COLD INDUCIBLE RNA-BINDING PROTEIN WITH AN ANTIGEN FOR THE

ACTIVATION OF DENDRITIC CELLS

FIELD OF THE INVENTION 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.

BACKGROUND OF THE INVENTION

Pathogens and cancer remain the leading causes of death worldwide. The development of vaccines to prevent diseases for which no vaccine currently exists, such as AIDS or malaria, or to treat chronic infections or cancers, as well as the improvement of efficacy and safety of existing vaccines, remains a high priority. In most cases, the development of such vaccines requires strategies capable of specifically stimulating CD8+ T-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. Dendritic cells (DC) 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. Moreover, 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. Thus, development of therapeutic strategies based on the use of molecularly defined components is a main goal in tumour immunology.

For these reasons, several vaccination strategies incorporate ligands for pattern recognition receptors (PRRs), agonistic antibodies against co-stimulatory molecules or cytokines able to trigger maturation of DC. However, if a DC encounters the maturation stimuli before seeing the antigen, it will undergo an activation program which will reduce its antigen capture capacity, processing and presentation and will fail to present subsequently encountered antigens. Thus, a strategy of targeting the antigen to the same DC which is receiving the toll-like receptor (TLR) stimulation may have advantages over the use of mixtures of antigens and TLR ligands not associated within the same particle or molecule. Moreover, 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. Interestingly, 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.

It is known in the art that 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).

However, there is still a need of additional strategies for the strengthening the immune response to an antigen. SUMMARY OF THE INVENTION

In a first aspect, the invention relates to a conjugate comprising

(i) CIRP or a functionally equivalent variant thereof conjugated directly or indirectly to;

(ii) 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.

In another aspect, the invention 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.

In another aspect, the invention relates to a veterinary or pharmaceutical composition that comprises the conjugate of the invention and a pharmaceutically acceptable carrier or adjuvant. In a further aspect, 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. In further aspects, 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.

In another aspect, 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

DESCRIPTION OF THE FIGURES

Figure 1. Antigen coupling to the MD2/TLR4 binding region of CIRP does not confer immunogenicity. C57BL/6 mice (n=3/group) were immunized with 50 nmoles of peptide SIINFEKLTEW-CIRP(106-125) and one week later animals were sacrificed, splenocytes stimulated with 1 μg/ml of SIINFEKL or left unstimulated (Neg) and the number of IFN-γ -producing cells measured by ELISPOT.

Figure 2. 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. As controls LPS (1 μg/ml), SIIN-CIRP (2 μΜ) treated with proteinase K or untreated cells were also included. One day later expression of CD86 and CD54 was analyzed by flow cytometry. (E) Supernatants of DC treated as in C were harvested and cytokine content was measured by ELISA. (F) DC untreated or treated with 2 μΜ SIIN-CIRP were incubated in transwell plates and their in vitro migration towards CCL21 was determined 2 h later by flow cytometry. Results are representative of 2-3 independent experiments.

Figure 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.

Figure 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 μg/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-γ released to the supernatants was determined by ELISA. Results are representative of two independent experiments.

Figure 5. Antigen targeting by CIRP enhances in vivo induction of T cell responses. (A) C57BL/6 mice (n=4/group) were immunized with 2 nmoles of SIIN- CIRP, untargeted SIIN or OVA protein. (B) C57BL/6 mice (n=4/group) were immunized with 2 nmoles of SIIN-CIRP or SIIN-ACIRP. (C) C57BL/6 mice (n=4/group) were immunized with 2 nmoles of SIIN-CIRP or a mixture of equimolar amounts of SIIN and CIRP. In all cases, one week later animals were sacrificed, splenocytes stimulated with 1 μg/ml SIIN and the number of IFN-y-producing cells measured by ELISPOT. Values obtained in the absence of stimulation with SIIN were always below 20 SFC for all groups. Results are representative of 2-3 independent experiments.

Figure 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 μΜ), LPS (1 μg/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. (D) C57BL/6 and IFNAR KO mice (n=4/group) were immunized with 2 nmoles of SIIN-CIRP and responses against SUN were determined by ELISPOT. Results are representative of two independent experiments.

Figure 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.

Figure 8. Combination with blockade of immunosuppressive elements and additional adjuvants enhances CIRP-dependent vaccination resulting in higher antitumour effect. (A) C57BL/6 mice (n=4/group) were immunized with 2 nmoles of SIIN-CIRP alone or combined with adjuvants poly(I:C), CpG, anti-CD40 antibodies, Imiquimod or a multiple adjuvant combination (MAC) which contains poly(I:C), Imiquimod and anti-CD40. One week later immune responses against SUN were determined by ELISPOT. (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. (C-E) C57BL/6 mice (n=4/group) were immunized with 2 nmoles of SIIN-CIRP plus i.p injection of isotype control antibody, antibodies against IL-10R, PD-1 or both (C), anti-CTLA-4 (D) or anti-CTLA-4, anti- PD-1 or both antibodies (E). Responses against SUN were measured as above. (F) 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. injections of SIIN-CIRP in combination with weekly administration of isotype control or anti-PD-1 antibodies. Tumour growth and animal survival were monitored. (G-H) B16-OVA tumour -bearing mice were treated as in F combining injections of SIIN-CIRP with isotype control or anti-IL-lOR plus anti-PD-1 antibodies (G) or anti- CTLA-4 plus anti-PD-1 (H). Results are representative of 2 independent experiments. Figure 9. CIRP induces human DC maturation and enhances of T cell activation.

(A) Human monocyte-derived DC from 11 independent donors (Dl to Dl l) were incubated with 2 μΜ CIRP, LPS (1 μ§/ηι1) or left untreated. Next day expression of maturation markers CD86 and CD54 was measured by flow cytometry. (B) Cytokine production by DC corresponding to representative donors #1 to #4 was measured by ELISA. (C) Human DC were differentiated from two individuals and treated as above. They were incubated for four days with T cells from allogeneic donors and T cell proliferation measured by tritiated thymidine incorporation.

Figure 10. Comparison of induced responses between SIINFEKL-CIRP and EDA- SIINFEKL. C57BL/6 mice (n=4/group) were immunized with 2 nmoles of EDA-SIIN or SIIN-CIRP. One week later animals were sacrificed, splenocytes stimulated with 1 μg/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

Figure 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). One week later, 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). Cells without any antigen were cultured as a negative control (Neg). The immune response was then determined by ELISPOT.

DETAILED DESCRIPTION OF THE INVENTION 1. Conjugates of the invention The authors of the present invention have observed that 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. In addition, 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. As observed in Example 2 of the present invention, 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.

Thus, in a first aspect, the invention relates to a conjugate (hereinafter, conjugate of the invention) comprising:

(i) a cold-inducible RNA-binding protein (CIRP) or a functional variant thereof, conjugated directly or indirectly to

(ii) 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.

It will be understood that the terms "peptide bond", "peptide", "polypeptide" and protein are known to the person skilled in the art. From here on, "peptide" and "polypeptide" will be used indistinctly. It will be understood that the term "conjugate", as used herein, 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). In a preferred embodiment, the conjugate is a fusion protein wherein:

(i) the proteins forming part of the conjugate are directly connected and the connection is via a peptide bond or

(ii) 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.

In the specific case that 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.

In some other embodiments, 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.

It will be understood that 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. Thus, in a preferred embodiment, 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.

The terms "Cold-Inducible RNA-binding protein", "CIRP", or "CIRBP" are indistinctly used herein and refer to the molecule resulting from the transcription/translation of the cirp gene and which is capable of specifically binding to Toll-like receptor 4 (TLR4). In one embodiment, 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). In another embodiment, 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). In an additional embodiment, 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).

In yet another embodiment 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). In yet another additional embodiment, 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 on the 18 ^th of January of 2017), 'RNA-binding protein 3 ' with GenPept accession number NP 006734.1 (released on the 7 ^th of October of 2016), 'RNA-binding motif (RNPl, RRM) protein 3' with GenPept accession number AAH06825.1 (released on the 15 ^m of July of 2006), 'RNA binding motif (RNP1 , RRM) protein 3 isoform CRA c' with GenPept accession number EAW50767.1 (released on the 23 ^rd of March of 2015), 'RNA binding motif (RNPl, RRM) protein 3 isoform CRA c' with GenPept accession number EAW50768.1 (released on the 23 ^rd of March of 2015), 'peroxisome proliferator-activated receptor gamma coactivator 1-alpha isoform 2' with GenPept accession number NP 037393.1 (released on the 21 ^st of September of 2016) and 'protein phosphatase 1G' with GenPept accession number NP 817092.1 (released on the 1 ^st of September of 2016). In a preferred embodiment, 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.

The terms "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. In one embodiment, 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%. Additionally, 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).

In another embodiment, 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.

In an additional embodiment, 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. In a further additional embodiment, 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. In another embodiment, 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. In yet another embodiment, 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.

In a preferred embodiment, 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 person skilled in the art will understand that amino acid sequences referred to in this description can be chemically modified, for example, by means of chemical modifications which are physiologically relevant, such as, phosphorylations, acetylations, etc. Thus, as used herein, 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. For example, by way of a simple illustration, 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. In one embodiment, 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).

