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WO2018162450A1 - Nouvelles compositions immunostimulatrices comprenant une entité protéine de liaison à l'arn inductible à froid (cirp)-antigène pour l'activation des cellules dendritiques - Google Patents

Nouvelles compositions immunostimulatrices comprenant une entité protéine de liaison à l'arn inductible à froid (cirp)-antigène pour l'activation des cellules dendritiques Download PDF

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WO2018162450A1
WO2018162450A1 PCT/EP2018/055409 EP2018055409W WO2018162450A1 WO 2018162450 A1 WO2018162450 A1 WO 2018162450A1 EP 2018055409 W EP2018055409 W EP 2018055409W WO 2018162450 A1 WO2018162450 A1 WO 2018162450A1
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cancer
protein
cirp
antigen
peptide
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PCT/EP2018/055409
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Juan José LASARTE SAGASTIBELZA
Pablo Sarobe Ugarriza
Lorea VILLANUEVA LEGARDA
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Fundación Para La Investigación Médica Aplicada
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/77—Ovalbumin
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to the Cold-Induced R A-binding Protein (CIRP), a natural ligand for Toll-like Receptor 4 (TLR4) and Myeloid Differentiation factor 2 (MD2), as a means for antigen (Ag) delivery to TLR4 expressing cells.
  • CIRP is capable of inducing appropriate selection and maturation of antigen presenting cells (APCs) while delivering the antigen of choice to antigen presenting cells, leading to an effective antigen specific CD8+ T-cell immune response.
  • APCs antigen presenting cells
  • CD8+ T-cells are activated by the presentation to T-cell receptors (TCRs) of short peptides associated with MHC class I molecules. 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 CD8+ T-cell activation.
  • TCRs T-cell receptors
  • 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 CD 8+ T-cells. These cells orchestrate a repertoire of immune responses from tolerance to self-antigens to resistance to infectious pathogens depending on their maturation status. 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.
  • Characterization of DC-activating ligands has allowed the development of vaccination strategies combining antigens with well-known adjuvant molecules, acting as immunostimulants and/or targeting vectors, and avoiding thus the use of microorganisms containing undefined adjuvant mixtures.
  • These activating molecules have been shown to increase the immunostimulatory properties of DC by signalling through different activation pathways, and in some cases, synergistic effects have been observed in vitro and in vivo for adjuvant combinations.
  • NK cells and T cells Besides their effects on DC it has been also shown that they may act on other cells with anti-tumour effects, including NK cells and T cells.
  • ligands for pattern recognition receptors PRRs
  • PRRs pattern recognition receptors
  • agonistic antibodies against co-stimulatory molecules or cytokines able to trigger maturation of DC.
  • TLR toll-like receptor
  • TLR 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.
  • antigens can be delivered to DCs using the extra domain A from fibronectin. This molecule is also capable of activating DCs by way of its binding to TLR4 on the surface of DCs (Lasarte, JJ, et al. Journal of Immunology 2007. 178:748-56; Durantez M, et al, Vaccine, 2010. 28:7146-54).
  • the invention relates to a conjugate comprising
  • a peptide, protein antigen or antigenic entity wherein said peptide or protein antigen is not the Cold-Inducible R A-binding protein (CIRP cold-inducible RNA-binding protein (CIRP), the Toll-like receptor 4 (TLR4), the myeloid differentiation factor 2 (MD2) or a peptide derived from TLR4 or MD2.
  • CIRP cold-inducible RNA-binding protein CIRP
  • TLR4 Toll-like receptor 4
  • MD2 myeloid differentiation factor 2
  • the invention in another aspect, relates to a composition comprising a plurality of different conjugates of the invention, wherein the different conjugates differ from at least part of the remaining conjugates in the sequence of the peptide or protein antigens.
  • the invention in another aspect, relates to a veterinary or pharmaceutical composition that comprises the conjugate of the invention and a pharmaceutically acceptable carrier or adjuvant.
  • 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 containing the polynucleotide or the vector according to the invention.
  • the invention relates to the conjugate of the invention, the pharmaceutical composition of the invention, the polynucleotide of the invention, the vector of the invention, the cell of the invention or the dendritic cell of the invention for use in medicine or for use in the treatment of diseases which require the generation of an immune response towards an antigen.
  • the invention relates to a kit-of-parts comprising CIRP or a functionally equivalent variant thereof linked to the first member of a binding pair, and more peptide or protein antigens each linked to the other member of the binding
  • SIIN-CIRP protein induces DC maturation, cytokine production and migration.
  • A Schematic representation of protein construct containing the CIRP moiety bound to the antigenic CD8 epitope SIINFEKL plus flanking sequences and a 6 His tail for purification purposes.
  • B Representative gel with Coomassie staining corresponding to SIIN-CIRP after protein purification.
  • C 293 cells expressing TLR4 or lacZ were incubated for one day with different doses of SIIN-CIRP or LPS and cell activation was determined by the production of IL-8.
  • D DC were incubated with graded concentrations of SIIN-CIRP, recombinant OVA, untargeted SIIN.
  • FIG. 3 Full-length CIRP sequence is necessary for DC activation.
  • A Schematic representation of SIIN-ACIRP construct, spanning SIINFEKL peptide bound to a truncated version of CIRP containing the first 100 amino acids.
  • B 293 cells expressing TLR4 were incubated for one day with SIIN-CIRP or the truncated version SIIN-ACIRP and cell activation was determined by the production of IL-8.
  • DC were stimulated with graded doses of SIIN-CIRP or SIIN-ACIRP and phenotypic maturation (C) and cytokine production (D) were determined.
  • FIG. 4 Linkage of SIIN CD8 epitope to CIRP protein enhances antigen presentation.
  • A DC were incubated overnight with graded doses of SIIN-CIRP, LPS (1 ⁇ g/ml) or left untreated. Next day they were harvested, washed and co-cultured with 10 5 purified CD8 T from OT-I mice at different ratios and T cell proliferation was determined after two more days.
  • B DC (4xl0 3 ) treated as above with SIIN-CIRP, SIIN or OVA were co-cultured with OT-I CD8 cells (10 4 ) and IFN- ⁇ released to the supernatants was determined by ELISA. Results are representative of two independent experiments.
  • FIG. 5 Antigen targeting by CIRP enhances in vivo induction of T cell responses.
  • FIG. 6 SIIN-CIRP activates MyD88- and TRIF-dependent pathways and induces type I IFN-dependent T cell responses.
  • A DC were incubated with SIIN- CIRP (2 ⁇ ), LPS (1 ⁇ g/ml) or left untreated and cells were harvested at different time- points. Expression of representative MyD88-dependent (left column) and TRIF- dependent (right column) genes was measured by RT-PCR. Results are expressed as fold-change with respect untreated DC.
  • DC from IFNAR KO or WT mice were stimulated with graded doses of SIIN-CIRP and phenotypic maturation (B) and IL-12 production (C) were determined.
  • FIG. 7 Vaccination with SIIN-CIRP has therapeutic anti-tumour effect.
  • A C57BL/6 mice were injected with 10 5 E.G7-OVA tumour cells. One week later, when the tumour diameter was about 5 mm, they were treated for 3 weeks with 2 weekly i.t. injections of SIIN-CIRP, CIRP, peptide SUN or PBS.
  • B Mice bearing 5 mm MC38- OVA tumours were treated as in A with SIIN-CIRP or PBS.
  • C Mice with E.G7-OVA tumours were depleted of CD8 cells or given control antibodies and then treated with the SIIN-CIRP vaccine. Tumour volume (left) and animal survival (right) were evaluated in all cases. Results correspond to 2 independent experiments with 6-8 mice/group in each experiment.
  • FIG. 8 Combination with blockade of immunosuppressive elements and additional adjuvants enhances CIRP-dependent vaccination resulting in higher antitumour effect.
  • B DC were incubated with SIIN-CIRP or left untreated and next day PD-L1 expression was determined by flow cytometry.
  • Results are expressed as mean fluorescence index (MFI) of PD-L1.
  • C57BL/6 mice were injected with 105 B16-OVA tumour cells. One week later, when the tumour diameter was about 5 mm, they were vaccinated for 3 weeks with 2 weekly i.t.
  • FIG. 10 Comparison of induced responses between SIINFEKL-CIRP and EDA- SIINFEKL.
  • One week later animals were sacrificed, splenocytes stimulated with 1 ⁇ g/ml SUN and the number of IFN-y-producing cells measured by ELISPOT. Values obtained in the absence of stimulation with SUN were always below 20 SFC for all groups. p ⁇ 0,001
  • FIG. 11 Conjugation of protein antigens to CIRP results in an immunogenic response to the antigens.
  • C57BL/6 mice were immunized with 2 nanomoles of OVA protein alone (OVA), or in combination with 2 or 10 nanomoles of CIRP (+2 CIRP or +10 CIRP) or with 2 nanomoles of the OVA-CIRP conjugate (OVA-CIRP).
  • mice were sacrificed and the spleen cells were stimulated with OVA peptide (amino acid 257-264) (SIIN; to analyze the response to CD8 T cells), or with OVA peptide (amino acid 323-339) (ISQ) or with the full protein OVA (OVA) (to measure the response to CD4 T cells).
  • OVA OVA peptide
  • Cells without any antigen were cultured as a negative control (Neg). The immune response was then determined by ELISPOT.
  • a fusion protein comprising a region of the Cold-Induced RNA-binding Protein (CIRP) and a peptide or protein antigen when used to treat DC is capable of inducing their maturation, expression of pro-inflammatory citokines, and improving antigen-presentation to T cells.
  • this same fusion protein comprising CIRP and a peptide or protein antigen, when administrated to mice with a tumour expressing said peptide or protein antigen, is capable of reducing the growth rate of the tumour in treated animals as opposed to animals left untreated or treated only with the antigen.
  • the administration of said fusion protein to naive animal models is capable of generating an immune response that is higher than the immune response generated only by the antigen.
  • conjugate of the invention relates to a conjugate (hereinafter, conjugate of the invention) comprising:
  • CIRP cold-inducible RNA-binding protein
  • a peptide or protein antigen or an antigenic entity wherein said peptide or protein antigen is not cold-inducible RNA-binding protein (CIRP), the Toll-like receptor 4 (TLR4), the myeloid differentiation factor 2 (MD2) or a peptide derived from TLR4 or MD2.
  • CIRP cold-inducible RNA-binding protein
  • TLR4 Toll-like receptor 4
  • MD2 myeloid differentiation factor 2
  • MD2 myeloid differentiation factor 2
  • conjugate refers to molecules comprising two protein moieties that may be directly connected or connected by an intervening moieity (also referred to as spacer or linker).
  • the conjugate is a fusion protein wherein:
  • the proteins forming part of the conjugate are connected by an intervening molecule, the intervening sequence is of peptidic nature and the linkage between both ends of the intervening sequence and the ends of the proteins forming part of the conjugate is also effected via peptide bonds.
  • the conjugate is a fusion protein
  • the protein may be obtained by translation of an mRNA sequence encoding the proteins forming part of the conjugate and, as the case may be, the peptidic linker or intervening sequence.
  • the proteins forming part of the conjugate are connected by a linkage which is not of peptidic nature or are connected by an intervening molecule which is not of peptidic nature or are connected by an intervening molecule which is of peptidic nature but wherein the bonds connecting the intervening molecule and the ends of the proteins forming part of the conjugate are not peptide bonds.
  • Conjugates can comprise all functional domains of the original proteins, or only part of them, or only portions of the proteins not maintaining the original function of the parental protein.
  • the term "recombinant protein” is a protein resulting from the expression of a recombinant DNA consisting on DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in the genome.
  • the fusion protein is a recombinant protein. l .l .Cold-Inducible RNA-binding Protein and functionally equivalent variants thereof
  • the first element of conjugate of the invention is the Cold-Inducible RNA-binding protein (CIRP) or a functionally equivalent variant thereof.
  • CIRP Cold-Inducible RNA-binding protein
  • CIRP Cold-Inducible RNA-binding protein
  • CLR4 Toll-like receptor 4
  • the CIRP used in the conjugates of the invention is human CIRP (UniProt Q14011, integrated into the UniProt database on the 15 th of July of 1999, release of the 18 th of January of 2017).
  • the CIRP used in the conjugates of the invention is murine CIRP (UniProt P60824, integrated into the UniProt database on the 13 th of April of 2004, release of the 2 nd of November of 2016).
