WO2011101332A1

WO2011101332A1 - Compositions based on the fibronectin extracellular domain a for the treatment of melanoma

Info

Publication number: WO2011101332A1
Application number: PCT/EP2011/052186
Authority: WO
Inventors: Fernando Aranda Vega; Juan José LASARTE SAGASTIBELZA; Jesús María PRIETO VALTUEÑA; Pablo Sarobe Ugarriza
Original assignee: Proyecto De Biomedicina Cima, S.L.; Llopiz Khatchikian, Diana Isabel
Priority date: 2010-02-16
Filing date: 2011-02-15
Publication date: 2011-08-25

Abstract

The invention relates to conjugates adequate for the generation of an immune response against melanoma or a metastasis thereof which comprise a melanoma antigenic polypeptide or peptide and the extracellular domain A of fibronectin. The invention relates as well to compositions comprising the conjugate of the invention and one or more adjuvants including poy(I:C), imiquimod and/or anti-CD40. The invention relates as well to methods for the treatment of melanoma or of a metastasis thereof using the conjugates or the compositions of the invention.

Description

COMPOSITIONS BASED ON THE FIBRONECTIN EXTRACELLULAR DOMAIN A FOR THE TREATMENT OF MELANOMA

FIELD OF THE INVENTION

The invention relates to the field of cancer immunotherapy and, more particularly, to methods for the treatment of melanoma by specifically directing an antigen to antigen- presenting cells by the use of a conjugate comprising the antigen and a ligand which binds specifically to said antigen-presenting cells.

BACKGROUND OF THE INVENTION

Melanoma is a malignant tumor originating from melanocytes, the cells that produce the pigment melanin (Balch CM et al (Eds.), Cutaneous melanoma. Quality Medical

15 Publishing 2003 (4th edition)). During the first trimester of fetal life, precursor melanocytes arise in the neural crest (ectoderm). As the fetus develops, these cells migrate to different body areas, such as the skin, uvea, leptomeninges and mucous membranes (e.g. upper oesophagus, vulva, anus). Accordingly, melanoma can arise in all these sites, although cutaneous melanoma is by far the most frequent type of

20 melanoma.

Although it represents approximately 5% to 7% of all skin malignancies, melanoma is the most lethal skin cancer and accounts for about 75% of all deaths from skin tumors: together with its increasing incidence and resistance to conventional antineoplastic 25 therapy, this makes melanoma one of the most challenging issues in oncology (Beddingfield FC, Oncologist 2003, 8: 459-65, Rigel DS et al,. CA Cancer J Clin 2000, 50: 215-36).

Surgery is the mainstay of treatment for localized melanoma. Radiotherapy and 30 systemic chemotherapy have a limited (if any) survival impact on the control of metastatic disease, due to the resistance of melanoma cells to the cytotoxic effects of these conventional antineoplastic agents at the maximum tolerable doses. To some extent, biotherapy (i.e. interferon-a, IFN) appears to improve patients survival under certain clinical circumstances (Kirkwood JM et al, Clin Cancer Res 2004, 10: 1670-7).

Tumor immunotherapy is aimed at inducing immune responses able to destroy tumor cells (Finn et al, N Engl J Med 2008;358:2704-15). In the case of T-cell response, tumors usually behave as poorly immunogenic, because they express low levels of antigens, MHC and co-stimulatory molecules (Smith et al, Proc Natl Acad Sci U S A 1989;86:5557-61), as opposed to the levels found on professional antigen presenting cells such as dendritic cells (DC). Besides the low tumor immunogenicity and an immunosuppressive environment, endogenous danger signals released by tumor cells (Van der Most et al, Cell Death Differ 2008;15: 13-20) are not sufficient to induce DC maturation (Melief et al, Immunity 2008;29:372-83). This activation process, which involves upregulation of co-stimulatory, adhesion and antigen presenting molecules as well as the production of cytokines and chemokines, is necessary for correct T cell activation, and is usually activated by danger signals associated to pathogens, like tolllike receptor (TLR) ligands, or to inflammation (cytokines or surface molecules such as CD40L expressed on activated CD4 T-cells) (Banchereau et al, Nature 1998;392:245- 52). Therefore, to bypass the low tumor immunogenicity and induce T-cell responses, new therapeutic strategies are based on the use of adjuvants (molecules able to induce DC maturation) with or without exogenously added tumor antigens, which will activate DC to properly present tumor antigens to T-cells and trigger their effector functions (Celis et al, Cancer Res 2007;67:7945-7).

