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WO2012123269A1 - Compositions immunigènes et procédés pour leur utilisation - Google Patents

Compositions immunigènes et procédés pour leur utilisation Download PDF

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Publication number
WO2012123269A1
WO2012123269A1 PCT/EP2012/053633 EP2012053633W WO2012123269A1 WO 2012123269 A1 WO2012123269 A1 WO 2012123269A1 EP 2012053633 W EP2012053633 W EP 2012053633W WO 2012123269 A1 WO2012123269 A1 WO 2012123269A1
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composition
cell
kit
parts
antigen
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Inventor
Beatriz Amorena Zabalza
Laura ARRIBILLAGA ARANGOA
Damián DE ANDRÉS CARA
María Jesús GRILLO DOLSET
Juan José LASARTE SAGASTIBELZA
Jesús María PRIETO VALTUEÑA
Beatriz SAN ROMÁN ABERASTURI
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Consejo Superior de Investigaciones Cientificas CSIC
Proyecto de Biomedicina CIMA SL
Universidad Publica de Navarra
Digna Biotech SL
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Consejo Superior de Investigaciones Cientificas CSIC
Proyecto de Biomedicina CIMA SL
Universidad Publica de Navarra
Digna Biotech SL
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001154—Enzymes
    • A61K39/001156—Tyrosinase and tyrosinase related proteinases [TRP-1 or TRP-2]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001166—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
    • A61K39/001168—Mesothelin [MSLN]
    • 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
    • A61K39/02—Bacterial antigens
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • A61K39/0275—Salmonella
    • 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
    • A61K39/12—Viral antigens
    • 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
    • A61K39/12—Viral antigens
    • A61K39/29—Hepatitis virus
    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • 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
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/625—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier binding through the biotin-streptavidin system or similar
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the fibronectin Extra Domain A (EDA), a natural ligand for TLR4, as a means for antigen (Ag) delivery to TLR4 expressing cells.
  • EDA is capable of inducing appropriate selection and maturation of antigen presenting cells (APCs) while delivering the antigen of choice to antigen presenting cells finally leading to an effective specific cytotoxic T lymphocytes (CTLs)-mediated response.
  • APCs antigen presenting cells
  • CTLs cytotoxic T lymphocytes
  • CTLs are activated by the presentation to T-cell receptors (TCRs) of short peptides associated with MHC class I molecules.
  • TCRs T-cell receptors
  • These peptide-MHC class I complexes are present on the surface of APCs, which are also capable of providing co-stimulatory signals required for optimal CTL activation.
  • Dendritic cells are the most potent APCs, with a unique capacity to interact with naive T lymphocytes and initiate primary immune responses, activating helper CD4+ and cytotoxic CD8+ T lymphocytes.
  • Antigen presentation and T cell stimulation by DC is reviewed by Guermonprez et al. ("Antigen presentation and T cell stimulation by DC”. Annu Rev Immunol 2002, 20: 621-627), which is here included by reference. These cells orchestrate a repertoire of immune responses from tolerance to self-antigens to resistance to infectious pathogens depending on their maturation status (Reis e Sousa, C, Nat Rev Immunol 2006;6(6):476-83). Thus, it is generally accepted that efficient activation of T-cell immune responses are dependent on DC maturation triggered by a combination of stimuli derived from microbial products or inflammatory signals.
  • a strategy of targeting the antigen to the same DC which is receiving the toll-like receptor (TLR) stimulation may have advantages over the use of mixtures of antigens and TLR ligands not associated within the same particle or molecule.
  • engagement of TLR on DC loaded with the antigen may induce DC activation, expression of cytokines and DC migration to draining lymph nodes for an efficient presentation of the processed antigen to T cells.
  • this TLR engagement may modify the maturation of the phagosome containing the antigen in a way which allows antigen presentation in a highly immunogenic manner (Blander JM, et al, Nature, 2006; 440: 808-1227).
  • WO2006134190 describes that a fusion protein comprising an antigen and the extra domain A of fibronectin (EDA) leads to antigen targeting to TLR4-expressing DC, enhancing cross-presentation and immunogenicity.
  • EDA extra domain A of fibronectin
  • HCV hepatitis C virus
  • the invention relates to a composition or kit-of-parts comprising
  • the invention relates to an avidin or streptavidin oligomer comprising a plurality of avidin or streptavidin monomers wherein each of the avidin or streptavidin monomers is conjugated to a fibronectin EDA domain or a functionally equivalent variant thereof and wherein at least one of the monomers in the oligomer is connected to a biotinylated antigenic entity through the biotin binding site in said at least one monomer.
  • the invention relates to a method for the generation of a composition according to the invention which comprises the steps of
  • fibronectin EDA domain i. the fibronectin EDA domain or a functionally equivalent variant thereof
  • the contacting is carried out under conditions adequate for the formation of complexes between the conjugate and the antigenic entity via the interaction between said first and second members of the binding pair and
  • the invention relates to a method for obtaining an immunogenic antigen presenting cell specific for a given antigenic entity comprising the steps of:
  • the invention relates to a composition comprising an antigen presenting cell according to the invention.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a composition, a kit-of-parts, oligomer or immunogenic composition according to the invention and a pharmaceutically acceptable carrier and to a composition, kit-of-parts, oligomer, immunogenic composition or pharmaceutical composition according to the invention for use in medicine and for use in the treatment of diseases which require the generation of an immune response towards said antigenic entity.
  • the invention relates to a conjugate comprising
  • the invention relates to a polynucleotide encoding a conjugate according to the invention wherein said conjugate is a fusion protein, to a vector comprising said polynucleotide and to a host cell comprising a conjugate, a polynucleotide according or a vector according to the invention.
  • FIG. 1 Recombinant EDAvidin tetramerizes and binds biotinylated proteins with high affinity.
  • A Recombinant EDAvidin was produced, purified, refolded, detoxified and analyzed by SDS-PAGE. Boiled and not boiled protein samples were run in loading buffer and gels were stained with Coomasie Blue.
  • B Biomolecular interaction analysis: Biotinylated BSA (Pierce) was immobilized covalently on a GLM chip (Biorad) and different concentrations of EDAvidin were injected three times over the protein bound surfaces and analyzed by using a surface plasmon resonance optical biosensor.
  • the sensor chip was regenerated with a pulse of biotin and them different concentrations of streptavidin were passed throw the surface. Results represented as RU (relative units) after the subtraction of the signal obtained in the reference channel.
  • C ELISA-based binding assay of EDAvidin to biotinylated proteins: biotinylated OVA or BSA protein-coated wells were incubated with EDAvidin or EDA, washed, incubated with anti-EDA antibodies and developed by using anti-rabbit IgG-HPRO antibodies.
  • EDAvidin SDS-PAGE-based binding assay of EDAvidin to biotinylated OVA protein: EDAvidin, biotinylated OVA (OVA-Biot) or EDAvidin incubated for 15 minutes at room temperature with biotinylated OVA were loaded into a SDS-PAGE and stained with Coomassie Blue.
  • E Binding of EDAvidin to biotinylated proteins in Western- Blot. A molecular weight marker containing biotinylated proteins (Biot) or the High- Range Rainbow Molecular Weight Marker (RB), were loaded into a 10% SDS-PAGE followed by electrophoretic transfer to nitrocellulose membranes. The membranes were incubated with EDAvidin, EDA or streptavidin horseradish peroxidase and subsequently with anti-EDA polyclonal antibodies. The bands were detected with ECL chemoluminescence.
  • FIG. 1 Recombinant EDAvidin improves antigen capture by bone marrow derived dendritic cells (BMDC) and activates monocyte THP-1 cells
  • BMDC bone marrow derived dendritic cells
  • A BMDC obtained from C57/B16 mice were incubated during 15 minutes with or without 5 mg/ml of the indicated products. Cells were washed and analyzed by flow cytometry for visualization of GFP.
  • THP-1 cells were incubated for 15 hours with 0, 1 ⁇ g/ml LPS, 0,25 ⁇ EDAvidin or culture medium (Neg). After culturing, supernatants were harvested and the released T F- ⁇ was measured by ELISA.
  • EDAvidin binds to NS3-biotinylated protein and induces anti NS3 protein immune responses in vivo.
  • A Biotinylated or not biotinylated NS3 proteins from HCV were coated onto an ELISA plate, incubated with EDAvidin or EDA, and developed as described in methods.
  • B, C HHD mice were immunized i.v. with 2 nmols of EDAvidin plus biotinylated NS3 (NS3Biot), EDANS3, biotinylated NS3 protein, EDA plus biotinylated NS3 protein or streptavidin plus biotinylated NS3 protein.
  • mice spleens were obtained and the number of IFN- ⁇ producing spots in response to CTL epitope pi 073 from NS3 or NS3 protein (B) or the CTL activity against target cells loaded with pi 073 (C) were measured by ELISPOT and by in vivo killing assay, respectively.
  • EDAvidin binds to TRP2(180- 188)-biotinylated peptides and induces anti-TRP2(180-188) immune responses in vivo.
  • C57BL/6 mice were immunized sc. with 2 nmols of EDAvidin plus melanoma TRP- 2(180-188) peptide biotinylated at the amino or carboxy terminus.
  • mice spleens were obtained and the number of IFN- ⁇ producing spots in response to CTL epitope TRP-2Q80-188) were measured by ELISPOT.
  • HS Hot-Saline antigenic extracts
  • B-SEwt formalin inactivated bacterins obtained from S. Enteritidis rough mutants
  • SEAw L and SEAGal mice were intraperitoneally immunized with the corresponding antigenic preparation, i . e. Hot Saline (HS)
  • mice Four weeks after vaccination, all mice were intraperitoneally (i.p.) infected with 2.3xl0 2 CFU/animal of SE-wt virulent strain and protection determined by logio CFU/ spleen of the challenging strain, at day 4 after challenge.
  • Statistical comparisons were performed by ANOVA and Bonferroni's test. ⁇ P ⁇ 0.01 with respect to the homologous HS extract. * P ⁇ 0.0005 with respect to B-SEAGal administered alone.
  • FIG. 5 Detection of biotinylated bacterins by EDAvidin in ELISA. Biotinylated and not biotinylated bacterins were used to coat ELISA plates and, then, incubated with EDAvidin or EDA (as a negative control). The binding was monitored using a rabbit polyclonal anti-EDA antibody followed by an anti-rabbit whole IgG horseradish- peroxidase-conjugated antibody (Sigma). The optical density ( ⁇ . ⁇ .405) is represented. Figure 6.
  • Enteritidis wild type (wt) parental strain (B-SEwt) or PBS were used as controls.
  • All mice were challenged i.p. with 2.3 > ⁇ 10 2 CFU/animal and spleen bacterial counts spleen determined 4 days later. Protection is expressed as counts (log 10 CFU/spleen) of the SE- wt challenging strain.
  • the invention relates to a conjugate comprising
  • conjugate refers to two or more compounds which are covalently linked together so that the function of each compound is retained in the conjugate.
  • conjugate refers to two or more compounds which are covalently linked together so that the function of each compound is retained in the conjugate.
  • extracellular domain A fibronectin extra domain A
  • fibronectin EDA fibronectin EDA
  • EDA extracellular domain A
  • fibronectin is understood as a multifunctional high molecular weight glycoprotein present in blood and in the extracellular matrix of tissues. Fibronectin is a dimer formed by two identical polypeptide chains bound by C-terminal disulfide bonds. Each monomer has an approximate molecular weight of 230-250 kDa. Each monomer contains three types of modules: type I, type II and type III. Each of these modules is formed by two anti-parallel ⁇ -helices.
  • EDA is understood as all those polypeptides derived from the EDA sequence by means of modification, insertion and/or deletion of one or more amino acids, provided that the function of binding to TLR4 receptors, of delivering antigens to dendritic cells and/or of activating dendritic cells is substantially preserved.
  • Functionally equivalent variants are those showing a degree of identity with respect to the fibronectin EDA domain higher than at least 25%, at least 40%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.
  • % sequence identity is determined by comparing two optimally aligned sequences over a comparison window, where the fragment of the polypeptide or polynucleotide sequence in the comparison window may comprise additions or deletions (e.g., gaps or overhangs) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical amino acid residue or nucleic acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window and multiplying the result by 100 to provide the percentage of sequence identity. Algorithms to align sequences are known in the art.
  • Exemplary algorithms include, but are not limited to, the local homology algorithm of Smith and Waterman (Add APL Math, 2:482, 1981); the homology alignment algorithm of Needleman and Wunsch J Mol Biol 1970;48:443); the search for similarity method of Pearson and Lipman (Proc Natl Acad Sci USA 1988:85:2444); and computerized implementations of these algorithms (GAP, BESTFIT, BLAST, PASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.).
  • two sequences may be aligned using the "Blast 2 Sequences" tool at the NCBI website at default settings (Tatusova and Madden. FEMS Microbiol Lett 1999;174:247-250).
  • amino acid sequences or nucleic acids sequences may be aligned by human inspection.
  • the capacity of the functionally equivalent variant to interact with TLR4 can be determined by means of using conventional methods known by the person skilled in the art.
  • the capacity of the fibronectin EDA region variant to bind to TLR4 can be determined using co-immunoprecipitation experiments, in which the protein of interest (e.g. EDA variant) is isolated with a specific antibody and the molecules which interact with the protein (e.g. TLR4) are subsequently identified by means of a western blot.
  • Assays for determining the capacity of the functionally equivalent variants of EDA to promote the maturation of dendritic cells are known by a person skilled in the art, such as for example incubation of BMDC with the recombinant proteins in vitro during 24-48 h and then testing by flow cytometry the upregulation of maturation markers, such as surface molecules CD54 and CD86, or by measuring the production of proinflammatory cytokines (such as IL-12 or TNF-alpha) by commercial ELISA.
  • the assay described in Example 2 of the present application based on determining the production of TNF-a by the THP1 monocyte cell line in response to the treatment the fibronectin EDA variant could also be used.
  • Assays for determining the capacity of the functionally equivalent variants of EDA to deliver antigens to dendritic cells are known by a person skilled in the art, such as for example the assay described in Example 2 of the present application based on determining the amount of marker protein (e.g. GFP) incorporated into dendritic cells contacted with a fusion protein comprising the EDA variant and the marker protein.
  • marker protein e.g. GFP
  • the fibronectin EDA of the conjugate of the invention corresponds to amino acids 1,631 to 1,721 of human fibronectin as shown in the UniProt database with accession number FINC HUMAN and which corresponds to the polypeptide of sequence SEQ ID NO: l .
  • first member of a binding pair refers to a molecule which has affinity for and “binds” to another (hereinafter known as “second member of the binding pair") under certain conditions, referred to as “binding conditions".
  • second member of the binding pair can be of a peptide (protein) or non-peptide nature.
  • binding refers to the interaction between affinity binding molecules or specific binding pairs (e.g., between biotin as an affinity tag molecule and streptavidin as an affinity-tag-binding molecule) as a result of non-covalent bonds, such as, but not limited to, hydrogen bonds, hydrophobic interactions, van der Waals bonds, and ionic bonds. Based on the definition of "binding,” and the wide variety of affinity binding molecules or specific binding pairs, it is clear that "binding conditions" vary for different specific binding pairs.
  • binding pair does not involve any particular size or any other technical structural characteristic other than that said binding pair can interact and bind to the other member of the binding pair resulting in a conjugate wherein the first and second components are bound to each other by means of the specific interaction between the first and second member of a binding pair.
  • the binding pair includes any type of immune interaction such as antigen/antibody, antigen/antibody fragment, hapten/anti-hapten as well as non-immune interactions such as avidin/biotin, avidin/biotinylated molecules, folic acid/folate- binding protein, hormone/hormone receptor, lectin/carbohydrate, lectin/molecule modified with carbohydrates, enzyme/enzyme substrate, enzyme/enzyme inhibitor, protein A/antibody, protein G/antibody, complementary nucleic acids (including s e q u e n c e s o f D N A , RN A a n d p p t i d e n u c l e i c a c i d s ( P N A ) ) , polynucleotide/polynucleotide-binding protein and the like.