In an embodiment, 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. Preferably, 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. Assays for determining the capacity of the functionally equivalent variants of CIRP to promote the maturation of dendritic cells are known by a person skilled in the art, such as for example the assay described in Example 2 of the present application based on determining the expression levels of different mature dendritic cell markers such as CD86.

In an embodiment, 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. Preferably, 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.

The person skilled in the art understands that the mutations in the nucleotide sequence encoding CIRP sequences which give rise to conservative substitutions of amino-acids in non-critical positions for the functionality of the protein are evolutively neutral mutations which do not affect its overall structure or its functionality.

1.2.Peptide or protein antigen

Component (ii) of the conjugate of the invention is at least one peptide or protein antigen.

As used herein, the expressions "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. As a result of contacting the antigenic peptide with the suitable cells in a subject, 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. In a preferred embodiment the peptide or protein antigen comprises at least a CD8+ T-cell epitope.

In a preferred non limiting embodiment, 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. 1.2.1. Tumour -associated Antigens

In an embodiment, the peptide or protein antigen of component (ii) of the conjugate may be a tumour -associated peptide or protein antigen. In that case, the tumour - associated peptide or protein antigen may refer to "tumour-specific antigens" and "tumour -associated antigens".

As a person skilled in the art would know, "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:

1. Mutated Oncogenes and Tumour Suppressor Genes-derived antigens

2. Products of Other Mutated Genes

3. Overexpressed or Aberrantly Expressed Cellular Proteins-derived antigens

4. Tumour Antigens Produced by Oncogenic Viruses, including neoantigens 5. Altered Cell Surface Glycoproteins

6. Cell Type-Specific Differentiation Antigens

"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.

Examples of proteins from which the tumour antigens derive 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-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE- Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE-family of tumour antigens (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE- 8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21ras, RCAS1, a- -fetoprotein, E-cadherin, a-catenin,13-catenin, γ-catenin, pl20ctn, gplOOPmel 117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, Connexin 37, Immunoglobuline-idiotype (Ig-idiotype), pl5, gp75, GM2 and GD2 gangliosides, viral products such as human papilloma virus proteins, Smad family of tumour antigens, lmp-1, PI A, EBV-encoded nuclear antigen (EBNA)-l, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-3, SSX-4, SSX-5, SCP-1 and CT- 7, and c-erbB-2, acute lymphoblastic leukemia (etv6, amll, cyclophilin b), B cell lymphoma (Ig-idiotype), glioma (E-cadherin, a-catenin,13-catenin, 7-catenin, pl20ctn), bladder cancer (p21ras), biliary cancer (p21ras), breast cancer (MUC family, HER2/neu, c-erbB-2), cervical carcinoma (p53, p21ras), colon carcinoma (p21ras, HER2/neu, c- erbB-2, MUC family), colorectal cancer (Colorectal associated antigen (CRC)-0017- 1A/GA733, APC), choriocarcinoma (CEA), epithelial cell cancer (cyclophilin b), gastric cancer (HER2/neu, c-erbB-2, ga733 glycoprotein), hepatocellular cancer (GPC- 3, a-fetoprotein), Hodgkins lymphoma (lmp-1, EBNA-1), lung cancer (CEA, MAGE-3, NY-ESO-1), lymphoid cell-derived leukemia (cyclophilin b), melanoma (pi 5 protein, gp75, oncofetal antigen, GM2 and GD2 gangliosides, Melan-A/MART-1, cdc27, MAGE-3, p21ras, gplOOPmel 117), myeloma (MUC family, p21ras), non-small cell lung carcinoma (HER2/neu, c-erbB-2), nasopharyngeal cancer (lmp-1, EBNA-1), ovarian cancer (MUC family, HER2/neu, c-erbB-2), prostate cancer (Prostate Specific Antigen (PSA) and its antigenic epitopes PSA-1, PSA-2, and PSA-3, PSMA, HER2/neu, c-erbB- 2, ga733 glycoprotein), renal cancer (HER2/neu, c-erbB-2), squamous cell cancers of the cervix and esophagus (viral products such as human papilloma virus proteins), testicular cancer (NY-ESO-1), and T cell leukemia (HTLV-1 epitopes).

In another preferred embodiment, 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.

In another preferred embodiment, 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. As used herein, the term "idiotype," refers to an epitope in the hypervariable region of an immunoglobulin. Typically, 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.

1.2.2. Viral Antigens

In another embodiment, the peptide or protein antigen of component (ii) of the conjugate is a viral antigen. As a person skilled in the art would know, "viral antigens" are peptide or protein antigens expressed by the virus host's cells and which form part of the viral particle. In a preferred embodiment, 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.

The term "virus", as used herein, 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 of the virus host.

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 Hepatitis B Surface antigen or Hepatitis core antigen), hepatitis C virus (for example core, El, E2, NS3, NS4 or NS5 antigens), from paramyxoviruses such as Respiratory Syncytial virus (such as F and G proteins or derivatives thereof), from parainfluenza virus, from rubella virus (such as proteins El and E2), measles virus, mumps virus, human papilloma viruses (for example HPV6, 11, 16, 18, eg LI, L2, El, E2, E3, E4, E5, E6, E7), flaviviruses (e.g. Yellow Fever Virus, Dengue Virus, Tick-borne encephalitis virus, Japanese Encephalitis Virus) or Influenza virus cells, such as HA, NP, NA, or M proteins, or combinations thereof), rotavirus antigens (such as VP7sc and other rotaviral components), and the like (see Fundamental Virology, Second Edition, eds. Fields, B. N. and Knipe, D. M. (Raven Press, New York, 1991) for additional examples of viral antigens).

1.2.3. Bacterial Antigens

Still in another embodiment, the peptide or protein antigen of component (ii) of the conjugate is a bacterial antigen.

As a person skilled in the art would know, "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. It will be understood that 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.

The term "bacteria", as used herein, 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 Yersinia.

The invention contemplates the use of bacterial antigens such as antigens from Neisseria spp, including N. gonorrhea and N. meningitidis (transferrin-binding proteins, lactoferrin binding proteins, PilC and adhesins); antigens from Streptococcus pyogenes (such as M proteins or fragments thereof and C5A protease); antigens from Streptococcus agalactiae, Streptococcus mutans; Haemophilus ducreyi; Moraxella spp., including M. catarrhalis, also known as Branhamella catarrhalis (such as high and low molecular weight adhesins and invasins); antigens from Bordetella spp., including B. pertussis, B. parapertussis and B. bronchiseptica (such as pertactin, pertussis toxin or derivatives thereof, filamenteous hemagglutinin, adenylate cyclase, fimbriae); antigens from Mycobacterium spp. , including M. tuberculosis, M. bovis, M. leprae, M. avium, M. paratuberculosis, M. smegmatis; Legionella spp, including L. pneumophila; (for example ESAT6, Antigen 85A, -B or -C, MPT 44, MPT59, MPT45, HSPIO,HSP65, HSP70, HSP 75, HSP90, PPD 19kDa [Rv3763], PPD 38kDa [Rv0934] ); antigens from Escherichia spp., including enterotoxic E. coli (for example colonization factors, heat- labile toxin or derivatives thereof, heat-stable toxin or derivatives thereof), antigens from enterohemorragic E. coli and enteropathogenic E. coli (for example shiga toxin- like toxin or derivatives thereof); antigens from Vibrio spp, including V. 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. difficile (for example 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. 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; 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. 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). 1.2.4. Fungal Antigens

In yet another embodiment, the peptide or protein antigen of component (ii) of the conjugate is a fungal antigen. A person skilled in the art would recognise that "fungal antigens" are peptide or protein antigens expressed by Eukaryotes of the kingdom Fungi, whether unicellular or multicellular. In a preferred embodiment, 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.

The term "fungus", as used herein, 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

In even a further embodiment, the peptide or protein antigen of component (ii) of the conjugate is a protozoan antigen. A person skilled in the art would acknowledge that "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. In a preferred embodiment, 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.

The term "protozoa", as used herein, refers to unicellular eukaryotic individuals of the traditional phylum Protozoa and presently excluded from the present taxonomical classification of the kingdoms Fungi and Plantae. Non- limiting examples of protozoan genera that may be used in the method of the present invention 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. and 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; and 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. cruzi; antigens from Giardia spp., including G. lamblia; antigens from Pneumocystis spp. , including P. carinii; antigens from Trichomonas spp. , including T. vaginalis. It will be observed that 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. Alternatively, any two adjacent segments can be bound by a spacer amino acid or a spacer peptide.

In a preferred embodiment, 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. I an even yet more preferred embodiment the peptide or protein antigen of the conjugate of the invention is not CIRP or TLR4 or MD2 or a fragment derived thereof.

1.2.6. Allergens or environmental antigens

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. 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. herbs of the genera Ambrosia, Artemisia, and Parietaria. Other allergen antigens that may be used include allergens 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). 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.

1.2.7. Antigenic entity

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. Thus in a preferred non-limiting embodiment, 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. In a preferred embodiment, the antigenic entity is a microorganism. As used herein, 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.

In a preferred embodiment, the microorganism is a pathogenic microorganism. "Pathogenic microorganism", as used herein, 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.