  • the CIRP used in the conjugates of the invention is any of the isoforms of the human protein defined in the UniProt database with accession numbers Ql 4011-1 corresponding to SEQ ID NO: 1, Q 14011-2 and Q 14011-3 (integrated into the UniProt database on the 15 th of July of 1999, release of the 18 th of January of 2017).
  • the protein used in component (i) of the conjugates of the invention is any of the predicted isoforms XI and X3 (accessible in the UniProt database under accession numbers: XP 011525970.1 and XP 016881726.1 in the GenPept database, release of 6 th June 2016) and the isoform cold-inducible RNA- binding protein isoform 2 with GenPept accession number NP 001287744.1 (released on the 28 th of August of 2016).
  • the protein used in component (i) of the conjugates of the invention is any of the following peptides sharing a common sequence with CIRP:, 'unnamed protein product' with GenPept accession number BAG65284.1 (released of the 24 th of July of 2008), 'Chain A, Solution Structure of Rrm Domain in A18 Hnrnp' with GenPept accession number 1X5S A (released on the 10 th of October of 2012), 'unnamed protein product' with GenPept accession number BAG65065.1 (released on the 24 th of July of 2008), 'RNPL' with GenPept accession number AAB17212.1 (released on the 21 st of October of 1996), 'unnamed protein product' with GenPept accession number CBB98488.1 (released on the 5 th of September of 2009), 'RNA-binding protein 3/RNA-binding motif protein 3/RNPL' with GenPept accession number P98179.1 (released
  • the CIRP of the conjugate of the invention corresponds to the amino-acid sequence of human CIRP as shown in the UniProt database with accession number Q14011. In a more particularly preferred embodiment, it corresponds to the polypeptide of sequence SEQ ID NO: 1. In another preferred embodiment, the CIRP of the conjugate of the invention corresponds to the amino-acid sequence of murine CIRP as shown in the UniProt database with accession number P60824. In another particularly preferred embodiment, it corresponds to the polypeptide of sequence SEQ ID NO: 2.
  • functional variant and “functionally equivalent variant” are interchangeable and are herein understood as all those peptides derived from the CIRP protein by means of modification, insertion and/or deletion of one or more amino acids, provided that the function of binding to TLR4, MD2, or the complex formed between TLR4 and MD2, and of activating dendritic cells are substantially maintained.
  • functionally equivalent variants are those showing a degree of identity with respect to human CIRP according to SEQ ID NO: 1 greater than at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.
  • functionally equivalent variants are also those showing a degree of identity with respect to murine CIRP according to SEQ ID NO: 2 greater than at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.
  • the degree of identity between two amino acid sequences can be determined by conventional methods, for example, by means of standard sequence alignment algorithms known in the state of the art, such as, for example BLAST (Altschul S.F. et al, J. Mol. Biol, 1990 Oct 5; 215(3):403-10).
  • the functionally equivalent variant of CIRP is a fragment of at least 50 contiguous aminoacids of human CIRP according to SEQ ID NO: 1. In yet another embodiment, the functionally equivalent variant of CIRP is a fragment of at least 50 contiguous aminoacids of murine CIRP according to SEQ ID NO: 2.
  • the functionally equivalent variant of CIRP is a polypeptide comprising SEQ ID NO: 3, which corresponds to amino acids 101 to 125 of human CIRP of sequence SEQ ID NO: 1.
  • the functionally equivalent variant of CIRP is a polypeptide comprising SEQ ID NO: 4, which corresponds to amino acids 101 to 125 of murine CIRP of sequence SEQ ID NO: 2.
  • the functionally equivalent variant of CIRP is a polypeptide comprising SEQ ID NO: 3, which comprises at least amino acids 91 to 126, at least 81 to 126, at least 71 to 126, at least 61 to 126, at least 51 to 126, at least 41 to 126, at least 31 to 126, at least 21 to 126, at least 11 to 126, or at least 1 to 126 of human CIRP of sequence SEQ ID NO: 1.
  • the functionally equivalent variant of CIRP is a polypeptide comprising SEQ ID NO: 4, which comprises at least amino acids 91 to 126, at least 81 to 126, at least 71 to 126, at least 61 to 126, at least 51 to 126, at least 41 to 126, at least 31 to 126, at least 21 to 126, at least 11 to 126, or at least 1 to 126 of human CIRP of sequence SEQ ID NO: 2.
  • the functionally equivalent of CIRP is different from a variant of CIRP consisting of amino acids 1-100 of SEQ ID NO. 1 or amino acids 1-100 of SEQ ID NO. 2.
  • the expression "functionally equivalent variant” means that the polypeptide or protein in question maintains at least one of the functions of CIRP, preferably at least one function related to the immune response, in particular, which maintains the capacity to interact with TLR4, with MD2, and/or with the complex formed by TLR4 and MD2, and to promote the maturation of dendritic cells.
  • the capacity of the functionally equivalent variant to interact with TLR4, with MD2, and/or with the complex formed by TLR4 and MD2 can be determined by means of using conventional methods known by the persons skilled in the art.
  • the capacity of CIRP variant to bind to TLR4, MD2, and or the TLR4-MD2 complex can be determined using co-immunoprecipitation experiments, in which the protein of interest (e.g. the CIRP variant) is isolated with a specific antibody and the molecules which interact with the protein (e.g. TLR4 or MD2) are subsequently identified by means of a western blot.
  • the capacity of the CIRP variant to interact with TLR4, with MD2 or with the complex formed between these two proteins is the binding assay described by Qiang et al. (Nat. Med. 2013, 19: 1489-1495).
  • the functionally equivalent variant of CIRP preserves at least 60% of the binding affinity towards TLR4, towards MD2 or towards the TLR4/MD2 complex with respect to the full-length human or murine CIRP.
  • the functionally equivalent variant of human or murine CIRP preserves at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the binding affinity to TLR4, to MD2 or to the TLR4/MD2 DC complex of the full length murine or human CIRP.
  • the functionally equivalent variant of human or murine CIRP preserves at least 60% of the DC activating capacity of the full-length human or murine CIRP, respectively.
  • the functionally equivalent variant of human or murine CIRP preserves at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the DC activating capacity of the full length murine or human CIRP.
  • Component (ii) of the conjugate of the invention is at least one peptide or protein antigen.
  • peptide antigen and protein antigen refer respectively to a peptide or protein molecule that comprises one or more epitopes capable of stimulating the immune system of an organism to generate an antigen- specific cell or humoral 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.
  • the antigenic protein or peptide may comprise one or more epitopes capable of generating an antibody response, one or more CD8+ T-cell determinant(s), one or more CD4+ T-cell determinant(s) or a combination thereof.
  • the peptide or protein antigen comprises at least a CD8+ T-cell epitope.
  • the peptide or protein antigen is selected from the group consisting of a tumour antigen, a viral antigen, a bacterial antigen, a fungal antigen, a protozoan antigen, an allergen, or combinations thereof.
  • Tumour -associated Antigens are selected from the group consisting of a tumour antigen, a viral antigen, a bacterial antigen, a fungal antigen, a protozoan antigen, an allergen, or combinations thereof.
  • the peptide or protein antigen of component (ii) of the conjugate may be a tumour -associated peptide or protein antigen.
  • the tumour - associated peptide or protein antigen may refer to "tumour-specific antigens" and "tumour -associated antigens”.
  • tumour -specific antigens are peptide or protein antigens expressed by tumour cells which are present only on tumour cells and not on any other cell, thus in a preferred embodiment the tumour -associated antigen is a "tumour specific antigen”.
  • Tumours also express peptide and protein antigens which are present on some tumour cells and also some normal cells (hereinafter referred to as a "tumour -associated antigens").
  • the tumour -associated peptide or protein antigen also comprises:
  • “Mutated Oncogenes and Tumour Suppressor Genes-derived antigens” are any antigen from a mutated protein which is produced in a tumour cell, that has an abnormal structure and that due to mutation can act as a tumour antigen. Such abnormal proteins are produced due to mutation of the concerned gene. Mutation of protooncogenes and tumour suppressors which lead to abnormal protein production are the cause of the tumour. Examples include the abnormal products of ras and p53 genes.
  • “Overexpressed or Aberrantly Expressed Cellular Proteins-derived antigens” are such antigens derived from proteins that are normally produced in very low quantities but whose production is dramatically increased in tumour cells, and trigger an immune response.
  • An example of such a protein is the enzyme tyrosinase, which is required for melanin production. Normally tyrosinase is produced in minute quantities but its levels are very much elevated in melanoma cells.
  • Cell Type-Specific Differentiation Antigens are those antigens derived from proteins that are cell-lineage specific.
  • tumour antigens examples include MAGE, MART- 1/Melan-A, gplOO, Dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophilin b, Colorectal associated antigen (CRC)-0017-1A/GA733, Carcinoembryonic Antigen (CEA) and its antigenic epitopes CAP-1 and CAP -2, etv6, amll, Prostate Specific Antigen (PSA) and its antigenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), T cell receptor/CD3 ⁇ chain, MAGE-family of tumour antigens (e.g., MAGE-A1, MAGE-A2, MAGE -A3, MAGE-A4, MAGE-A5 , MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-family of tumour antigen
  • the tumor antigen comprises or consists of the protein GPC-3 as defined in NCBI protein database with Reference Sequence: NP 001158089.1 (integrated in NCBI on Sep 9, 2009; last updated on Feb 26, 2018), or with UniProt accession number P51654-1 (integrated in UniProt on October 1, 1996) or any antigenic fragment derived thereof.
  • the tumour antigen is an immunogenic molecule capable of inducing an immune response against a tumour 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 tumour cell idiotype is an Id protein or a mixture thereof isolated from a sample of the patient or a molecule comprising the CD3 region of a hybridoma obtained resulting from the tumour cells of the patient.
  • the peptide or protein antigen of component (ii) of the conjugate is a viral antigen.
  • viral antigens are peptide or protein antigens expressed by the virus host's cells and which form part of the viral particle.
  • the peptide or protein is displayed "on the surface of a virus". As used herein this term refers to any peptide or protein that is accessible to reagents, such as antibodies, without the need of disrupting the virus structure. It will be understood that the peptide or protein displayed on the surface may be a capsid peptide or protein for not-enveloped viruses or an envelope peptide or protein for enveloped viruses.
  • virus refers to a small infectious agent that can replicate only inside the living cells of organisms.
  • Non- limiting examples of viral families that may be used in the method of the present invention include Adenoviridae, African swine fever-like viruses, Arenaviridae, Arteriviridae, Astroviridae, Baculoviridae, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Deltavirus, Filoviridae, Flaviviridae, Hepadnaviridae, Hepeviridae, Herpesviridae, Orthomyxoviridae, Paramyxoviridae, Picomaviridae, Poxyviridae, Reoviridae, Retroviridae and Rhabdoviridae. Since the virus co-opts the cell machinery of the cells it infects in order to replicate, the viral antigen may also be present in the membrane of the infected cells
  • 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 peptide or protein antigen of component (ii) of the conjugate is a bacterial antigen.
  • bacterial antigens are peptide or protein antigens expressed by Prokaryotes of the domain Bacteria. Individual Prokaryotes of the domain Bacteria are denominated bacterium. In a preferred embodiment, the peptide or protein is displayed "on the surface of the bacterium". As used herein this term refers to any peptide or protein that is accessible to reagents, such as antibodies, without the need of disrupting the bacterium's structure.
  • the peptide or protein displayed on the surface may be a cell wall or cell membrane peptide or protein for most bacteria, or a cell membrane peptide or protein for bacteria of the class Mollicutes (such as bacteria from the genus Mycoplasma), which lack a cell wall.
  • bacteria refers to Prokaryotes of the domain Bacteria.
  • Non- limiting examples of bacterial genera that may be used in the method of the present invention include: Actinomyces, Bacillus, Bacteroides, Bartonella, Bordetella, Borrelia, Brucella, Burkholderia, Campylobacter, Chlamydia, Clostridium, Corynebacterium, Coxiella, Ehrlichia, Enter ococcus, Eschericia, Francis ella, Haemophilus, Helicobacter, Klebsiella, Legionella, Leptospira, Listeria, Moraxella, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Nocardia, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptobacillus, Streptococcus, Treponema, Ureaplasma, Vibrio and Yersini
  • 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.
  • 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
  • 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 toxin- like 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 to
  • 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); Corymb acterium 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), PstSl, (Rv0932), SodA (Rv3846), Rv2031c 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, cho line-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 peptide or protein antigen of component (ii) of the conjugate is a fungal antigen.
  • fungal antigens are peptide or protein antigens expressed by Eukaryotes of the kingdom Fungi, whether unicellular or multicellular.