  • antigen/antibody antigen/antibody fragment
  • the expression "specific binding” refers to the capacity of a first molecule to bind specifically to a second molecule by means of the existence of complementarity between the three-dimensional structures of the two molecules with a substantially higher affinity for non-specific binding such that the binding between said first and second molecule preferably takes place before the binding of any of said molecules with respect to the other molecules present in the reaction mixture.
  • KD dissociation constant
  • bond and binding are used indistinctly to refer to an interaction between two or more entities. In those cases in which two entities are bound to one another, they can be directly bound (for example, by means of covalent bonds, ionic forces, hydrogen bonds, electrostatic interactions, Van der Waals forces or a combination of the above) or they can be indirectly bound, for example, by means of a linker.
  • the first member of a binding pair is a biotin-binding molecule. More preferably, the biotin-binding molecule is avidin.
  • the term "avidin” refers to a glycoprotein found in egg white and in tissues of birds, reptiles and amphibian and which has the capacity to bind to biotin with high affinity as well as any expressed or engineered form of the avidin biotin-binding molecule, such as streptavidin, neutravidin and the like.
  • avidin includes both avidin found naturally in the eggs of Gallus gallus (NCBI accession numbers NM_205320.1 / GL45384353en) as well as the orthologues of said protein in other species.
  • streptavidin corresponds to the protein from Streptomyces avidinii (accession number CAA00084.1 in GenBank), as well as the orthologues, homologues and fragments of streptavidin defined in the same manner as avidin.
  • Streptavidin comprises 4 subunits each of which contains a binding site for biotin.
  • Streptavidin or avidin fragments which retain substantial binding activity for biotin, such as at least 50 percent or more of the binding affinity of native streptavidin or avidin, respectively, may also be used.
  • the affinity of the avidin variant for biotin is of at least 10 15 M "1 , 10 14 M "1 , 10 13 M "1 , 10 12 M “1 , 10 10 M “1 or 10 9 M "1 .
  • avidin and “streptavidin” as used herein are intended to encompass biotin-binding fragments, mutants and core forms of these binding pair members.
  • Avidin and streptavidin are available from commercial suppliers.
  • nucleic acid sequences encoding streptavidin and avidin and the streptavidin and avidin amino acid sequences can be found, for example, in GenBank Accession Nos. X65082; X03591 ; M --205320; X05343; Z21611 ; and Z21554.
  • Avidin and streptavidin variants suitable for use in the present invention include, without limitation
  • Core streptavidin which is a truncated version of the full-length streptavidin polypeptide which may include streptavidin residues 13- 138, 14-138, 13-139 and 14-139. See, e.g., Pahler et al, (J Biol Chem 1987:262: 13933-37).
  • Mutants with reduced immunogenicity such as mutants modified by site-directed mutagenesis to remove potential T cell epitopes or lymphocyte epitopes. See Meyer et al, Protein Sci 2001;10:491-503. Mutants of avidin and core forms of avidin which retain substantial biotin binding activity or increased biotin binding activity also may be used. See Hiller et al., J Biochem 1991;278:573-85; Livnah et al Proc Natl Acad Sci USA 1993;90:5076-80.
  • Variants resulting from the chemical modification of avidin such as those resulting from the complete or partial modification of glycosylation and fragments thereof as well as the completely deglycosylated avidin variant known as neutravidin.
  • protei n with bi otin binding capacity as describ ed in WO04018509,
  • the first member of the binding pair comprises the sequence
  • biotin has an extremely high affinity for both streptavidin (10 13 M "1 ) and avidin (10 15 M "1 ).
  • streptavidin and avidin are tetrameric polypeptides that each binds four molecules of biotin. Conjugates comprising streptavidin or avidin therefore have a tendency to form tetramers and higher structures. As a result, they can cross-link their corresponding immune cell receptors for more potent signal transduction, such as through aggregation of receptors.
  • the fibronectin EDA or a functionally equivalent variant thereof and the first member of a binding pair may be directly connected, i.e. by means of a specific direct interaction between both components.
  • the fibronectin EDA or a functionally equivalent variant thereof and the first member of a binding pair may be indirectly connected i.e. by means of using a "connector".
  • the first and second components of the conjugate form a single polypeptide chain (hereinafter the fusion protein of the invention).
  • the different elements of the fusion protein of the invention can be placed in any order provided that the fibronectin EDA maintains its dendritic cell activating properties and that the first member of the binding pair maintains its capacity of binding to the second member of the binding pair.
  • the fibronectin EDA region is connected to the C- terminal end of the first member of the binding pair.
  • the invention also contemplates fusion proteins comprising more than one fibronectin EDA as well as more than one first member of a binding pair.
  • fusion proteins may also contain a variety of arrangements, which are shown in the N- to C-terminal regions, such as:
  • the conjugate of the invention further comprises a "tag".
  • tag as used herein, relates to any amino acid sequence for which specific binding molecules are available, thus allowing the detection/purification of any polypeptide carrying said tag.
  • the tag is generally placed at the amino- or the carboxyl- terminus of the polypeptide. The presence of such tag allows the adapter molecule to be detected using an antibody against the tag polypeptide. Also, the provision of the tag enables the adapter polypeptide to be readily purified by affinity purification using an anti-tag antibody or another type of affinity reagent that binds to the epitope tag.
  • Various tag polypeptides and their respective antibodies are well known in the art.
  • poly-histidine poly-his
  • poly-histidine-glycine poly-his-glycine tags
  • flu HA tag polypeptide and its antibody 12CA5 Fluorescence Activated Cell Biol, 1988;8:2159-2165
  • c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto
  • gD Herpes Simplex virus glycoprotein D
  • tag polypeptides include the Flag-peptide (Hopp et al, BioTechnology 1988;6: 1204-1210); the KT3 epitope peptide [Martin et al., Science 1993;255: 192-194); tubulin epitope peptide (Skinner et al., J Biol Chem 1991;266: 15163-15166); and the T7 gene 10 protein peptide tag (Lutz-Freyermuth et al, Proc Natl Acad Sci USA 1990,;87:6393-6397).
  • the purification tag is a polyhistidine tag.
  • the purification tag is an hexahistidine tag.
  • the fusion protein of the present invention has the sequence:
  • the invention relates to a polynucleotide encoding a conjugate of the invention.
  • a polynucleotide encoding a conjugate of the invention.
  • the polynucleotides of the invention will only encode the conjugates in which component (ii) has a peptide nature and which forms a single peptide chain with component (i), regardless of both the relative orientation and the fact that both components are directly connected or separated by a spacer region.
  • the polynucleotide according to the invention has the sequence
  • the invention in another aspect, relates to a gene construct comprising a polynucleotide of the invention.
  • the construct preferably comprises the polynucleotide of the invention located under the operative control of sequences regulating the expression of the polynucleotide of the invention.
  • promoters suitable for the embodiment of the present invention include, without being necessarily limited to, constitutive promoters such as the derivatives of the genomes of eukaryotic viruses such as the polyoma virus, adenovirus, SV40, CMV, avian sarcoma virus, hepatitis B virus, the promoter of the metallothionein gene, the promoter of the herpes simplex virus thymidine kinase gene, retrovirus LTR regions, the promoter of the immunoglobulin gene, the promoter of the actin gene, the promoter of the EF-1 alpha gene as well as inducible promoters in which the expression of the protein depends on the addition of a molecule or an exogenous signal, such as the tetracycline system, the NFKB/UV light system, the Cre/Lo
  • the promoter used for expressing the polynucleotides of the invention are promoters functional in dendritic cells such as the fascin gene promoter as described by Bros et al. (J Immunol 2003;171 : 1825-1834), the DC-CK1, DC-STAMP and DC-SIGN gene promoters, the Dectin-2 promoter described in Morita et al, ⁇ Gene Ther 2001;8: 1729- 37), the CDl lc gene promoter as described in Masood, R., et al. (Int J Mol Med 2001;8:335-343) and Somia, N. V., et al. (Proc Acad Sci USA 1995;92:7570-7574).
  • the fascin gene promoter as described by Bros et al. (J Immunol 2003;171 : 1825-1834)
  • the DC-CK1, DC-STAMP and DC-SIGN gene promoters the Dectin-2 promoter described in Morita e
  • promoters which are tissue-specific include the promoter of the albumin gene (Miyatake et al, J Virol 1997;71 :5124-32), the core promoter of hepatitis virus (Sandig et al, Gene Ther 1996;3 : 1002-9), the promoter of the alpha- fetoprotein gene (Arbuthnot et al., Hum.GeneTher 1996;7: 1503-14), and the promoter of the globulin-binding protein which binds to thyroxine (Wang, L., et al, Proc Natl Acad Sci USA 1997;94: 11563-11566).
  • the polynucleotides of the invention or the gene constructs forming them can form part of a vector.
  • the invention relates to a vector comprising a polynucleotide or a gene construct of the invention.
  • a person skilled in the art will understand that there is no limitation as regards the type of vector which can be used because said vector can be a cloning vector suitable for propagation and for obtaining the polynucleotides or suitable gene constructs or expression vectors in different heterologous organisms suitable for purifying the conjugates.
  • suitable vectors include expression vectors in prokaryotes such as pUC18, pUC19, Bluescript and their derivatives, mpl8, mpl9, pBR322, pMB9, CoIEl, pCRl, RP4, phages and shuttle vectors such as pSA3 and pAT28, expression vectors in yeasts such as vectors of the type of 2 micron plasmids, integration plasmids, YEP vectors, centromeric plasmids and the like, expression vectors in insect cells such as the pAC series and pVL series vectors, expression vectors in plants such as vectors of expression in plants such as pIBI, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pORE series vectors and the like and expression vectors in superior eukaryotic cells based on viral vectors (adenoviruses, viruses
  • the vector of the invention can be used to transform, transfect or infect cells which can be transformed, transfected or infected by said vector.
  • Said cells can be prokaryotic or eukaryotic.
  • the vector wherein said DNA sequence is introduced can be a plasmid or a vector which, when it is introduced in a host cell, is integrated in the genome of said cell and replicates together with the chromosome (or chromosomes) in which it has been integrated.
  • Said vector can be obtained by conventional methods known by the persons skilled in the art (Sambrook et al, Molecular Cloning: A Laboratory Manual, 2001), mentioned above).
  • the invention relates to a cell comprising a conjugate, a polynucleotide, a gene construct or a vector of the invention, for which said cell has been able to be transformed, transfected or infected with a construct or vector provided by this invention.
  • the transformed, transfected or infected cells can be obtained by conventional methods known by persons skilled in the art (Sambrook et al, 2001, mentioned above).
  • said host cell is an animal cell transfected or infected with a suitable vector.
  • Host cells suitable for the expression of the conjugates of the invention include, without being limited to, mammal, plant, insect, fungal and bacterial cells.
  • Bacterial cells include, without being limited to, Gram-positive bacterial cells such as species of the Bacillus, Streptomyces, Listeria and Staphylococcus genus and Gram-negative bacterial cells such as cells of the Escherichia, Salmonella and Pseudomonas genus.
  • Fungal cells preferably include cells of yeasts such as Saccharomyces, Pichia pastoris and Hansenula polymorpha.
  • Insect cells include, without being limited to, Drosophila and Sf9 cells.
  • Plant cells include, among others, cells of crop plants such as cereals, medicinal, ornamental or bulbous plants.
  • Suitable mammal cells in the present invention include epithelial cell lines (human, ovine, porcine, etc.), osteosarcoma cell lines (human, etc.), neuroblastoma cell lines (human, etc.), epithelial carcinomas (human, etc.), glial cells (murine, etc.), hepatic cell lines (from monkey, etc.), CHO (Chinese Hamster Ovary) cells, COS cells, BHK cells, HeLa cells, 911, AT1080, A549, 293 or PER.C6, NTERA-2 human ECC cells, D3 cells of the mESC line, human embryonic stem cells such as HS293, BGV01, SHEF1, SHEF2, HS181, NIH3T3 cells, 293 T, REH and MCF-7 and hMSC cells.
  • epithelial cell lines human, ovine, porcine, etc.
  • the conjugate of the invention can be obtained using any method known for a person skilled in the art. It is thus possible to obtain the EDA peptide or the variant of said protein by any standard method.
  • the EDA peptide can be obtained from cDNA by means of expression in a heterologous organism such as, for example, Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris or from tobacco chloroplasts as described by Farran et al. (Planta 2010;231 :977-90).
  • the latter must be conjugated to the first member of the binding pair.
  • the conjugation of said first member of the binding pair to the EDA molecule can be carried out in different ways.
  • One possibility is the direct conjugation of a functional group to the therapeutically active component in a position which does not interfere with the activity of said component.
  • functional groups refer to a group of specific atoms in a molecule which are responsible for a characteristic chemical reaction of said molecule.
  • Examples of functional groups include, without limitation, hydroxy, aldehyde, alkyl, alkenyl, alkynyl, amide, carboxamide, primary, secondary, tertiary and quaternary amines, aminoxy, azide, azo (diimide), benzyl, carbonate, ester, ether, glyoxylyl, haloalkyl, haloformyl, imine, imide, ketone, maleimide, isocyanide, isocyanate, carbonyl, nitrate, nitrite, nitro, nitroso, peroxide, phenyl, phosphine, phosphate, phosphono, pyridyl, sulfide, sulfonyl, sulfinyl, thioester, thiol and oxidized 3,4-dihydroxyphenylalanine (DOPA) groups.
  • DOPA 3,4-dihydroxyphenylalanine
  • Examples of said groups are maleimide or glyoxylyl groups, which react specifically with thiol groups in the Apo A molecule and oxidized 3,4-dihydroxyphenylalanine (DOPA) groups which react with primary amino groups in the EDA molecule and of component (ii).
  • DOPA 3,4-dihydroxyphenylalanine
  • conjugates can first be conjugated to the therapeutically active compound and, then, conjugated to the EDA peptide or, alternatively, it is possible to conjugate the bifunctional group to the EDA peptide and, then, conjugate the latter to component (ii).
  • conjugates include the conjugates known as ketone- oxime (described in US20050255042) in which the first component of the conjugate comprises an aminoxy group which is bound to a ketone group present in a heterobifunctional group which, in turn, is bound to an amino group in the second component of the conjugate.
  • the agent used to conjugate components (i) and (ii) of the conjugates of the invention can be photolytically, chemically, thermically or enzymatically processed.
  • the use of linking agents which can be hydrolyzed by enzymes that are in the target cell, such that the therapeutically active compound is only released into the cell is of interest. Examples of linking agent types that can be intracellularly processed have been described in WO04054622, WO06107617, WO07046893 and WO07112193.
  • the first member of a binding pair is a compound of a peptide nature (e.g., streptavidin), including both oligopeptides, peptides and proteins
  • a polypeptide chain using widely known methods to the person skilled in the art so that the protein can be covalently coupled to a second polypeptide.
  • suitable methods for the covalent coupling of two polypeptides include methods based on the conjugation through the thiol groups present in the cysteine moieties, methods based on the conjugation through the primary amino groups present in the lysine moieties (US6809186), methods based on the conjugation through the N- and C-terminal moieties can be used.
  • Reagents suitable for the modification of polypeptides to allow their coupling to other compounds include: glutaraldehyde (allows binding compounds to the N-terminal end of polypeptides), carbodiimide (allows binding the compound to the C-terminal end of a polypeptide), succinimide esters (for example MBS, SMCC) which allow activating the N-terminal end and cysteine moieties, benzidine (BDB), which allows activating tyrosine moieties, and periodate, which allows activating carbohydrate moieties in those proteins which are glycosylated.
  • glutaraldehyde allows binding compounds to the N-terminal end of polypeptides
  • carbodiimide allows binding the compound to the C-terminal end of a polypeptide
  • succinimide esters for example MBS, SMCC
  • BDB benzidine
  • BDB benzidine
  • the conjugate in a single step using a gene construct of the invention encoding said conjugate, for which said construct is introduced in a vector suitable for its expression in a heterologous organism together with transcription and, optionally, translation control elements.