In another embodiment, the microorganism is an inactivated microorganism. As used herein, the term inactivated form of a microorganism refers to a dead or inactivated cell of such a microorganism which is no longer capable to form a single colony on a plate specific for said microorganism. The term "inactivated form of the microorganism", as described herein, also encompasses lysates, fractions or extracts of the microorganism.

In a preferred embodiment, 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. 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.

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.

In a preferred embodiment, the bacterin has been obtained by formalin treatment of the bacteria. In those cases wherein the antigenic entity is an attenuated pathogen, 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.

In another preferred embodiment, the antigenic entity is a virus, which can be either an inactivated virus or an attenuated virus. Examples of 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, Marburg virus and influenza virus.

In another preferred embodiment, 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.

In another preferred embodiment, 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. In another preferred embodiment, 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. Other cancer cells that can be used in the present invention include sarcomas, such as osteosarcoma or osteogenic sarcoma (bone), chondrosarcoma (cartilage), leiomyosarcoma (smooth muscle), rhabdomyosarcoma (skeletal muscle), mesothelial sarcoma or mesothelioma (membranous lining of body cavities), fibrosarcoma (fibrous tissue), angiosarcoma or hemangioendothelioma (blood vessels), liposarcoma (adipose tissue), glioma or astrocytoma (neurogenic connective tissue found in the brain), myxosarcoma (primitive embryonic connective tissue), melanoma (melanocytes), an mesenchymous or mixed mesodermal tumour (mixed connective tissue types).

In addition cells from liquid tumour are also susceptible to treatment. A "liquid tumour," which refers to neoplasia that is diffuse in nature, as they do not typically form a solid mass. Particular examples include neoplasia of the reticuloendothelial or hematopoetic system, such as lymphomas, myelomas and leukemias. Non- limiting examples of leukemias include acute and chronic lymphoblastic, myeolblastic and multiple myeloma. Typically, such diseases arise from poorly differentiated acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia. Specific myeloid disorders include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML). 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). 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. In a preferred embodiment, 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. Thus, 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.

The skilled person will appreciate that when the antigenic entity is formed by different types of molecules, only part of the molecules of the antigenic entity may be modified by the second member of the binding pair. In this case, the expression "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 .

1.3.Directly coupled conjugate

In an embodiment of the invention, 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. The term "directly coupled conjugate", as used herein, means that conjugate is provided as a single polypeptide chain comprising component (i) and component (ii).

The term "single polypeptide chain", as used herein 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. In a preferred embodiment component (i) and component (ii) and the optional peptide or polypeptide spacers are linked by peptide bonds, thus forming a "fusion protein". In another preferred embodiment 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.

According to the invention, 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). In this sense, a preferred intermediate amino acid sequence according to the invention would be a hinge region characterized by a structural ductility allowing this movement. In a particular embodiment, 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). It is thus assured that the secondary and tertiary structure of component (i) is not affected by the presence of component (ii) and vice versa. 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. Similarly, the three-dimensional structure of the spacer is preferably linear or substantially linear.

The preferred examples of 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. Preferred examples of 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. The possible examples of 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). Preferably, said linker comprises or consists of amino acid sequence GGGVEGGG (SEQ ID NO: 11).

Alternatively, 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. A person skilled in the art will appreciate that 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. This extends to the positions of the spacers or linkers relative to components (i) and (ii), meaning that they can be placed in any position relative to elements (i) and (ii) as long as the functional properties of components (i) and (ii) are maintained.

Thus, in an embodiment of the conjugate of the invention, the component (ii) of the conjugate is placed or conjugated at the N-terminal end of the component (i). In another embodiment of the conjugate, the component (ii) is placed or conjugated at the C- terminal end of component (i). In an additional embodiment of the conjugate, 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). In a further additional embodiment of the conjugate, 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). In yet another embodiment, the conjugate of the invention comprises at least two conjugated repeats of any of the conjugates embodiments previously described.

The terms "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. In another embodiment of the invention, 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. et al, Procedures of the National Academy of Sciences, 2012. 109(12):E690-7) and comprises two peptides derived from the second immunoglobulin-like collagen adhesin domain (CnaB2) of the Streptococcus pyogenes fibronectin-binding protein. These peptides form an isopeptide amide bond with one another, as stated in Zakeri et al., through an autocatalytic reaction. Several uses of the SpyTag/SpyCatcher system are described in Reddington & Howarth (Reddington, S.C. & Howarth, M., Current Opinion on Chemical Biology, 2015. 29:94-99). The SpyTag polypeptides are described in SEQ ID NO: 5 and SEQ ID NO: 6, while the SpyCatcher polypeptide is described in SEQ ID NO: 7.

Accordingly, in another preferred embodiment, 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 person skilled in the art will appreciate that the conjugates of the invention, when they are connected by the polypeptides of 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:

Component (i) - SpyTag

Component (i) - SpyCatcher

Component (ii) - SpyTag

Component (ii) - SpyCatcher Hence, in one embodiment of the invention, 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. Thus, examples of arrangement of the elements of the conjugate of the invention, always referring to the placement of elements in the N-terminal to C- terminal direction, are:

Component (i) - SpyTag - SpyCatcher - component (ii)

Component (i) - SpyCatcher - SpyTag - component (ii)

Component (ii) - SpyTag - SpyCatcher - component (i)

- Component (ii) - SpyCatcher - SpyTag - component (i) In one embodiment, 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

In another embodiment of the invention, 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). In this embodiment, component (i) is linked to one member of a binding pair and component (ii) is linked to the other member of the biding pair.

The term "first member of a binding pair", as used herein, 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". The first and/or second members of the binding pair can be of a peptide (protein) or non- peptide nature.

Without being bound by theory, it is believed in the art that these kinds of non-covalent bonds result in binding, in part due to complementary shapes or structures of the molecules involved in the binding pair. The term "binding" according to the invention 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. Those skilled in the art can easily determine conditions whereby, in a sample, binding occurs between the affinity binding molecules. In particular, those skilled in the art can easily determine conditions whereby binding between affinity binding molecules that would be considered in the art to be "specific binding" can be made to occur. As understood in the art, such specificity is usually due to the higher affinity between the affinity binding molecules than for other substances and components (e.g., vessel walls, solid supports) in a sample. In certain cases, the specificity might also involve, or might be due to, a significantly more rapid association of affinity binding molecules than with other substances and components in a sample.

The term "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.

As used in the present invention, 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. It is understood that there is high affinity in the binding of two molecules when the complex resulting from said binding has a dissociation constant (¾) 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. In a preferred embodiment of the invention, the biding pair will have a dissociation constant (¾) of less than 10 ^~9 M under physiological conditions. The terms "bond" and "binding", in the case of the indirect conjugate, are used indistinctly to refer to an interaction between two or more entities. In those cases in which two entities are bound to one another, they can be directly bound (for example, by means of covalent bonds, ionic forces, hydrogen bonds, electrostatic interactions, Van der Waals forces or a combination of the above) or they can be indirectly bound, for example, by means of a linker.

In a preferred embodiment, the first member of a binding pair is a biotin-binding molecule. More preferably, the biotin-binding molecule is avidin. As used herein, 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. The term 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. The term streptavidin, as used herein, 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. Preferably, 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.

For convenience, in the instant description, the terms "avidin" and "streptavidin" as used herein are intended to encompass biotin-binding fragments, mutants and core forms of these binding pair members. Avidin and streptavidin are available from commercial suppliers. Moreover, the 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);

Truncated forms of streptavidin and avidin that retain strong binding to biotin (See, e.g. Sano et al, (J Biol Chem 1995; 270:28204-09) (describing core streptavidin variants 16-133 and 14-138) (U.S. Pat. No. 6,022,951);

- Mutants of streptavidin and core forms of streptavidin which retain substantial biotin binding activity or increased biotin binding activity. See Chilcoti et al, Proc Natl Acad Sci USA 1995;92(5): 1754-8; Reznik et al, Nat Biotechnol 1996;14(8): 1007-1011;

Mutants with reduced immunogenicity, such as mutants modified by site- directed mutagenesis to remove potential T cell epitopes or lymphocyte epitopes. See Meyer et al, Protein Sci 2001; 10:491-503;

Mutants of avidin and core forms of avidin which retain substantial biotin binding activity or increased biotin binding activity also may be used. See Hiller et al, J Biochem 1991;278:573-85; Livnah et al Proc Natl Acad Sci USA 1993;90:5076-80;

Variants resulting from the chemical modification of avidin such as those resulting from the complete or partial modification of glycosylation and fragments thereof as well as the completely deglycosylated avidin variant known as neutravidin;

- Avidin mutants as described in WO05047317A1 ;

Avidin- like proteins as described in WO06045891 ;

Recombinant avidin as described in WOO 198349;

Avidin variants as described in WO0027814;

Monomeric streptavidin as described in WO06084388;

- Modified streptavidin dimers such as those described in WO06058226;

The protein with biotin binding capacity as described in WO04018509;

Streptavidin having a higher affinity for biotin as described in WO9840396; The modified streptavidin and avidin molecules as described in WO9640761 ;

The streptavidin mutants as described in W09711183;

The 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).

In a preferred embodiment, the first member of the binding pair comprises the sequence of SEQ ID NO: 14.