  • the peptide or protein is displayed "on the surface of the fungus". As used herein this term refers to any peptide or protein that is accessible to reagents, such as antibodies, without the need of disrupting the fungus's structure. It will be understood that the peptide or protein displayed on the surface may be a cell wall or a cell membrane peptide or protein.
  • fungus refers to unicellular and multicellular eukaryotic individuals of the kingdom Fungi.
  • Non-limiting examples of fungal genera that may be used in the method of the present invention include: Apophysomyces, Aspergillus, Basidiobolus, Blastomyces, Candida, Coccidioides, Conidiobolus, Cryptococcus, Encephalitozoon, Enter ocytozoon, Epidermophyton, Exophiala, Fonsecaea, Fusarium, Geotrichum, Histoplasma, Hortaea, Lacazia, Lichteimia, Malassezia, Microsporum, Paracoccidioides, Penicillum, Phialophora, Piedraia, Pneumocystis, Pseudallescheria, Rhinosporidium, Rhizopus, Sporothrix, Syncephalastrum, Trichophyton, and Trichosporon.
  • 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; Coccidioides fungal antigens such as spherule antigens and other Coccidioides fungal antigen components; and Tinea fungal antigens such as trichophyton and other Tinea fungal antigen components. 1.2.5. Protozoan Antigens
  • the peptide or protein antigen of component (ii) of the conjugate is a protozoan antigen.
  • protozoan antigens are peptide or protein antigens expressed by unicellular Eukaryotes of the traditional phylum Protozoa, which comprised the subphyla Sarcomastigophora, Sporozoa, Cnidospora and Ciliophora.
  • the peptide or protein is displayed "on the surface of the protozoa". As used herein this term refers to any peptide or protein that is accessible to reagents, such as antibodies, without the need of disrupting the protozoa's structure. It will be understood that the peptide or protein displayed on the surface may be cell membrane peptide or protein.
  • protozoan genera refers to unicellular eukaryotic individuals of the traditional phylum Protozoa and presently excluded from the present taxonomical classification of the kingdoms Fungi and Plantae.
  • protozoan genera include: Acanthamoeba, Babesia, Balamuthia, Balantidium, Blastocystis, Cryptosporidium, Cyclospora, Dientamoeba, Entamoeba, Giardia, Leishmania, Naegleria, Plasmodium, Prototheca, Pythium, Sappinia, Toxoplasma, Trichomonas, and Trypanosoma.
  • Protozoan 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, P032, 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, sporozoit
  • T. gondii for example SAG2, SAGS, Tg34, p30 and other toxoplasmal 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. histolytica
  • antigens from Babesia spp. including B. microti
  • antigens from Trypanosoma spp. including T.
  • antigens from Giardia spp. including G. lamblia
  • antigens from Pneumocystis spp. including P. carinii
  • antigens from Trichomonas spp. including T. vaginalis.
  • the antigen of component (ii) of the conjugate can be the complete protein, as well as isolated domains of said protein, peptide fragments or polyepitopes, fusion proteins comprising multiple epitopes (for example from 5 to 100 different epitopes).
  • the polypeptide can optionally include additional segments, for example, it can include at least 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 75, 90 or even 100 or more segments, each being a part of the naturally occurring protein and/or of a naturally occurring tumour, viral, bacterial or protozoan antigen which can be the same or different from the protein or proteins from which the other segments are derived.
  • Each of these segments can have a length of at least 8 amino acids, and each contains at least one epitope (preferably two or more) different from the epitopes of the other segments.
  • At least one (preferably at least two or three) of the segments in the hybrid polypeptide can contain, for example, 3, 4, 5, 6, 7 or even 10 or more epitopes, particularly epitopes of binding to MHC class I or class II.
  • Two, three or more of the segments can be contiguous in the hybrid polypeptide, i.e., they can be bound end-to-end, without a spacer between them.
  • any two adjacent segments can be bound by a spacer amino acid or a spacer peptide.
  • the peptide or protein antigen of the conjugate of the invention is at least one peptide or protein antigen or a fragment of said protein or peptide, wherein the peptide or protein or the fragment thereof is selected from the group consisting of a tumour antigen, a viral antigen, a bacterial antigen, a fungal antigen or a protozoan antigen.
  • the peptide or protein antigen of the conjugate of the invention is not CIRP or TLR4 or MD2 or a fragment derived thereof.
  • 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, but not limited to, 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 not contemplated already as fungal antigens, such antigens from the genus Alternaria and Cladosporium.
  • Component (ii) is the conjugate can be an antigenic entity.
  • an “antigenic entity” is herein defined to encompass any cell, microorganism that are at least capable of binding to an antibody, or preferably, of generating a T-cell response, more preferably a CD4 T-cell response, even more preferably a CD8 T-cell response, thus contributing to the development of an stronger immune response.
  • the antigenic entity forming part of the conjugates of the invention may be a microorganism (bacterial, virus, fungi, protozoa and the like), a tumour cell, an allergenic source, or combinations thereof.
  • the antigenic entity is selected from the group consisting of a bacteria, a virus, a fungus, a protozoan, a tumour cell, an allergenic source, or a combination thereof.
  • the antigenic entity is a microorganism.
  • the terms "microorganism” or “microbe” refer to an organism of microscopic size, to a single-celled organism, and/or to any virus particle.
  • the term, as used herein, includes Bacteria, Archaea, single-celled Eukaryotes (protozoa, fungi, and ciliates), and viral agents.
  • 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.
  • inactivated form of the microorganism also encompasses lysates, fractions or extracts of the microorganism.
  • 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
  • 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. In addition, 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.
  • a compound such as merthiolate, formalin, formaldehyde, diethylamine, binary ethylenamine (BEI), beta propiolactone (BPL), and glutaraldehyde. Combinations of these compounds may be used. In addition, it is possible to kill the bacteria by sterilizing radiation, heat, ultrasounds (e.g. sonication), cell rupture (e
  • 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. 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. enter ocolitica, 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. difficile; 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, C. pneumoniae and C. psittaci; Leptospira spp., including L. interrogans; Treponema spp., including T. pallidum, T. denticola and T. hyodysenteriae;
  • Streptococcus spp. including S. pneumonia, and Haemophilus spp., including H. influenzae.
  • the bacterin has been obtained by formalin treatment of the bacteria.
  • 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 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, Mar.
  • the antigenic entity is a fungus.
  • 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.
  • the antigenic entity is a protozoan.
  • 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; Leishmania major, Entamoeba spp., including E. histolytica; Babesia spp., including B. microti; Trypanosoma spp., including T. cruzi; Giardia spp., including G. lamblia; Pneumocystis spp., including P. carinii; Trichomonas spp., including T. vaginalis.
  • the antigenic entity is a tumour cell.
  • Representative tumour 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, thymus, 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 tumour 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 tumour, cancer, malignancy or neoplasia cell may derived from a tumour in any stage, e.g., early or advanced, such as a stage I, II, III, IV or V tumour.
  • the tumour may have been subject to a prior treatment or be stabilized (non-progressing) or in remission.
  • the antigenic entity is indirectly conjugated to component (i) of the conjugate of the invention (CIRP or the functional variant of CIRP) via two members of a binding pair as it is further described below.
  • 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 which is coupled to the antigenic entity.
  • 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 component (i) containing coupled with the first member of the binding pair, becomes "decorated" with said components.
  • the whole cell may be a cell of a microorganism or a tumour cell.
  • antigenic entity modified by a second member of the binding pair has to be understood as the fraction of the the components of the antigenic entity which are modified by a second member of the binding pair and are therefore available to bind to the first member of the binding pair in the first component .
  • the components (i) (CIRP or a functionally equivalent variant thereof as previously defined) and (ii) (the peptide or protein antigen) of the conjugate of the invention are bound by direct conjugation.
  • direct conjugate means that conjugate is provided as a single polypeptide chain comprising component (i) and component (ii).
  • single polypeptide chain means that component (i) and component (ii), can be conjugated end-to-end but also may include one or more optional peptide or polypeptide "linkers” or “spacers” intercalated between them, linked by a covalent bond.
  • component (i) and component (ii) and the optional peptide or polypeptide spacers are linked by peptide bonds, thus forming a "fusion protein”.
  • component (i) and component (ii) are linked by an isopeptide bond, as described below.
  • An isopeptide bond is an amide bond that is not present on the main chain of a protein.
  • the bond forms between the carboxyl terminus of one protein and the amino group of a lysine residue on another (target) protein.
  • Isopeptide bonds can occur between the side chain amine of lysine and the side chain carboxyl groups of either glutamate or aspartate.
  • the spacer or linker amino acid sequences can act as a hinge region between components (i) and (ii), allowing them to move independently from one another while maintaining the three-dimensional form of the individual domains, such that the presence of peptide spacers or linkers does not alter the functionality of any of the components (i) and (ii), nor the DC activating properties or component (i) neither the antigenic properties of component (ii).
  • a preferred intermediate amino acid sequence according to the invention would be a hinge region characterized by a structural ductility allowing this movement.
  • said intermediate amino acid sequence is a flexible linker. The effect of the linker region is to provide space between the component (i) and component (ii).
  • the spacer is of a polypeptide nature.
  • the linker peptide preferably comprises at least 2 amino acids, at least 3 amino acids, at least 5 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 30 amino acids, at least 40 amino acids, at least 50 amino acids, at least 60 amino acids, at least 70 amino acids, at least 80 amino acids, at least 90 amino acids or approximately 100 amino acids.
  • the spacer or linker can be bound to components flanking the two components of the conjugates of the invention by means of covalent bonds, preferably by peptide bonds; and also preferably the spacer is essentially non-immunogenic, and/or is not prone to proteolytic cleavage, and/or does not comprise any cysteine residue.
  • the three-dimensional structure of the spacer is preferably linear or substantially linear.
  • spacer or linker peptides include those that have been used to bind proteins without substantially deteriorating the function of the bound proteins or at least without substantially deteriorating the function of one of the bound proteins. More preferably the spacers or linkers have been used to bind proteins comprising coiled coil structures.
  • linker peptides comprise 2 or more amino acids selected from the group consisting of glycine, serine, alanine and threonine.
  • a preferred example of a flexible linker is a polyglycine linker.
  • linker/spacer sequences include SGGTSGSTSGTGST (SEQ ID NO: 8), AGSSTGSSTGPGSTT (SEQ ID NO: 9) or GGSGGAP (SEQ ID NO: 10). These sequences have been used for binding designed coiled coils to other protein domains (Muller, K.M., Arndt, K.M. and Alber, T., Meth. Enzimology, 2000, 328: 261-281).
  • said linker comprises or consists of amino acid sequence GGGVEGGG (SEQ ID NO: 11).
  • a suitable linker peptide can be based on the sequence of 10 amino acid residues of the upper hinge region of murine IgG3.
  • This peptide (PKPSTPPGSS, SEQ ID NO: 12) has been used for the production of dimerized antibodies by means of a coiled coil (Pack, P. and Pluckthun, A., 1992, Biochemistry 31 : 1579-1584) and can be useful as a spacer peptide according to the present invention. Even more preferably, it can be a corresponding sequence of the upper hinge region of human IgG3.
  • the sequences of human IgG3 are not expected to be immunogenic in human beings.
  • Additional linker peptides that can be used in the conjugate of the invention include the peptide of sequence APAETKAEPMT (SEQ ID NO: 13), the peptide of sequence GAP, the peptide of sequence AAA and the peptide of sequence AAALE.
  • the different components (i) and (ii) of the conjugate of the invention can be placed in any order provided that the component (i) (CIRP or a functionally equivalent variant thereof as previously defined) maintains its dendritic cell activating properties and that the component (ii) (the peptide or protein antigen or the antigenic entity) maintains the antigenic properties.
  • the component (ii) of the conjugate is placed or conjugated at the N-terminal end of the component (i).
  • the component (ii) is placed or conjugated at the C- terminal end of component (i).
  • two portions of the component (ii) are placed or conjugated to the component (i), one of them at the N-terminal end, and the other at C-terminal end of the component (i).
  • two molecules of the component (i) are placed or conjugated to the component (ii), one of them at the N-terminal end, and the other at C-terminal end of the component (ii).
  • the conjugate of the invention comprises at least two conjugated repeats of any of the conjugates embodiments previously described.
  • N-terminal end and C-terminal end do not mean that component (i) and component (ii), or viceversa, need to be directly conjugated end-to-end, but that they maintain that relative order of component (i) and component (ii) positions regardless of the presence of additional elements at the end of either component (i) or (ii) or intercalated between them, such as linkers/spacers or members of a binding pair.