Several approaches have been used to induce immune responses able to reject B16 melanoma, based on use of DC pulsed or expressing different types of antigens (Boczkowski et al, J Exp Med 1996;184:465-72) (Ashley et al, J Exp Med 1997;186: 1177-82) (Bellone et al, J Immunol 2000;165:2651-6) or combined with complex immunogens such as recombinant viruses (Tormo et al, Cancer Res 2006;66:5427-35) or virus-like particles (Song et al, Cancer Lett 2007;256:90-100) which contain many DC-activating signals. Characterization of DC-activating ligands has allowed the development of vaccination strategies combining antigens with well-known adjuvant molecules, acting as immuno stimulants and/or targeting vectors, and avoiding thus the use of microorganisms containing undefined adjuvant mixtures (Celis et al, Cancer Res 2007;67:7945-7). These activating molecules have been shown to increase the immuno stimulatory 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 (Ahonen et al, J Exp Med 2004;199:775-84. Epub 2004 Mar 814-16; Napolitani et al., Nat Immunol 2005;6:769-76; Ouyang et al, Biochem Biophys Res Commun 2007;354: 1045-51). Moreover, besides their effects on DC (Scarlett et al, Cancer Res 2009;69:7329-37), it has been also shown that they may act on other cells with anti-tumor effects, including NK cells and T-cells (Ahonen et al, Blood 2008;111 :3116-25). Thus, development of therapeutic strategies based on the use of molecularly defined components is a main goal in tumor immunology.

DESCRIPTION OF THE FIGURES

Figure 1. Immunization with OVA plus poly(I:C) and antiCD40 does not protect mice against the challenge with B16-OVA cells. C57BL/6 mice (n=6 per group) were immunized i.v. with OVA plus poly(I:C) and anti-CD40 or left untreated. Six days later they were challenged s.c. with 5 x 10⁵ B16-OVA tumor cells and evolution of tumor growth was monitored twice a week. Graphs represent the average tumor volume per group of animals studied (A) and survival (B). (C) OVA expression in tumor cells (n= six culture wells) as measured by real time RT-PCR. Results are normalized with actine. (D) Analysis by flow cytometry of tumor cells stained with anti-MHC class I K^b molecules (open histogram) or control isotype (grey histogram).

Figure 2. Immunotherapy based on adjuvant administration has a poor effect of B16-OVA tumor bearing mice. (A) C57BL/6 mice (n=6) were injected s.c. with 10⁵ B16-OVA tumor cells and when tumor diameter reached 5 mm they were treated 5 times in a 20-day period with Imiquimod + antiCD40 or left untreated. Graph represents the average tumor volume per group of animals studied. (B) Na^'ive mice (n=3) were injected with Imiquimod plus antiCD40 and NK cell activation was measured one day later in the spleen as CD69 upregulation in CD3^"NK1.1⁺ cells (filled bars) and as IFN-γ production against the NK-cell sensitive YAC-1 cell line (open bars). (C) Activation of splenic DC in animals shown in B was measured as % of CD86 cells in the CDl lc population. (D) ELISPOT assay measuring recognition of peptide OVA(257-264) and OVA protein by splenocytes from mice tumor-bearing mice (n=3) treated twice two days apart with imiquimod plus antiCD40 or left untreated.

Figure 3. Innate immune responses in B16-OVA-tumor bearing mice after Imiquimod + anti-CD40 administration. C57BL/6 mice (n=3) were injected s.c. with 10⁵ B16-OVA tumor cells and when tumor diameter reached 5 mm they were administered with Imiquimod plus antiCD40 or left untreated. (A) One day later spleen cells were stimulated with YAC-1 cells and IFN-y-producing cells were enumerated in ELISPOT assays. (B) Alternatively, splenocytes were stained with anti-CD 1 lc and anti- CD86 antibodies and the proportion of CD86-expressing DC was analyzed by flow cytometry.

Figure 4. Combination of adjuvants imiquimod plus anti-CD40 with the tumor antigen OVA induces adaptive immunity with better antitumor effects. C57BL/6 mice (n=3) received a single immunization with OVA plus Imiquimod and anti-CD40 or were left untreated. Six days later, they were sacrificed and the number of IFN-γ producing cells after stimulation with OVA antigens was measured by ELISPOT (A). C57BL/6 mice (n=6) were injected s.c. with 10⁵ B 16. OVA tumor cells and when tumor diameter reached 5 mm they were treated every two days during a 20-day period with OVA plus Imiquimod and anti-CD40. Average tumor volume per group of animals studied (B) and survival (C) are represented. Mice (n=2) bearing 5 mm tumors received two administrations of OVA plus imiquimod and anti-CD40 and six days after the first injection their splenocytes were stimulated with OVA antigens and IFN-y-producing cells were measured by ELISPOT (D). Results are representative of two independent experiments. Figure 5. Administration of OVA with poly(I:C) plus anti-CD40 antibodies delays the growth of established B16-OVA tumors. C57BL/6 mice (n=6) were injected s c. with 10⁵ B 16. OVA tumor cells and when tumor diameter reached 5 mm they were treated every two days during a 20-day period with OVA plus poly(I:C) and anti-CD40. Average tumor volume per group of animals studied (A) and survival (B) are represented. (**; p<0.01)