The interaction between biotin and its binding partner, avidin or streptavidin, offers several advantages in the context of the present invention. For example, biotin has an extremely high affinity for both streptavidin (10 ^~13 M) and avidin (10 ^~15 M). Additionally, 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. Alternatively, 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. The same applies to the peptide or protein antigen (component (ii)) and the second member of the binding pair: their interaction may be direct or using a linker. The features specified above for linkers in the direct conjugate apply to the linkers of indirect conjugates. A person skilled in the art will appreciate that 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. Thus, non-limitative examples of arrangement of the elements of the indirect conjugate of the invention, always referring to the placement of elements in the N-terminal to C-terminal direction, and wherein the first member of the binding pair would be avidin and the second member of the binding pair would be biotin, are:

Component (i) - avidin— biotin - component (ii)

- Component (ii) - avidin— biotin - component (i)

Component (i) - biotin— avidin - component (ii)

Componene (ii) - biotin— avidin - component (i)

The same would apply, mutatis mutandis, to streptavidin instead of avidin. Further additional aspects of the invention also refer to a second polypeptide that comprises

a) 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;

wherein 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:

a) Component (i) of the conjugate of the invention, as previously described; and b) A polypeptide wherein the aminoacid sequence is selected from the group consisting of:

a. The aminoacid sequences of SEQ ID NO: 5, SEQ ID NO: 6; SEQ ID NO: 7; or b. The aminoacid sequence of the polypeptide of the first binding pair, as previously described;

wherein a) and b) are linked by peptide bond. In an embodiment of the invention, the second single-chain polypeptide comprises additional elements such as linkers, spacers or tags. Also, in a further additional embodiment of the invention, 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.

1.5.Tagging of the conjugate For the purpose of facilitating the isolation and purification of the conjugate of the invention, 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). By way of a non- limiting illustration, 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. Alternatively, 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. Other tag peptides include the Flag peptide and the KT3 epitope. The tag peptide is generally arranged at the amino- or carboxy- terminal end. In a preferred embodiment, the tag peptide is a His tag, more preferably a hexahistidine tag.

2. Methods for obtaining the conjugate of the invention

The conjugate of the invention can be obtained using any method known for a person skilled in the art. It is thus possible to obtain CIRP or its functionally equivalent variant (component (i)) by any standard method. For example, 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. Accordingly, 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. Further details are given below when describing the polynucleotides, vectors and host cells of the invention. Depending on the nature and particular embodiment of the conjugate to be obtained and the nature of their components, once a sufficient amount of the purified component (i) is available, the latter must be conjugated to the first member of the binding pair. The conjugation of said first member of the binding pair to the 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. As understood in the present invention 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. 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).

Another possibility is to conjugate the first member of the binding pair to the component (i) molecule by means of the use of homo- or hetero- bifunctional groups. 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.

In another embodiment, the agent used to conjugate components (i) and (ii) of the conjugates of the invention can be photolytically, chemically, thermically or enzymatically processed. In particular, 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.

In a preferred embodiment, wherein the first member of a binding pair is a compound of a peptide nature (e.g., strep tavidin), including both oligopeptides, peptides and proteins, it is possible to chemically modify 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. Thus, 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.

In order for the conjugates between the CIRP component and the antigenic entity to be formed, 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. In a preferred embodiment, the second member of a binding pair is biotin.

Incorporation of biotin into an antigenic entity may be carried out using biotinylating agents. As used herein a "biotinylating agent" 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. Another example of a preferred 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. Alternatively, 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.

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. To overcome any such problem, 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. Alternatively, 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.

3. Compositions of the invention

In another aspect, the invention 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. Prefereably, 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.

In one embodiment, 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. In one embodiment, 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.

In a preferred non-limiting embodiment, 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. Furthermore, in another non-limiting embodiment, 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. In a preferred embodiment, 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.

In another preferred embodiment, 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.

In another preferred embodiment, 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.

In another preferred embodiment, 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. In another preferred embodiment, 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. Wherein 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.

Wherein 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. Typically, 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. Offering the low-denaturing and net-zero charge characteristics of non-ionic detergents, 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. As an example, 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).

Commercial sources of tumour lysates are also available. For example, Protein Biotechnologies (www.proteinbiotechnologies.com) sells lung, breast, colon, uterine, cervical, ovarian, and stomach tumour lysates.

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. In another embodiment, the different conjugates forming part of the composition differ in the nature of the sequence connecting component (i) and the plurality of components (ii). In another embodiment, 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). In another embodiment, 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).

In the specific case of compositions wherein the conjugates are forming part of a single polypeptide chain, the polypeptide linking components (i) and (ii) may differ among the conjugates. In one embodiment, in some 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.

It will be understood that the 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).

4. Polynucleotides, vectors and host cells of the invention

In another aspect, the invention relates to a polynucleotide encoding a conjugate of the invention. A person skilled in the art will understand that 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. In a preferred embodiment, 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 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), 'R PL' 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 on the 18 ^th of January of 2017), 'RNA-binding protein 3 ' with GenPept accession number NP 006734.1 (released on the 7 ^th of October of 2016), 'RNA-binding motif (RNPl, RRM) protein 3' with GenPept accession number AAH06825.1 (released on the 15 ^th of July of 2006), 'RNA binding motif (RNPl, RRM) protein 3 isoform CRA c' with GenPept accession number EAW50767.1 (released on the 23 ^rd of March of 2015), 'RNA binding motif (RNP1, RRM) protein 3 isoform CRA c' with GenPept accession number EAW50768.1 (released on the 23 ^rd of March of 2015), 'peroxisome proliferator-activated receptor gamma coactivator 1-alpha isoform 2' with GenPept accession number NP 037393.1 (released on the 21 ^st of September of 2016) and 'protein phosphatase 1G' with GenPept accession number NP 817092.1 (released on the 1 ^st of September of 2016). In a more preferred embodiment, the polynucleotide encoding the conjugate of the invention comprises a nucleotide sequence that encodes human CIRP according to SEQ ID NO: 1 or murine CIRP according to SEQ ID NO: 2.

In the same manner, the 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.

In another aspect, the invention 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. A person skilled in the art will understand that the polynucleotides of the invention must access the nucleus of a target tissue and there be transcribed and translated to give rise to the biologically active conjugate.

In principle, any promoter can be used for the gene constructs of the present invention provided that said promoter is compatible with the cells in which the polynucleotide is to be expressed. Thus, 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/Lox system and the promoter of heat shock genes, the regulatable promoters of RNA polymerase II described in WO/2006/135436 as well as tissue-specific promoters.

Other examples of 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. Thus, in another aspect, 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. Thus, suitable vectors according to the present invention 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 vectors (adenoviruses, viruses associated to adenoviruses as well as retroviruses and lentiviruses) as well as non-viral vectors such as pSilencer 4.1-CMV (Ambion), pcDNA3, pcDNA3.1/hyg pHCMV/Zeo, pCR3.1, pEFl/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2, pTRACER- HCMV, pUB6/V5-His, pVAXl, pZeoSV2, pCI, pSVL and pKSV-10, pBPV-1, pML2d and pTDTl. 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. By way of example, 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).

Therefore, in another aspect, 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). In a particular embodiment, 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.

A further additional aspect of the invention is drawn to a polynucleotide (second polynucleotide of the invention), said polynucleotide encoding a polypeptide comprising:

a) Component (i) of the conjugate of the invention, as previously described; and b) A polypeptide, the amino acid sequence of which is selected from the group consisting of:

a. The aminoacid sequences of SEQ ID NO: 5, SEQ ID NO: 6; SEQ ID NO: 7; or

b. The aminoacid sequence of the polypeptide of the first binding pair, as previously described; In additional embodiment of the invention, the second polynucleotide of the invention encodes a single-chain polypeptide comprising additional elements such as linkers, spacers or tags. Also, in a further additional embodiment of the invention, 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.

The previously described second polynucleotide and the construct, the vector, and the cell comprising said second polynucleotide 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

The authors of the present invention have observed that the combination of the conjugate according to the present invention and a Toll-like receptor (TLR) agonist results in the generation of an immune response which is greater than that obtained when each of said components was separately administered. For instance, 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.

Thus, in another aspect the invention relates to a pharmaceutical or veterinary composition, combination, package or kit-of-parts comprising, together or separately:

(i) a conjugate of the invention, a composition according to the invention, a polynucleotide or gene construct of the invention, a vector of the invention, a cell according to the invention and

(ii) at least one pharmacologically acceptable carrier or adjuvant.

As a person skilled in the art understands, components (i) and (ii) can be formulated as a single preparation, or as separate preparations.

The term "pharmaceutical or veterinary composition", as used herein, 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. Accordingly, the term "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. The terms "package" and "kits-of-parts", as used herein, 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. Component (i) of the pharmaceutical or veterinary composition, combination, package or kit-of-parts 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. In a preferred particular embodiment, said first component comprises the direct conjugate between CIRP or a functionally equivalent variant thereof and the peptide or protein antigen. In another preferred embodiment, said first component comprises the indirect conjugate between CIRP or a functionally equivalent variant thereof and the peptide or protein antigen.