  • the components (i) and (ii) of the conjugate of the invention are directly conjugated using the "SpyTag/SpyCatcher system".
  • the SpyTag/SpyCatcher system was recently described by Zakeri et al. (Zakeri, B.
  • the components (i) (CIRP or its functionally equivalent variant) and (ii) (the peptide or protein antigen) of the conjugate are directly conjugated via a polypeptide pair comprising a) SEQ ID NO: 5 and/or SEQ ID NO: 6 (SpyTag); and b) SEQ ID NO: 7 (SpyCatcher).
  • a polypeptide pair comprising a) SEQ ID NO: 5 and/or SEQ ID NO: 6 (SpyTag); and b) SEQ ID NO: 7 (SpyCatcher).
  • SEQ ID NO: 5 or SEQ ID NO: 6 and SEQ ID NO: 7 have been obtained by contacting fusion proteins comprising each a component of the conjugate and a component of SpyTag/Spy Catcher system.
  • the possible fusion proteins to be used in the preparation of the conjugates of the invention are as follows:
  • the direct conjugates may be formed by the reaction between the fusion proteins with SpyTag and SpyCatcher polypeptide regions arranged in any order, provided that CIRP or its functionally equivalent variant maintains its dendritic cell-activating properties and that the peptide or protein antigen keeps its antigenicity.
  • CIRP or its functionally equivalent variant maintains its dendritic cell-activating properties and that the peptide or protein antigen keeps its antigenicity.
  • the directly coupled conjugate as a whole is not human CIRP, murine CIRP or any of the human isoforms described above. l AIndirectly coupled conjugate
  • the conjugate may comprise the components (i) (CIRP or its functionally equivalent variant) and (ii) (the peptide or protein antigen) linked through an indirect conjugation, meaning a non-covalent bond between component (i) and component (ii).
  • component (i) is linked to one member of a binding pair and component (ii) is linked to the other member of the biding pair.
  • first member of a binding pair refers to a molecule which has affinity for and "binds" to another molecule (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 sequences of DNA, RNA and peptide nucleic acids (PNA)), polynucleotide/polynucleotide-binding protein and the like.
  • 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/
  • 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.
  • the complex resulting from said binding has a dissociation constant (3 ⁇ 4) of less than 10 "6 M, less than 10 "7 M, less than 10 "8 M, less than 10 ⁇ 9 M, less than 10 "10 M, less than 10 "11 M, less than 10 "12 M, less than 10 "13 M, less than 10 "14 M or less than 10 "15 M.
  • the biding pair will have a dissociation constant (3 ⁇ 4) of less than 10 ⁇ 9 M under physiological conditions.
  • two entities 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, 10 "14 M, 10 "13 M, 10 "12 M, 10 "10 M or 10 "9 M.
  • avidin and streptavidin 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; NM_205320.1; 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;
  • Streptavidin having a higher affinity for biotin as described in WO9840396;
  • streptavidin with modified affinity as described in WO9624606.
  • Different avidin variants are commercially available, such as Extravidin (Sigma- Aldrich), NeutrAvidin (Thermo Scientific), NeutrAvidin (Invitrogen) and NeutraLite (Belovo).
  • the first member of the binding pair comprises the sequence of SEQ ID NO: 14.
  • biotin has an extremely high affinity for both streptavidin (10 ⁇ 13 M) and avidin (10 ⁇ 15 M).
  • both 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 CIRP or a functionally equivalent variant thereof (component (i)) and the first member of a binding pair may be directly connected, i.e. by means of a specific direct interaction between both elements.
  • the CIRP or a functionally equivalent variant thereof and the first member of a binding pair may be indirectly connected e.g. by means of using a spacer or linker.
  • linkers in the direct conjugate apply to the linkers of indirect conjugates.
  • the different elements of the indirect conjugate of the invention can be placed in any order provided that the CIRP or its functionally equivalent variant (component (i)) 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, and that the peptide or protein antigen (component (ii)) maintains its antigenicity and the second binding pair maintains its capacity of binding to the first member of the binding pair.
  • component (i) of the conjugate of the invention as described above, and b) a polypeptide whose amino acid is SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, or a polypeptide which is first member of a binding pair, as described above;
  • sequence a) and sequence b) forms a single polypeptide chain; and wherein components are linked by peptide bonds. Regardless of their relative order or insertion of any other polypeptide -connector, -tag, -spacer, or alike; a construct comprising second polynucleotide herein described.
  • a further additional aspect of the invention is drawn to a second polypeptide comprising:
  • the second single-chain polypeptide comprises additional elements such as linkers, spacers or tags.
  • the elements of the second single chain polypeptide from the N-terminal to the C-terminal ends are placed in any relative position as long as the functional properties of the component (i) are preserved.
  • Yet another embodiment of the invention corresponds to a polynucleotide construct encoding the second single-chain polypeptide described previously.
  • said conjugate can contain, if desired, an additional peptide which can be used for the purposes of isolating or purifying the conjugate, such as a tag peptide.
  • Said tag peptide can be located in any position of the conjugate which does not alter the functionality of any of the polypeptides of components (i) and (ii).
  • said tag peptide can be located in the N-terminal position of the conjugate of the invention such that the C-terminal end of the tag peptide is bound to the N-terminal end of the conjugate of the invention.
  • the tag peptide can be located in the C-terminal position of the conjugate of the invention such that the N- terminal end of the tag peptide is bound to the C-terminal end of the conjugate of the invention.
  • Virtually any peptide or peptide sequence allowing the isolation or purification of the conjugate can be used, for example, polyhistidine sequences, peptide sequences which can be recognized by antibodies which can serve to purify the resulting fusion protein by immunoaffinity chromatography, such as tag peptides, for example, influenza virus hemagglutinin (HA)-derived epitopes, C-myc and the antibodies 8F9, 3C7, 6E10, G4, B7 and 9E10 against it; the Herpes Simplex virus D (gD) tag protein and the antibodies thereof.
  • HA hemagglutinin
  • tag peptides include the Flag peptide and the KT3 epitope.
  • the tag peptide is generally arranged at the amino- or carboxy- terminal end.
  • the tag peptide is a His tag, more preferably a hexahistidine tag.
  • component (i) can be obtained from coding DNA (like a cDNA) by means of expression in a heterologous organism such as, for instance, Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris or from tobacco chloroplasts as described by Farran et al. (Planta, 2010. 231 : 977-90).
  • This coding DNA can comprise additional coding sequences.
  • the expression of the coding DNA in the heterologous organism results in a single polypeptide chain which comprises the whole conjugate of the invention (component (i) and component (ii)), component (i) bound to the first member of the binding pair (when it has a peptide nature, e.g. avidin or streptavidin), or also component (i) bound to SpyTag- or SpyCatcher- polypeptide.
  • component (i) and component (ii) when it has a peptide nature, e.g. avidin or streptavidin
  • component (i) bound to the first member of the binding pair when it has a peptide nature, e.g. avidin or streptavidin
  • component (i) bound to SpyTag- or SpyCatcher- polypeptide when it has a peptide nature, e.g. avidin or streptavidin
  • component (i) bound to SpyTag- or SpyCatcher- polypeptide when it has a peptide nature, e.g. avid
  • the conjugation of said first member of the binding pair to the component (i) 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, sulfmyl, 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 component (i) molecule and of component (ii).
  • DOPA 3,4-dihydroxyphenylalanine
  • the bifunctional group can first be conjugated to the therapeutically active compound and, then, conjugated to component (i) or, alternatively, it is possible to conjugate the bifunctional group to component (i) and, then, conjugate the latter to component (ii).
  • Illustrative examples of this type of 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., strep tavidin), 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 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-NHS, biotin-sulfoNHS, biotin-LC-NHS, biotin-LC-sulfoNHS, biotin-LC-LC- NHS, and biotin-LC-LC-sulfoNHS (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).
  • Biotin can also be incorporated into a protein or peptide antigen through the use of biotin-containing resins or adding biotinylated amino-acids at the end of the protein synthesis process.
  • 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.
  • PBS phosphate buffered saline
  • DMSO dimethylsulfoxide
  • biotin can be added to the antigenic moiety by enzymatic means using, for instance, the Biotin AviTag technology from Avidity, Inc. (Denver, Colorado).
  • 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 labelling 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. (Clinical and Diagnostic Laboratory Immunology, 2003. 10:339-44), can be used to produce a genetically engineered biotinylated protein.
  • the invention in another aspect, relates to a composition (hereinafter composition of the invention) comprising a plurality of different conjugates according to the invention and as defined under paragraph 1 above, wherein the different conjugates differ from at least part of the other conjugates in the composition in the sequence of the peptide or protein antigens.
  • the plurality of conjugates in the compositions of the invention contain peptide or protein antigens derived from the same source, wherein the source can be any antigenic entity as defined above, i.e.
  • any cell or microorganism at least capable of binding to an antibody, or preferably, of generating a T-cell response, more preferably a CD4 T-cell, even more preferably a CD8 T-cell response, thus contributing to the development of an stronger immune response.
  • the plurality of conjugates forming part of the compositions according to the invention are characterized in that they contain different antigens derived from a microorganism (bacterial, virus, fungi, protozoa and the like), from a tumour cell, from an allergenic source, or combinations thereof.
  • the plurality of conjugates contain a collection of protein or peptide antiges which are found in the extract of a microorganism (bacterial, virus, fungi, protozoa and the like), of a tumour cell, of an allergenic source, or combinations thereof, and, in case it is an extract, it may comprise one or more epitopes capable of stimulating the immune system of an organism to generate an antigen-specific cell or humoral response.
  • the composition of the invention is formed by conjugates which contain different peptide or protein antigens derived from the same source, said source being selected from the group consisting of any antigenic entity as defined above, including bacteria, a virus, a fungus, a protozoan, a tumour cell, an allergenic source, or a combination thereof.
  • the composition of the invention is formed by conjugates which contain different peptide or protein antigens derived from the same source, said source being selected from the group consisting of a bacterial extract, a virus extract, a fungal extract, a protozoan extract, a tumour cell extract, an allergenic source extract, or combinations thereof.
  • any of the preferred microorganisms as defined above as suitable antigenic entities can be used as a source for obtaining a mixture of protein or peptide antigens that, when conjugated to the first component of the conjugates, will result in a plurality of conjugates and thereby, in the compositions according to the invention.
  • the microorganism is a pathogenic microorganism.
  • Suitable bacteria that can be used as source of the protein or peptide antiges forming part of the plurality of conjugates which form the ccompositions of the invention are as defined above as suitable antigenic entities.
  • the source of for obtaining a mixture of protein or peptide antigens that, when conjugated to the first component of the conjugates, will result in a plurality of conjugates and thereby, in the compositions according to the invention is a virus.
  • Suitable viruses that can be used as source of the protein or peptide antiges forming part of the plurality of conjugates which form the ccompositions of the invention are as defined above as suitable antigenic entities.
  • the source of for obtaining a mixture of protein or peptide antigens that, when conjugated to the first component of the conjugates, will result in a plurality of conjugates and thereby, in the compositions according to the invention is a fungus.
  • Suitable fungi that can be used as source of the protein or peptide antiges forming part of the plurality of conjugates which form the ccompositions of the invention are as defined above as suitable antigenic entities.
  • the source of for obtaining a mixture of protein or peptide antigens that, when conjugated to the first component of the conjugates, will result in a plurality of conjugates and thereby, in the compositions according to the invention is a protozoan.
  • Suitable protozoae that can be used as source of the protein or peptide antiges forming part of the plurality of conjugates which form the ccompositions of the invention are as defined above as suitable antigenic entities.
  • the source of for obtaining a mixture of protein or peptide antigens that, when conjugated to the first component of the conjugates, will result in a plurality of conjugates and thereby, in the compositions according to the invention is a tumor cell.
  • Suitable tumor cells that can be used as source of the protein or peptide antiges forming part of the plurality of conjugates which form the ccompositions of the invention are as defined above as suitable antigenic entities.
  • the plurality of protein and peptide antigens forming part of the conjugates of the compositions of the invention 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 plurality of protein and peptide antigens forming part of the conjugates of the compositions of the invention is a tumour cell extract, the term includes tumour cell extracts, tumour cell sonicates, tumour cell hot water extracts and tumour subcellular 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.
  • tumour 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 XI 14, Tween 20 and Tween 80.
  • a tumour 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, Journal of Chromatography, 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 ('Protein Purification. Principles and Practice', Springer Verlag, 1994), and Coligan, J.E. et al. ('Current Protocols in Protein Science', John Wiley and sons, 2000).