  • the transcription and, optionally, translation control elements present in the expression cassette of the invention include promoters, which direct the transcription of the nucleotide sequence to which they are operatively linked and other sequences which are necessary or suitable for the transcription and its suitable regulation in time and place, for example, initiation and termination signals, cleavage sites, polyadenylation signal, replication origin, transcriptional enhancers, transcriptional silencers, etc.
  • Said elements, as well as the vectors used for constructing the expression cassettes and the recombinant vectors according to the invention are generally chosen according to the host cells to be used.
  • the conjugates of the invention can be contacted with an antigenic entity which is modified with a second member of a binding pair. This will lead to the formation of a complex between the conjugates and the antigenic entity via the specific interaction between said first and second member of the binding pair. This complex may then be used as immunogenic composition in order to obtain an immune response against said antigenic entity.
  • the invention relates to a composition or kit-of- parts comprising
  • composition refers to a material composition that comprises the above-mentioned components, that is, a conjugate comprising the fibronectin EDA or a functionally equivalent variant thereof and a first member of a binding pair; and an antigenic entity which is coupled to a second member of said binding pair as well as any product resulting, directly or indirectly, from the combination of the different components in any quantity thereof.
  • the composition may be formulated as a single formulation or may be presented as separate formulations of each of the components, which may be combined for joint use as a combined preparation.
  • the composition may be a kit-of-parts wherein each of the components is individually formulated and packaged.
  • compositions according to the invention are described in detail below. 4.
  • A. Conjugate comprising the fibronectin EDA or a functionally equivalent variant thereof and a first member of a binding pair
  • fibronectin EDA or a functionally equivalent variant thereof and a first member of a binding pair has been described in detail above.
  • fibronectin EDA or the functionally equivalent variant thereof and the first member of a binding pair form a single polypeptide chain.
  • the first member of the binding pair is a biotin-binding molecule. Even more preferably, the biotin-binding molecule is avidin or streptavidin. 4.B. Antigenic entity modified by a second member of the binding pair
  • the second component of the composition or kit-of-parts according to the invention is an antigenic entity modified by a second member of a binding pair.
  • modified means that the antigenic entity contains one or more covalently or high-affinity coupled second members of the binding pair. It will be understood that, when the antigenic entity is formed by a plurality of different molecules, a substantial percentage of the molecules will be modified by a second member of the binding pair.
  • second member of a binding pair refers to a molecule which is capable of binding with high affinity to the first member of the binding pair.
  • the first and second members of the binding pair are, respectively, a biotin binding molecule and biotin. Even more preferably, the biotin-binding molecule is avidin or streptavidin.
  • an “antigenic entity” is herein defined to encompass any cell, microorganism, soluble molecule, mixture of molecules or cell-surface bound molecule (including a protein, lipid or carbohydrate), that are at least capable of binding to an antibody or of generating a T-cell response and may also contribute to the development of an immune response.
  • the present invention is not particularly limiting with regard to the type of antigenic entity that can be modified with the second member of the binding pair.
  • the antigenic entity can be a whole cell which has been modified on its surface by the second member of the binding pair and that, upon being contacted with the conjugate containing the first member of the binding pair, becomes "decorated" with said conjugates.
  • the whole cell may b e a cell of a microorganism or a tumour cell.
  • the antigenic entity may be a heterogeneous composition comprising different antigenic molecules such as a cell lysate or extract.
  • the antigenic entity may be an isolated molecule.
  • the antigenic entity is a microorganism.
  • microorganism or “microbe” refer to an organism of microscopic size, to a single-celled organism, and/or to any virus particle.
  • the microorganism is a pathogenic microorganism.
  • Pathogenic microorganism refers to any disease-causing microorganism as defined above.
  • the pathogen may be an "attenuated pathogen", which refers to a live microorganism that is less virulent in its natural host but which preferably, when introduced said host, causes a protective immunological response such that resistance to infection will be enhanced and/or the clinical severity of the disease reduced.
  • Suitable pathogens for use in the present invention include, without limitation, bacteria, viruses, protozoa, fungi and the like.
  • the microorganism is an inactivated microorganism.
  • inactivated form of a microorganism refers to a dead or inactivated cell of such a microorganism which is no longer capable to form a single colony on a plate specific for said microorganism.
  • the term "inactivated form of the microorganism”, as described herein, also encompasses lysates, fractions or extracts of the microorganism. 4.B.III.1. Bacteria
  • the microorganism is a bacterial cell, which can be, without limitation, a whole-inactivated bacterial cell (known as bacterin), a subcellular bacterial fraction of a mixture of subcellular bacterial fractions or a live attenuated bacterial cell.
  • bacterin a whole-inactivated bacterial cell
  • subcellular bacterial fraction of a mixture of subcellular bacterial fractions or a live attenuated bacterial cell.
  • a bacterin useful in vaccines may be obtained by culturing the bacterium of interest, and then killing the bacterium to produce a bacterin containing a variety of bacterial components, including cell wall components.
  • the bacteria may be killed by a variety of methods including those to expose them to a compound such as merthiolate, formalin, formaldehyde, diethylamine, binary ethylenamine (BEI), beta propiolactone (BPL), and glutaraldehyde. Combinations of these compounds may be used.
  • it is possible to kill the bacteria by sterilizing radiation, heat, ultrasounds (e.g. sonication), cell rupture (e.g. French press) or other procedures. Combinations of these crude, purified or structurally modified compounds as well as synthesized fractions may be used, individually or combined.
  • Suitable bacteria that can be used as antigenic entities either as whole- inactivated bacteria or as attenuated live bacteria include, without limitation, Neisseria spp., including N gonorroheae and N meningitidis; Streptococcus spp., including S. pyogenes; Bordetella spp., including B. pertussis; Mycobacterium spp., including M. tuberculosis, M. bovis, M. leprae, M. avium, M. paratuberculosis, M. smegmatis; Legionella spp., including L. pneumophila,Escherichia spp, including enterotoxic E.
  • E. coli enterohemorragic E. coli and enteropathogenic E. coli Vibrio spp, including V. cholera , Shigella spp., including S. sonnei, S. dysenteriae, S. flexnerii; Yersinia spp., including Y. enterocolitica, Y. pestis, Y. pseudotuberculosis; Campylobacter spp., including C. jejuni, Salmonella spp., including S. bongori, and S. enterica subspp.
  • enterica (serogroups A, B, C, D and E), salamae, arizonae, diarizonae, houtenae, and indica; Listeria spp., including L. monocytogenes; Helicobacter spp., including H.pylori; Pseudomonas spp, including P. aeruginosa; Staphylococcus spp., including S. aureus and S. epidermidis; Enterococcus spp., including E. faecalis and E. faecium; Clostridium spp., including C. tetani, C. botulinum and C.
  • Bacillus spp. including B. anthracis; Corynebacterium spp., including C. diphtheriae; Borrelia spp., including B. burgdorferi , B. garinii, B. afzelii, B. andersonfi and B. hermsii; Ehrlichia spp., including E. equi , Rickettsia spp, including R. rickettsii; Chlamydia spp., including C. trachomatis, Chlamydia C. pneumonia and C. psittaci; Leptospira spp., including L.
  • Treponema spp. including T. pallidum, T. denticola and T. hyodysenteriae
  • Streptococcus spp. including S. pneumonia, and Haemophilus spp.
  • the bacterin has been obtained by formalin treatment of the bacteria.
  • the bacterin is a Salmonella bacterin.
  • said attenuated pathogen can be obtained by numerous methods including but not limited to chemical mutagenesis, genetic insertion, deletion (Miller, J., 1972, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) or recombination using recombinant DNA methodology (Maniatis, T., et al, 1982, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), laboratory selection of natural mutations, etc.
  • the antigenic entity is an attenuated Salmonella strain.
  • Methods for obtaining attenuated Salmonella strains which are non-reverting non- virulent auxotrophic mutants suitable for use as live vaccines are described in U.S. Pat. No. 4,735,801 issued on Apr. 5, 1988 and copending U.S. patent application Ser. No. 798,052, filed Nov. 14, 1985, by Stocker, which are incorporated by reference herein in their entirety.
  • a reliable method to achieve attenuation of Salmonella has been described (Hoiseth, S. K., and Stocker, B. A. D., Nature 1981 ;291 :238; Stocker B. A. D., et al, Develop Biol Standard 1982;53 :47; and U. S. Pat. No. 4,550,081) and can be used in a particular embodiment of the invention.
  • Attenuated Salmonella which can be used in the live vaccine formulations of the invention include but are not limited to S. typhi, S. enterica sbsp. enterica serotypes typhimurium (S. Typhimurium), enteritidis (S. Enteritidis), paratyphi (S. Paratyphi).
  • Salmonella may be attenuated by modification of the genome structure of the bacteria, such as by deletion of part or parts of a Salmonella gene, by insertion of heterologous nucleotide sequence into at least one Salmonella gene, and/or by substitution of part or parts of a Salmonella gene by heterologous nucleotide sequence. It is possible to attenuate Salmonella by introducing mutations that (i) confer auxotrophy, (ii) interfere with sugar metabolism and lipopolysaccharide biosynthesis or (iii) affect some global means of regulating genes needed for a full display of virulence. Attenuation can be determined by performing virulence studies in recipient and determining lethality and splenic infections in surviving mice.
  • Salmonella strains harboring non-reverting mutations in genes involved in the pre-chorismate biosynthetic pathway which make excellent oral vaccines triggering strong humoral, local and cellular immune responses in the host (Chatfield S, N. et al, Vaccine, 1989;7(6): 495-8; Chatfield S, N.
  • Salmonella bacteria that have been attenuated by chromosomal deletion of gene(s) for aromatic compound biosynthesis (aro), mutation in the galE gene, or that are cva-, crp- vir plasmid-, etc., deletion or inactivation in a gene located within the Salmonella pathogenicity island 2 (SPI2), deletion or inactivation of at least one gene associated with pathogenesis selected from ssaV, ssaJ, ssaU, ssaK, ssaL, ssaM, ssaO, ssaP, ssaQ, ssaR, ssaS, ssaT, ssaU, ssaD, ssaE, ssaG, ssaL, ssaC (spiA) and ssaH genes, by deletion or inactivation of at least one gene selected from sseA, sseB, sseC
  • Salmonella vaccines and some inactivated Salmonella vaccines are commercially available (see U.S. Pat. Nos. 7,045,122; 6,923,957; 6,905,691; 6,605,285; 5,843,426; 5,733,760; 5,424,065; 5,389,368; and 6,592,869 relate to Salmonella vaccines, including attenuated and inactivated vaccines).
  • the Salmonella is attenuated as a result of a defect in the LPS.
  • Strains carrying a deletion in LPS are known in the art and include, inter alia, strains carrying a deletion or inactivation in the wbaP gene, encoding the phosphogalactosyltransferase starting O-antigen biosynthesis as described in Ilg et al. ⁇ Infect Immun 2009;77: 2568-75); strains carrying a deletion or inactivation of the wzy gene which codes for the O-antigen polymerase as described by Piao et al. ⁇ J Microbiol 2010;48:486-95), strains carrying a deletion or an inactivation in the waaL gene encoding the O-antigen ligase as described by Nagy et al.
  • the microorganism is a virus, which can be either an inactivated virus or an attenuated virus.
  • infectious pathogens include viruses such as, but not limited to dengue virus, rotavirus, viral meningitis virus, rhinovirus, respiratory syncytial virus (RSV), parainfluenza virus, rotavirus, tick borne encephalitis virus, coronaviridae, rhabodoviridiae, VZV, human papilloma virus (HPV), hepatitis B virus (HBV), hepatitis C virus (HCV), retroviruses such as human immunodeficiency virus (HIV- 1 and HIV-2), herpes viruses such as Epstein Barr Virus (EBV), cytomegalovirus (CMV), Herpesvirus type 8 (Kaposi sarcoma agent), HSV-1 and HSV-2, SARS, EDBV, FeLV, FIV, HTLV-I, HTL V-II, Ebola virus,
  • viruses such
  • Fungi for use with the compositions and methods of the invention include, but are not limited to, Candida species (including C.albicans, C.glabrata and C.tropicalis), Aspergillus, Fusarium, Basidiomycetes, Blastomyces, Coccidioides, Cryptococcus, Histoplasma, Microsporum, Trichophyton, Zygomycetes, and Scedosporium.
  • Candida species including C.albicans, C.glabrata and C.tropicalis
  • Aspergillus Fusarium, Basidiomycetes, Blastomyces, Coccidioides, Cryptococcus, Histoplasma, Microsporum, Trichophyton, Zygomycetes, and Scedosporium.
  • Protozoa for use with the compositions and methods of the present invention include, but are not limited to, Plasmodium spp., including P. falciparum, Toxoplasma spp. and T. gondii; schistosomae, Leishmania major, Trypanosoma cruzi; Entamoeba spp., including E. histolytica; Babesia spp., including B. microti; Trypanosoma spp., including T. cruzi; Giardia spp., including G. lamblia; leishmania spp., including L. major; Pneumocystis spp., including P. carinii; Trichomonas spp., including T. vaginalis; Schisostoma spp., including & mansoni.
  • the antigenic entity is a tumor cell.
  • tumor cells which can be incorporated in the composition of the invention include, without limitation, carcinomas, which may be derived from any of various body organs including lung, liver, breast, skin, bladder, stomach, colon, pancreas, and the like.
  • Carcinomas may include adenocarcinoma, which develop in an organ or gland, and squamous cell carcinoma, which originate in the squamous epithelium.
  • sarcomas such as osteosarcoma or osteogenic sarcoma
  • liquid tumor which refers to neoplasia that is diffuse in nature, as they do not typically form a solid mass.
  • neoplasia of the reticuloendothelial or hematopoetic system such as lymphomas, myelomas and leukemias.
  • leukemias include acute and chronic lymphoblastic, myeolblastic and multiple myeloma.
  • diseases arise from poorly differentiated acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia.
  • Lymphoid malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocyte leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM).
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • PLL prolymphocyte leukemia
  • HLL hairy cell leukemia
  • W Waldenstrom's macroglobulinemia
  • Specific malignant lymphomas include non- Hodgkin ' s lymphoma and variants, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T- cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed- Sternberg ' s disease.
  • the tumor, cancer, malignancy or neoplasia cell may derived from a tumor in any stage, e.g., early or advanced, such as a stage I, II, III, IV or V tumor.
  • the tumor may have been subject to a prior treatment or be stabilized (non-progressing) or in remission.
  • the antigenic entity is a heterogeneous mixture of molecules derived from an extract of a microorganism (bacterial, virus, fungi, protozoa and the like) or from a tumor cell.
  • a microorganism bacterial, virus, fungi, protozoa and the like
  • the second member of the binding pair modifies a substantial percentage of the molecules found in the antigenic entity and that, therefore, the expression "antigenic entity modified by a second member of the binding pair" has to be understood as the components of the antigenic entity which are modified by a second member of the binding pair and not the antigenic entity.
  • the antigenic entity is a bacterial extract
  • it may be prepared by bacteriological culture followed by heat inactivation, concentration and harvest of biomass, alkaline lysis of single bacterial biomass or alkaline lysis of mixtures of bacterial biomass under defined conditions.
  • the alkaline lysates under different conditions may be mixed prior to purification by filtration.
  • the obtained filtrate may be further purified, such as to remove particulate matter, and may also be lyophilized and/or formulated.
  • Suitable bacterial preparations for obtaining an extract or lysate for use in the present invention are essentially those as described above in the context of the antigenic entity being a bacterial cell.
  • the lysate is from a bacterium of the Salmonella genus.
  • the bacterial extract is a hot saline extract (HS). This type of treatment releases the antigen complex in a saline medium and with heat.
  • This HS contains phospholipids, surface proteins and lipopolysaccharide and can be obtained essentially as described in the prior art (Gamazo et al, Infect Immun 1989;57: 1419-1426).