The term "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. Preferably, 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, Avridine™, 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 toxin (PT), cholera toxin (CT), E.coli heat- labile toxins (LT1 and LT2), Pseudomonas Endotoxin A and exotoxin, B. 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. aureus, Salmonella toxin, EDIM and mutants of mutant toxins such as CRM- 197, non- toxic mutants of diphtheria toxin; saponins such as ISCOMs (immunostimulating complexes), chemokines, quimiokines and cytokines such as interleukins (IL-I IL-2, IL- 4, IL-5, IL-6, IL-7, IL-8, IL-12, etc), interferons (such as the interferon gamma) macrophage colony stimulating factor (M-CSF), tumour necrosis factor (TNF), defensins 1 or 2, RANTES, MlPl-alpha, and MEP-2, muramyl peptides such as N- acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl- D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- ( -2'-dipalmitoyl-s-n-glycero-3-hydroxyphosphoryloxy)-ethylami ne (MTP-PE) etc; adjuvants derived from the family of CpG molecules, CpG dinucleotides and synthetic oligonucleotides which comprise CpG motifs, and synthetic adjuvants such as PCPP, , alum and the like, aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine, MTP-PE and RIBI, containing three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a squalene emulsion at 2% Tween 80. Other examples of adjuvants include DDA (dimethyl dioctadecyl ammonium bromide) and QuilA.

In a preferred embodiment, the adjuvant of the pharmaceutical or veterinary composition, combination, package or kit-of-parts of the invention is a TLR ligand. 5.1.TLR ligand In the present invention "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.

Toll-like receptors (or TLRs) are a family of type I transmembrane proteins forming part of the innate immune system. In vertebrates they also enable the adaptation of the immune system. TLRs together with interleukin receptors form a superfamily known as the Interleukin- 1 /toll-like receptor superfamily. All the members of this family have in common the domain called the Toll-IL-1 receptor (TIL) domain.

In a particular embodiment, 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.

As the person skilled in the art understands, there is a large variety of immune assays available to detect the activity of agonist ligands and generally ligands of the TLR receptor, such as the in vitro co-stimulation of dendritic cells. Briefly, 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. It is thus possible to detect the agonistic activity of a TLR agonist ligand by means of detecting changes in the expression levels of said proteins in the dendritic cells after being exposed to said ligand, as described for example by Chen X.Z. et al. (Arch Dermatol Res. 2010; 302(1): 57-65).

In one embodiment of the present invention, the TLR agonist is capable of causing a signalling response through TLR-1. Non-limiting examples of 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.

In an alternative embodiment, the TLR agonist is capable of causing a signalling response through TLR-2. Non-limiting examples of TLR-2 agonists include, without limitation, lipopeptides from M. tuberculosis, B. burgdorferi, T. pallidum, glycoinositolphospholipids from Trypanosoma species, glycolipids from Treponema maltophilum, porins from Neisseria, atyptical LPS from Leptospira species, and 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.

In an alternative embodiment, 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.).

In an alternative embodiment, 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. In one embodiment the TLR agonist is HSP 60, 70 or 90. In an alternative embodiment, 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:

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. For example, purified monophosphoryl lipid A is described in Ribi et al, 1986 (supra), and 3-0-Deacylated monophosphoryl or diphosphoryl lipid A derived from Salmonella sp. is described in GB 2220211 and US 4912094. Other purified and synthetic lipopolysaccharides have been described (US 6,005,099 and EP 0 729 473 Bl; Hilgers et al, 1986, International Archives of Allergy Immunology, 79(4):392-6; Hilgers et al, 1987. Immunology. 60(l): 141-6; and EP 0 549 074 Bl). Bacterial Iipopolysaccharide adjuvants may be 3D-MPL and the f3(l-6) glucosamine disaccharides described in US 6,005,099 and EP 0 729 473 Bl . Accordingly, other 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. In another aspect of the present invention 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:

wherein R2 may be H or P03H2; R3 may be an acyl chain or 8-hydroxymyristoyl or a 3- acyloxyacyl residue having the formula:

wherein R4 is

O

i

-C-(OU _x -CH3 and X and Y have a value of 0 up to 20. A yet further non-toxic derivative of LPS, which shares little structural homology with LPS and is purely synthetic is that described in WO 00/00462, the contents of which are fully incorporated herein by reference. In an alternative embodiment, the TLR agonist is capable of causing a signalling response through TLR-5, such as bacterial flagellin.

In an alternative embodiment, the TLR agonist is capable of causing a signalling response through TLR-6 such as mycobacterial lipoprotein, di-acylated LP, and phenol- soluble modulin. Further TLR6 agonists are described in W02003043572.

In an alternative embodiment, 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. Further TLR7 agonists are described in W00285905. In another embodiment, the adjuvant does not comprise a TLR7 agonist. In another embodiment, the adjuvant does not comprise Imiquimod.

In an alternative embodiment, 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); Other TLR-8 agonists which may be used include those described in W02004071459 and US20090298863 such as the compound with the formula

wherein:

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;

or a pharmaceutically acceptable salt thereof; or alternatively is a compound with the formula:

wherein:

^■ 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;

or a pharmaceutically acceptable salt or stereoisomer thereof.

In an alternative embodiment, 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. In one embodiment, CpG nucleotides are CpG oligonucleotides. In an alternative embodiment, the TLR agonist is capable of causing a signalling response through TLR- 10. In an alternative embodiment, the TLR agonist is capable of causing a signalling response through TLR-11 such as Profilin from Toxoplasma gondii.

Alternatively, the TLR agonist is capable of causing a signalling response through any combination of two or more of the above TLRs.

The authors of the present invention have observed that the conjugates of the invention are capable of improving the antitumour response obtained by means of the use of TLR ligands and of CD40 agonists. Specifically, 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. Moreover, 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). Additionally, as it is shown in Example 6, 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.

Thus, in a preferred embodiment, the pharmaceutical or veterinary composition, combination, package or kit-of-parts of the invention further comprises a CD40 agonist as an adjuvant.

The term "CD40 Agonist", as used herein, 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. 26: 17478-17485). Alternatively, the determination of whether a compound is a CD40 agonist can be carried out for example, in macrophages that expressed CD40 on the membrane. In said 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.

In a preferred embodiment, 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:

(i) a conjugate, a polynucleotide, a gene construct, a vector or a host cell according to the invention;

(ii) at least a TLR ligand; and

(iii) a CD40 agonist. In a preferred embodiment, wherein the pharmaceutical or veterinary composition, combination, package or kit-of-parts comprises the conjugate of the invention, a TLR ligand and a CD40 agonist, 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. In a more preferred embodiment, the TLR3 ligand is poly(LC) (polyinosinic-polycytidylic acid or polyinosinic-polycytidylic acid sodium salt) and the TLR9 agonist ligand is CpG oligonucleotides. In yet another particular embodiment, wherein 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.

In another embodiment, the pharmaceutical or veterinary composition, combination, package or kit-of-parts of the invention further comprise one or more inhibitors of an immunosuppressive molecule. As it is known by the skilled person, 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". Whenever a pair ligand-receptor is responsible for the immunosuppressive effect, the immunosuppressive molecule can be either the receptor or the ligand. In the context of the invention, 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.

In a preferred embodiument, 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. In a preferred embodiment, 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. In another preferred embodiment, 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. Accordingly, in the context of the invention, 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. As non- limitative examples, 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. Therefore, the term "neutralising antibody", as used herein, is any antibody or antigen-binding fragment thereof that binds to an antigen and interferes with the efector ability of said antigen. Typically, 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 efector activity of the specific target molecule and would be known by the person skilled in the art. As a non- limitative example, 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.

Therefore, in an embodiment of the invention, the pharmaceutical or veterinary composition, combination, package or kit-of-parts of the invention may also comprise one or more inhibitors of immunosuppressive molecules. In a preferred embodiment of the invention, the inhibitors are one or more neutralising antibodies. In an even more preferred embodiment of the invention, 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. In an embodiment, 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. In another preferred embodiment, 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.

In a preferred embodiment, wherein 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. In another embodiment, 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. In a more preferred embodiment, the TLR3 ligand is poly(LC) (polyinosinic-polycytidylic acid or polyinosinic-polycytidylic acid sodium salt) and the TLR9 agonist ligand is CpG oligonucleotides. In yet another particular embodiment, wherein 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. In another embodiment, 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. In an even more preferred embodiment of the invention, 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.

Accordingly, in one embodiment, the pharmaceutical or veterinary composition, combination, package or kit-of-parts according to the invention comprises:

(i) a conjugate as defined in the invention;

(ii) 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 (iii) a IL-10 receptor inhibitor, preferably an anti-IL-lOR (Receptor of IL-10) antibody.

In another embodiment, the pharmaceutical or veterinary composition, combination, package or kit-of-parts according to the invention comprises:

(i) a conjugate as defined in the invention;

(ii) 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

(iii) a CTLA-4 inhibitor, preferably a neutralizing anti-CTLA-4 antibody.

In another embodiment, the pharmaceutical or veterinary composition, combination, package or kit-of-parts according to the invention comprises:

(i) a conjugate as defined in the invention;

(ii) 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

(iii) a PD-1 inhibitor, preferably an anti-PD-1 neutralizing antibody or an anti-PD- Ll neutralizing antibody.