  • tumour lysates are also available.
  • Protein Biotechnologies www.proteinbiotechnologies.com
  • Suitable tumour 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 tumour cell.
  • the different conjugates forming part of the composition differ in the nature of the sequence connecting component (i) and the plurality of components (ii).
  • the conjugates forming part of the compositions of the invention are single polypeptide chains which differ from at least part of the other conjugates of the composition in the sequence of the peptide or protein antigens and in the sequence of the region connecting components (i) and (ii).
  • the different conjugates forming part of the composition differ in the nature of the sequence connecting component (i) and in the nature of components (ii).
  • the polypeptide linking components (i) and (ii) may differ among the conjugates.
  • CIRP or the functional variant thereof and the peptide or protein antigen or antigenic entity are connected via a polypeptide comprising SEQ ID NO: 5 and SEQ ID NO: 7 whereas in other conjugates within the same composition CIRP or the functional variant thereof and the peptide or protein antigen or antigenic entity are connected via a polypeptide comprising SEQ ID NO:6 and SEQ ID NO:7.
  • compositions of the invention may also contain a mixture of conjugates wherein part of the conjugates are single polypeptide chains (with the same or different antigenic entities or the same or different linking regions) and part of the conjugates are conjugates wherein components (i) and (ii) are indirectly conjugated (wherein the antigenic entities may be the same or different).
  • the invention relates to 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 polypeptide chain or fusion protein with component (i), regardless of both the relative order and the fact that both components are directly connected or separated by a spacer region, wherein the link between the components of the conjugate (components (i), (ii), and other additional peptide elements) is by peptide bonds.
  • the polynucleotide sequence encoding component (i) of the polynucleotide encoding the conjugate of the invention can be any polynucleotide sequence that encodes CIRP or any functional variant of CIRP according to the embodiments previously described.
  • the polynucleotide according to the invention comprises a polynucleonucleotide sequence that encodes human CIRP, murine CIRP; or a polypeptide selected from the group consisting of any of the isoforms of the human protein defined in the UniProt database with accession numbers Ql 4011-1, Q14011-2 and Q 14011 -3 (integrated into UniProt on the 15 th of July of 1999) as well as any of the predicted isoforms XI and X3 (accessible in the UniProt database under accession numbers: XP 011525970.1 and XP 016881726.1 in the GenPept database, release of 6 th June 2016) and the iso form cold-inducible RNA-binding protein iso form 2 with GenPept accession number NP_001287744.1 (released on the 28 th of August of 2016), the 'unnamed protein product' with GenPept accession number BAG65284.1 (released on the 24 th
  • polynucleotide sequence encoding component (ii) of the polynucleotide encoding the conjugate of the invention can be any polynucleotide sequence that encodes a peptide or protein antigen according to any of the embodiments previously described.
  • the invention in another aspect, relates to a gene construct comprising a polynucleotide encoding the conjugate 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
  • promoters which are tissue-specific include the promoter of the albumin gene (Miyatake et al., Journal of Virology, 1997. 71 :5124-32), the core promoter of hepatitis virus (Sandig et al, Gene Therapy, 1996. 3: 1002-9), the promoter of the alpha-fetoprotein gene (Arbuthnot et al, Human Gene Therapy, 1996. 7: 1503- 14), and the promoter of the globulin-binding protein which binds to thyroxine (Wang, L., et al, Procedures of the National Academy of Sciences, 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 pET (such as pET14b), pUC18, pUC19, Bluescript and their derivatives, mpl8, mpl9, pBR322, pMB9, ColEl, 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
  • 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 ah, 2001, "Molecular cloning, to Laboratory Manual", 2nd ed., Cold Spring Harbor Laboratory Press, N.Y. Vol 1-3 a).
  • 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 genera.
  • Fungal cells preferably include cells of yeasts such as Saccharomyces cereviseae, 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, 293T, REH and MCF-7 and hMSC cells.
  • epithelial cell lines human, ovine, porcine, etc.
  • a further additional aspect of the invention is drawn to a polynucleotide (second polynucleotide of the invention), said polynucleotide encoding a polypeptide comprising:
  • the second polynucleotide of the invention encodes a single-chain polypeptide comprising additional elements such as linkers, spacers or tags.
  • the elements of the single chain polypeptide encoded by the second polypeptide are placed in any relative position from the N-terminal to the C-terminal ends as long as the functional properties of the component (i) are preserved.
  • Another additional embodiment of the invention comprises a construct comprising the aforementioned second polynucleotide.
  • An additional embodiment of the invention comprises a vector comprising the aforementioned second polynucleotide.
  • Yet another embodiment of the invention comprises a cell comprising the aforementioned second polynucleotide or the aforementioned vector.
  • compositions of the invention can be produced as has already been described for the construction and production of the first polynucleotide encoding the conjugate of the invention, the construct, the vector, and the cell comprising said first polynucleotide. 5.
  • Pharmaceutical and veterinary compositions of the invention can be produced as has already been described for the construction and production of the first polynucleotide encoding the conjugate of the invention, the construct, the vector, and the cell comprising said first polynucleotide. 5.
  • Example 6 of the present invention shows that the administration of a conjugate according to the present invention in combination with a TLR agonist is capable of inducing innate and adaptive responses providing better antitumour effects than the combination of the conjugate with a single TLR agonist.
  • the invention relates to a pharmaceutical or veterinary composition, combination, package or kit-of-parts comprising, together or separately:
  • components (i) and (ii) can be formulated as a single preparation, or as separate preparations.
  • pharmaceutical or veterinary composition refers to any composition comprising at least one pharmaceutically active ingredient and at least one other ingredient, as well as any product which results, directly or indirectly, from combination, complexation, or aggregation of any two or more of the ingredients, from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients.
  • pharmaceutical composition as used herein may encompass, inter alia, any composition made by admixing a pharmaceutically active ingredient and one or more pharmaceutically acceptable carriers.
  • kits-of-parts refers to preparations wherein the each of the components or parts is formulated separately but packaged in a single container, optionally together with other components.
  • the molar concentrations of the components forming part of the pharmaceutical or veterinary composition, combination, package or kit-of-parts of the invention can vary, but preferably include ratios of the two components between 50: 1 and 1 :50, more preferably between 20: 1 and 1 :20, between 1 : 10 and 10: 1, between 5: 1 and 1 :5.
  • compositions of the invention has been described in detail in the context of the conjugate of the invention and in the context of the compositions of the invention.
  • said first component comprises the direct conjugate between CIRP or a functionally equivalent variant thereof and the peptide or protein antigen.
  • said first component comprises the indirect conjugate between CIRP or a functionally equivalent variant thereof and the peptide or protein antigen.
  • 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 choice of a pharmaceutically acceptable carrier is determined in principle by the manner in which the inventive vaccine is administered.
  • the pharmaceutical or veterinary composition, combination, package or kit-of-parts of the invention can be administered, for example, systemically or locally.
  • Routes for systemic administration in general include, for example, transdermal, oral, parenteral routes, including subcutaneous, intravenous, intramuscular, intraarterial, intradermal and intraperitoneal injections and/or intranasal administration routes.
  • Routes for local administration in general include, for example, topical administration routes but also intradermal, transdermal, subcutaneous, or intramuscular injections or intralesional, intracranial, intrapulmonal, intracardial, and sublingual injections. More preferably, the pharmaceutical or veterinary composition, combination, package or kit-of-parts may be administered by an intradermal, subcutaneous, or intramuscular route.
  • the pharmaceutical or veterinary composition, combination, package or kit-of-parts are therefore preferably formulated in liquid or solid form.
  • the suitable amount of the pharmaceutical or veterinary composition, combination, package or kit-of-parts to be administered can be determined by routine experiments with animal models. Such models include, without implying any limitation, rabbit, sheep, mouse, rat, dog and non- human primate models.
  • Preferred unit dose forms for injection include sterile solutions of water, physiological saline or mixtures thereof. The pH of such solutions should be adjusted to about 7.4.
  • Suitable carriers for injection include hydrogels, devices for controlled or delayed release, polylactic acid and collagen matrices.
  • Suitable pharmaceutically acceptable carriers for topical application include those which are suitable for use in lotions, creams, gels and the like.
  • the pharmaceutically acceptable carriers for the preparation of unit dose forms which can be used for oral administration are well known in the prior art. The choice thereof will depend on secondary considerations such as taste, costs and storability, which are not critical for the purposes of the present invention, and can be made without difficulty by a person skilled in the art.
  • 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 tox
  • 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.
  • the adjuvant of the pharmaceutical or veterinary composition, combination, package or kit-of-parts of the invention is a TLR ligand.
  • TLR 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 or 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
  • 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.
  • 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 TLR agonist is capable of causing a signalling response through TLR-1.
  • TLR- 1 agonists include tri-acylated lipopeptides (LPs); phenol-soluble modulins; Mycobacterium tuberculosis LP; S-(2,3-bis(palmitoyloxy)-(2-RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser-(S)- Lys(4)-OH, trihydrochloride (Pam3Cys) LP which mimics the acetylated amino terminus of a bacterial lipoprotein and OspA LP from Borrelia burgdoferi.
  • LPs tri-acylated lipopeptides
  • phenol-soluble modulins include S-(2,3-bis(palmitoyloxy)-(2-RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser-(S
  • the TLR agonist is capable of causing a signalling response through TLR-2.
  • TLR-2 agonists include, without limitation, lipopeptides from M. tuberculosis, B. burgdorferi, T.
  • glycoinositolphospholipids from Trypanosoma species glycolipids from Treponema maltophilum
  • porins from Neisseria atyptical LPS from Leptospira species
  • Porphyromonas species lipoarabinomannan from mycobacteria
  • peptidoglycans from Staphylococcus species including Staphylococcus aureus zymosan
  • heat shock proteins HSPs
  • lipoteichoic acid from gram-positive bacteria phenol-soluble modulin from Staphylococcus species
  • mannuronic acids Yersina virulence factors
  • CMV virions measles haemagglutinin, HSP70 and zymosan from yeast and variants thereof.
  • the TLR agonist is capable of causing a signalling response through TLR-3, such as double stranded RNA, polyinosinic-polycytidylic acid (Poly I:C), or Poly ICLC (it consists of carboxymethylcellulose, polyinosinic- polycytidylic acid, and poly-L-lysine double-stranded RNA; available as Hiltonol ® from Oncovir Inc.).
  • a signalling response through TLR-3 such as double stranded RNA, polyinosinic-polycytidylic acid (Poly I:C), or Poly ICLC (it consists of carboxymethylcellulose, polyinosinic- polycytidylic acid, and poly-L-lysine double-stranded RNA; available as Hiltonol ® from Oncovir Inc.).
  • the TLR agonist is capable of causing a signalling response through TLR-4, such as one or more of the EDA domain of fibronectin, a lipopolysaccharide (LPS) from gram-negative bacteria, or fragments thereof; heat shock protein (HSP) 10, 60, 65, 70, 75 or 90; surfactant Protein A, hyaluronan oligosaccharides, heparan sulphate fragments, fibronectin fragments, fibrinogen peptides and b-defensin-2.
  • the TLR agonist is HSP 60, 70 or 90.
  • the TLR agonist capable of causing a signalling response through TLR-4 is a non -toxic derivative of LPS such as monophosphoryl lipid A (MPL) as described by Ribi et al. (1986, Immunology and Immunopharmacology of bacterial endotoxins, Plenum Publ. Corp., NY, p407-419) and having the structure:
  • MPL monophosphoryl lipid A
  • a further detoxified version of MPL results from the removal of the acyl chain from the 3- position of the disaccharide backbone, and is called 3-0-deacylated monophosphoryl lipid A (3D-MPL).
  • the non-toxic derivatives of LPS, or bacterial lipopolysaccharides, which may be used as TLR agonists in the present invention, may be purified and processed from bacterial sources, or alternatively they may be synthetic.
  • purified monophosphoryl lipid A is described in Ribi et al, 1986 (supra)
  • 3-0-Deacylated monophosphoryl or diphosphoryl lipid A derived from Salmonella sp. is described in GB 2220211 and US 4912094.
  • LPS derivatives that may be used as TLR agonists in the present invention are those immunostimulants that are similar in structure to that of LPS or MPL or 3D-MPL.
  • the LPS derivatives may be an acylated monosaccharide, which is a sub-portion to the above structure of MPL.