Figure 6. A multiple adjuvant combination plus antigen targeting strategy enhances NK cell activity and induces polyfunctional high-avidity T-cell responses. C57BL/6 mice (n=3) received a single administration of OVA plus Imiquimod and anti- CD40, OVA plus multiple adjuvant combination (MAC; Imiquimod, anti-CD40 and poly(I:C), EDA-OVA plus Imiquimod and anti-CD40 or EDA-OVA plus MAC. One day later they were sacrificed and activation of NK cells was measured as IFN-γ production against YAC-1 cells (A). In equivalent groups, six days after immunization of mice (n=3) the number of IFN-γ producing cells was measured by ELISPOT after stimulation with OVA antigens (B). IFN-γ content, as determined by ELISA, of cell culture supernatants of splenocytes obtained from groups shown in B and stimulated with different concentrations of OVA(257-264) (C). Percentage of CD107⁺IFN- γ⁺ and IFN-Y⁺TNF-y⁺ IL-2⁺ CD8 cells in splenocytes from mice shown in B after stimulation with 1 ng/ml of OVA(257-264) (D). Results are representative of two independent experiments.

Figure 7. Conjugation of OVA antigen to EDA enhances the induction of T-cell responses. C57BL/6 mice (n=3) received a single administration of MAC (Imiquimod, poly(LC) and anti-CD40) plus OVA (MAC + OVA) , MAC plus EDA and free OVA (MAC + EDA + OVA) or MAC plus EDA conjugated to OVA (MAC + EDA-OVA). Six days after immunization the number of IFN-γ producing cells was measured by ELISPOT after stimulation with OVA(257-264) peptide. Figure 8. Therapeutic administration of EDA-OVA+MAC induces tumor rejection. C57BL/6 mice (14-17 per group) were injected s.c. with 10⁵ B16.0VA tumor cells and when tumor diameter reached 5 mm they were treated every two days during a 20-day period with EDA-OVA+MAC or left untreated. Tumor volume (A) and mice survival (B) was monitored twice a week. Results correspond to the sum of two independent experiments. (C) C57BL/6 mice (n=3) received a single administration of OVA plus Imiquimod and anti-CD40 or EDA-OVA plus multiple adjuvant combination (MAC; Imiquimod, anti-CD40 and poly(LC); EDA-OVA + MAC). Six days after immunization the number of IFN-γ producing cells was measured by ELISPOT after stimulation with irradiated B16.F 10 or B16-OVA tumor cells.

Figure 9. Administration of EDA-OVA+MAC to tumor bearing mice induces T- cell responses against different tumor antigens. Mice (n=5-6 per group) which rejected B16-OVA tumors after immunotherapy with EDA-OVA+MAC or na^'ive mice were challenged with 10⁵ B16-OVA or B16.F10 tumor cells 60 days after completing the treatment. Their survival was monitored twice a week (A). Immune responses against tumor antigens were measured by IFN-γ ELISPOT in mice cured after immunotherapy with EDA-OVA+MAC (B) .

Figure 10. Production of TNF-a by THP-1 cells stimulated with EDA-TRP2(59- 257). THP-1 cells were incubated for 15 hours with 1 μΜ EDA-TRP2(59-257), EDA- TRP2(59-257) digested with Proteinase K (prot K), 0, 1 ug/ml LPS or culture medium (Neg). After culture, supematants were harvested and the released TNF-a was measured by ELISA.

Figure 11. Immune response induced after immunization of mice with EDA- TRP2(59-257) plus MAC. C57BL/6 mice (n=3) received a single administration of EDA-TRP2(59-257)+MAC. Six days after immunization the number of IFN-γ producing cells was measured by ELISPOT after stimulation with TRP-2(180-188) (10 μg/ml) peptide or no antigen (A) or with proteins EDA-TRP2(59-257), EDA-OVA or EDA (all at 2 μΜ). Figure 12. Therapeutic effect of EDA-TRP2(59-257) plus MAC administration on mice bearing B16.F10 tumors. C57BL/6 mice (n=10/group) with 4-5 mm B16.F10 tumors were treated for three weeks by biweekly administrations of EDA-TRP2(59- 257) + MAC or left untreated. Tumor growth (panel A) and animal survival (panel B) was monitored every 2-3 days.

DETAILED DESCRIPTION OF THE INVENTION

Conjugate of the invention

The authors of the present invention have observed that a recombinant protein comprising the fibronectin EDA region and a melanoma-associated antigen when administrated to mice with a melanoma, is capable of inducing the tumor rejection in half of treated animals. As observed in Example 3 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) the fibronectin extracellular domain A or a functionally equivalent variant thereof and

(ii) at least one melanoma-associated antigenic protein or peptide or an antigenic fragment of said protein or peptide

wherein components (i) and (ii) are covalently coupled.

The first element of the conjugate is the fibronectin EDA region or a functionally equivalent variant thereof.

The terms "extracellular domain A", "EDA region" or "extra type III" are indistinctly used herein and refer to a region of the fibronectin molecule resulting from the transcription/translation of an exon of the fibronectin gene and which is capable of specifically binding to Toll-like receptors 4 (TLR4). This domain was originally described by Muro A. F. et al ; (J. Cell. Biol., 2003, 162: 149-160). The EDA region may be derived from fibronectin obtained from different species such as human (SwissProt P02751), mouse (SwissProt PI 1276), bovine (SwissProt P07589) or rat (SwissProt P04937).