In another embodiment, the pharmaceutical or veterinary composition, combination, package or kit-of-parts according to the invention comprises:

(i) a conjugate as defined in the invention;

(ii) a CD40 agonist, preferably an agonistic anti-CD40 antibody; and

(iii) a IL-10 receptor inhibitor, preferably an anti-IL-lOR (Receptor of IL-10) antibody.

In another embodiment, the pharmaceutical or veterinary composition, combination, package or kit-of-parts according to the invention comprises:

(i) a conjugate as defined in the invention;

(ii) a CD40 agonist, preferably an agonistic anti-CD40 antibody; and

(iii) a CTLA-4 inhibitor, preferably a neutralizing anti-CTLA-4 antibody. In another embodiment, the pharmaceutical or veterinary composition, combination, package or kit-of-parts according to the invention comprises:

(i) a conjugate as defined in the invention;

(ii) a CD40 agonist, preferably an agonistic anti-CD40 antibody; and

(iii) a PD-1 inhibitor, preferably an anti-PD-1 neutralizing antibody or an anti-PD- Ll neutralizing antibody.

Accordingly, in one embodiment, the pharmaceutical or veterinary composition, combination, package or kit-of-parts according to the invention comprises:

(i) a conjugate as defined in the invention;

(ii) a TLR agonist and a CD40 agonist, wherein 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; and

(iii) a IL-10 receptor inhibitor, preferably an anti-IL-lOR (Receptor of IL-10) antibody.

In another embodiment, the pharmaceutical or veterinary composition, combination, package or kit-of-parts according to the invention comprises:

(i) a conjugate as defined in the invention;

(ii) a TLR agonist and a CD40 agonist, wherein 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; and

(iii) a CTLA-4 inhibitor, preferably a neutralizing anti-CTLA-4 antibody.

In another embodiment, the pharmaceutical or veterinary composition, combination, package or kit-of-parts according to the invention comprises:

(i) a conjugate as defined in the invention;

(ii) a TLR agonist and a CD40 agonist, wherein 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;

(iii) a PD-1 inhibitor, preferably an anti-PD-1 neutralizing antibody or an anti-PD- Ll neutralizing antibody.

In another embodiment, the pharmaceutical or veterinary composition, combination, package or kit-of-parts according to the invention comprises:

(i) a conjugate as defined in the invention;

(ii) a PD-1 inhibitor, preferably an anti-PD-1 neutralizing antibody or an anti-PD- Ll neutralizing antibody; and

(iii) an IL-10 inhibitor, preferably a neutralizing anti-IL-lOR antibody.

In another embodiment, the pharmaceutical or veterinary composition, combination, package or kit-of-parts according to the invention comprises:

(i) a conjugate as defined in the invention;

(ii) a PD-1 inhibitor, preferably an anti-PD-1 neutralizing antibody or an anti-PD- Ll neutralizing antibody; and

(iii) a CTLA-4 inhibitor, preferably a neutralizing anti-CTLA-4 antibody.

6. Methods for obtaining antigen presenting cells (APC) and APCs obtainable by said method

Another therapeutic approach is to take advantage of the normal role of antigen presenting cells as immune educators. 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.

The term "antigen presenting cell" (APC), as used herein, refers to a class of cells capable of presenting one or more antigens in the form of peptide-MHC complex recognizable by specific effector cells of the immune system, and thereby inducing an effective cellular immune response against the antigen or antigens being presented. 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. In a preferred embodiment, the APC are dendritic cells. Preferably, 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.

The term "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. These cells have to be distinguished from tolerogenic APCs, which are cells that, in the absence of deliberate exposure to maturation signals, can tolerize peripheral CD4+ and CD8+ T cells by inducing deletion, anergy or regulation, depending on the model system studied.

The term "dendritic cells (DCs)" 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. Alternatively, 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. Alternatively, the antigen-presenting cells (APCs), 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). In another aspect, 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

(i) contacting an inmature antigen-presentig cell with the conjugate the invention, the pharmaceutical or veterinary composition, combination, package or kit-of- parts of the invention, the polynucleotide of the invention, the construct of the invention, the vector of the invention or the cell of the invention in conditions suitable for the stimulation and/or the maturation of the APC, and

(ii) recovering the mature APC. In a preferred embodiment, the APC is a dendritic cell. APCs and, in particular, dendritic cells, obtained in this way characteristically express the cell surface marker CD83. In addition, 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.

Optionally, standard techniques, such as morphological observation and immunochemical staining, can be used to verify the presence of APCs and, in particular, dendritic cells. For example, 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.

Once 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. In one preferred approach to culture of APC precursors, 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.

Preferred aspects of 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.

In 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. Typically, 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. In one embodiment, 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.

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.

Once the APC have been sensitized with the antigen, 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. For example, 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. Thus, in further aspects, 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. It will be understood that, for the purposes of medical uses, the cells may originate from the same individual which is to be treated (autologous transplantation) or from a different individual (allogeneic transplantation). In allogeneic transplantation, the donor and recipient are matched based on similarity of HLA antigens in order to minimize the immune response of both donor and recipient cells against each other.

The 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:

- ⁵¹ Cr-release lysis assay (or equivalent) for CTL function,

Assay for cytokines released by T cells upon contacting modified APCs

In vitro T cell education

Transgenic animal models

Proliferation Assays which measures the capacity of T cells to proliferate in response to reactive compositions.

Monitoring TCR Siqnal Transduction Events.

7. Therapeutic uses of the conjugates, polynucleotides, vectors, host cells and dendritic cells of the invention

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. Thus, 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. 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. 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.

Accordingly, in one aspect, 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.

In one aspect, 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. In another aspect, 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.

In another aspect, the invention 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.

In another aspect, 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.

In a particular embodiment of the uses and methods for the treatment of cancer described above, 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.

In a preferred embodiment, 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 Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Oral Cavity and Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer, Pancreatic Cancer, Penile Cancer, Pituitary Tumors, Prostate Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoma - Adult Soft Tissue Cancer, Skin Cancer, Small Intestine Cancer, Stomach Cancer, Testicular Cancer, Thymus Cancer, Thyroid Cancer, Uterine Sarcoma, Vaginal Cancer, Vulvar Cancer, Waldnstrom Macroglobulinemia, and Wilms Tumor.

In a further preferred embodiment, the cancer is selected from the group consisting of a Thymus cancer, a Colon/Rectal cancer, a melanoma and a hepatocellular carcinoma. In another aspect, 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.

In another aspect, the invention 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. In another aspect, 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.

In a preferred embodiment, 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.

For use in medicine or veterinary medicine, the pharmaceutical compositions of the invention can be administered by any route, including, without limitation, oral, intravenous, intramuscular, intrarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal route. A review of the different forms for the administration of active ingredients, of the excipients to be used and of the processes for manufacturing them can be found in Tratado de Farmacia Galenica, C. Fauli i Trillo, Luzan 5, S.A. de Ediciones, 1993 and in Remington ^' s Pharmaceutical Sciences (A.R. Gennaro, Ed.), 20th edition, Williams & Wilkins PA, USA (2000). Examples of pharmaceutically acceptable carriers are known in the state of the art and include phosphate buffered saline (PBS) solutions, water, emulsions, such as oil/water emulsions, different types of wetting agents, sterile solutions, etc. The compositions comprising said carriers can be formulated by conventional methods known in the state of the art.

Formulations suitable for parenteral administration, such as, for example, intravenous 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.

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. After the first treatment, 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. For administration, 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. In some regimens, patients may optionally receive in addition a suitable dosage of a biological response modifier including but not limited to the cytokines IFN-β, IFN-γ, IL-2, IL-4, IL-6, TNF or other cytokine growth factor, antisense TGF-β, antisense IL-10, and the like. In addition, wherein 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. These target cells are typically cancer cells, or pathogen-infected cells which express unique antigenic peptides on their MHC class I surfaces. Similarly, 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.

Generally, 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.

Preferably, 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. The term "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. In preferred embodiments, 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. 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. In one embodiment, the DC (e.g. isolated from the patient or autologous dendritic cells) are pulsed with the compositions of the invention and then injected back into the subject where they present and activate immune cells in vivo. In yet another embodiment, 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. Similarly, 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. Thus, 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.

In the case that the immunogenic cells are used to treat a tumour, 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. As indicated above, 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.

8. Vaccine compositions

In another aspect, 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. In a further aspect, the invention also refers to the manufactured vaccine or immunotherapeutic composition per se. As used herein, 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. As used herein, 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.

As it will be understood, the immune response generated by the vaccine or immunotherapeutic composition may be a humoral or a cellular immune response. In a preferred embodiment, 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.

In another aspect, 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.

In another aspect, 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.

The expression "cellular immune response", is used herein to describe an immune response against foreign antigen(s) that is mediated by T cells and their secretion products. The "humoral immune response", is used herein to describe an immune response against foreign antigen(s) that is mediated by antibodies produced by B cells.

Throughout the description and claims the word "comprise"; and variations of the word, are not intended to exclude other technical features, additives, elements, components, or steps. Furthermore, the word "comprise" encompasses the case of "consisting of. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention.

EXAMPLES

The invention is described herein by way of the following examples which are to be construed as merely illustrative and not limitative of the scope of the invention.

