ ID NO: 126) and 42-2 (5'-CTAGGTAATC AATTACTCGA GCAGCTGCAGAAGCTTGAATTCGATATCCA-S'; SEQ ID NO: 127), giving rise to plasmid pET-42S(+).
  • plasmid pET-42S(+) was linearized by digestion with restriction enzymes Xhol and Avrll, and ligated to the complementary annealed oligonucleotides 42T- 1 (5 ' -TCGAGCACCACCACCACCACCACCACGGTGGTT
  • Table 3 Overview over IL-I muteins, expression vectors and oligonucleotides used for their construction.
  • mice Biological activity of human IL-l ⁇ n6-269 and human IL-l ⁇ n6-269 muteins in mice [00237] Three female C3H/HeJ mice per group were injected intravenously with 10 ⁇ g of either the wild type human IL- l ⁇ i 19.209 protein or one of the human IL- l ⁇ i 19.209 protein muteins of EXAMPLE 10. Serum samples were withdrawn before and 3 h after injection and analysed for the relative increase in the concentration of the pro-inflammatory cytokine IL-6. As shown in table 4, mice injected with the wild type human IL- l ⁇ i 1 9 .20 9 protein showed an increase of 2.38 ng/ml in serum IL-6 concentrations.
  • PBMCs Peripheral blood mononuclear cells
  • Table 5 shows that with the exception of muteins hIL- l ⁇ i I 6-269 (D54R) and hIL- l ⁇ i I 6-269 (K63S/K65S), much higher amounts of all mutants were necessary to induce the same IL-6 secretion as wild type human IL- l ⁇ i I 9-269 , indicating a reduction in bioactivity.
  • the factor by which biological activity was reduced ranged from 13 fold for mutein hIL-l ⁇ n 6-269 (Rl IG) to 381 fold for mutein hIL-l ⁇ ii 6-269 ( ⁇ SND 52"54 ).
  • Table 5 Biological activity of human IL-l ⁇ n6-269 and human IL-l ⁇ n6-269 muteins in human PBMC.
  • ELISA plates were coated either with the wild type hIL-l ⁇ n6-26 9 protein or the respective hIL-l ⁇ n6-26 9 mutein at a concentration of 1 ⁇ g/ml. The plates were blocked and then incubated with serially diluted mouse sera from day 35. Bound antibodies were detected with enzymatically labeled anti-mouse IgG antibody. Antibody titers of mouse sera were calculated as the average of those dilutions which led to half maximal optical density at 450 nm, and are shown in Table 6.
  • mice immunized with Q ⁇ coupfl to either wild type hIL-l ⁇ n6-26 9 protein or to one of the hIL-l ⁇ n6-26 9 muteins were tested for their ability to inhibit the binding of human IL-l ⁇ protein to its receptor.
  • ELISA plates were therefore coated with a recombinant human IL-lreceptorl-hFc fusion protein at a concentration of 1 ⁇ g/ml, and co-incubated with serial dilutions of the above mentioned sera and 100 ng/ml of hIL-l ⁇ n6-269 protein.
  • Human PBMCs were therefore prepared as described in EXAMPLE HB and incubated with 10 ng/ml wild type hIL-l ⁇ n6-269, which had been premixed with titrating concentrations of the sera described above. After over night incubation the cell culture supernatants were analyzed for the presence of IL-6. The neutralizing capacity of the sera was expressed as those dilutions which lead to half maximal inhibition of IL-6 secretion.
  • Table 7 Neutralizing titer determined in sera of mice immunized with various IL-I beta muteins.
  • mice As readout of the inflammatory activity of the injected hIL-l ⁇ n6 -2 6 9 , serum samples are withdrawn immediately before and 3 h after injection and analysed for the relative increase in the concentration of the pro-inflammatory cytokine IL-6. Whereas naive mice show a strong increase in serum IL-6 concentrations 3h after injection of hIL-l ⁇ n6 -2 6 9 , all mice immunized with Q ⁇ coupled to the wild type hIL-l ⁇ n6 -2 69 protein or to one of the hIL-l ⁇ n6 -2 69 muteins do not show any increase in serum IL-6, indicating that the injected hIL-l ⁇ n6 -2 6 9 is efficiently neutralized by the antibodies induced by the vaccines.