  • the extract is a chaotropic extract (ChE) obtained essentially as described by Altman et al. (Biochem J 1982;201 :505-513).
  • the antigenic entity is a tumor cell extract
  • the term includes tumor cell extracts, tumor cell sonicates, tumor cell hot water extracts and tumor sub-cellular fractions.
  • Extracts can be obtained by any method known to disrupt the cells such as by mechanical disruption with glass beads, a Dounce homogenizer, French press, sonication, freeze-thawing, shearing, osmotic disruption, irradiation or exposure to microwaves or a combination of these methods.
  • tumor cells are treated with collagenase in order to dissociate them prior to the extraction.
  • a variety of detergents may be used to solubilize cells, including anionic, cationic, zwitterionic and non-ionic detergents. By virtue of their amphipathic nature, detergents are able to disrupt bipolar membranes. In selecting a detergent, consideration will be given to the nature of the target antigen(s), and the fact that anionic and cationic detergents are likely to have a greater effect on protein structure than zwitterionic or non-ionic detergents. However, non-ionic detergents tend to interfere with charge-bases analyses like mass spectroscopy, and are also suspectible to pH and ionic strength. Zwitterionic detergents provide intermediate properties that, in some respects, are superior to the other three detergent types.
  • zwitterionics also efficiently disrupt protein aggregation without the accompanying drawbacks.
  • Exemplary anionic detergents include chenodeoxycholic acid, N- lauroylsarconsine sodium salt, lithium dodecyl sulfate, 1-octanesulfonic acid sodium salt, sodium cholate hydrate, sodium deoxycholate, sodium dodecyl sulfate and glycodeoxycholic acid sodium salt.
  • Cationic detergents include cetylpyridinium chloride monohydrate and hexadecyltrimethylammonium bromide.
  • Zwitterionic detergents include CHAPS, CHAPSO, SB3-10 and SB3-12.
  • Non-ionic detergents may be selected from N- decanoyl-N- methylglucamine, digitonin, n-dodecyl beta -D-maltoside, octyl a-D- glucopyranoside, Triton X- 100, Triton X-l 14, Tween 20 and Tween 80.
  • a tumor cell extract useful in the invention can be a fractionated extract.
  • an extract can be fractionated by centrifugation to remove insoluble material such as membranes and large cellular structures.
  • Fractionation of the extract can include, without limitation, centrifugation, protein precipitation, liquid-liquid extraction, solid-phase extraction, or chromatography such as reverse phase chromatography, ion pairing chromatography or ion exchange chromatography, as described, for example, in Rubino, J. Chromatog 2001;764:217-254. Additional methods that can be used to obtain and fractionate cellular extracts are well known in the art, as described, for example, in Scopes, supra, 1994, and Coligan et al, supra, 2000.
  • Biotechnologies sells lung, breast, colon, uterine, cervical, ovarian and stomach tumor lysates.
  • Suitable tumor cells for obtaining an extract or lysate for use in the present invention are essentially those as described above in the context of the antigenic entity being a tumor cell.
  • the antigenic entity is an isolated antigenic molecule.
  • the antigenic molecule can be, for example, but is not limited to, a viral antigen, a bacterial antigen, a fungal antigen, a protozoal antigen, an allergen or environmental antigen, a differentiation antigen, a tumor antigen, an embryonic antigen, an antigen of oncogenes and mutated tumor-suppressor genes, a unique tumor antigen resulting from chromosomal translocations and/or derivatives thereof.
  • the antigenic polypeptide is an immunogenic fragment of a viral antigen, bacterial antigen, a fungal antigen, a protozoal antigen, an allergen or environmental antigen, a differentiation antigen or a tumor antigen.
  • the expression "immunogenic fragment” refers to a peptide molecule which comprises one or more epitopes capable of stimulating the immune system of an organism to generate an antigen- specific cell or humoral immune response.
  • the antigen generates a state of sensitivity or capacity for immune response in said subject such that both antibodies and immune cells obtained from said subject are capable of specifically reacting with the antigen.
  • Viral antigens which are capable of eliciting an immune response against the virus include animal and human retro- and lentiviral antigens such as those of HIV- 1, namely HIV-1 antigens, (such as tat, nef, gpl20 or gpl60, gp40, p24, gag, env, vif, vpr, vpu, rev), human herpes viruses, (such as gH, gL gM gB gC gK gE or gD or derivatives thereof or Immediate Early protein such as ICP27, ICP47, ICP4, ICP36 from HSVl or HSV2, cytomegalovirus, especially Human, (such as gB or derivatives thereof), Epstein Barr virus (such as gp350 or derivatives thereof), Varicella Zoster Virus (such as gpl, II, 111 and IE63), or from a hepatitis virus such as hepatitis B virus (for example
  • Influenza virus cells such as HA, NP, NA, or M proteins, or combinations thereof
  • rotavirus antigens such as VP7sc and other rotaviral components
  • the antigenic peptide derives from hepatitis C virus. In a more preferred embodiment, the peptide derives from the hepatitis C virus NS3 protein. In a still more preferred embodiment, the antigenic peptide corresponds to the amino acids 1073-1081 of the HLA-A2-restricted NS3 peptide, whose sequence is CVNGVCWTV (SEQ ID NO: 5). In a preferred embodiment, the antigenic peptide derives from human papillomavirus. In a more preferred embodiment, the antigenic peptide derives from human papillomavirus of a serotype selected from the groups 6, 11, 16 and 18. In a still more preferred embodiment, the peptide derives from the human papillomavirus E6 or E7 proteins.
  • HPV E7 protein-derived antigenic peptide or "HPV E6 protein-derived antigenic peptide” means a fragment of the HPV E7 or E6 proteins which is capable of stimulating the immune system of a mammal such that an immune response against said protein capable of inhibiting the growth of tumors caused by the expression of E7 or E6 or of inhibiting the proliferation of HPV is generated.
  • the antigen or the antigens can be the complete E6 or E7 proteins, as well as isolated domains of said protein, peptide fragments of the E6 or E7 protein or polyepitope fusion proteins comprising multiple epitopes (for example from 5 to 100 different epitopes).
  • the latter comprises an HPV16 E7 protein-derived antigenic peptide c o r r e s p o n d i n g t o o r c o n t a i n i n g a m i n o a c i d s 1 t o 2 9 o f E 7 (MHGDTPTLHEYMLDLQPETTDLYCYEQLNH SEQ ID NO: 6), a peptide corresponding to or containing amino acids 43 to 98 of E7
  • a peptide comprises the sequence RAHYNIVTF (SEQ ID NO:8).
  • the viral antigenic entity can be individual or combined preparations of one or more of the above viral protein as well as fragments thereof prepared as synthetic peptides, purified pepidic or proteic extracts or recombinant proteins of the virus.
  • the invention contemplates the use of bacterial antigens such as antigens from Neisseria spp, including N. gonorrhea and N. meningitidis (transferrin-binding proteins, lactoferrin binding proteins, PilC and adhesins); antigens from Streptococcus, pyogenes (such as M proteins or fragments thereof and C5A protease); antigens from Streptococcus agalactiae, Streptococcus mutans; Haemophilus ducreyi; Moraxella spp, including M catarrhalis, also known as Branhamella catarrhalis (such as high and low molecular weight adhesins and invasins); antigens from Bordetella spp., including B.
  • pertussis B. parapertussis and B. bronchiseptica (such as pertactin, pertussis toxin or derivatives thereof, filamenteous hemagglutinin, adenylate cyclase, fimbriae); antigens from Mycobacterium spp., including M. tuberculosis, M. bovis, M. leprae, M. avium, M. paratuberculosis, M. smegmatis; Legionella spp, including L.
  • pneumophila for example ESAT6, Antigen 85A, -B or -C, MPT 44, MPT59, MPT45, HSPIO,HSP65, HSP70, HSP 75, HSP90, PPD 19kDa [Rv3763], PPD 38kDa [Rv0934] ); antigens from Escherichia spp, including enterotoxic E. coli (for example colonization factors, heat- labile toxin or derivatives thereof, heat-stable toxin or derivatives thereof), antigens from enterohemorragic E. coli and enteropathogenic E. coli (for example shiga toxinlike toxin or derivatives thereof); antigens from Vibrio spp, including V.
  • enterotoxic E. coli for example colonization factors, heat- labile toxin or derivatives thereof, heat-stable toxin or derivatives thereof
  • enterohemorragic E. coli and enteropathogenic E. coli for example shiga toxin
  • cholera for example cholera toxin or derivatives thereof
  • antigens from Shigella spp including S. sonnei, S. dysenteriae, S. flexnerii
  • Yersinia spp including Y. enterocolitica (for example a Yop protein)
  • antigens from Y. pestis, Y. pseudotuberculosis Campylobacter spp, including C. jejuni (for example toxins, adhesins and invasins)
  • antigens from Salmonella spp including S. typhi, S. enterica and S. bongori
  • Listeria spp. including L. monocytogenes
  • Helicobacter spp including H.
  • pylori for example urease, catalase, vacuolating toxin
  • antigens from Pseudomonas spp including P. aeruginosa
  • Staphylococcus spp. including S. aureus, S. epidermidis
  • Enterococcus spp. including E. faecalis, E. faecium
  • Clostridium spp. including C. tetani (for example tetanus toxin and derivative thereof); antigens from C. botulinum (for example botulinum toxin and derivative thereof), antigens from C.
  • Clostridium toxins A or B and derivatives thereof antigens from Bacillus spp., including B. anthracis (for example anthrax toxin and derivatives thereof); Corynebacterium spp., including C. diphtheriae (for example diphtheria toxin and derivatives thereof); antigens from Borrelia spp., including B. burgdorferi (for example OspA, OspC, DbpA, DbpB); antigens from B. garinii (for example OspA, OspC, DbpA, DbpB), B.
  • B. burgdorferi for example OspA, OspC, DbpA, DbpB
  • B. garinii for example OspA, OspC, DbpA, DbpB
  • afzelii for example OspA, OspC, DbpA, DbpB
  • antigens from B. andersonfi for example OspA, OspC, DbpA, DbpB and antigens from B. hermsii; antigens from Ehrlichia spp., including E. equi and the agent of the Human Granulocytic Ehrlichiosis; Rickettsia spp, including R. rickettsii; Chlamydia spp., including C.
  • trachomatis for example MOMP, heparin-binding proteins
  • antigens from Chlamydia pneumoniae for example MOMP, heparin-binding proteins
  • antigens from C. psittaci for example MOMP, heparin-binding proteins
  • Leptospira spp. including L. interrogans
  • Treponema spp. including T. pallidum (for example the rare outer membrane proteins), antigens from T. denticola, T. hyodysenteriae, antigens from M.
  • tuberculosis such as Rv2557, Rv2558, RPFs: Rv0837c, Rvl884c, Rv2389c, Rv2450, Rvl009, aceA (Rv0467), PstS l , (Rv0932), SodA (Rv3846), Rv2031 c 16kDal., Tb Ral2, Tb H9, Tb Ra35, Tb38-1, Erd 14, DPV, MTI, MSL, mTTC2 and hTCCl); antigens from Chlamydia (such as the High Molecular Weight Protein (HWMP), ORF3 (EP 366 412), and putative membrane proteins (Pmps); antigens from Streptococcus spp, including S.
  • HWMP High Molecular Weight Protein
  • ORF3 ORF3
  • Pmps putative membrane proteins
  • pneumoniae PsaA, PspA, streptolysin, choline-binding proteins, the protein antigen Pneumolysin, and mutant detoxified derivatives thereof); antigens derived from Haemophilus spp., including H. influenzae type B (for example PRP and conjugates thereof); antigens from non typeable H. influenzae (such as OMP26, high molecular weight adhesins, P5, P6, protein D and lipoprotein D, and fimbrin and fimbrin derived peptides, or multiple copy variants or fusion proteins thereof).
  • H. influenzae type B for example PRP and conjugates thereof
  • antigens from non typeable H. influenzae such as OMP26, high molecular weight adhesins, P5, P6, protein D and lipoprotein D, and fimbrin and fimbrin derived peptides, or multiple copy variants or fusion proteins thereof).
  • the bacterial antigens may be purified or recombinant bacterial proteins or synthetic bacterial peptides as well as combinations and mixtures of any of the above.
  • Fungal antigens for use with the compositions and methods of the invention include, but are not limited to, e.g., antigens from Candida spp., including C. albicans,; histoplasma fungal antigens such as heat shock protein 60 (HSP60) and other histoplasma fungal antigen components; antigens from Cryptococcus spp., including C. Neoformans such as capsular polysaccharides and other cryptococcal fungal antigen components; coccidiodes fungal antigens such as spherule antigens and other coccidiodes fungal antigen components; and tinea fungal antigens such as trichophytin and other coccidiodes fungal antigen components.
  • antigens from Candida spp. including C. albicans
  • histoplasma fungal antigens such as heat shock protein 60 (HSP60) and other histoplasma fungal antigen components
  • antigens from Cryptococcus spp. including C. Neoformans
  • Protozoal antigens include, but are not limited to, antigens from Plasmodium spp., including P. falciparum such as merozoite surface antigens, sporozoite surface antigens, circumsporozoite antigens, gametocyte/gamete surface antigens, blood-stage antigen pf, 55/RESA and other plasmodial antigen components (for example RTS.S, TRAP, MSP1, AMA1, MSP3, EBA, GLURP, RAP1, RAP2, Sequestrin, PfEMPl, Pf332, LSA1, LSA3, STARP, SALSA, PfEXPl, Pfs25, Pfs28, PFS27/25, Pfsl6, Pfs48/45, Pfs230 and their analogues in Plasmodium spp.); antigens from Toxoplasma spp.
  • P. falciparum such as merozoite surface antigens, sporozo
  • T. gondii for example SAG2, SAGS, Tg34, p30 and other toxoplasmal antigen components
  • schistosomae antigens such as glutathione- S- transferase, paramyosin, and other schistosomal antigen components
  • leishmania major and other leishmaniae antigens such as gp63 , lipophosphoglycan and its associated protein and other leishmanial antigen components
  • Trypanosoma cruzi antigens such as the 75-77 kDa antigen, the 56 kDa antigen and other trypanosomal antigen components, antigens from Entamoeba spp., including E.
  • the antigen can be an allergen or environmental antigen, such as, but not limited to, an antigen derived from naturally occurring allergens such as pollen allergens (tree-, herb, weed-, and grass pollen allergens), insect allergens (inhalant, saliva and venom allergens), animal hair and dandruff allergens, and food allergens.
  • an antigen derived from naturally occurring allergens such as pollen allergens (tree-, herb, weed-, and grass pollen allergens), insect allergens (inhalant, saliva and venom allergens), animal hair and dandruff allergens, and food allergens.
  • Important pollen allergens from trees, grasses and herbs originate from the taxonomic orders of Fagales, Oleales, Pinoles and platanaceae including La birch (Betula), alder (Alnus), hazel (Corylus), hornbeam (Carpinus) and olive (Olea), cedar (Cryptomeria and Juniperus), Plane tree (Platanus), the order of Poales including i.e. grasses of the genera Lolium, Phleum, Poa, Cynodon, Dactylis, Holcus, Phalaris, Secale, and Sorghum, the orders of Asterales and Urticales including i.a.
  • allergen antigens from house dust mites of the genus Dermatophagoides and Euroglyphus, storage mite e.g Lepidoglyphys, Glycyphagus and Tyrophagus, those from cockroaches, midges and fleas e.g.
  • Blatella, Periplaneta, Chironomus and Ctenocepphalides those from mammals such as cat, dog and horse, birds, venom allergens including such originating from stinging or biting insects such as those from the taxonomic order of Hymenoptera including bees (superfamily Apidae), wasps and ants (superfamily Formicoidae).
  • venom allergens including such originating from stinging or biting insects such as those from the taxonomic order of Hymenoptera including bees (superfamily Apidae), wasps and ants (superfamily Formicoidae).
  • Still other allergen antigens that may be used include inhalation allergens from fungi such as from the genus Alternaria and Cladosporium.