Materials and Methods

Recombinant protein and peptide antigens

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). Before protein elution, 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 (C57BL/6-Tg(TcraTcrb)l lOOMjb/J) mice were purchased from Harlan (Barcelona) and Jackson Laboratories, respectively. IFNAR KO (C57BL/6-IFN- a/bRo/o) 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.

Cell lines

HEK293/human TLR4 (hTLR4)-MD2-CD14- or HEK293/LacZ-expressing cells (Invivogen) were grown in complete DMEM medium (supplemented with 10% FCS, 2 mM glutamine and 1% Penicillin/Streptomycin) as well as 5 μg/ml blasticidin and 25 μ^πιΐ 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 (a kind gift of Dr. G. Kroemer; Paris, France) were cultured in complete RPMI medium (RPMI 1640 supplemented with 10% FCS, 2 mM glutamine and 1% Penicillin/Streptomycin).

Dendritic cell stimulation experiments

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 μg/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).

Flow cytometry

To analyze DC phenotype, murine cells were stained with FITC-labelled anti-CD54, PE-anti-CDl lc, PE-Cy5-anti-CD86 and PE-anti-PD-Ll antibodies, whereas for human DC, FITC-labelled anti-CD86 and PE-labelled anti-CD54 antibodies and their corresponding control isotypes (all from BD-Biosciences; San Diego, CA) were used. After 30 min, cells were washed and surface expression of the different molecules was analyzed by using a FACScanto flow cytometer (BD-Biosciences).

DC migration assays

DC migration assays were carried out in 24-well plates with transwell inserts of 5 μιη 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.

In vitro antigen presentation assays

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-γ production and cells pulsed overnight with 0.5 μθ of tritiated thymidine to determine cell proliferation.

Cell activation experiments with 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.

Analysis by real-time PCR

Total RNA extraction from DC and real-time PCR were performed as described in Aranda F et al (Cancer Res 2011;71 :3214-24), using primers shown in Table 1. Results were normalized according to β-actin. The amount of each transcript was expressed by the formula: 2ACt (ACt = Ct(P-actin)-Ct(gene)).

Cytokine determination by ELISA

Cytokines produced by murine or human DC (IL-12, TNF-D and IL-10), HEK293 cells (IL-8) or CD8 T cells (IFN-γ) were determined by ELISA sets (BD-Biosciences).

Immunization of mice and analysis of T cell responses

C57BL/6 or IFNAR KO 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. In some experiments, mice received i.p. 500 μg of anti-IL-lOR (1B1.3A; BE0050, BioXcell), 100 μg of anti-CTLA-4 (9D9; BE0164, BioXcell), 50 μg of anti-PD-1 (RMP1-14; BE0146, BioXcell) or the corresponding isotype control antibodies (BioXcell) on day 0. In adjuvant combination experiments mice also received Imiquimod cream (Meda Aldara™; topical application; 2.5 mg/mouse), poly(LC) (Amersham; 50 μg/mouse s.c), CpG 1668 (Sigma; 50 μg/mouse s.c), agonistic FGK45.5 anti-CD40 antibodies (BioXcell; 50 μg/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. One week later animals were sacrificed and T cell responses were measured by enumerating IFN-γ- producing cells by ELISPOT as described in Aranda F et al (Cancer Res 2011 ;71 :3214- 24) using a kit from BD-Biosciences. For these experiments splenocytes were stimulated with 1 μg/ml of OVA(257-264) or left unstimulated. Tumour treatment experiments

C57BL/6 mice were injected s.c. with 10 ⁵ tumour cells (E.G7-OVA, MC38-OVA or B16-OVA). One week later, when the tumour diameter was about 5 mm, they were treated for 3 weeks with 2 weekly i.t. injections of CIRP-containing immunogens, CIRP, peptide antigens or PBS. In combination experiments, vaccine was accompanied by three weekly i.p. injections of 100 μg of anti-PD-1, 500 μg of anti-IL-lOR plus 100 μg of anti-PD-1 or the corresponding isotype control antibodies. Tumour volume was calculated according to the formula: V= (length x width2)/2. For depletion experiments, mice received i.p. injections of 200 μg of anti-CD8 (H35.17.2; a kindly gift of Dr. C. Leclerc; Paris, France) or isotype control (BE0088, BioXcell) antibodies on days -1, 0, 1 and 6, being 0 the day when treatment starts. Mice were killed when tumour diameter reached 17 mm. Statistical analysis

Survival curves of animals treated with different protocols were plotted according to the Kaplan-Meier method and were compared using the log-rank test. Immune responses were analyzed using nonparametric Kruskal-Wallis and Mann- Whitney U tests. P<0.05 was taken to represent statistical significance.

EXAMPLE 1

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. In order to study its capacity to target antigens to DC, activate these cells and improve the induction of T cell responses against targeted antigens, the inventors first designed a peptide immunogen containing the MD2/TLR4- binding region of CIRP (amino acids 106-125) fused to amino acids 257-267 (SIINFEKLTEW) from ovalbumin, which includes the T cell epitope OVA(257-264) (SIINFEKL) (recognized by CD8 T cells from H-2 ^b mice) and their corresponding C- terminal three amino-acid flanking region. Mice were immunized with this immunogen and T cell responses against peptide SIINFEKL analyzed by ELISPOT. No T-cell responses were induced by this immunogen (Figure 1).

EXAMPLE 2

SIIN-CIRP protein induces DC maturation, cytokine production and improves antigen presentation to T cells

Since the use of the MD2/TLR4 binding region of CIRP did not confer immunogenicity to SIINFEKL antigen, it was decided to design a fusion protein containing amino acids 254-267 (QLESIINFEKLTEW) from ovalbumin, which includes SIINFEKL and their corresponding three amino-acid flanking regions, linked to the N-terminus of the full sequence of murine CIRP (from now on SIIN-CIRP). A 6-His tail was also added to the N-terminus for protein purification (Figure 2A). Protein was expressed in bacteria and highly purified (Figure 2B) until endotoxin levels were below 10 ng endotoxin/mg of protein. In a first set of experiments, it was analyzed the capacity of the SIIN-CIRP protein to induce TLR4-mediated activation. Experiments carried out in 293 cells confirmed that in those cells transfected with TLR4, IL-8 was induced by SIIN-CIRP and by LPS, whereas in control cells expressing lacZ, neither SIIN-CIRP nor LPS, induced cytokine production (Figure 2C).

Once demonstrated the TLR4 binding capacity of SIIN-CIRP, its effect on murine DC was analyzed. Regarding phenotypic maturation, it was found that SIIN-CIRP and LPS, but not untargeted CD8 epitope or the original antigen OVA, upregulated expression of maturation markers CD86 and CD54 (Figure 2D). Importantly, treatment of the protein with proteinase K completely abolished marker upregulation, indicating that the stimulatory effect observed was due to the protein and not to potential bacterial endotoxin derived from protein production. Functional properties of SIIN-CIRP -treated DC were also studied by measuring cytokine production. Incubation of DC with graded doses of SIIN-CIRP showed a dose- response (in the range of that induced by 1 μg/ml of LPS) in the production of IL-12, TNF-a and IL-10 (Figure 2E), confirming the stimulatory capacity of the protein. Finally, it was also tested in vitro the migratory capacity of SIIN-CIRP -treated DC. As shown in Figure 2F, incubation of DC with SIIN-CIRP greatly increased DC migration.

To demonstrate that linking of SUN peptide to a protein moiety is not sufficient for DC activation, SUN was included in a new construct. Thus it was synthesized as a control protein a truncated version of SIIN-CIRP (SIIN-ACIRP) containing only amino acids 1- 100 from CIRP (Figure 3 A), and lacking the MD2/TLR4 binding region. Activation of TLR4-expressing 293 cells was much lower with SIIN-ACIRP than with SIF bound to full-length CIRP (Figure 3B). Accordingly, incubation of DC with the truncated protein did not induce DC maturation, measured as up-regulation of CD86 (Figure 3C). When DC were stimulated with the truncated SIIN-ACIRP construct, as occurred with phenotypic maturation, no cytokine production was observed (Figure 3D).

After characterization of the DC activating capacity of SIIN-CIRP, we next tested its ability to improve antigen presentation by these cells. To this end, DC pre-incubated in different conditions were co-cultured with OT-I CD8 T cells, specific for SUN peptide. DC pre-incubated with SIIN-CIRP strongly stimulated the proliferation of OT-I T cells in a dose-dependent manner (Figure 4A), as opposed to untreated DC or those mature DC incubated with LPS but without any antigen. Due to the importance of IFN-γ production by CD8 T cells in antitumour responses, we measured the production of this cytokine in equivalent experiments. As in proliferation experiments, a clear production of IFN-γ was induced by DC treated with SIIN-CIRP (Figure 4B). In order to compare SIIN-CIRP with other SIIN-containing antigens, DC incubated with peptide SUN or the whole OVA protein were also included. Interestingly, when DC were pulsed with equimolecular amounts of SUN peptide or OVA, lower or no activation of CD8 responses were observed. All together, these in vitro results indicate that SIIN-CIRP is able to induce DC activation in a TLR4-dependent manner, favouring antigen presentation to T cells. EXAMPLE 3

Antigen targeting by CIRP enhances in vivo induction of T cell responses Once demonstrated the activation and targeting properties of CIRP we next tested the in vivo capacity of SIIN-CIRP to induce T cell responses. Mice received a single immunization with SIIN-CIRP in saline in the absence of any additional adjuvant and one week later CD8 responses induced against SIIN were compared with those induced by immunization with the same molar amounts of untargeted SIIN or the SIIN- containing protein OVA. These experiments showed that while SIIN-CIRP clearly primed SIIN-specific IFN-y-producing cells, untargeted SIIN or OVA did not induce appreciable T cell responses (Figure 5 A). However, coupling of SIIN to the truncated CIRP protein (SIIN-ACIRP construct) led to poor T cell responses (Figure 5B). Finally, to demonstrate that besides the DC-activating effect of CIRP, targeting was also required, equivalent immunization experiments were carried out comparing SIIN-CIRP with SIIN co-administered with a CIRP protein lacking the SIIN moiety. We found that administration of SIIN and CIRP as a single molecule clearly improved in vivo induction of anti-SIIN T cell responses (Figure 5C). In summary, the CIRP -containing immunogen described induces T cell responses due to its DC activating and targeting effects.