  • Gout is a painful inflammatory disorder caused by the precipitation of monosodium urate (MSU) crystals in joints and periarticular tissues. MSU crystals have been shown to activate the so called NALP3 inflammasome, resulting in the production of active IL-l ⁇ , which is mainly responsible for initiating and promoting the inflammatory response characteristic of the disease.
  • C57BL/6 mice (5 per group) are immunized subcutaneously three times at two weeks intervals with 50 ⁇ g Q ⁇ -mIL-l ⁇ i I 9-269 or 50 ⁇ g of Q ⁇ VLPs alone.
  • mice are challenged intraperitoneally with 1.5 mg MSU crystals.
  • Six hours after the challenge mice are sacrificed and neutrophil numbers as well as the concentrations of the neutrophil chemoattractants KC and MIP-2 are measured in peritoneal exsudates.
  • Q ⁇ -mIL-l ⁇ ii 9 .26 9 -immunized mice show markedly reduced neutrophilia and MIP-2 and KC concentrations, when compared to Q ⁇ -immunized controls.
  • mice In a mouse model for multiple sclerosis, C57BL/6 mice (8 per group) are immunized subcutaneously three times at two weeks intervals with 50 ⁇ g Q ⁇ -mIL-l ⁇ i 19.209 or 50 ⁇ g of Q ⁇ VLPs alone. One week after the last immunization all mice are injected subcutaneously with 100 ⁇ g MOG peptide (MEVGWYRSPFSRVVHLYRNGK, SEQ ID NO: 191) mixed with complete Freund's adjuvant. On the same day and two days later all mice are injected intraperitoneally with 400 ng of pertussis toxin.
  • MOG peptide MEVGWYRSPFSRVVHLYRNGK, SEQ ID NO: 191
  • mice are scored on a daily basis for development of neurological symptoms according to the following scheme: 0, no clinical disease; 0.5, end of tail limp; 1, tail completely limp; 1.5, limp tail and hind limb weakness (unsteady gait and poor grip of hind legs); 2, unilateral partial hind limb paralysis; 2.5, bilateral partial hind limb paralysis; 3, complete bilateral hind limb paralysis; 3.5, complete bilateral hind limb paralysis and unilateral front limb paralysis; 4, total paralysis of hind and front limbs.
  • Q ⁇ -mIL-l ⁇ ii 9 .26 9 -immunized mice show clearly reduced clinical symptoms when compared to Q ⁇ -immunized mice.
  • mouse IL-ICC115-270 A Cloning, expression and purification of mouse IL-ICC115-270 and mouse IL-ICC115-270
  • nucleotide sequence encoding amino acids 115-270 of wild type murine IL- l ⁇ was amplified by PCR from a library of TNF ⁇ -activated murine macrophages using oligonucleotides ILl ⁇ lC (5'-ATATATCATA TGTCTGCCCC TTACACCTAC CAGAGTG-3': SEQ ID NO: 196) and ILl ⁇ 2 (5'-ATATATCTCG AGTGATATCT GGAAGTCTGT CATAGAG-3'; SEQ ID NO:25).
  • the DNA fragment was digested with Nhel and Xhol, and cloned into the expression vector pET42T(+), giving rise to the expression plasmid pET42T-mIL-l an 5.270.
  • the nucleotide sequence encoding amino acids 119-271 of wild type human IL- l ⁇ was amplified by PCR from a LPS-activated human B cell cDNA library using oligonucleotides HIL-3 (5'-ATATATCATA TGCTGAGCAA TGTGAAATAC AACTTTATG-3'; SEQ ID NO:141) and HIL-4 (5'-ATATATCTCG AGCGCCTGGT TTTCCAGTAT CTGAAAG-3'; SEQ ID NO:142).