  • a disaccharide agonist may be a purified or synthetic lipid A of the following formula:
  • R2 may be H or P03H2;
  • R3 may be an acyl chain or 8-hydroxymyristoyl or a 3- acyloxyacyl residue having the formula:
  • TLR agonist is capable of causing a signalling response through TLR-5, such as bacterial flagellin.
  • the TLR agonist is capable of causing a signalling response through TLR-6 such as mycobacterial lipoprotein, di-acylated LP, and phenol- soluble modulin.
  • TLR-6 such as mycobacterial lipoprotein, di-acylated LP, and phenol- soluble modulin.
  • TLR6 agonists are described in W02003043572.
  • the TLR agonist is capable of causing a signalling response through TLR-7 such as loxoribine, a guanosine analogue at positions N7 and C8, or an imidazoquinoline compound, or derivative thereof.
  • TLR-7 such as loxoribine, a guanosine analogue at positions N7 and C8, or an imidazoquinoline compound, or derivative thereof.
  • TLR7 agonists are described in W00285905.
  • the adjuvant does not comprise a TLR7 agonist.
  • the adjuvant does not comprise Imiquimod.
  • the TLR agonist is capable of causing a signalling response through TLR-8 such as an imidazoquinoline molecule with anti-viral activity, for example resiquimod (R848);
  • TLR-8 agonists which may be used include those described in W02004071459 and US20090298863 such as the compound with the formula
  • each Rl is independently H, or a substituted or unsubstituted alkyl, alkenyl, or alkynyl, which may be interrupted by one or more O, S, or N heteroatoms, or a substituted or unsubstituted aryl or heteroaryl;
  • R2 is H, OH, SH, halo, or a substituted or unsubstituted alkyl, alkenyl, or alkynyl, which may be interrupted by one or more O, S, or N heteroatoms, or a substituted or unsubstituted O-(alkyl), O - (aryl), O- (heteroaryl), -S-(alkyl), S-(aryl), S-(heteroaryl), aryl, or heteroaryl;
  • ⁇ Rl is H, or a substituted or unsubstituted alkyl, alkenyl, or alkynyl, which may be interrupted by one or more O, S, or N heteroatoms, or a substituted or unsubstituted aryl or heteroaryl;
  • ⁇ R2 is H, OH, SH, halo, or a substituted or unsubstituted alkyl, alkenyl, or alkynyl, which may be interrupted by one or more O, S, or N heteroatoms, ora substituted or unsubstituted O-(alkyl), O-(aryl), O-(heteroaryl), S-(alkyl), -S- ( ar yl)i > S-(heteroaryl), aryl, or heteroaryl;
  • ⁇ R7 is independently H or a substituted or unsubstituted -C(0)(C1-18 alkyl) or - C(0)2(C1-18 alkyl), -0C02(C1-18 salkyl);
  • ⁇ R8 is H, -OH ,0-(alkyl), -OC02 (CI -18 alkyl), -OC(O) (Cl-18 alkyl), or aracemic, L- or D-amino acid group -OC(0)CHNH2Rl;
  • the TLR agonist is capable of causing a signalling response through TLR-9 such as s DNA containing unmethylated CpG nucleotides, in particular sequence contexts known as CpG motifs.
  • CpG-containing oligonucleotides induce a predominantly Thl response.
  • Such oligonucleotides are well known and are described, for example, in WO 96/02555, WO 99/33488 and U.S. Patent Nos. 6,008,200 and 5,856,462.
  • CpG nucleotides are CpG oligonucleotides.
  • the TLR agonist is capable of causing a signalling response through TLR- 10.
  • the TLR agonist is capable of causing a signalling response through TLR-11 such as Profilin from Toxoplasma gondii.
  • the TLR agonist is capable of causing a signalling response through any combination of two or more of the above TLRs.
  • Figure 6 A shows how the administration of said conjugate with a combination of TLR agonists and CD40 agonist to naive animal models is capable of inducing a higher rate of IFNy production by T cells.
  • the previously mentioned conjugate causes in this mice a potent antitumour activity mediated by CD 8+ T cells against tumours expressing said protein ( Figure 8).
  • the administration of the combination of said fusion protein with TLR agonists and CD40 agonist to a tumour bearing mice induce an innate and adaptive immune response and the tumour growth is completely blocked.
  • the pharmaceutical or veterinary composition, combination, package or kit-of-parts of the invention further comprises a CD40 agonist as an adjuvant.
  • CD40 Agonist refers to a compound that binds to the CD40 receptor and triggers signalling 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 the increase in the expression of more CD40 and TNF receptors in macrophages or to 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. (Journal of Biological Chemistry, 1991.
  • the determination of whether a compound is a CD40 agonist can be carried out for example, in macrophages that expressed CD40 on the membrane.
  • macrophages when a CD40-agonist-bearing-Tcell interacts with the macrophage, the macrophage express more CD40 and TNF receptors on its surface which helps 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 the ones described in US2008286289, US2007292439, US2005136055). It will be understood that the invention also refers to pharmaceutical or veterinary compositions, combinations, packages or kits-of-parts comprising more than one adjuvant and, in particular, to a pharmaceutical or veterinary composition, combination, package or kit-of-parts composition comprising both a TLR agonist and a CD40 agonist. Accordingly in one embodiment, the pharmaceutical or veterinary composition, combination, package or kit-of-parts according to the invention may comprise at least three components, namely:
  • the TLR ligand is selected from the group consisting of a TLR3 agonist, a TLR9 agonist or a combination of both and the CD40 agonist is a CD40 agonistic antibody.
  • the TLR3 ligand is poly(LC) (polyinosinic-polycytidylic acid or polyinosinic-polycytidylic acid sodium salt) and the TLR9 agonist ligand is CpG oligonucleotides.
  • the pharmaceutical or veterinary compositions comprises the conjugate of the invention, a TLR ligand and a CD40 agonist
  • the TLR ligand is not a TLR7 ligand or is not imiquimod.
  • the pharmaceutical or veterinary composition, combination, package or kit-of-parts of the invention further comprise one or more inhibitors of an immunosuppressive molecule.
  • an immunosuppressive molecule molecules that downregulate the response of the immune system through the T cell activation state or the antigen presentation process would be an "immunosuppressive molecule".
  • the immunosuppressive molecule can be either the receptor or the ligand.
  • the activity of these immunosuppressive molecules in the organism may result in an undesirable outcome of the therapy with the conjugates or compositions of the invention, nullifying the immunogenic effect of the treatment. Therefore, it would desirable to inhibit any immunosuppressive molecule.
  • the inhibitors of the immunosuppressive molecule can be an inhibitor of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), an inhibitor of programmed cell death -1 (PD-1), an inhibitor of the ligand of PD-1 (PD-L1), an inhibitor of the IL-10 receptor or a combination thereof.
  • CTLA-4 cytotoxic T lymphocyte-associated antigen 4
  • PD-1 programmed cell death -1
  • PD-L1 inhibitor of the ligand of PD-1
  • the pharmaceutical or veterinary composition, combination, package or kit-of-parts of the invention comprises an inhibitor of PD-1 and an inhibitor of CTLA-4.
  • the pharmaceutical or veterinary composition, combination, package or kit-of-parts of the invention comprises an inhibitor of PD-1 and an inhibitor of IL-10 receptor.
  • an “inhibitor” refers to the inhibitor of an immunosuppressive molecule.
  • Non-limitative examples of such inhibitors would be neutralising antibodies generated against such immunosuppressive molecules.
  • the binding of the antibody to the immunosuppressive molecule would occur in such a way that the immunosuppressive molecule would be unable and trigger its effect.
  • neutralising antibody is any antibody or antigen-binding fragment thereof that binds to an antigen and interferes with the effector ability of said antigen.
  • the neutralizing antibodies used in the preparations of the present invention can bind to the immunosuppressive molecule and are able to inhibit or reduce the immunosuppressive effect of the molecule relative to the immunosuppressive effect in the absence of said antibody(ies) or in the presence of a negative control.
  • Methods for confirming whether an antibody is a neutralising antibody would be based on the determination of the reduction in effector activity of the specific target molecule and would be known by the person skilled in the art.
  • one possible method to assess the neutralising activity an antibody against an immunosuppressive molecule would comprise contacting said antibody with the target molecule, contacting the antibody-molecule complex with antigen-stimulated dendritic cells, quantifying after an appropriate lapse of time the maturation of the dendritic cells, for instance through flow cytometry, and comparing the proportion of mature dendritic cells relative to those treated with the immunosuppressive molecule and the antigen only.
  • the pharmaceutical or veterinary composition, combination, package or kit-of-parts of the invention may also comprise one or more inhibitors of immunosuppressive molecules.
  • the inhibitors are one or more neutralising antibodies.
  • the neutralising antibodies are antibodies selected from the group consisting of anti-IL-lOR (Receptor of IL-10) antibody, an anti- CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-Ll antibody or a combination thereof.
  • the pharmaceutical or veterinary composition, combination, package or kit-of-parts of the invention comprises an anti-PD-1 antibody and an anti- CTLA-4 antibody.
  • the pharmaceutical or veterinary composition, combination, package or kit-of-parts of the invention comprise an anti- PD-1 antibody and an anti-IL-lOR antibody. It will be understood that the invention also refers to compositions comprising at least one adjuvant and at least one inhibitor of an immunsuppressive molecule.
  • the pharmaceutical or veterinary composition, combination, package or kit-of-parts comprises the conjugate of the invention, an adjuvant and an inhibitor of an immunosuppressive molecule
  • the adjuvant is a TLR ligand or a CD40 agonist or a combination thereof.
  • the TLR ligand is selected from the group consisting of a TLR3 agonist, a TLR9 agonist or a combination of both and the CD40 agonist is a CD40 agonistic antibody.
  • the TLR3 ligand is poly(LC) (polyinosinic-polycytidylic acid or polyinosinic-polycytidylic acid sodium salt) and the TLR9 agonist ligand is CpG oligonucleotides.
  • the pharmaceutical or veterinary compositions comprises the conjugate of the invention, a TLR ligand and a CD40 agonist, the TLR ligand is not a TLR7 ligand or is not imiquimod.
  • the inhibitors of the immunosuppressive molecule can be an inhibitor of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), an inhibitor of programmed cell death -1 (PD-1) or an inhibitor of the IL-10 receptor.
  • CTLA-4 cytotoxic T lymphocyte-associated antigen 4
  • PD-1 programmed cell death -1
  • the neutralising antibodies are antibodies selected from the group consisting of anti-IL-lOR antibody, an anti-CTLA-4 antibody, an anti-PD-1 antibody, and anti-PD-Ll antibody.
  • the pharmaceutical or veterinary composition, combination, package or kit-of-parts according to the invention comprises:
  • a TLR agonist preferably a TLR3 agonist, a TLR9 agonist or a combination of both and even more preferably being different from a TLR7 agonist or from imiquimod; and
  • a IL-10 receptor inhibitor preferably an anti-IL-lOR (Receptor of IL-10) antibody.
  • the pharmaceutical or veterinary composition, combination, package or kit-of-parts according to the invention comprises:
  • a TLR agonist preferably a TLR3 agonist, a TLR9 agonist or a combination of both and even more preferably being different from a TLR7 agonist or from imiquimod;
  • CTLA-4 inhibitor preferably a neutralizing anti-CTLA-4 antibody.
  • the pharmaceutical or veterinary composition, combination, package or kit-of-parts according to the invention comprises:
  • a TLR agonist preferably a TLR3 agonist, a TLR9 agonist or a combination of both and even more preferably being different from a TLR7 agonist or from imiquimod;
  • a PD-1 inhibitor preferably an anti-PD-1 neutralizing antibody or an anti-PD- Ll neutralizing antibody.
  • the pharmaceutical or veterinary composition, combination, package or kit-of-parts according to the invention comprises:
  • a CD40 agonist preferably an agonistic anti-CD40 antibody
  • IL-10 receptor inhibitor preferably an anti-IL-lOR (Receptor of IL-10) antibody.
  • the pharmaceutical or veterinary composition, combination, package or kit-of-parts according to the invention comprises:
  • a CD40 agonist preferably an agonistic anti-CD40 antibody
  • compositions, combination, package or kit-of-parts according to the invention comprises:
  • a CD40 agonist preferably an agonistic anti-CD40 antibody
  • a PD-1 inhibitor preferably an anti-PD-1 neutralizing antibody or an anti-PD- Ll neutralizing antibody.