As used herein, "fibronectin" is understood as a multifunctional high molecular weight glycoprotein present in blood and in the extracellular matrix of tissues. Fibronectin is a dimer formed by two identical polypeptide chains bound by C-terminal disulfide bonds. Each monomer has an approximate molecular weight of 230-250 kDa. Each monomer contains three types of modules: type I, type II and type III. Each of these modules is formed by two anti-parallel β-helices. "Functionally equivalent variant" is understood as all those peptides derived from the EDA sequence by means of modification, insertion and/or deletion of one or more amino acids, provided that the function of binding to TLR4 receptors and of activating dendritic cells is substantially maintained. Functionally equivalent variants are those showing a degree of identity with respect to the fibronectin EDA domain greater than at least 25%, at least 40%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%. 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. Basic local alignment search tool. J Mol Biol. 1990 Oct 5; 215(3):403-10).

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 the fibronectin EDA region, preferably at least one function related to the immune response, in particular, which maintains the capacity to interact with TLR4 and to promote the maturation of dendritic cells. The capacity of the functionally equivalent variant to interact with TLR4 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 the fibronectin EDA region variant to bind to TLR4 can be determined using co-immunoprecipitation experiments, in which the protein of interest (e.g. EDA variant) is isolated with a specific antibody and the molecules which interact with the protein (e.g. TLR4) are subsequently identified by means of a western blot. A yeast two-hybrid assay or electrophoresis assays in native conditions can also be used. The latter methodology is based on the migration of the protein complexes in polyacrylamide gels based on their molecular weight. Given that the migration is also defined by the charge, a solution containing Coomassie blue, conferring a net negative charge to the proteins without denaturing or breaking their interactions with other proteins, is used as cathode buffer. A second denaturing dimension in SDS-PAGE gels allows separating the spots and subsequently identifying the identity of the subunits forming the complex by means of mass spectrometry. Assays for determining the capacity of the functionally equivalent variants of EDA to promote the maturation of dendritic cells are known by a person skilled in the art, such as for example the assay described in Example 3 of the present application based on determining the expression levels of different mature dendritic cell markers such as CD86.

The person skilled in the art understands that the mutations in the nucleotide sequence encoding the EDA domain 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.

In a preferred embodiment, the fibronectin EDA region of the conjugate of the invention corresponds to amino acids 1,631 to 1,721 of human fibronectin as shown in the UniProt database with accession number FINC HUMAN and which corresponds to the polypeptide of sequence SEQ ID NO: 1.

NIDRPKGLAFTDVDVDS IKIAWESPQGQVSRYRVTYSSPEDGIRELFPAPDGEDDTAEL QGLRPGSEYTVSVVALHDDMESQPLIGIQST (SEQ ID NO : 1 ) Component (ii) of the conjugate of the invention is at least one melanoma-associated antigenic protein or peptide or an antigenic fragment of said protein or peptide. As used herein, the expressions "melanoma-associated antigenic protein" and "melanoma-associated antigenic peptide" refer respectively to a protein or peptide molecule that is associated with or specific to a melanoma and which comprises one or more epitopes capable of stimulating the immune system of an organism to generate a antigen-specific cell or humoral response which results in the inhibition of the growth of melanoma tumors or of the metastasis of melanoma tumors. 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. Acceptable clinical toxicity may occur if, for example, a melanoma-associated antigen is expressed at low levels in a non-melanoma cell (defined as any somatic or germ cell other than a melanoma cell, including, but not limited to, melanocytes and other pigment-containing cells) but at higher levels in a melanoma cell. Chemically, an epitope may either be composed of a carbohydrate, a peptide, a fatty acid, a anorganic substance or derivatives thereof and any combinations thereof. If an epitope is a polypeptide, it will usually include at least 3 amino acids, preferably 8 to 50 amino acids, and more preferably between about 10-20 amino acids in the peptide. There is no critical upper limit to the length of the peptide, which could comprise nearly the full length of the polypeptide sequence. Epitopes can be either linear or conformational epitopes. A linear epitope is comprised of a single segment of a primary sequence of a polypeptide chain. Linear epitopes can be contiguous or overlapping. Conformational epitopes are comprised of amino acids brought together by folding of the polypeptide to form a tertiary structure and the amino acids are not necessarily adjacent to one another in the linear sequence.

The melanoma-associated antigenic protein or peptides comprise not only epitopes capable of generating an antibody response, but also cytotoxic T cell determinant, T helper determinants or a combination thereof.

The melanoma-associated antigenic protein or peptide may refer to "melanoma tumor specific antigen" and "Tumor-associated Antigens". As a person skilled in the art would know "tumor-specific antigens" are antigenic proteins expressed by tumor cells which are present only on tumor cells and not on any other cell, thus in a preferred embodiment the melanoma-associated antigen is a "melanoma tumor specific antigen". Tumors also express antigenic proteins which are present on some tumor cells and also some normal cells (hereinafter referred to as a "Tumor- Associated Antigens").