  • tumor antigens examples include MAGE, MART-l/Melan-A, gp l OO,
  • DPPIV Dipeptidyl peptidase IV
  • ADAbp adenosine deaminase-binding protein
  • CRC Colorectal associated antigen
  • CEA Carcinoembryonic Antigen
  • PSMA prostate-specific membrane antigen
  • T-cell receptor/CD3 ⁇ chain MAGE-family of tumor antigens (e.g., MAGE-A1, MAGE-A2, MAGE- A3, MAGEA4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE- All, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-A
  • the tumor antigen is mesothelin or an immunogenic fragment thereof.
  • Reference to mesothelin herein refers to both the isolated full-length polypeptide and isolated polypeptide fragments of at least 10 contiguous amino acids from the full-length sequence wherein the fragment binds to antisera raised against the full-length polypeptide.
  • the tumor antigen is a melanoma-associated antigen such as
  • TRPl/gp75, TRP2, Tyrosinase, gplOO (Pmell7), Melan- A/MART- 1 , COA1, RAB38/NY-MEL-1, a Melanoma Antigen Gene (MAGE) family member, in particular, MAGE-1, -2, -3, -4, -6 or -12), a B Melanoma Antigen (BAGE) family member, a GAGE family member (GAGE-1 to 7, 7b and 8), a LAGE-l/NY-ESO-1 family member, GnTV, CDK4 and catenin or an immunogenic fragment thereof.
  • MAGE Melanoma Antigen Gene
  • BAGE B Melanoma Antigen
  • GAGE-1 to 7, 7b and 8 GAGE family member
  • LAGE-l/NY-ESO-1 family member GnTV, CDK4 and catenin or an immunogenic fragment thereof.
  • the melanoma-associated antigen is TRP2.
  • TRP2 or "tyrosinase-related protein 2" is used herein to refer to a protein showing specific expression in melanocytes and having DOPAchrome tautomerase activity.
  • the TRP2-derived antigen is selected from the group of SVYDFFVWL (SEQ ID NO: 9), TLDSQVMSL (SEQ ID NO: 10), LLGPGRPYR (SEQ ID NO: 11), LLGPGRPYR, (SEQ ID NO: 12), ANDPIFVVL (SEQ ID NO: 13), QCTEVRADTRPWSGP (SEQ ID NO: 14) and ALPYWNFATG (SEQ ID NO: 15).
  • the TRP2-derived antigen corresponds to amino acids 59 to 257 of the TRP2 protein and has the sequence
  • the tumor antigen is an immunogenic molecule capable of inducing an immune response against a tumor cell idiotype derived from the same subject to which it is administered.
  • idiotype refers to an epitope in the hypervariable region of an immunoglobulin.
  • an idiotype or an epitope thereof is formed by the association of the hypervariable or complementarity determining regions (CDRs) of VH and VL domains.
  • the immunogenic molecule capable of inducing an immune response against a tumor cell idiotype is a Id protein or a mixture thereof isolated from a sample of the patient or a molecule comprising the CD3 region of an hybridoma obtained resulting from the tumor cells of the patient.
  • the antigenic entity is coupled to a second member of binding pair.
  • the antigenic entity may contain a second member of a binding pair or may be reacted in the presence of a modifying agent which couples said second member of the binding pair to the antigenic entity.
  • the second member of a binding pair is biotin.
  • biotinylating agents refers to any molecule which is capable of adding a biotin molecule to a reactive group in a target molecule.
  • a biotinylating agent can react with amino groups, carboxyl groups or thiol groups in the target molecule.
  • Suitable biotinylating agents include, without limitation, Biotin-PEO- Amine (reactive with carboxyl groups), PEO-Iodoacetyl-Biotin (reactive with thiol groups), biotin- HS, biotin-sulfo HS, biotin-LC- HS, biotin-LC-sulfo HS, biotin- LC-LC- HS, and biotin-LC-LC-sulfo HS (reactive with amino groups).
  • LC stands for "long chain,” which represents a seven atom spacer between biotin and the NHS ester.
  • biotinylating agent biotin derivative is tetrafluorophenyl polyethylene oxide biotin (TFP-PEO-biotin).
  • the active ester reagents are preferably used at about 0.05 - 0.5 M concentrations, and more preferably at 0.1 M concentrations, in phosphate buffered saline (“PBS”) or in a solution of 9: 1 PBS to dimethylsulfoxide (“DMSO”), if necessary for solubilization.
  • biotin can be added to the antigenic moiety by enzymatic means using, for instance, the Biotin AviTag technology from Avidity, Inc. (Denver, Colo.).
  • the Biotin AviTag is comprised of a unique 15 amino acid peptide that is recognized by biotin ligase, BirA that attaches biotin to a lysine residue in the peptide sequence. (Schatz, Biotechnology 1993; 11 : 1138-43.
  • the Biotin AviTag can be genetically fused to any protein of interest, allowing the protein to be tagged with a biotin molecule.
  • Biotin AviTag technology One potential drawback to the Biotin AviTag technology is the possibility of a low degree of biotinylation, because the system biotinylates the protein at a single, unique lysine residue in the tag region.
  • the purified tagged proteins can be modified in vitro using purified biotin ligase. Because the biotinylation is performed enzymatically, the reaction conditions are gentler, the labeling is highly specific, and the reaction is more efficient than chemical modification of the protein using biotin derivatives.
  • the methods described in Jordan, et al. (Clin Diag Lab Immunol 2003; 10: 339-44), can be used to produce a genetically engineered biotinylated protein.
  • compositions or kits-of-parts according to the invention may further comprise at least one immune co-estimulatory molecule.
  • immune costimulatory molecule includes any molecule which is able to either enhance the stimulating effect of an antigen- specific primary T cell stimulant or to raise its activity beyond the threshold level required for cellular activation, resulting in activation of naive T cells and the resulting increase in an individual's immune response against the first component.
  • Suitable immune co-stimulatory molecule for use in the compositions according to the present invention include, without limitation, TLR ligands, soluble forms of any of B7, CD137-L, CD134-L, GITR-L and CD40-L.
  • the soluble form of a co-stimulatory molecule derived from an antigen presenting cell retains the ability of the native co- stimulatory molecule to bind to its cognate receptor/ligand on T cells and stimulate T cell activation.
  • the immune co-stimulatory agent is a TLR ligand.
  • TLR receptor ligand is understood as a molecule which specifically binds to at least one of the TLR (toll-like receptor) receptors and which upon binding is capable of stimulating some of the signals of co-stimulation signals characteristic of the binding of said receptor with its natural ligand or other signals which result from the binding of said receptor with a TLR agonist.
  • TLRs Toll-like receptors
  • TIL Toll-like receptors
  • TLRs TLRs
  • TLR ligands induce several immune responses depending on the cells in which the TLR is expressed as well as depending on the origin of TLR ligand. For example, in the case of microbial ligands, immune cells can produce cytokines that will cause inflammation. In the case of a viral factor, the cells can undergo apoptosis.
  • the ligands are agonist ligands.
  • Agonist ligands of TLR receptors are (i) natural ligands of the actual TLR receptor, or a functionally equivalent variant thereof which conserves the capacity to bind to the TLR receptor and induce co-stimulation signals thereon, or (ii) an agonist antibody against the TLR receptor, or a functionally equivalent variant thereof capable of specifically binding to the TLR receptor and, more particularly, to the extracellular domain of said receptor, and inducing some of the immune signals controlled by this receptor and associated proteins.
  • the binding specificity can be for the human TLR receptor or for a TLR receptor homologous to the human one of a different species.
  • TLR 1 Multiple triacyl Lipopeptides.
  • TLR 2 Multiple glycopeptides, lipopeptides and lipoproteins. Lipoteichoic acid, HSP70, zymosan from host cells.
  • TLR 3 double-stranded RNA poly(LC) (polyinosinic-polycytidylic acid or polyinosinic-polycytidylic acid sodium salt)
  • TLR 4 Lipopolysaccharides (Gram-negative bacteria), different heat shock proteins (bacteria and host cells), fibrinogen from host cells, heparan sulfate fragments from host cells, hyaluronic acid fragments from host cells, many others.
  • TLR 6 multiple diacyl lipopeptides (mycoplasmas)
  • TLR 7 Imidazoquinoline, loxoribine (guanosine analog), small synthetic compounds of bropirimine and single-stranded RNA.
  • TLR 8 Small synthetic compounds, single-stranded RNA.
  • said assay consists of contacting a culture of dendritic cells with a TLR agonist ligand and measuring the activation of said cells. Said activation can be determined by means of the detection of any marker, for example poly(LC) in the event that the receptor is TLR3.
  • the activated dendritic cells express different proteins such as CD80 (B7.1), CD86 (B7.2) and CD40.
  • the co-stimulatory molecule is a CD40 agonist.
  • CD40 agonist refers to a compound that binds to the CD40 receptor and triggers signaling in a manner similar to the endogenous CD40 ligand.
  • Assays adequate for determining whether a compound is capable of acting as a CD40 ligand are those based on the detection of either the increase in the expression of more CD40 and TNF receptors in macrophages or the activation of B cells and their transformation into plasma cells.
  • the activation of B-cells in response to a CD40 ligand can be assayed by measuring the increase in Inositol 1,4,5-Trisphosphate levels or the activation of tyrosine kinases as described by Uckun et al. (J Biol Chem 1991;26: 17478-17485).
  • the determination of whether a compound is a CD40 agonist can be carried out for example, in macrophages that expressed CD40 on the membrane.
  • the macrophage when a CD40-agonist-bearing-Tcell interacts with the macrophage, the macrophage expresses more CD40 and TNF receptors on its surface which helps to increase the level of activation.
  • the increase in activation results in the introduction of potent microbicidal substances in the macrophage, including reactive oxygen species and nitric oxide.
  • Suitable CD40 agonists for use in the present invention include, without limitation, soluble CD40 Ligand (CD40L), a functionally equivalent variant of the CD40 ligand, CD40L fragments (such as the ones described in WO2009141335), conjugates and derivatives thereof such as oligomeric CD40L polypeptides, e.g., trimeric CD40L polypeptides, the C4BP Core protein (the C-terminal domain of the alpha chain of C4BP) as described in WO05051414 and a CD40 agonistic antibody.
  • CD40L soluble CD40 Ligand
  • CD40L fragments such as the ones described in WO2009141335
  • conjugates and derivatives thereof such as oligomeric CD40L polypeptides, e.g., trimeric CD40L polypeptides
  • the C4BP Core protein the C-terminal domain of the alpha chain of C4BP
  • the CD40 agonist is a CD40 agonistic antibody (such as those described in US2008286289, US2007292439, US2005136055).
  • compositions and kits-of-parts according to the present invention may be used alone or may be delivered in combination with other antigens or with other compounds such as cytokines that are known to enhance immune stimulation of CTL responses, such as, GM-CSF, IL-12, IL-2, TNF, IFN, IL-18, IL-3, IL-4, IL-8, IL-9, IL- 13, IL-10, IL-14, IL-15, G-SCF, IFN alpha, IFN beta, IFN gamma, TGF-a, TGF- ⁇ , and the like.
  • cytokines are known in the art and are readily available in the literature or commercially.
  • the co-stimulatory molecule is an universal DR binding peptide (a PADRE peptide) as described in W09726784 and having the structure selected from the group consisting of aAXAAAKTAAAAa, aAXAAAATLKAAa, aAXVAAATLKAAa, aAXVAAATLKAAa, aAXIAAATLKAAa, aKXVAAWTLKAAa, and aKFVAAWTLKAAa wherein a is D-Alanine, A is L- Alanine, X is cyclohexylalanine, K is lysine, T is threonine, L is leucine, V is valine, I is isoleucine, W is tryptophan, and F is phenylalanine.
  • a PADRE peptide universal DR binding peptide
  • the co-stimmulatory molecules may also be modified with the second member of the binding pair which may form complexes with the conjugate of the invention by means of the interaction between the first member of the binding pair in the conjugate according to the invention and the second member of the binding pair in the co-stimulatory molecule.
  • the compositions of the invention may be formed by a mixture of different complexes, wherein some of the complexes are formed by the conjugate of the inventon and the antigenic entity modified with a second member of the binding pair and part of the complexes are formed by the conjugate of the inventon and the co-stimulatory molecule modified with a second member of the binding pair.
  • the ratio of the two different kinds of complexes may vary depending on the specific circunstamces.
  • the first member of the binding pair is avidin or a functionally equivalent variant thereof, thus resulting in that the conjugate of the invention appears as a tetramer carrying four biotin binding regions.
  • part of the biotin-binding sites may be occupied by the biotinylated antigenic entity and part of the biotin-binding sites may be occupied by the biotinylated co- stimmulatory molecule.
  • the tetrameric EDAvidin is bound to two biotinylated antigenic entities and to two biotinylated co-stimmulatory molecules. 5.
  • the invention relates to a method for the generation of a composition according to the invention which comprises the steps of
  • fibronectin EDA domain i. the fibronectin EDA domain or a functionally equivalent variant thereof
  • the contacting is carried out under conditions adequate for the formation of complexes between the conjugate and the antigenic entity via the interaction between said first and second members of the binding pair and
  • the invention relates to an immunogenic composition obtainable using the method according to the invention or "immunogenic composition of the invention".
  • oligomers may further contain biotinylated antigenic entities connected to the different monomers by the biotin binding sites giving rise to oligomers which are another aspect of the present invention.
  • the invention relates to an avidin or streptavidin oligomer comprising a plurality of avidin or streptavidin monomers wherein each of the avidin or streptavidin monomers is conjugated to a fibronectin EDA domain or a functionally equivalent variant thereof and wherein at least one of the monomers in the oligomer is connected to a biotinylated antigenic entity through the biotin binding site in said at least one monomer.
  • oligomer refers to a protein complex with multiple components.
  • the oligomer is formed by four identical subunits in which case it is a homotetramer.
  • biotin binding sites in the oligomers of the invention need not be all of them occupied by the biotinylated antigenic entity.
  • the biotinylated antigenic entity is connected to one, to two, to three or to four monomers.
  • the oligomers according to the invention may also be modified, in addition by one or more biotinylated antigenic entities, by one or more biotinylated immune coestimulatory molecule or molecules.
  • the biotinylated coestimulatory molecule is selected from the group consisting of a biotinylated TLR ligand, a biotinylated CD40 agonist and a biotinylated PADRE peptide.
  • the oligomer according to the invention comprises two monomers which are connected to biotinylated antigenic entities and two monomers which are connected to biotinylated immune coestimulatory molecules.
  • the oligomers according to the present invention can be prepared by contacting the monomers under conditions adequate for the formation of the oligomers followed by contacting with the biotinylated antigenic entities, or with a mixture of the biotinylated antigenic entities and the biotinylated immune coestimulatory molecules.
  • the purified monomers can be contacted in separate batches with the biotinylated antigenic entities and the biotinylated immune coestimulatory molecules and then contacted in the adequate ratios so that the oligomers are formed with the desired proportion of monomers connected to biotinylated antigenic entities to monomers connected to biotinylated immune coestimulatory molecules.
  • compositions and oligomers of the invention are capable of inducing the activation of an immune response in vivo against the antigenic entity forming part of the composition.
  • in vivo administration of EDAvidin mixed with biotinylated NS3 protein was able to induce strong anti-NS3 T cell immune responses and that in vivo administration of EDAvidin mixed with biotinylated bacterins of Salmonella rough mutants improved significantly (p ⁇ 0.001) the efficacy of the corresponding bacterin administered alone (see Examples 3 to 6 of the invention).
  • EDAvidin can be used to prepare immunoreactive complexes with biotinylated antigens which could be included in prophylactic or therapeutic vaccine formulations against viral infections such as HCV, against bacterial infections such as salmonellosis or against cancer.
  • the invention relates to a pharmaceutical composition, or pharmaceutical composition of the invention, comprising an immunologically effective amount of a composition, a kit-of-parts, an oligomer or an immunogenic composition of the invention and at least one pharmacologically acceptable carrier or adjuvant.