  • the pharmaceutical or veterinary composition, combination, package or kit-of-parts according to the invention comprises:
  • TLR agonist a TLR agonist and a CD40 agonist
  • the TRL agonist is preferably a TLR3 agonist, a TLR9 agonist or a combination of both and even more preferably being different from a TLR7 agonist or from imiquimod and wherein the CD40 agonist is an agonistic anti-CD40 antibody;
  • IL-10 receptor inhibitor preferably an anti-IL-lOR (Receptor of IL-10) antibody.
  • the pharmaceutical or veterinary composition, combination, package or kit-of-parts according to the invention comprises:
  • TLR agonist a TLR agonist and a CD40 agonist
  • the TRL agonist is preferably a TLR3 agonist, a TLR9 agonist or a combination of both and even more preferably being different from a TLR7 agonist or from imiquimod and wherein the CD40 agonist is an agonistic anti-CD40 antibody;
  • CTLA-4 inhibitor preferably a neutralizing anti-CTLA-4 antibody.
  • the pharmaceutical or veterinary composition, combination, package or kit-of-parts according to the invention comprises:
  • TLR agonist a TLR agonist and a CD40 agonist
  • the TRL agonist is preferably a TLR3 agonist, a TLR9 agonist or a combination of both and even more preferably being different from a TLR7 agonist or from imiquimod and wherein the CD40 agonist is an agonistic anti-CD40 antibody;
  • a PD-1 inhibitor preferably an anti-PD-1 neutralizing antibody or an anti-PD- Ll neutralizing antibody.
  • the pharmaceutical or veterinary composition, combination, package or kit-of-parts according to the invention comprises:
  • a PD-1 inhibitor preferably an anti-PD-1 neutralizing antibody or an anti-PD- Ll neutralizing antibody
  • an IL-10 inhibitor preferably a neutralizing anti-IL-lOR antibody.
  • the pharmaceutical or veterinary composition, combination, package or kit-of-parts according to the invention comprises:
  • a PD-1 inhibitor preferably an anti-PD-1 neutralizing antibody or an anti-PD- Ll neutralizing antibody
  • CTLA-4 inhibitor preferably a neutralizing anti-CTLA-4 antibody.
  • 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 cell with a product of the invention (conjugate, pharmaceutical or veterinary composition, combination) 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 peptide or protein antigen, or the antigenic entity and the presentation thereof in the surface associated to the major histocompatibility antigen. These cells thus activated and maturated can be administered to the patient, such that the presentation of the antigens to the immune system of the patient occurs, which eventually results in the generation of an immune response mediated by T cells.
  • 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 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.
  • dendritic cells refers to a diverse population of morphologically similar cell types found in a variety of lymphoid and non-lymphoid tissues (Steinman Annual Review of Immunology, 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.
  • 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- labelled cells, or by using anti-CD83 MAb magnetic beads.
  • a high yield of a relatively homogenous population of DCs can be obtained by treating monocytes present in blood samples or progenitors from 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.
  • 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. (Journal of Experimental Medicine, 1992. 176: 1693-1702).
  • the invention relates to an APC presenting an antigenic entity or at least one peptide or protein antigen which is obtainable by a method comprising
  • the APC is a dendritic cell.
  • 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 CD1 alpha, CD40, CD86, CD54, and CD80, but lose expression of CD14.
  • 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 RII), 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-labelled antibodies directed against one or more of the characteristic cell surface markers noted above, e.g., CD83, HLA- ABC, HLA-DR, CD1 alpha, CD40, and/or CD54.
  • This technique can also be used to distinguish between immature and mature DCs, using fluorochrome-labelled 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 tumour 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 conjugate, vector, composition or kit-of-parts according to the invention.
  • the contacting step comprises the contacting/incubating of the immature APC with the conjugates, with the vectors, with the compositions, or with the components of the kit-of-parts for sufficient time.
  • sensitization may be increased by contacting the APCs with heat shock protein(s) (HSP) non-covalently bound to the composition. It has been demonstrated that HSPs non-covalently bound to antigenic molecules can increase APC sensitization.
  • HSP heat shock protein
  • 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 cells, usually by measuring the incorporation of a labelled nucleotide analogue.
  • 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.
  • Labelling 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 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 veterinary 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).
  • autologous 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 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 response to reactive compositions.
  • the conjugates, compositions, polynucleotides, vectors, cells, pharmaceutical or veterinary composition, or combinations of the invention can be administered to a patient in order to induce an specific immune response against the antigen provided in the conjugate.
  • the administration 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.
  • the conjugates, the compositions, the polynucleotides or vectors, the veterinary or pharmaceutical compositions, or the cells of the invention are administered to a patient in an amount sufficient to elicit an effective CD8+ T cell response to the tumour, virus, bacterial, fungal or protozoan antigen and/or to alleviate, reduce, cure or at least partially arrest symptoms and/or complications from the disease or infection.
  • a therapeutically effective dose 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 quantity of the conjugates, number of copies of the polynucleotides or vectors, amount of the veterinary or pharmaceutical compositions, or of the number of cells of the invention 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, CD8+ T cell 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 invention relates to the conjugate, the composition, the polynucleotide, the vector, the cell, the pharmaceutical or veterinary composition, the combination, the package, the kit-of-parts or the APC of the invention for use in medicine.
  • the invention relates to the conjugate, the composition, the polynucleotide, the vector, the cell, the pharmaceutical or veterinary composition, the combination, the package, the kit-of-parts or the APC of the invention for simultaneous, concurrent, separate or sequential use in combination therapy with an adjuvant and/or one or more inhibitors, preferably one or more antibodies or inhibitors of an immunosuppressive molecule.
  • the invention relates to the conjugate, the composition, the polynucleotide, the construct, the vector, the cell, the pharmaceutical or veterinary composition, the combination, the package, the kit-of-parts, or the APC of the invention wherein the peptide or protein antigen is a tumor antigen or the antigenic entity is a tumor cell, for use in the treatment of a cancer expressing said tumor antigen or tumor antigenic entity.
  • the invention in another aspect, relates to a method for the treatment of cancer expressing a tumor antigen or tumor antigenic entity in a subject in need thereto comprising the administration to the patient of the conjugate, the composition, the polynucleotide, the construct, the composition, the vector, the cell, the pharmaceutical or veterinary composition, the combination, the package, the kit-of-parts, or the APC of the invention, wherein the peptide or protein antigen is a tumor antigen or the antigenic entity is a tumor cell from said cancer.
  • the invention relates to the use of the conjugate, the composition, the polynucleotide, the construct, the vector, the cell, the pharmaceutical or veterinary composition, the combination, the package, the kit-of-parts, or the APC of the invention, wherein the peptide or protein antigen is a tumor antigen or the antigenic entity is a cell expressing a tumor antigen for the manufacture of a medicament for the the treatment of cancer expressing said tumor antigen.
  • the conjugate, the composition, the polynucleotide, the construct, the vector, the cell, the pharmaceutical or veterinary composition, the combination, the package, the kit-of-parts, or the APC of the invention can be used or administered in simultaneous, concurrent, separate, or sequential combination therapy with an adjuvant and/or one or more inhibitors, preferably one or more antibodies or inhibitors of an immunosuppressive molecule.
  • the cancer is preferably selected from the group consisting of Adrenal Cancer, Anal Cancer, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain/CNS, Tumors, Breast Cancer, Cancer of Unknown Primary, Castleman Disease, Cervical Cancer, Colon/Rectum Cancer, Endometrial Cancer, Esophagus Cancer, Ewing Family Of Tumors, Eye Cancer, Gallbladder Cancer, Gastrointestinal Carcinoid Tumors, Gastrointestinal Stromal Tumor (GIST), Gestational Trophoblastic Disease, Hodgkin Disease, Kaposi Sarcoma, Kidney Cancer, Laryngeal and Hypopharyngeal Cancer, Leukemia, Liver Cancer or Hepatocellular carcinoma, Lung Cancer, Lymphoma, Lymphoma of the Skin, Melanoma, Malignant Mesothelioma, Multiple Myeloma, Myelodysplasia Syndrome, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal
  • the cancer is selected from the group consisting of a Thymus cancer, a Colon/Rectal cancer, a melanoma and a hepatocellular carcinoma.
  • the invention relates to the conjugate, the composition, the polynucleotide, the construct, the vector, the cell, the pharmaceutical or veterinary composition, the combination, the package, the kit-of-parts, or the APC of the invention wherein the peptide or protein antigen or antigenic entity is an infectious agent, for use in the treatment of an infectious disease caused by said infectious agent.
  • the invention in another aspect, relates to a method for the treatment of an infectioius disease caused by an infectious agent expressing an antigen or antigenic entity in a subject in need thereto comprising the administration to the patient of the conjugate, the composition, the polynucleotide, the construct, the vector, the cell, the pharmaceutical or veterinary composition, the combination, the package, the kit-of-parts, or the APC of the invention wherein the peptide or protein antigen is an antigen expressed by said infectious agent or wherein the antigenic entity is said infectious agent.
  • the invention relates to the use of the conjugate, the composition, the polynucleotide, the vector, the cell, the pharmaceutical or veterinary composition, the combination, the package, the kit-of-parts, or the APC of the invention wherein the peptide or protein antigen is an antigen from an infectious agent or wherein the antigenic entity is an infectious agent for the manufacture of a medicament for the the treatment an infectious disease caused by said infectious agent.
  • the peptide or protein antigen or the antigenic entity is a viral antigen or antigenic entity and the infectious disease is caused by a virus expressing the viral antigen, and wherein the virus is preferably a virus causing a chronic viral infection.
  • 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.
  • 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.
  • 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- ⁇ , IFN- ⁇ , IL-2, IL-4, IL-6, TNF or other cytokine growth factor, antisense TGF- ⁇ , antisense IL-10, and the like.
  • a biological response modifier including but not limited to the cytokines IFN- ⁇ , IFN- ⁇ , IL-2, IL-4, IL-6, TNF or other cytokine growth factor, antisense TGF- ⁇ , antisense IL-10, and the like.
  • the therapeutic agent is an APC according to the present invention, by means of their capacity to induce CD4 and CD8-mediated cell responses.
  • 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 stimulate an immune response against a target cell. As with cytotoxic T cells, helper T cells are stimulated with the antigen-loaded APCs in vitro or in vivo.
  • 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 have been described previously and therefore, the cells are suitable for the treatment of infectious diseases, allergic diseases or neoplastic diseases.
  • 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 methods of treatment or prevention of the present invention, or the treatments or prevention methods wherein the different products of the present invention are used 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 tumour 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.
  • the DC are pulsed with the compositions of the invention and then used to stimulate peripheral blood lymphocytes or tumour -infiltrating lymphocytes (TIL) in culture and activate CTLs targeted against the antigenic entity that are then infused into the patient.
  • TIL peripheral blood lymphocytes or tumour -infiltrating lymphocytes
  • fibroblasts, and other APCs, or tumour cells are pulsed with the compositions of the invention and used to activate tumour 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 subject, in some cases preceded by the administration of diphenhydramine and hydrocortisone. See, e.g., Korbling et ah, Blood, 1986. 67:529-532 and Haas et ah, Experimental Hematology, 1990. 18:94-98.
  • 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.
  • the invention relates to the use of a conjugate, the composition, the polynucleotide, the construct, the vector, the cell, the pharmaceutical or veterinary composition, the combination, the package, the kit-of-parts, or the APC of the invention for the manufacture of a vaccine or of an immunotherapeutic composition.
  • the invention also refers to the manufactured vaccine or immunotherapeutic composition per se.
  • the term "vaccine” refers to a formulation or preparation which is in a form that is capable of being administered to a vertebrate and which induces an 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 term “vaccine” also encompasses a “cancer vaccine” or “tumoral vaccine”, which is intended to prevent cancer from developing in healthy people, and/or to treat and/or inhibit the progression of an existing cancer by strengthening the immune response against the cancer.
  • the immune response generated by the vaccine or immunotherapeutic composition may be a humoral or a cellular immune response.
  • the immune response generated by the vaccine or immunotherapeutic composition is an antigen specific T cell immune response, more preferably a CD4+ T cell immune response, even more preferably a CD8+ T cell immune response.
  • the vaccine or immunotherapeutic composition 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.
  • the invention relates to the use of a conjugate, the composition, the polynucleotide, the construct, the vector, the cell, the pharmaceutical or veterinary composition, the combination, the package, the kit-of-parts, or the APC of the invention for the manufacture of vaccine or immunotherapeutic composition for inducing dendritic cell maturation in vitro or in vivo.
  • the invention relates to the use of a conjugate, the composition, the polynucleotide, the construct, the vector, the cell, the pharmaceutical or veterinary composition, the combination, the package, the kit-of-parts, or the APC of the invention for the manufacture of vaccine or immunotherapeutic composition for stimulating an antigen specific CD4+ or CD8+ T cell 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.