  • HIL-3 5'-ATATATCATA TGCTGAGCAA TGTGAAATAC AACTTTATG-3'
  • HIL-4 5'-ATATATCTCG AGCGCCTGGT TTTCCAGTAT CTGAAAG-3'; SEQ ID NO:142.
  • the DNA fragment was digested with Nhel and Xhol, and cloned into the expression vector pET42T(+), giving rise to the expression plasmid pET42T-hIL-
  • EXAMPLE 16 A. Biological activity of human IL-l.a119.271, human IL-l ⁇ n9-27i (D145K), mouse IL-
  • PBMC from a healthy donor (5x10 5 cells per well) were incubated with titrating amounts of either the wild type human IL- Ia 1 ⁇ -271 protein, the human IL- Ia 1 ⁇ -271 (D145K) mutein, the wild type mouse IL-Ia 115-270 protein, or the mouse IL-IaH 5-27 O (D145K) mutein. After over night incubation the amount of IL-6 in the cell culture supernatant was measured by Sandwich ELISA as readout of the biological activity of the different proteins.
  • Table 8 shows that 21 fold higher amounts of the mouse IL-Ia 115-270 (D145K) mutein were required to induce the same amount of IL-6 as the corresponding wild type mouse IL-I an 5.270 protein.
  • the human IL- Ia 1 ⁇ -271 (D145K) mutein 46-fold higher amounts than the wild type human IL- Ia 1 ⁇ -271 protein were required.
  • both the human IL- Ia 1 ⁇ -271 (D 145K) mutein and the mouse IL-I an 5.270 (D 145K) mutein have reduced bioactivity in human cells as compared to their wild type counterparts.
  • Table 8 Biological activity of IL-I ⁇ wild type proteins and muteins in human PBMC as determined by IL-6 induction.
  • mice 2 7 o protein, and mouse IL-l ⁇ s-270 (D145K) in mice
  • mice per group Four female Balb/c mice per group were injected intravenously with 10 ng of either the wild type human IL-Ia 11 ⁇ 271 protein, the human IL-Ia 119-271 (D145K) mutein, the wild type mouse IL- Ian 5-270 protein, or the mouse IL- Ian 5-270 (D 145K) mutein.
  • SAA serum amyloid A
  • mice IL-Ia 115-270 (D145K) mutein induced 53 % less SAA than the corresponding wild type mouse IL- l ⁇ i 15-270 protein (p ⁇ 0.05, Student t-test) and the human IL-Ia 119-271 (D145K) mutein induced 67% less SAA than the corresponding wild type human IL- l ⁇ i 1 9 -271 protein (p ⁇ 0.001 Student t-test).
  • Table 9 Biological activity of IL-I ⁇ wild type proteins and muteins in mice determined by SAA.
  • Mouse IL-l ⁇ ng.26 9 muteins and mouse IL-I an 5.270 muteins carrying the mutations of the corresponding human muteins of SEQ ID NO:131 to 140 and SEQ ID NO:205 to 218 are created according to table 10 and coupled to Q ⁇ .
  • the efficacy of mIL-l ⁇ i 1 9 .20 9 and mIL-1 ⁇ i 15-270 muteins coupled to Q ⁇ is tested in the murine collagen-induced arthritis model (CIA).
  • mice Male DBA/1 mice are immunized subcutaneously three times (days 0, 14 and 28) with 50 ⁇ g of either Q ⁇ -mIL-l ⁇ i 1 9 .20 9 mutein, Q ⁇ -mIL- l ⁇ i 15-270 mutein or Q ⁇ alone, and then injected intradermally at day 42 with 200 ⁇ g bovine type II collagen mixed with complete Freund's adjuvant. After a booster injection of 200 ⁇ g bovine type II collagen mixed with incomplete Freund's adjuvant at day 63 mice were examined on a daily basis for the development of arthritis symptoms.
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