  • the invention relates to a composition, kit-of-parts, oligomer, immunogenic composition or pharmaceutical composition according to the invention for use in medicine.
  • immunogenically effective amount has its usual meaning in the art, i.e. an amount of an immunogen which is capable of inducing a significant immune response against pathogenic agents that share immunological features with the immunogen.
  • adjuvant is understood as any substance intensifying the effectiveness of the pharmaceutical composition of the invention.
  • Suitable adjuvants include, without limitation, adjuvants formed by aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc, formulations of oil-in-water or water-in-oil emulsions such as complete Freund' s Adjuvant (CFA) as well as the incomplete Freund' s Adjuvant (IF A); mineral gels; block copolymers, AvridineTM, SEAM62, adjuvants formed by components of the bacterial cell wall such as adjuvants including liposaccharides (e.g., lipid A or Monophosphoryl Lipid A (MLA), trehalose dimycolate (TDM), and components of the cell wall skeleton (CWS), heat shock proteins or the derivatives thereof, adjuvants derived from ADP-ribosylating bacterial toxins, which include diphtheria toxin (DT), pertussis
  • cereus exoenzyme B B. sphaericus toxin, C. botulinum toxins C2 and C3, C. limosum exoenzyme as well as the toxins of C. perfringens, C. spiriforma and C. difficile, S.
  • limosum exoenzyme and synthetic adjuvants such as PCPP, the cholera toxin, Salmonella toxin, alum and the like, aluminum hydroxide, N- acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L- alanyl-D-isoglutamine, MTP-PE and RIBI, containing three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a squalene emulsion at 2% Tween 80.
  • adjuvants include DDA (dimethyl dioctadecyl ammonium bromide) and QuilA.
  • carrier refers to a diluent or excipient with which the active ingredient is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • Water or aqueous solutions of saline solution and aqueous dextrose and glycerol solutions, particularly for injectable solutions, are preferably used as carriers.
  • Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E.W. Martin, 1995.
  • the carriers of the invention are approved by a regulatory agency of the Federal or a state government or listed in the United States Pharmacopoeia or another generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • the pharmaceutical compositions of the invention can be administered by any route, including, without limitation, oral, intravenous, intramuscular, intrarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal route.
  • routes including, without limitation, oral, intravenous, intramuscular, intrarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal route.
  • compositions comprising said carriers can be formulated by conventional methods known in the state of the art.
  • the invention relates to a composition, kit-of-parts, oligomer, immunogenic composition or pharmaceutical composition according to the invention for use in a method of treatment or prevention of a disease which requires the generation of an immune response against the antigenic entity.
  • the invention relates to the use of a composition, kit-of-parts, oligomer, immunogenic composition or pharmaceutical composition according to the invention for the manufacture of a medicament for the treatment or prevention of a disease which requires the generation of an immune response against the antigenic entity.
  • the invention relates to a method for the treatment or prevention of a disease which requires the generation of an immune response against an antigenic entity in a subject in need thereof which comprises the administration to said subject a composition, kit-of-parts, oligomer, immunogenic composition or pharmaceutical composition according to the invention.
  • the disease to be treated or prevented will require an adequate composition, kit-of-parts, oligomer, immunogenic composition or pharmaceutical composition which comprises the antigenic entity derived from the agent which is causing the disease (pathogenic microorganism or tumor cell) and against which an immune response is desired.
  • the invention relates to therapeutic or prophylactic uses of the compositions, kits-of-parts, oligomers, immunogenic composition and pharmaceutical composition according to the invention wherein:
  • the antigenic entity is selected from the group consisting of a pathogenic microorganism, an extract of a pathogenic microorganism or an antigen from a pathogenic microorganism, then the disease which requires the generation of an immune response against said antigenic entity is an infectious disease caused by the pathogenic microorganism;
  • the antigenic entity is selected from the group consisting of a pancreatic tumor cell, a pancreatic tumor cell extract, mesothelin or an immunogenic fragment thereof, then the disease which requires the generation of an immune response against said antigenic entity is pancreatic cancer;
  • the antigenic entity is selected from the group consisting of a pancreatic tumor cell, a pancreatic tumor cell extract, mesothelin or an immunogenic fragment thereof, then the disease which requires the generation of an immune response against said antigenic entity is pancreatic cancer;
  • the antigenic entity is selected from the group consisting of a leukemia cell, a leukaemia cell extract, an Ig-idiotype or an immunogenic fragment thereof, then the disease which requires the generation of an immune response against said antigenic entity is leukemia;
  • the antigenic entity is selected from the group consisting of a melanoma cell, a melanoma cell extract, TRP2 or an immunogenic fragment thereof then the disease which requires the generation of an immune response against said antigen is melanoma;
  • the antigenic entity is selected from the group consisting of HCV, an HCV extract, HCV NS3 or an immunogenic fragment thereof, then the disease which requires the generation of an immune response against said antigenic entity is a disease caused by HCV infection;
  • the antigenic entity is selected from the group consisting of HPV, an HPV extract, HPV E7 or an immunogenic fragment thereof, then the disease which requires the generation of an immune response against said antigen is a disease caused by HPV infection.
  • infectious disease refers to diseases caused by pathogens such as viruses, bacteria, fungi, protozoa, and parasites. Infectious diseases may be caused by viruses including adenovirus, cytomegalovirus, dengue, Epstein-Barr, hanta, hepatitis A, hepatitis B, hepatitis C, herpes simplex type I, herpes simplex type II, human immunodeficiency virus (HIV), human papilloma virus (HPV), influenza, measles, mumps, papova virus, polio, respiratory syncytial virus, rinderpest, rhinovirus, rotavirus, rubella, SARS virus, smallpox, viral meningitis, and the like.
  • viruses including adenovirus, cytomegalovirus, dengue, Epstein-Barr, hanta, hepatitis A, hepatitis B, hepatitis C, herpes simplex type I, herpe
  • Infections diseases may also be caused by bacteria including Bacillus antracis, Borrelia burgdorferi, Campylobacter jejuni, Chlamydia trachomatis, Clostridium botulinum, Clostridium tetani, Diptheria, E. coli, Legionella, Helicobacter pylori, Mycobacterium rickettsia, Mycoplasma nesisseria, Pertussis, Pseudomonas aeruginosa, S. pneumonia, Streptococcus, Staphylococcus, Vibria cholerae, Yersinia pestis, and the like.
  • bacteria including Bacillus antracis, Borrelia burgdorferi, Campylobacter jejuni, Chlamydia trachomatis, Clostridium botulinum, Clostridium tetani, Diptheria, E. coli, Legionella, Helicobacter pylori, Mycobacterium
  • Infectious diseases may also be caused by fungi such as Aspergillus fumigatus, Blastomyces dermatitidis, Candida albicans, Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum, Penicillium marneffei, and the like. Infectious diseases may also be caused by protozoa and parasites such as chlamydia, kokzidioa, leishmania, malaria, rickettsia, trypanosoma, and the like.
  • the infectious disease is caused by a bacterium.
  • the infectious disease is caused by a bacterium of the family Enterobacteriaceae.
  • Enterobacteriaceae and “enterobacteria” refer to a large family of bacteria, including many of the more familiar pathogens, such as Salmonella and Escherichia coli. Genetic studies place them among the Proteobacteria, and they are given their own order (Enterobacteriales).
  • Enterobacteriaceae examples include, but are not limited to, Alishewanella, Alterococcus, Aquamonas, Aranicola, Arsenophonus, Azotivirga, Blochmannia, Brenneria, Buchnera, Budvicia, Buttiauxella, Cedecea, Citrobacter, Dickeya, Edwardsiella, Enterobacter, Erwinia (e.g. Erwinia amylovora), Escherichia (e.g. Escherichia coli), Ewingella, Grimontella, Hafnia, Klebsiella (e.g.
  • Klebsiella pneumoniae Kluyvera, Leclercia, Leminorella, Moellerella, Morganella, Obesumbacterium, Pantoea, Pectobacterium, Candidatus Phlomobacter, Photorhabdus (e.g. Photorhabdus luminescens), Plesiomonas (e.g. Plesiomonas shigelloides), Pragia Proteus (e.g. Proteus vulgaris), Providencia, Rahnella, Raoultella, Salmonella (e.g Salmonella enterica), Samsonia, Serratia (e.g.
  • Serratia marcescens Serratia marcescens
  • Shigella Sodalis
  • Tatumella Trabulsiella
  • Wigglesworthia Xenorhabdus
  • Yersinia e.g. Yersinia pestis
  • Yokenella Yokenella
  • enterobacterial infections include, but are not limited to, Anthrax (by the bacterium Bacillus anthracis), bacterial acute grastroenteritis (caused by a variety of bacteria including, among others, Salmoenell enterica, Listeria monocytogene and Campylobacter spp), Bacterial Meningitis (caused by a variety of bacteria, including, but not limited to, Neisseria meningitides, Streptococcus pneumoniae, Listeria monocytogenes, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus agalactiae and Haemophilus influenzae), Botulism (caused by bacterium Clostridium botulinum), Brucellosis (caused by bacteria of the genus Brucella), Campylobacteriosis (caused by bacteria of the genus Campylobacter), Cat Scratch Disease (caused by Bartonella henselae
  • pancreatic cancer refers to a group of malignant or neoplastic cancers originating in the pancreas of an individual and includes primary and metastatic cancers of the exocrine pancreas, including adenocarcinoma, serous and mucinous cystadenocarcinomas, acinar cell carcinoma, undifferentiated carcinoma, pancreatoblastoma and endocrine pancreatic cancers such as insulinoma.
  • leukemia refers to neoplastic diseases of the blood and blood forming organs and include, without limitation, acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblasts, promyelocytic, myelomonocytic, monocytic and erythroleukemia) and chronic leukemias (such as chronic myelocytic -granulocytic- leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia).
  • acute leukemias such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblasts, promyelocytic, myelomonocytic, monocytic and erythroleukemia
  • chronic leukemias such as chronic myelocytic -granulocytic- leukemia, chronic myelogenous
  • melanoma includes, but is not limited to, melanomas, metastatic melanomas, melanomas derived from either melanocytes or melanocyte related nevus cells, melanocarcinomas, melanoepitheliomas, melanosarcomas, melanoma in situ, superficial spreading melanoma, modular melanoma, lentigo malignant melanoma, acral lentiginous melanoma, invasive melanoma and familial atypical mole and melanoma (FAM-M) syndrome.
  • melanomas metastatic melanomas
  • melanocarcinomas melanoepitheliomas
  • melanosarcomas melanosarcomas
  • melanoma in situ superficial spreading melanoma
  • modular melanoma lentigo malignant melanoma
  • melanoma refers not only to primary melanomas but also to "melanoma metastasis" which, as used herein, refers to the spread of melanoma cells to regional lymph nodes and/or distant organs. This event is frequent given that melanomas contain multiple cell populations characterized by diverse growth rates, karyotypes, cell-surface properties, antigenicity, immunogenicity, invasion, metastasis, and sensitivity to cytotoxic drugs or biologic agents. Melanoma shows frequent metastasi s to brain, lungs, lymph nodes, and skin. Thus, the compositions of the present invention are also adequate for the treatment of melanoma metastasis.
  • disease caused by HCV infection includes, without limitation, progressive liver fibrosis, inflammation and necrosis leading to cirrhosis, end-stage liver disease, and hepatocellular carcinoma.
  • the term "disease caused by HPV infection” includes warts (such as foot warts), genital warts, recurrent respiratory papillomatosis (such as laryngeal papillomas) and cancers associated with papilloma infections.
  • Cancers that have been associated with the papilloma virus include anogenital cancers (e.g. cervical, perianal, vulvar, vaginal, penile cancers, etc), head and neck cancers (e.g. oropharyngeal cavity cancer, esophageal cancer, etc) and skin cancers (e.g., basal cell carcinoma, squamous cell carcinoma).
  • the invention relates to the use of a composition, kit-of-parts of the invention, an oligomer of the invention or a pharmaceutical composition of the invention for the manufacture of a vaccine.
  • the term "vaccine” refers to a formulation which contains a composition according to the present invention and which is in a form that is capable of being administered to a vertebrate and which induces a protective immune response sufficient to prevent and/or ameliorate an infection and/or to reduce at least one symptom of an infection and/or to enhance the efficacy of another dose of composition of the invention.
  • the immune response generated by the vaccine may be a humoral or a cellular immune response.
  • cellular immune response is used herein to describe an immune response against foreign antigen(s) that is mediated by T-cells and their secretion products.
  • the "humoral immune response”, is used herein to describe an immune response against foreign antigen(s) that is mediated by antibodies produced by B-cells.
  • the vaccine is systemically or locally administered.
  • the vaccine can be administered by means of a single administration, or with a boost by means of multiple administrations as has been previously described for the administration of the compositions of the invention.
  • compositions and vaccines according to the invention can be presented as a single formulation (for example, as a tablet or capsule comprising a fixed amount of each component) or, otherwise, can be presented as separate formulations for subsequent combination for joint, sequential or separate administration.
  • compositions of the invention also contemplate the formulation as a kit of parts wherein the components are formulated separately but are packaged in the same container.
  • the formulation of the first and second component of the compositions of the invention can be similar, in other words, formulated in a similar way (for example, in tablets or in pills), allowing administration by the same route.
  • the two components can be presented in a blister pack. Each blister will contain the medicaments to be consumed throughout the day. If the medicaments need to be admini stered several times a day, the medicaments corresponding to each administration can be arranged in separate sections of the blister pack, preferably noting on each section of the blister pack the time of day when they need to be administered.
  • the components of the composition of the invention can be formulated in a different manner so that the different components are administered differently.
  • the first component to be formulated as a tablet or capsule for oral administration and for the second component to be formulated for intravenous administration.
  • the invention relates to a composition (hereinafter second composition of the invention) comprising
  • fibronectin EDA functionally equivalent variant of fibrionectin EDA
  • an antigenic entity have been described in detail above and are used in the context of the present compositions in the same meaning as the compositions of the invention.
  • the antigenic entity is a bacterial extract.
  • the bacterial extract is a Salmonella extract.
  • the bacterial extract has been obtained from an attenuated bacterium.
  • the attenuated bacterium carries a defect in the LPS.
  • the antigenic entity is an extract of an attenuated Salmonella strain carrying a defect in LPS caused by a deletion in the galM-galK-galT-galE genes.
  • the bacterial extract can be a hot saline extract of the bacterial cell.
  • the second composition of the invention may be used in medicine for the prevention and treatment of diseases that require an immune response against an antigenic entity.
  • the invention relates to a second composition of the invention as well as to a pharmaceutical composition comprising said second composition for use in medicine.
  • the second composition of the invention may be used for the prevention and treatment of diseases caused by bacterial infections.
  • Diseases that can be treated with the second composition of the invention are mentioned above in the context of the pharmaceutical compositions and medical uses of the compositions of the invention.
  • the second component of the second composition of the invention is a bacterin from a Salmonella strain and the second composition of the invention is used for prevention and the treatment of a Salmonella infection.
  • the bacterin has been isolated from a Salmonella strain carrying a defect in the galM-galK-galT-galE genes.
  • antigen presenting cells capture virus antigens among others and present them to T cells to recruit their help in an initial T cell immune response. Due to the fact that an antigen alone may not be enough to generate an immune response, it is possible to contact an immature antigen presenting cells with a composition, kit-of-parts, oligomer or immunogenic composition according to the invention which results in the activation and maturation of the antigen presenting cells, the capture of the antigen or antigens found in the antigenic entity and the presentation thereof in the surface associated to the major histocompatibility antigen.
  • the invention relates to an in vitro method for obtaining an immunogenic antigen presenting cell specific for a given antigenic entity comprising the steps of:
  • APC antigen presenting cell
  • APC suitable for use in the present invention include both professional APC such as dendritic cells (DC), macrophages and B-cells as well as non-professional APC such as fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic beta cells and vascular endothelial cells.
  • DC dendritic cells
  • B-cells non-professional APC such as fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic beta cells and vascular endothelial cells.