The melanoma-associated antigenic protein or peptide also comprises:

1. Mutated Oncogenes and Tumor Suppressor Genes-derived antigens

2. Products of Other Mutated Genes

3. Overexpressed or Aberrantly Expressed Cellular Proteins-derived antigens

4. Tumor Antigens Produced by Oncogenic Viruses

5. Altered Cell Surface Glycoproteins

6. Cell Type-Specific Differentiation Antigens

"Mutated Oncogenes and Tumor Suppressor Genes-derived antigens" are any antigen from a mutated protein which is produced in a tumor cell, in this case in a melanoma, that has an abnormal structure and that due to mutation can act as a tumor antigen. Such abnormal proteins are produced due to mutation of the concerned gene. Mutation of protoonco genes and tumor suppressors which lead to abnormal protein production are the cause of the tumor. 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 tumor 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, in this case melanocyte lineage-specific antigens. In a preferred embodiment, the melanoma-associated antigenic protein or peptide is a polypeptide encoded by the genes defined in the first column of Tables I, II, III and IV. In a still more preferred embodiment, component (ii) is an antigenic peptide derived from a melanoma-associated polypeptide which is selected from the group consisting of the peptides shown in Tables I, II, III and IV.

Table I. Melanoma Differentiation Antigens

a e : are tumor-spec c antgens

Table III: Tumor antigens resulting from mutations

a e : nt gens over-expresse n tumors

In another particular embodiment, the melanoma-associated antigenic protein is selected from the group consisting of TRPl/gp75, TRP2, Tyrosinase, gplOO (Pmell7), Melan- A/MART- 1, COAl, RAB38/NY-MEL- 1 , a Melanoma Antigen Gene (MAGE) family member, in particular, MAGE-1, -2, -3, -4, -6 or -12), a B Melanoma Antigen (BAGE) family member, a GAGE family member (GAGE-1 to 7, 7b and 8), a LAGE-l/NY- ESO-1 family member, GnTV, CDK4 and catenin. The term a "Melanoma Antigen Gene (MAGE) family member" refers to any antigen showing a certain degree of structural identity to an antigen originally identified in a melanoma cell line which was lysed by a panel of autologous cytotoxic T lymphocytes ("CTLs"). Cells which did not express a MAGE-type antigen were not killed by the CTL, and by selecting these "antigen-loss" variants, six independent antigens were identified. Van den Eynde et al., Int. J. Cancer, 44: 634 (1989). Suitable MAGE family members include, without limitation, MAGE-1, -2, -3, -4, -6, -12, -B5, -B6, -C2, -C3, - D.

The term B Melanoma Antigen (BAGE) family member refers to any antigen encoded by a family of closely related genes originally identified as being specifically expressed in the MZ2-MEL melanoma cell line (Boel et al., Immunity 1995, 2: 167-75). Members of the BAGE family suitable for use in the present invention include, without limitation, BAGE1, BAGE la, BAGE lb, BAGElc, BAGE Id, BAGEle and BAGE If The term "GAGE family member" refers to any antigen encoded by a family of closely related genes originally identified as being specifically expressed in the MZ2-MEL.43 melanoma cell line (Van den Eynde B., et ah, J. Exp. Med., 182: 689-698, 1995). Members of the GAGE family suitable for use in the present invention include, without limitation, GAGE-1, -2, -3, -4, -5, -6, -7, -7b and -8.

The term a LAGE-l/NY-ESO-1 family member refers to any antigen encoded by a family of closely related genes originally identified as being specifically expressed in the LB373-MEL4.0 melanoma cell line but not in normal skin (Lethe et al., Int. J. Cancer, 76, 903-908, 1998). Members of the GAGE family suitable for use in the present invention include, without limitation, LAGE-1 and NY-ESO-1, which appear to result from alternative splicing from the same gene.

In a preferred embodiment, the melanoma-associated antigenic protein is TRP2. The term "TRP2" or "tyrosinase-related protein 2" is used herein to refer to a protein showing specific expression in melanocytes and having DOPAchrome tautomerase activity. TRP2 proteins suitable as melanoma-associated polypeptides include, without limitation, human TRP2 (accession number P40126 in SwissProt build of January, 19^th, 2010), bovine TRP2 (accession number Q95119 in SwissProt build of January, 19^th, 2010), pig (Sus scrofa) TRP2 (accession number Q4R1H1 in SwissProt build of January, 19^th, 2010) and mouse (Mus musculus) TRP2 (accession number P29812 in SwissProt build of January, 19^th, 2010). The human TRP2 corresponds to the polypeptide of sequence SEQ ID NO:336.