EXAMPLE 4

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). Thus, which pathways were activated by SIIN-CIRP in DC were analysed by measuring expression of genes described as representative of each of them. Time course experiments showed that both, genes dependent on MyD88 (Figure 6A, left panels) and TRIF (Figure 6A, right panels), were upregulated upon incubation of DC with SIIN-CIRP. Moreover, since it was found that IFN-beta upregulation by CIRP was maintained in DC, and induction of CD8 responses has been shown to depend in some settings on the production of type I IFN, the IFN dependency of the CIRP-induced T cell responses was tested. It was observed that SIIN-CIRP induced a lower phenotypic maturation (Figure 6B) and cytokine production (Figure 6C) in DC from mice lacking type I IFN receptor. Finally, immunization experiments showed that poorer responses were induced in these animals than in WT mice, suggesting that T-cell responses induced by CIRP -targeted antigens depend on type I IFN signalling pathway (Figure 6D). EXAMPLE 5

Vaccination with a CIRP-containing immunogen has CD8-dependent therapeutic anti-tumour effect

Once demonstrated the in vivo immunogenicity of SIIN-CIRP, its ability as an anti- tumour therapeutic vaccine was tested. Thus, 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. These experiments showed that SIIN-CIRP -treated mice, but not control groups, had a delay in tumour growth, as compared with untreated mice (Figure 7A). Moreover, 60% of mice vaccinated with SIIN-CIRP survived after treatment, whereas survival rates in remaining groups ranged around 15-25%. These results were confirmed in the MC-38- OVA tumour model, demonstrating that SIIN-CIRP was also able to induce rejection of tumours in this setting (Figure 7B). In order to characterize the effector cell subset responsible for the therapeutic antitumour effect vaccination experiments combined with depletion of CD8 cells in mice bearing E.G7-OVA tumour s were carried. As expected by the presence of CD8 T cell epitope SIIN peptide as the only antigen in the vaccine, depletion of CD8 cells completely abolished the therapeutic effect of vaccination (Figure 7C), confirming the essential role of these cells in our vaccination protocol.

EXAMPLE 6 CIRP as a vaccination platform for combination with other immune enhancing strategies

In order to enhance responses induced by CIRP -based vaccines, 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. In the case of TLR-based adjuvants, no enhancement was observed with Imiquimod (TLR7), whereas poly(LC) (TLR3) and CpG oligonucleotides (TLR9), increased SIIN-CIRP-induced responses. Finally, a multiple adjuvant combination (MAC) (Aranda, F. et al, supra) also enhanced CIRP-induced responses (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. et al., Oncoimmunology, 2016;5:el075113). Another relevant immunosuppressive molecule associated with DC activation is PD-L1, which inhibits T cells after engaging PD-1. Analysis of PD-L1 expression in DC showed that CIRP upregulated this molecule (Figure 8B) suggesting that CIRP -based vaccines would benefit from inhibition of the PD-l/PD-Ll pathway. Thus the effect of vaccination combined with single blockade of IL-10 or PD-1 was tested, as well as vaccination plus blockade of both immunosuppressive pathways. As shown in Figure 8C, although blockade of IL-10R or PD-1 enhanced vaccine potency, simultaneous blockade of these molecules in combination with SIIN-CIRP vaccine induced the strongest T cell responses. The other clinically targeted checkpoint is CTLA-4. Thus, it was also tested the efficacy of combined SIIN-CIRP immunization with anti-CTLA-4 antibodies. As occurred with PD-1 or IL-10 blockade, anti-CTLA-4 antibodies enhanced T cell responses induced by the SIIN-CIRP vaccine (Figure 8D). Moreover, combined blockade of PD-1 and CTLA- 4 have shown improved preclinical and clinical results. For these reasons, the combination of SIIN-CIRP vaccine with PD-1 and CTLA-4 blockade was tested. Interestingly, the triple combination not only induced enhanced responses in comparison with vaccination alone, but also improved results obtained using the vaccine plus single PD-1 or CTLA-4 blockade (Figure 8E). In order to test the therapeutic efficacy of combined strategies using CIRP -based vaccines in a more challenging tumour model, experiments in mice bearing 5 mm B16- OVA tumours were carried out. For these experiments it was initially chosen 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. Although SIIN-CIRP vaccination in the presence of control antibodies delayed tumour growth, none of the animals survived at the end of the experiment. However combination of SIIN-CIRP plus PD-1 blockade clearly induced a slower tumour growth, leading to tumour rejection and survival in 30% of mice (Figure 8F). Finally, since vaccination with combined IL-lO/PD-1 blockade gave more potent T cell responses than vaccine + PD-1 blockade, the therapeutic vaccination experiments were repeated using now the triple therapy. As above, SIIN-CIRP vaccination in the presence of control antibodies delayed tumour growth, similarly to administration of IL-10R and PD-1 -blocking antibodies in the absence of vaccine. However, combination of the SIIN- CIRP vaccine with antibodies against PD-1 and IL-10R almost abolished tumour growth during treatment period, which was later strongly delayed, enhancing tumour rejection and long term survival above 40% (Figure 8G). Similar results were obtained for the combination of the SIIN-CIRP vaccine with antibodies against PD-1 and CTLA- 4 , which also almost abolished tumor growth during the treatment period, which was also strongly delayed later, enhancing tumor rejection and long term survival above 80% (Figure 8H).

EXAMPLE 7

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). In additional experiments the T cell stimulatory capacity of CIRP -treated human DC in MLR assays was also tested. It was observed that human DC treated with CIRP stimulated allogeneic T cell proliferation more efficiently than untreated DC, confirming the mature phenotype of these cells (Figure 9C).

EXAMPLE 8

Comparison EDA vs. CIRP

Our group has been working in the development of vaccination strategies using the extra domain A from fibronectin (EDA), and we have shown that conjugation of peptide or protein antigens to EDA enhances antigen immunogenicity. This is due to the TLR4- binding capacity of EDA, which ultimately results in the induction of proinflammatory cytokines (Okamura, Y., et al. Journal of Biological Chemistry, 2001. 276: 10229- 10233). This way, EDA behaves as a targeting vector and immunostimulatory molecule. This effect has been demonstrated when inducing CD8 T cell responses in vivo (Lasarte, J.J. et al., Journal of Immunology, 2007. 178:748-756 (2007)), as well as when using viral antigens (e.g. NS3 protein from hepatitis C virus; Mansilla, C. et al. Journal of Hepatology, 2009. 51 : 520-527) and tumor antigens (E7 protein from human papilomavirus in a model of cervical cancer; Mansilla, C. et al. International Journal of Cancer, 2012. 131 :641-651). In order to compare the adjuvant effect of CIRP and EDA we carried out immunization experiments using constructs including the same antigen (peptide SIINFEKL) in immunogens SIINFEKL-CIRP o de EDA-SIINFEKL. Two weeks after subcutaneous administration of 2 nanomoles of these proteins to C57BL/6 mice, spleen were obtained and lymphocytes wer stimulated with 1 μg/ml of SUN peptide to measure the number of IFN-producing cells. As shown in Figure 10, responses induced by SIIN-CIRP were significantly higher than those induced by EDA- SIIN. EXAMPLE 9

CIRP conjugated to a protein antigen enhances in vivo induction of T cell responses against epitopes in the protein

An OVA-CIRP conjugate was designed, 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. For that purpose, 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). One week later, mice were sacrificed and the spleen cells were stimulated with OVA peptide (corresponding to amino acids 257-264 of ovoalbumin) (SUN; to analyze the response to T CD8 cells) or with OVA peptide (corresponding to amino acids 323-339 of ovoalbumin) (ISQ) or of the full protein OVA (OVA) (to measure the response to CD4 T cells). Cells without any antigen were cultured as a negative control (Neg). 24 hours later, the number of spots corresponding to IFN-γ producing cells was measured by ELISPOT. As shown in Fig. 11, the null or low immunogenicity of OVA protein was not increased upon addition of non- conjugated CIRP at different concentrations. However, the conjugation of OVA to CIRP (OVA-CIRP) provided an immunogenicity which was capable of inducing responses mediated by CD8 and CD4 T cells.