  • the APC are dendritic cells.
  • the APC for use in the methods of the inventions are dendritic cells, since these are the only APC having the capacity to present antigens in an efficient amount to activate naive T-cells for cytotoxic T-lymphocyte (CTL) responses.
  • CTL cytotoxic T-lymphocyte
  • immunogenic when used herein to refer to the APCs, refers to phenotypically mature antigen presenting cell with and effector function on of immunogenicity (Reis e Sousa C, Nature reviews, 2006; 6:476-483).
  • a phenotypically mature antigen presenting cell is an APC expressing high cell-surface levels of MHC molecules, CD40, CD80, CD83 and CD86.
  • An effector function of immunogenicity refers to the ability to prime an immune response.
  • dendriti c cell s refers to a diverse popul ation of morphologically similar cell types found in a variety of lymphoid and non- lymphoid tissues (Steinman Ann. Rev Immunol 1991 ;9:271-296). Dendritic cells constitute the most potent and preferred APCs in the organism. While the dendritic cells can be differentiated from monocytes, they possess distinct phenotypes. For example, a particular differentiating marker, CD 14 antigen, is not found in dendritic cells but is possessed by monocytes. Also, mature dendritic cells are not phagocytic, whereas the monocytes are strongly phagocytotic cells. It has been shown that mature DCs can provide all the signals necessary for T cell activation and proliferation.
  • Dendritic cells can be isolated or generated from blood or bone marrow, or secondary lymphoid organs of the subj ect, such as but not limited to spleen, lymph nodes, tonsils, Peyer's patches of the intestine, and bone marrow, by any of the methods known in the art.
  • DCs used in the methods of the invention are (or terminally differentiated) dendritic cells.
  • the source of dendritic cells is preferably human blood monocytes.
  • Immune cells obtained from such sources typically comprise predominantly recirculating lymphocytes and macrophages at various stages of differentiation and maturation.
  • Dendritic cell preparations can be enriched by standard techniques (see e.g., Current Protocols in Immunology, 7.32.1-7.32.16, John Wiley and Sons, Inc., 1997) such as by depletion of T cells and adherent cells, followed by density gradient centrifugation.
  • DCs may optionally be further purified by sorting of fluorescence- labeled cells, or by using anti-CD83 MAb magnetic beads.
  • a high yield of a relatively homogenous population of DCs can be obtained by treating DC progenitors present in blood samples or bone marrow with cytokines, such as granulocyte- macrophage colony stimulating factor (GM-CSF) and interleukin 4 (IL-4). Under such conditions, monocytes differentiate into dendritic cells without cell proliferation. Further treatment with agents such as TNF alpha stimulates terminal differentiation of DCs.
  • cytokines such as granulocyte- macrophage colony stimulating factor (GM-CSF) and interleukin 4 (IL-4).
  • GM-CSF granulocyte- macrophage colony stimulating factor
  • IL-4 interleukin 4
  • the antigen-presenting cells including but not limited to macrophages, dendritic cells and B-cells, can be obtained by production in vitro from stem and progenitor cells from human peripheral blood or bone marrow as described by Inaba et al. (J Exp Med; 1992 : 176 1693-1702).
  • APCs and, in particular, dendritic cells, obtained in this way characteristically express the cell surface marker CD83.
  • such cells characteristically express high levels of MHC class II molecules, as well as cell surface markers CD 1 alpha, CD40, CD86, CD54, and CD80, but lose expression of CD 14.
  • Other cell surface markers characteristically include the T cell markers CD2 and CD5, the B cell marker CD7 and the myeloid cell markers CD 13, CD32 (Fc gamma R II), CD33, CD36, and CD63, as well as a large number of leukocyte-associated antigens.
  • standard techniques such as morphological observation and immunochemical staining, can be used to verify the presence of APCs and, in particular, dendritic cells.
  • the purity of APCs and, in particular, dendritic cells can be assessed by flow cytometry using fluorochrome-labeled antibodies directed against one or more of the characteristic cell surface markers noted above, e.g., CD83, ULA- ABC, HLA-DR, CD1 alpha, CD40, and/or CD54.
  • This technique can also be used to distinguish between immature and mature DCs, using fluorochrome-labeled antibodies directed against CD 14, which is present in immature, but not mature, DCs.
  • APCs and, in particular, dendritic cell precursors may be obtained from a healthy subject or a subject known to be suffering from a disease associated with the expression of a particular antigen.
  • Such DC precursors may be allogeneic or autologous.
  • APCs precursors are obtained, they are cultured under appropriate conditions and for a time sufficient to expand the cell population and maintain the APCs in a state for optimal antigen uptake, processing and presentation.
  • APCs are generated from such APCs precursors by culture ex vivo in serum free or protein-free medium for 40 hours, in the absence of exogenously added cytokines, as detailed in WOO 127245.
  • APC isolation and culture include the use of culture medium lacking exogenously supplied cytokines and culture under serum-free conditions in a manner effective to result in the generation of antigen-loaded superactivated APCs, which are cells that have already processed an antigen and have the ability to present the antigen to the immune cells and quickly generate antigen-specific immune responses, e. g., CTL-mediated T cell responses to tumor antigens.
  • APCs can be preserved by cryopreservation either before or after exposure to the composition, kit-of-parts oligomer or immunogenic composition of the invention.
  • step (i) of the method of obtaining immunogenic APC of the invention the immature APC are contacted with a composition, kit-of-parts, oligomer or immunogenic composition according to the invention.
  • the contacting step comprises the contacting/incubating of the immature APC with the compositions, with the components of the kit-of-parts or the oligomer for sufficient time.
  • sensitization may be increased by contacting the APCs with heat shock protein(s) (hsp) noncovalently bound to the composition. It has been demonstrated that hsps noncovalently bound to antigenic molecules can increase APC sensitization.
  • the activation of the APC can be detected by contacting the APC with T cell clones expressing a T-cell receptor specific for the antigenic peptide present in the composition and measuring the proliferation of the T-cell s, usually by measuring the incorporation of a labeled nucleotide analog.
  • the cells are isolated in order to obtain the antigen-primed APC.
  • Cell surface markers can be used to isolate the cells necessary to practice the methods of this invention.
  • DCs express MHC molecules and costimulatory molecules (e.g., B7-1 and B7-2). The expression of surface markers facilitates identification and purification of these cells. These methods of identification and isolation include FACS, column chromatography and the like.
  • Labeling agents which can be used to label cell antigen include, but are not limited to monoclonal antibodies, polyclonal antibodies, proteins, or other polymers such as affinity matrices, carbohydrates or lipids. Detection proceeds by any known method, such as immunoblotting, western blot analysis, tracking of radioactive or bioluminescent markers, capillary electrophoresis, or other methods which track a molecule based upon size, charge or affinity.
  • the invention relates to an APC obtainable by the method previously mentioned.
  • the antigen-loaded immunogenic APC obtained using the method of the present invention can be used to activate CD8+ T cells and/or CD4+ T-cells in vitro or can be introduced directly in a subject to activate the T cells in vivo.
  • the invention relates to an APC obtainable by the method of the invention for use in medicine as well as to a vaccine or pharmaceutical composition comprising the APC obtainable by the method of the invention.
  • the cells may originate from the same individual which is to be treated (autologous transplantation) or from a different individual (allogeneic transplantation). In allogeneic transplantation, the donor and recipient are matched based on similarity of HLA antigens in order to minimize the immune response of both donor and recipient cells against each other.
  • the APCs according to the invention or the pharmaceutical compositions comprising said APCs or DCs can be used in a method for the prevention and treatment of a disease which requires the generation of an immune response against the antigenic entity.
  • the APCs can be used wherein the disease to be treated or prevented is pancreatic cancer in which case the antigenic entity used in the method for ontaining the immunogenic antigen presenting is selected from the group consisting of a pancreatic tumor cell, a pancreatic tumor cell extract, mesothelin or an immunogenic fragment thereof.
  • the APCs can also be used for the treatment of leukemia, in which case the antigenic entity used in the method for ontaining the immunogenic antigen presenting is selected from the group of a leukemia cell, a leukemia cell extract, an Ig- idiotype or an immunogenic fragment thereof.
  • the APCs according to the invention can also be used for the treatment of melanoma, in which case the antigenic entity used in the method for ontaining the immunogenic antigen presenting is selected from the group consisting of a melanoma cell, a melanoma cell extract, TRP2 or an immunogenic fragment thereof.
  • the APCs according to the invention can also be used for the treatment of a disease caused by HCV infection, in which case the antigenic entity used in the method for obtaining the immunogenic antigen presenting is selected from the group consisting of HCV, an HCV extract, HCV NS3 or an immunogenic fragment thereof; and the disease which requires the generation of an immune response against said antigenic entity is a disease caused by HCV infection.
  • the APCs according to the invention can also be used for the treatment of a disease caused by HPV infection, in which case the antigenic entity is selected from the group consisting of HPV, an HPV extract, HPV E7 or an immunogenic fragment thereof.
  • CD8 + T cells educated in vitro can be introduced into a mammal where they are cytotoxic against target cells bearing antigenic peptides corresponding to T cells are activated to recognize them on class I MHC molecules.
  • target cells are typically cancer cells, or pathogen-infected cells which express unique antigenic peptides on their MHC class I surfaces.
  • CD4 helper T cells which recognize antigenic peptides in the context of MHC class II, can also be stimulated by the APCs of the invention, which present antigenic peptides both in the context of class I and class II MHC. Helper T cells also 5 stimulate an immune response against a target cell.
  • helper T cells are stimulated with the antigen-loaded APCs in vitro or in vivo.
  • the invention relates to an antigen-presenting cell according to the invention, for use in a method for induction of a cytotoxic cell response against the cytotoxic antigen.
  • the invention relates to an antigeni c) presenting cell according to the invention for use in a method for induction of a cytotoxic cell response against an infectious, allergic or neoplastic disease.
  • the immunogenicity of the antigen-presenting cells or educated T cells produced by the methods of the invention can be determined by well known methodologies including, but not limited to the following:
  • Proliferation Assays which measures the capacity of T cells to proliferate in 0 response to reactive compositions.
  • the APC cells can be used for the treatment of different diseases depending on the type of antigenic entity which form part of the compositions used for the sensitization of the antigen-presenting cells. Suitable antigenic entities for sensitization 5 have been described previously and therefore, the cells are suitable for the treatment of infectious diseases, allergic diseases or neoplastic diseases.
  • the invention relates to the antigen-presenting cells of the invention for use in the treatment of a disease which requires the generation of an immune response against the antigenic entity.
  • the invention relates to the use of an antigen- 30 presenting cell of the invention in the manufacture of a medicament for the treatment of a disease which requires the generation of an immune response against the antigenic entity.
  • the invention relates to a method for the treatment of a disease that requires the generation of an immune response against the antigenic entity in a subject which comprises the administration to said subject of an antigen-presenting cell of the invention.
  • the methods of treatment or prevention of the present invention comprise the so-called adoptive immunotherapy.
  • adoptive immunotherapy refers to a therapeutic approach for treating cancer or infectious diseases in which immune cells are administered to a host with the aim that the cells mediate either directly or indirectly specific immunity to (i.e., mount an immune response directed against) the undesired cells.
  • the immune response results in inhibition of tumor and/or metastatic cell growth and/or proliferation and most preferably results in neoplastic cell death and/or resorption.
  • the immune cells can be derived from a different organism/host (exogenous immune cells) or can be cells obtained from the subject organism (autologous immune cells)
  • the immune cells are typically activated in vitro by a particular antigen (in this case the antigenic entity used in the compositions of the invention) applying any of the techniques mentioned above for the activation of APC in vitro.
  • a particular antigen in this case the antigenic entity used in the compositions of the invention
  • Methods of performing adoptive immunotherapy are well known to those of skill in the art (see, e g, US Pat Nos 5,081,029, 5,985,270, 5,830,464, 5,776,451, 5,229, 115, 690,915, and the like).
  • the invention contemplates numerous modalities of adoptive immunotherapy.
  • the DC e.g. isolated from the patient or autologous dendritic cells
  • the compositions of the invention are pulsed with the compositions of the invention and then injected back into the subject where they present and activate immune cells in vivo.
  • the DC are pulsed with the compositions of the invention and then used to stimulate peripheral blood lymphocytes or tumor-infiltrating lymphocytes (TIL) in culture and activate CTLs targeted against the antigenic entity that are then infused into the patient.
  • TIL tumor-infiltrating lymphocytes
  • fibroblasts, and other APCs, or tumor cells are pulsed with the compositions of the invention and used to activate tumor cells or PBLs ex vivo to produce CTLs directed against the antigenic entity that can then be infused into a patient.
  • Inoculation of the activated cells is preferably through systemic administration.
  • the cells can be administered intravenously through a central venous catheter or into a large peripheral vein. Other methods of administration (for example, direct infusion into an artery) are within the scope of the invention.
  • the APCs of the invention and, in particular, the dendritic cells of the invention can be provided in a formulation which is suitable for administration to a patient, e.g., intravenously.
  • APCs and, in particular, DCs of the invention, that are suitable for administration to a patient are referred to herein as a "vaccine", “APC vaccine” or “DC vaccine.”
  • a vaccine or DC vaccine may further comprise additional components to help modulate the immune response, or it may be further processed in order to be suitable for administration to a patient.
  • Methods of intravenous administration of dendritic cells are known in the art, and one of skill in the art will be able to vary the parameters of intravenous administration in order to maximize the therapeutic effect of the administered DCs.
  • APCs or DCs are administered to a subject in any suitable manner, often with at least one pharmaceutically acceptable carrier.
  • the suitability of a pharmaceutically acceptable carrier is determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Most typically, quality control tests (e.g., microbiological assays clonogenic assays, viability tests), are performed and the cells are reinfused back to the subj ect, in some cases preceded by the administration of diphenhydramine and hydrocortisone. See, e.g., Korbling et al. Blood 1986;67:529-532 and Haas et al. Exp. Hematol 1990;18:94-98.
  • Formulations suitable for parenteral administration include aqueous isotonic sterile injection solutions which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • aqueous isotonic sterile injection solutions which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient
  • aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • APCs of the invention can be administered to a subject at a rate determined by the effective dose, the toxicity of the cell type (e.g., the LD-50), and the side-effects of the cell type at various concentrations, as appropriate to the mass and overall health of the subject as determined by one of skill in the art. Administration can be accomplished via single or divided doses.
  • the APCs of the invention can supplement other treatments for a disease or disorder, including, for example, conventional radiation therapy, cytotoxic agents, nucleotide analogues and biologic response modifiers.
  • the dose of the APCs administered to a patient, in the context of the present invention should be sufficient to trigger a beneficial therapeutic response in the patient over time, or to inhibit growth of cancer cells, or to inhibit infection.
  • cells are administered to a patient in an amount sufficient to elicit an effective CTL response to the virus or tumor antigen and/or to alleviate, reduce, cure or at least partially arrest symptoms and/or complications from the disease or infection.
  • An amount adequate to accomplish this is defined as a "therapeutically effective dose. " The dose will be determined by the activity of the APC produced and the condition of the patient, as well as the body weight or surface area of the patient to be treated. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular cell in a particular patient.
  • the physician In determining the effective number of cells to be administered in the treatment or prophylaxis of diseases such as cancer (e.g., metastatic melanoma, prostate cancer, etc.), the physician needs to evaluate circulating plasma levels, CTL toxicity, progression of the disease, and the induction of immune response against any introduced cell type.
  • diseases such as cancer (e.g., metastatic melanoma, prostate cancer, etc.)
  • the physician needs to evaluate circulating plasma levels, CTL toxicity, progression of the disease, and the induction of immune response against any introduced cell type.
  • blood samples Prior to infusion, blood samples are obtained and saved for analysis. Generally at least about 10 4 to 10 6 and typically, between 10 8 and 10 10 cells are infused intravenously or intraperitoneally into a 70 kg patient over roughly 60-120 minutes. Preferably, cell numbers of at least 10 7 for each vaccination point are used.