  • humoral immune response is used herein to describe an immune response against foreign antigen(s) that is mediated by antibodies produced by B cells.
  • pET14b plasmids encoding CIRP and CIRP -bound antigens containing a 6x His tail at the N-terminus were purchased from Genscript and used to transform either BL21(DE3) (Novagen) or ClearColi BL21 (DE3) E. coli cells (Lucigen). After overnight induction with 0.4 mM IPTG and bacterial lysis, proteins were harvested from inclusion bodies and resuspended in buffer containing 8 M urea and 20 mM HEPES pH 7.2. They were purified by affinity chromatography (HisTrap; Pharmacia) using a fast protein liquid chromatography platform (AKTA; Pharmacia).
  • endotoxin was removed by extensive washing with buffer UTT (8 M urea, 20 mM HEPES, 0.4% Tween 20, 0.4% Triton X-100, pH 7.2), and then eluted with 500 mM imidazol. The eluted protein was desalted using HiTrap desalting columns (Pharmacia). The levels of endotoxin were always below 10 ng endotoxin/mg of protein as tested by Quantitative Chromogenic Limulus Amebocyte Lysate assay (Lonza). Recombinant endotoxin-free OVA protein (Endograde) was purchase from Hyglos (Germany). Peptide OVA(257- 264) SIINFEKL was purchased from Genecust (Luxemburg) whereas peptide SIIN- CIRP(106-125) was prepared in an Apex 396 automatic synthesizer (Aapptec).
  • mice C57BL/6 and OT-I mice were purchased from Harlan (Barcelona) and Jackson Laboratories, respectively.
  • IFNAR KO C57BL/6-IFN- a/bRo/o mice were obtained from Mathew Albert (Institute Pasteur, Paris, France). Experimental work with mice was conducted according to relevant national and international guidelines, after approval by the institutional review board.
  • HEK293/human TLR4 (hTLR4)-MD2-CD14- or HEK293/LacZ-expressing cells were grown in complete DMEM medium (supplemented with 10% FCS, 2 mM glutamine and 1% Penicillin/Streptomycin) as well as 5 ⁇ g/ml blasticidin and 25 ⁇ hygromycin.
  • E.G7-OVA thymoma cells (ATCC), MC38-OVA colorrectal carcinoma cells (a kind gift of Dr. I Melero; Pamplona, Spain) and B16-OVA melanoma cells were cultured in complete RPMI medium (RPMI 1640 supplemented with 10% FCS, 2 mM glutamine and 1% Penicillin/Streptomycin).
  • Bone marrow-derived DC were generated as described in Zabaleta, A, et al. (Mol Ther, 2008. 16:210-7). Human DC were differentiated from monocytes obtained from buffy coats from the Blood and Tissue Bank of Navarra. Samples were obtained after informed consent and all investigation was conducted according to the principles of the Declaration of Helsinki after approval by the institutional ethical review board. DC were differentiated using a 3-day protocol as described in Dauer, M et al. (J Immunol Methods, 2005. 302: 145-55). In both cases, DC were collected and cultured in 96-well plates (2 x 10 5 cells/well) with different concentrations of CIRP-containing protein, LPS (1 ⁇ g/ml) or left unstimulated. One day later supernatants were harvested to determine cytokine production and DC were analyzed by flow cytometry. In some experiments CIRP-containing proteins were previously digested for 30 min with proteinase K (20 mg/ml).
  • DC migration assays were carried out in 24-well plates with transwell inserts of 5 ⁇ pore size using a Transwell chamber (Costar Corning, Cambridge, MA).
  • DC (2 x 105) harvested one day after CIRP stimulation were cultured in the upper compartment, whereas the lower compartment contained culture medium with or without 30 ng/ml of CCL21 (Peprotech). After 2 hours, inserts were removed and 10 4 fluorochrome- conjugated beads (Cytognos) were added to the lower compartment.
  • Well contents were harvested and analyzed by flow cytometry. The amount of DC corresponding to 1400 collected fluorescent beads was calculated for each well. Results are expressed as chemotactic index, fold increase in the number of migrated cells in the presence vs. in the absence of CCL21 chemokine.
  • Splenic CD8 T cells were purified from OT-I mice by positive selection (Miltenyi) and cultured in 96-well plates (10 4 cells/well) with graded numbers of DC previously incubated for 12 h with antigens (proteins or peptides). After 24 h supernatants were harvested to measure IFN- ⁇ production and cells pulsed overnight with 0.5 ⁇ of tritiated thymidine to determine cell proliferation.
  • HEK293 cells Control HEK293 cells expressing LacZ or hTLR4-MD2-CD14-transduced cells (5 x 10 4 cells/well) were cultured in 96 well plates in the presence of different protein antigens or LPS. As in DC cultures, some experiments contained proteinase K-treated proteins. Next day supematants were harvested and cell activation was determined by measuring IL-8 production.
  • Cytokines produced by murine or human DC (IL-12, TNF-D and IL-10), HEK293 cells (IL-8) or CD8 T cells (IFN- ⁇ ) were determined by ELISA sets (BD-Biosciences).
  • mice were immunized s.c. with equimolar amounts (2 nanomoles) of CIRP-containing protein antigens, OVA protein, peptides or unbound mixtures of CIRP protein and antigenic peptide.
  • mice received i.p. 500 ⁇ g of anti-IL-lOR (1B1.3A; BE0050, BioXcell), 100 ⁇ g of anti-CTLA-4 (9D9; BE0164, BioXcell), 50 ⁇ g of anti-PD-1 (RMP1-14; BE0146, BioXcell) or the corresponding isotype control antibodies (BioXcell) on day 0.
  • mice also received Imiquimod cream (Meda AldaraTM; topical application; 2.5 mg/mouse), poly(LC) (Amersham; 50 ⁇ g/mouse s.c), CpG 1668 (Sigma; 50 ⁇ g/mouse s.c), agonistic FGK45.5 anti-CD40 antibodies (BioXcell; 50 ⁇ g/mouse s.c.) or a multiple adjuvant combination (MAC) as described in Aranda F et al (Cancer Res 2011;71 :3214-24) containing Imiquimod, poly(LC) and anti-CD40.
  • Imiquimod cream Meda AldaraTM; topical application; 2.5 mg/mouse
  • poly(LC) Amersham; 50 ⁇ g/mouse s.c
  • CpG 1668 Sigma
  • agonistic FGK45.5 anti-CD40 antibodies BioXcell; 50 ⁇ g/mouse s.c.
  • MAC multiple adjuvant combination
  • tumour cells E.G7-OVA, MC38-OVA or B16-OVA.
  • tumour diameter was about 5 mm.
  • vaccine was accompanied by three weekly i.p. injections of 100 ⁇ g of anti-PD-1, 500 ⁇ g of anti-IL-lOR plus 100 ⁇ g of anti-PD-1 or the corresponding isotype control antibodies.
  • CIRP Antigen coupling to the MD2/TLR4 binding region of CIRP does not confer immunogenicity CIRP has been described as a TLR4-binding protein which activates macrophages and induces the production of inflammatory cytokines (Qiang, X. et ah, supra). It has been reported that peptides spanning amino acids 100-125 bind the TLR4 partner protein MD2 (Qiang, X. et ah, supra), suggesting that CIRP biological properties may depend on interactions through this region.
  • SIIN-CIRP protein induces DC maturation, cytokine production and improves antigen presentation to T cells
  • CIRP activates MyD88 and TRIF pathways and induces type I IFN-dependent T cell responses
  • TLR4 ligands may signal through MyD88- and/or TRIF-dependent pathways (Yamamoto M, et al, Science, 2003. 301 :640-3, Mata-Haro V, et al, Science, 2007. 316: 1628-32).
  • TRIF TRIF-dependent pathways
  • Vaccination with a CIRP-containing immunogen has CD8-dependent therapeutic anti-tumour effect
  • mice with 5 mm established E.G7-OVA tumours were vaccinated over a three-week period with SIIN-CIRP, CIRP without any co-expressed antigen, untargeted peptide SIIN, or left untreated.
  • SIIN-CIRP -treated mice but not control groups, had a delay in tumour growth, as compared with untreated mice ( Figure 7A).
  • 60% of mice vaccinated with SIIN-CIRP survived after treatment, whereas survival rates in remaining groups ranged around 15-25%.
  • mice were initially immunized with SIIN-CIRP and adjuvants signalling through different DC receptors and activation routes, since it was have previously shown that vaccine efficacy can be improved by combination of different adjuvants (Aranda, F. et al., Cancer Research, 2011. 71 :3214-24). These experiments showed that combination with non-TLR adjuvants (agonistic anti-CD40 antibodies) clearly enhanced CIRP -induced T cell responses.
  • TLR-7 TLR-7
  • TLR3 poly(LC)
  • TLR9 TLR9
  • MAC multiple adjuvant combination
  • Figure 8A Analysis of cytokines produced by DC after CIRP stimulation showed important IL-10 production ( Figure 2E). It was recently demonstrated that blockade of adjuvant-induced IL-10 greatly improves anti -tumour efficacy of therapeutic vaccines (Llopiz, D.
  • mice bearing 5 mm B16- OVA tumours were carried out.
  • the vaccine + PD-1 blockade strategy because anti-PD-1 antibodies do not provide any therapeutic benefit in the B16 model when used as monotherapy, but in combination with the vaccine enhance T cell responses at the priming phase (as shown above), and may also promote T cell functions of tumour in filtrating lymphocytes expressing PD- 1.
  • SIIN-CIRP vaccination in the presence of control antibodies delayed tumour growth, none of the animals survived at the end of the experiment.
  • CIRP induces efficient human DC maturation Homology between human and murine CIRP is above 95% at the amino acid level. Moreover, it has been seen that murine CIRP could activate HEK293 cells expressing human TLR4 ( Figure 2C). Thus, advantage was taken of this high similarity across species and the effect of murine CIRP on human DC was tested. Incubation of human monocyte-derived DC with CIRP induced clear phenotypic upregulation of maturation- associated markers CD86 and CD54 ( Figure 9A). As for murine DC, CIRP -induced cytokine production was also measured. It was again observed that CIRP induced the production of IL-12, TNF-a and IL-10 ( Figure 9B).
  • EDA extra domain A from fibronectin
  • CIRP conjugated to a protein antigen enhances in vivo induction of T cell responses against epitopes in the protein
  • OVA-CIRP conjugate comprising the OVA protein fused to a CIRP sequence at its N-terminal side, preceded by a 6-Histidiin tag.
  • the protein was expressed in bacteria and was purified following the same protocols as in the pervious examples for the purificiaton of other CIRP constructs. Once purified, its immunogenicity was compared with that of protein OVA or that of protein OVA administered together with CIRP with no covalent bound.
  • mice 8 weeks old C57BL6 mice were immunizied with 2 nanomoles of OVA protein alone (OVA), or in addition to 2 or 5 nanomoles of CIRP (+2 CIRP or +10 CIRP) or with 2 nanomoles of the OVA-CIRP conjugate (OVA-CIRP).
  • OVA peptide corresponding to amino acids 257-264 of ovoalbumin
  • SUN to analyze the response to T CD8 cells
  • OVA peptide corresponding to amino acids 323-339 of ovoalbumin
  • ISQ OVA peptide
  • OVA full protein OVA

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Abstract

L'invention concerne de nouvelles compositions immunostimulatrices qui comprennent des conjugués à base d'une protéine CIRP et de polypeptides ou protéines pour la stimulation des cellules présentatrices d'antigène telles que les cellules dendritiques. Les conjugués peuvent être directs (p. ex., protéines de fusion) ou indirects (p. ex., par liaison biotine-avidine). Des compositions, des associations, des préparations et des kits constitués des parties constitutives des différents conjugués selon l'invention, à usage médical ou vétérinaire, sont en outre décrits.
PCT/EP2018/055409 2017-03-06 2018-03-06 Nouvelles compositions immunostimulatrices comprenant une entité protéine de liaison à l'arn inductible à froid (cirp)-antigène pour l'activation des cellules dendritiques WO2018162450A1 (fr)

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WO2020165425A1 (fr) * 2019-02-15 2020-08-20 Abera Bioscience Ab Corps d'inclusion décoré et utilisations associées
CN115010812A (zh) * 2022-04-12 2022-09-06 河南省龙星生物科技有限公司 一种非洲猪瘟抗原介导细胞免疫的多聚体及应用

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