1 MSPLWWGFLL SCLGCKILPG AQGQFPRVCM TVDSLVNKEC CPRLGAESAN VCGSQQGRGQ

61 CTEVRADTRP WSGPYILRNQ DDRELWPRKF FHRTCKCTGN FAGYNCGDCK FGWTGPNCER

121 KKPPVIRQNI HSLSPQEREQ FLGALDLAKK RVHPDYVITT QHWLGLLGPN GTQPQFANCS

181 VYDFFVWLHY YSVRDTLLGP GRPYRAIDFS HQGPAFVTWH RYHLLCLERD LQRLIGNESF

241 ALPYWNFATG RNECDVCTDQ LFGAARPDDP TLISRNSRFS SWETVCDSLD DYNHLVTLCN

301 GTYEGLLRRN QMGRNSMKLP TLKDIRDCLS LQKFDNPPFF QNSTFSFRNA LEGFDKADGT

361 LDSQVMSLHN LVHSFLNGTN ALPHSAANDP IFVVLHSFTD AIFDEWMKRF NPPADAWPQE

421 LAPIGHNRMY NMVPFFPPVT NEELFLTSDQ LGYSYAIDLP VSVEETPGWP TTLLWMGTL

481 VALVGLFVLL AFLQYRRLRK GYTPLMETHL SSKRYTEEA [TRP2, SEQ ID NO:336]. In a preferred embodiment, the melanoma-associated antigen is an antigenic fragment of TRP2. In a still more preferred embodiment, said TRP2-derived antigen is selected from the group of SVYDFFVWL (SEQ ID NO: 43), TLDSQVMSL (SEQ ID NO: 44), LLGPGRPYR (SEQ ID NO:45), LLGPGRPYR, (SEQ ID NO:46), ANDPIFVVL (SEQ ID NO: 47), QCTEVRADTRPWSGP (SEQ ID NO: 48) and ALPYWNFATG (SEQ ID NO: 49). In a preferred embodiment, the TRP2-derived antigen corresponds to amino acids 59 to 257 of the TRP2 protein and has the sequence

1 GQCTEVRADT RPWSGPYILR NQDDRELWPR KFFHRTCKCT GNFAGYNCGD CKFGWTGPNC

61 ERKKPPVI RQ NIHSLSPQER EQFLGALDLA KKRVHPDYVI TTQHWLGLLG PNGTQPQFAN 121 CSVYDFFVWL HYYSVRDTLL GPGRPYRAI D FSHQGPAFVT WHRYHLLCLE RDLQRLIGNE

1 81 SFALPYWNFA TGRNECDVC ( SEQ I D NO : 35 6 )

It will be observed that the antigen or the antigens can be the complete protein (for example the TRP2 protein as well as others from the above cited proteins), 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), without being limited to the peptides presented in tables I, II, III and IV. 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 tumor 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.

For the purpose of facilitating the isolation and purification of the fusion protein of the invention, said fusion protein can contain, if desired, an additional peptide which can be used for the purposes of isolating or purifying the fusion protein, such as a tag peptide. Said tag peptide can be located in any position of the fusion protein which does not alter the functionality of any of the polypeptides (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 fusion protein 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 immuno affinity chromatography, such as tag peptides, for example, influenza virus hemagglutinin (HA)-derived epitopes (Field et al, 1988, Mol. Cell. Biol, 8: 2159-2165), C-myc and the antibodies 8F9, 3C7, 6E10, G4, B7 and 9E10 against it (Evan et al, 1985, Molecular and Cellular Biology, 5:3610-3616); the Herpes Simplex virus D (gD) tag protein and the antibodies thereof (Paborsky et al, 1990, Protein Engineering, 3:547-553). Other tag peptides include the Flag peptide (Hopp et al, 1988, BioTechnology, 6: 1204-1210) and the KT3 epitope (Martin et al, 1993, Science, 255: 192-194). 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 an hexahistidine tag.

A person skilled in the art will appreciate that the different elements of the conjugate of the invention can be placed in any order provided that the fibronectin EDA maintains its dendritic cell activating properties and that the melanoma-associated antigenic peptide or protein or epitope thereof maintains the antigenic properties.

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, among others:

- melanoma-associated antigenic protein or peptide - fibronectin EDA,

- fibronectin EDA - melanoma-associated antigenic protein or peptide, Moreover, the invention also contemplates conjugates comprising more than one fibronectin EDA regions as well as more than one melanoma-associated antigenic protein or peptide. These conjugates may also contain a variety of arrangements such as the following which are shown in the N- to C-terminal regions:

- fibronectin EDA - melanoma-associated antigenic protein/peptide- fibronectin

EDA,

- melanoma-associated antigenic protein/peptide - fibronectin EDA - melanoma - associated antigenic peptide,

- repeats of the last two.

In a preferred embodiment, components (i) and (ii) of the conjugate form a single polypeptide chain.

The person skilled in the art will appreciate that the elements forming the conjugates of the invention, i.e., the EDA peptide and at least one melanoma-associated antigenic protein or peptide, can be conjugated directly or, alternatively, they can contain an additional amino acid sequence acting as a linker between said components. According to the invention, said intermediate amino acid sequence acts as a hinge region between said domains, allowing them to move independently from one another while maintaining the three-dimensional form of the individual domains. 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. In a preferred embodiment, said flexible linker is a flexible linker peptide with a length of 20 amino acids or less.

The effect of the linker region is to provide space between the EDA peptide and component (ii). It is thus assured that the secondary structure of the EDA peptide is not affected by the presence of component (ii) and vice versa. The spacer is preferably 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.