  • the injections may be e.g. 4 times repeated in a 2 weeks interval and should be given preferably near lymph nodes by intradermal or subcutaneous injections. Booster injections may be performed after a 4-week pause. Vital signs and oxygen saturation by pulse oximetry are closely monitored. Blood samples are obtained 5 minutes and 1 hour following infusion and saved for analysis. Cell reinfusion is repeated roughly every month for a total of 10-12 treatments in a one year period.
  • infusions can be performed on a outpatient basis at the discretion of the clinician. If the reinfusion is given as an outpatient, the participant is monitored for at least 4 hours following the therapy.
  • cells of the present invention can be administered at a rate determined by the LD-50 (or other measure of toxicity) of the cell type, and the side-effects of the cell type at various concentrations, as applied to the mass and overall health of the patient. Administration can be accomplished via single or divided doses.
  • patients may optionally receive in addition a suitable dosage of a biological response modifier including but not limited to the cytokines IFN-a, IFN- ⁇ , IL-2, IL-4, IL-6, TNF or other cytokine growth factor, anti sense TGF- ⁇ , anti sense IL-10, and the like.
  • a biological response modifier including but not limited to the cytokines IFN-a, IFN- ⁇ , IL-2, IL-4, IL-6, TNF or other cytokine growth factor, anti sense TGF- ⁇ , anti sense IL-10, and the like.
  • the cells can be used alone or in conjunction with other therapeutic regimens including but not limited to administration of IL-2, other chemotherapeutics (e.g. doxirubicin, vinblastine, vincristine, etc ), radiotherapy, surgery, and the like.
  • the cells may, optionally, be expanded in culture. This expansion can be accomplished by repeated stimulation of the T cells with the compositions of the invention with or without IL-2 or by growth in medium containing IL-2 alone.
  • Other methods of T cell cultivation for example with other lymphokines, growth factors, or other bioactive molecules are also within the scope of the invention.
  • Plasmid pET21a-Streptavidin-Alive (Howarth et al, Nat Methods 2006;3 :267- 273), expressing wild-type subunit of streptavidin with a 6His tag was used for the construction of the expression plasmid pET21a-ED A- Streptavidin.
  • the extra domain A from fibronectin was obtained from plasmid pET20b-EDA (Lasarte et al, J.Immunol., 2007, 178:748-756) by PCR amplification using primers CAT AT GAAC AT T GAT C G C C C T AAAG GAC T (SEQ ID NO: 17) (Upper EDA-Ndel) and CATATG TGTGGACTGGAT TCCAATCAGGGG (SEQ ID NO: 18) (Lower EDA-Ndel). The resulting PCR product was cloned in pCR2.1-TOPO using the TOPO TA cloning kit (Invitrogen Life Technologies).
  • This plasmid was digested with Ndel and the obtained DNA fragment was subcloned in the Ndel digested and dephosphorylated plasmid pET2 la- Streptavidin- Alive. All constructs were verified by DNA sequencing. The resulting plasmid expressing EDA in the C-terminal end of streptavidin was transformed into BL21(DE3) cells that were induced at OD600 0.9 with 0.4 mM isopropyl-P-D-thiogalactopyranoside (IPTG) for the expression of the recombinant protein.
  • IPTG isopropyl-P-D-thiogalactopyranoside
  • EDAvidin was purified from inclusion bodies by affinity chromatography (Histrap, GE Healthcare, Uppsala, Sweden) and eluted with an imidazol gradient. Resulting proteins were refolded in a sepharose G25 column using a urea gradient size- exclusion chromatography followed by dialysis. Purified recombinant proteins were analyzed by SDS-PAGE and stained with Coomassie blue (Bio-Safe Coomassie reagent, Bio-Rad, Hercules, CA). Non boiled samples were also loaded into the SDS- PAGE confirming the presence of tetrameric forms of the EDAvidin fusion protein.
  • a molecular weight marker containing biotinylated proteins (M.W. 6,500- 180,000, Sigma), or the High-Range Rainbow Molecular Weight Marker (12000- 225000, GE Healthcare) as negative control, were loaded into a 10% SDS-PAGE followed by electrophoretic transfer to nitrocellulose membranes. Membranes were blocked overnight with PBS containing 0.5% Tween 20 and 5% milk (blocking buffer). The detection of biotinylated proteins was carried out by incubating the membranes with 1.33 nmols of EDAvidin or EDA protein in blocking buffer, for 90 minutes. After washing, the membranes were incubated for 2 hours with blocking buffer containing a 1/2000 dilution of polyclonal anti-EDA antibody produced in rabbit at CIMA.
  • OVA protein Ovalbumin, chicken egg, Grade III, Sigma
  • NS3 protein from HCV were biotinylated using Sulfo- HS-SS-Biotin (Thermo Scientific). Briefly, 2 mg of protein were incubated with Sulfo-NHS-SS-Biotin for 30 minutes at room temperature and at a Biotimprotein ratio of 20: 1. Then, the non-reacted Sulfo-NHS-SS- Biotin was removed by dialysis using a Slide-a-Lyzer Dialyisis cassette (3,500 MWCO, Thermo Scientific). Immunopure biotinylated Bovine Serum Albumin (Biotin-LC-BSA) was purchased from Thermo Scientific.
  • Microtitre plates (Nunc maxisorp, Roskide, Denmark) were coated with 0, 1 ⁇ g
  • Binding capacity of EDAvidin to biotinylated proteins was also analyzed by surface plasmon resonance (SPR) using ProteOn XPR36 (Bio-Rad, Hercules CA, USA) optical biosensor.
  • SPR surface plasmon resonance
  • BSA and BSA-biotinylated proteins were covalently immobilized onto the surface of a GLC sensor chips (Bio-Rad) using the coupling reagents sulfo- HS and EDC (Bio-Rad). After protein immobilization, chip surface was treated with ethanolamine to deactivate the excess of reactive esters.
  • BMDC bone marrow-derived dendritic cells
  • Remaining cells were grown at 10 6 cells/ml in 12-well plates in CM (RPMI 1640 supplemented with 10% FCS, 2 mM glutamine, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin and 5x10 "5 M 2-mercaptoethanol) supplemented with 20 ng/ml of mGM-CSF and 20 ng/ml of mIL-4 (both from Peprotech; London, UK). Every two days, two thirds of medium was replaced with fresh medium containing cytokines. Non adherent dendritic cells were harvested at day 7 and cultured in the presence or absence of different stimuli at 37 °C and 5% C02. In vitro analysis on monocyte activation
  • THP-1 cells were plated at 2 x 10 5 cells/well and cultured in the presence of different concentrations of the indicated antigens in culture medium. After 15 hours of incubation, culture supernatants were harvested. The concentration of human T F-a released to the medium by THP-1 cells was quantified using a commercial ELISA assay (BD-Pharmingen), according to manufacturer's instructions.
  • mice were immunized i.v. with 200 ⁇ of a saline solution containing
  • T-cells producing IFN- ⁇ were enumerated by ELISPOT using a kit from BD-Biosciences (San Diego, CA) according to manufacturer ' s instructions culturing 8 x 10 5 splenocytes from the immunized mice in the absence/presence of the indicated peptides pl073 and pl038 (10 ⁇ ), NS3 protein (0, 1 ⁇ g/ml) or culture medium (negative control). The numbers of spots were counted using an automated ELISPOT reader (CTL, Aalen, Germany).
  • Salmonella Enteritidis 3934 (Solano et al, MolMicrobio. 2002;43 :793-808) was used as: (i) parental wild type (SE-wt) smooth strain for constructing the SEAwaaL and SEAGal rough mutants; (ii) virulent strain in mice experiments; and (iii) smooth control strain in all the experiments.
  • SEAGal mutant was constructed by deleting the 4378 bp galETKM operon using the plasmid pK03blue as reported previously (Proc Natl Acad Sci USA 2009;106:7997-8002) and the galE-Ew/galE-Rw and galM-Ew/galM-Rw primers described in Table 1.
  • the SEAwaaL mutant was obtained by replacing the waaL gene with a chloramphenicol resistance cassette using a previously described one step inactivation technique (Datsenko et al., Proc Natl Acad Sci USA 2000;97:6640- 6645) with some modifications (Garcia et al, Mol Microbiol 2004;54:264-277).
  • the chloramphenicol antibiotic resistance cassette was amplified by PCR from the MudQ transposon, using the waal-Clo Fw and waal-Clo Rv primers described in Table 2. Table 2. Oligonucleotides designed and used to generate the bacterial strains.
  • SEAGal HS-SEAGa/ and B-SEAGal
  • SEAwaaL HS-SEAWML and B- SEAwaaL
  • SE-wt HS-SEwt and B-SEwt strains were obtained as described previously by Estevan M. et al. (Vet Microbiol, 2006; 118: 124-32) and Grillo MJ. et al. (Vaccine 2006;24(15):2910-6).
  • EDA Antigenic preparations were mixed with EDA, either obtained in E.
  • EDA E. coli
  • MEDA plant chloroplasts
  • EDA immunomodulating recombinant proteins
  • EDA was expressed in E. coli BL21(DE3) cells (Amershan Pharmacia Biotech) by pET20bl-2 plasmid transfection, purified by HiTrap ® affinity chromatography system (Pharmacia), concentrated using an Amicon Ultra 4-5000 MWCO centrifugal filter device (Millipore), and purified from endotoxins, using Profos EndoTrap ® columns (Hyglos Gmbh), as described previously (Lasarte JJ, et al, J Immunol 2007;178(2):748-756).
  • the MEDA protein was obtained from leaves of transformed tobacco plants and purified to near homogeneity and characterized as described previously (Planta, 2010, 231 :977-90), filter-sterilized using a 20 ⁇ membrane (Millipore ). Absence of contamination was checked in both protein preparations by plating onto Luria-Bertani medium supplemented with 5% agar (LA).
  • bacterins were biotinylated and decorated with a recombinant fusion protein between EDA and streptavidin (named EDAvidin), in order to obtain BEDA antigenic preparations.
  • EDAvidin a recombinant fusion protein between EDA and streptavidin
  • the Sulfo- HS-SS-Biotin ® system (Thermo Scientific Pierce Protein Research Products) was used according to the manufacturer ' s instructions.
  • the non-reacted Sulfo-NHS-SS-Biotin molecules were removed by dialysis using a Slide-a-Lyzer ® Dialyisis cassette (3,500 MWCO, Thermo Scientific).
  • EDAvidin to bind biotinylated and non-biotinylated bacterins was assessed by ELISA in 96-well microtitre plates (Nunc Maxisorp ® ; eBioscience) coated with 0.1 ⁇ g/well of biotinylated bacterins or conventional bacterins as control.
  • mice were studied in 8 to 10-week old female BALB/c mice (Charles River International, France). Animals were accommodated in cages, with water and food ad libitum, at the animal facilities of the Institution (registration code ES/31-2016-000002-CR-SU-US). Mice handling and experimental procedures were carried out in compliance with the current European, national and local (RD 1201/2005) regulations, with the approval of the Institution animal experimentation Committee. In all cases, mice were inoculated i.p.
  • mice Four weeks after immunization, all mice were challenged i.p. with the optimal sub-lethal dose (i.e., the minimal dose able to induce a moderate and homogeneous level of splenic infection in all the infected mice), being 2- 2.3 x lO 2 CFU of SE-wt, as estimated in a previous dose-response experiment (data not shown).
  • BEDA-SEAGa/ and BEDA-SEAwaaL i.e. biotinylated bacterins obtained from SEAGal or SEAwaaL, allowing the reaction with EDAvidin
  • 40 ⁇ g of biotinylated bacterin protein bound to EDAvidin/mouse were used for immunization.
  • SEAwaaL colonies and SE- wt challenging strain were distinguished from each other by double plating in LA and LA supplemented with chloramphenicol (20 mg/L).
  • Kolmogorov-Smirnov test was applied to assess the normal distribution of data obtained in each experiment. Then, statistical comparisons of means were performed by a one-way ANOVA test, followed by the Fisher's Protected Least Significant Difference (PLSD) test (when four or less groups were compared) or Bonferroni's test (when more than four groups were compared). Results on protection were graphically represented by the box-plot method, where boxes represent 50% of central data, line inside boxes represents the median of logio CFU/spleen and whiskers delimit at least 90% of the data obtained.
  • PLSD Fisher's Protected Least Significant Difference
  • Recombinant protein EDAvidin was produced by linking EDA to the C-terminal end of streptavidin.
  • the recombinant EDAvidin protein was expressed in E.coli as 6xHis fusion protein, purified from inclusion bodies by affinity chromatography, desalted and contaminant endotoxins were removed as described in methods.
  • the resulting protein was characterized by SDS-PAGE and western blot using anti-His antibodies (data not shown). A band corresponding to the putative molecular weight of an EDAvidin tetramer was observed after the Coomassie blue staining ( Figure 1A, lane 1).
  • EDAvidin The binding capacity of EDAvidin to bind to biotinylated proteins was also studied by SDS-PAGE. Accordingly, when EDAvidin was mixed with biotinylated ovalbumin (OVABiot) the tetrameric EDAvidin was converted in a larger molecular complex which may correspond to the putative EDAvidin-OVABiot association (Fig. ID). Binding of EDAvidin to biotinylated proteins was also studied by western-blot using a molecular weight marker mixture consisting in biotinylated proteins. Thus, the biotinylated protein mixture was run is SDS-PAGE, transferred to nitrocellulose membranes and incubated with EDAvidin or with EDA as control. Membranes were then incubated with anti-EDA antibodies and developed using a secondary anti-IgG antibody conjugated with HRP. It was found that only EDAvidin was able to bind to the biotinylated proteins (Fig. IE).
  • EDAvidin retains the proinflammatory activity of EDA and targets biotinylated antiogens to dendritic cells.
  • GFP green fluorescence protein
  • EDAvidin plus biotinylated NS3 induce strong anti-NS3 cellular immune responses in vivo.
  • NS3 protein was biotinylated (NS3Biot) and the capacity of EDAvidin to bind to NS3Biot by ELISA was studied. It was found that EDAvidin, but not free EDA was able to associate with NS3Biot coated onto the ELISA plate ( Figure 3A). We then tested in vivo the capacity of a mixture of EDAvidin and NS3Biot to induce anti-NS3 cellular immune responses.
  • HHD mice were injected intravenously (i.v.) with (i) EDAvidin plus NS3Biot, (ii) EDA-NS3, (iii), NS3Biot, (iv) EDA plus NS3Biot or (v) streptavidin plus NS3Biot.
  • EDAvidin plus NS3Biot was as good as EDA-NS3 protein to induce anti-NS3 specific T cell immune responses.
  • EDAvidin was incubated with melanoma TRP-2(180-188) peptide biotinylated at the amino or carboxy terminus.
  • C57/BL6 mice were immunized sc. with 2 nmols of EDAvidin plus the biotinylated melanoma TRP-2(180-188) peptides and seven days after immunization, mice were sacrificed and the number of IFN-y producing spots in response to CTL epitope TRP- 2(180-188) were measured by ELISPOT.
  • mice were immunized with S. Enteritidis HS or bacterins, alone or mixed with EDA (HS and bacterins) or MEDA (only bacterins), and challenged with a sub-lethal dose of smooth virulent SE-wt.
  • EDA HS and bacterins
  • MEDA only bacterins

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Abstract

L'invention concerne des compositions immunogènes et des trousses d'éléments comprenant un premier conjugué comprenant la fibronectine EDA et un premier élément d'une paire de liaison et un second conjugué comprenant une entité antigénique et un second élément d'une paire de liaison. L'invention concerne également des méthodes thérapeutiques à l'aide desdites compositions et trousses d'éléments ainsi que des cellules présentatrices d'antigènes matures et activées obtenues à l'aide des compositions immunogènes et des trousses d'éléments de l'invention.
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WO2023213969A1 (fr) * 2022-05-05 2023-11-09 Juno Therapeutics Gmbh Protéine de liaison virale et réactifs, articles et méthodes d'utilisation associés
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