In a more preferred embodiment, the linker peptide comprises 2 or more amino acids selected from the group consisting of glycine, serine, alanine and threonine. In a preferred embodiment of the invention, said flexible linker is a polyglycine linker. The possible examples of linker/spacer sequences include SGGTSGSTSGTGST (SEQ ID NO:337), AGSSTGSSTGPGSTT (SEQ ID NO:338) or GGSGGAP (SEQ ID NO:339). 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:340).

The linker can be bound to components flanking the two components of the conjugates of the invention by means of covalent bonds and 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.

The linker can include tetranectin residues 53-56, which in tetranectin forms a β-sheet, and residues 57-59 forming a turn in tetranectin (Nielsen, B.B. et al., FEBS Lett. 412: 388-396, 1997). The sequence of the segment is GTKVHMK (SEQ ID NO:341). This linker has the advantage that when it is present in the native tetranectin, it is binding the trimerization domain with the CRD domain, and therefore it is suitable for connecting the trimerization domain to another domain in general. Furthermore the resulting construct is not expected to be more immunogenic than the construct without a linker. 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: 342) 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:343), the peptide of sequence GAP, the peptide of sequence AAA and the peptide of sequence AAALE (SEQ ID NO:344).

Alternatively, the two components of the conjugates of the invention can be connected by a peptide the sequence of which contains a cleavage target for a protease, thus allowing the separation of the EDA peptide of component (ii). The protease cleavage sites suitable for their incorporation in the polypeptides of the invention include the enterokinase target site (sequence DDDDK, SEQ ID NO:345), factor Xa target site (cleavage site IEDGR, SEQ ID NO:346), thrombin target site (cleavage site LVPRGS, SEQ ID NO:347), protease TEV target site (cleavage site ENLYFQG, SEQ ID NO:348), PreScission protease target site (cleavage site LEVLFQGP, SEQ ID NO:349) and intein target site and the like.

In another preferred embodiment, the conjugate of the invention is the polypeptide referred to herein as M-EDA-Linker-TRP2-Linker-6xHist (SEQ ID NO:350) and having the sequence 1 MNIDRPKGLA FTDVDVDSIK IAWESPQGQV SRYRVTYSSP EDGIRELFPA PDGEDDTAEL

61 QGLRPGSEYT VSVVALHDDM ESQPLIGIQS TAAAMSPLWW GFLLSCLGCK ILPGAQGQFP

121 RVCMTVDSLV NKECCPRLGA ESANVCGSQQ GRGQCTEVRA DTRPWSGPYI LRNQDDRELW

181 PRKFFHRTCK CTGNFAGYNC GDCKFGWTGP NCERKKPPVI RQNIHSLSPQ EREQFLGALD

241 LAKKRVHPDY VITTQHWLGL LGPNGTQPQF ANCSVYDFFV WLHYYSVRDT LLGPGRPYRA

301 IDFSHQGPAF VTWHRYHLLC LERDLQRLIG NESFALPYWN FATGRNECDV CTDQLFGAAR

361 PDDPTLISRN SRFSSWETVC DSLDDYNHLV TLCNGTYEGL LRRNQMGRNS MKLPTLKDIR

421 DCLSLQKFDN PPFFQNSTFS FRNALEGFDK ADGTLDSQVM SLHNLVHSFL NGTNALPHSA

481 ANDPIFWLH SFTDAIFDEW MKRFNPPADA WPQELAPIGH NRMYNMVPFF PPVTNEELFL

541 TSDQLGYSYA IDLPVSVEET PGWPTTLLW MGTLVALVGL FVLLAFLQYR RLRKGYTPLM

601 ETHLSSKRYT EEAAAALEHH HHHH [SEQ ID NO:350].

The polypeptide of SEQ ID NO:350 comprises an N-terminal methionine, the fibronectin EDA (underlined), a trialaline linker, the complete human TRP2 (double underlined), a AAALE linker and an hexahistidine tag.

In another preferred embodiment, the conjugate of the invention is the polypeptide referred to herein as M-EDA-Linker-TRP2(59-257)-Linker-6xHist (SEQ ID NO:351) and having the sequence

1 MNIDRPKGLA FTDVDVDSIK IAWESPQGQV SRYRVTYSSP EDGIRELFPA PDGEDDTAEL

61 QGLRPGSEYT VSVVALHDDM ESQPLIGIQS TAAAGQCTEV RADTRPWSGP YILRNQDDRE

121 LWPRKFFHRT CKCTGNFAGY NCGDCKFGWT GPNCERKKPP VIRQNIHSLS PQEREQFLGA

181 LDLAKKRVHP DYVITTQHWL GLLGPNGTQP QFANCSVYDF FVWLHYYSVR DTLLGPGRPY

241 RAIDFSHQGP AFVTWHRYHL LCLERDLQRL IGNESFALPY W FATGRNEC DVCAAALEHH

301 HHHH [SEQ ID NO: 351]

The polypeptide of SEQ ID NO:351 comprises an N-terminal methionine, the fibronectin EDA (underlined), a trialaline linker, the amino acids 59-257 of human TRP2 (double underlined), a AAALE linker and an hexahistidine tag.

The conjugates of the invention are capable of generating an innate as well as an adaptive immune response towards the antigenic peptide or peptides.

The terms "innate immunity" and "innate immune response", which are used herein interchangeably, refer to the innate immune system, which, unlike the "adaptive immune system", uses a set of germline-encoded receptors for the recognition of conserved molecular patterns present in microorganisms. The innate immune system provides the body with a first line defence against invading pathogens. In an innate immune response, an invading pathogen is recognized by a germline-encoded receptor, the activation of which initiates a signaling cascade that leads to the induction of cytokine expression. Innate immune system receptors have broad specificity, recognizing molecular structures that are highly conserved among different pathogens. The generation of an innate immune response can be monitored by using any of the assays described in examples 1 to 4 of the present invention, namely, by measuring the NK cell activity (for instance, by measuring CD69 up-regulation in CD3⁺ NK1.1⁺ cells or by measuring IFN-γ production against the NK-sensitive YAC-1 cells.

The terms "adaptive immunity" and "adaptive immune response", which are used herein interchangeably, refer to the response of antigen-specific lymphocytes to antigen and the development of immunological memory which is mediated by the clonal selection of lymphocytes.

Methods for obtaining the conjugates of the invention The conjugates of the invention can be obtained using any method known for a person skilled in the art. It is thus possible to obtain the EDA peptide or the variant of said protein by any standard method. For example, the EDA peptide can be obtained from cDNA by means of expression in a heterologous organism such as, for example, Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris.

Once a sufficient amount of the purified EDA peptide is available, the latter must be conjugated to the melanoma-associated antigenic peptide or peptides. The conjugation of component (ii) to the EDA molecule can be carried out in different ways. One possibility is the direct conjugation of a functional group to the therapeutically active component in a position which does not interfere with the activity of said component. 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 groups maleimide or glyoxylyl which react specifically with thiol groups in the Apo A molecule and oxidized 3,4-dihydroxyphenylalanine (DOPA) groups which react with primary amino groups in the EDA molecule and of component (ii).

Another possibility is to conjugate component (ii) to the EDA molecule by means of the use of homo- or heterobifunctional groups. The bifunctional group can first be conjugated to the therapeutically active compound and, then, conjugated to the EDA peptide or, alternatively, it is possible to conjugate the bifunctional group to the EDA peptide and, then, conjugate the latter to component (ii). 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 which 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 which can be intracellularly processed have been described in WO04054622, WO06107617, WO07046893 and WO07112193. In a preferred embodiment, since component (ii) of the conjugate of the invention is a compound of a peptide nature, 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, periodate, which allows activating carbohydrate moieties in those proteins which are glycosylated. Polynucleotides, gene constructs, vectors and host cells of the invention

In the particular case that the EDA component and the melanoma-associated antigenic protein or peptide form a single peptide chain, it is possible to obtain the conjugate in a single step by expressing in a host cell a polynucleotide encoding said conjugate. Thus, in another aspect, the invention relates to a polynucleotide encoding the conjugate of the invention. The person skilled in the art will appreciate that the polynucleotides of the invention will encode only those conjugates in which component (ii) and the EDA polypeptide or its functionally equivalent variant form a single peptide chain, independently of the relative orientation and independently of the fact that both components are directly linked or separated by a spacer region.

As used in the present invention, the term "polynucleotide" refers to a polymeric form of nucleotides of any length and formed by ribonucleotides and/or deoxyribonucleotides. The term includes both single-stranded and double-stranded polynucleotides, as well as modified polynucleotides (methylated, protected and the like). In another aspect, the invention relates to a gene construct, hereinafter gene construct of the invention, which comprises a polynucleotide of the invention. The construct preferably comprises the polynucleotide of the invention operatively bound to sequences regulating the expression of the polynucleotide of the invention. 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 performing the present invention include, without necessarily being limited to, constitutive promoters such as those derived from the genomes of eukaryotic viruses such as the polyoma virus, adenovirus, SV40, CMV, avian sarcoma virus, hepatitis B virus, the metallothionein gene promoter, the herpes simplex virus thymidine kinase gene promoter, retrovirus LTR regions, the immunoglobulin gene promoter, the actin gene promoter, the EF-1 alpha gene promoter as well as inducible promoters in which the expression of the protein depends on adding a molecule or an exogenous signal, such as the tetracycline system, the NFKB/UV light system, the Cre/Lox system and the heat shock gene promoter, the regulatable RNA polymerase II promoters described in WO/2006/135436 as well as tissue-specific promoters.

Other examples of promoters which are tissue-specific include the albumin gene promoter (Miyatake et al, 1997, J. Virol, 71 :5124-32), the hepatitis virus core promoter (Sandig et al., 1996, Gene Ther., 3: 1002-9); the alp ha- fetoprotein gene promoter (Arbuthnot et al, 1996, Hum. GeneTher., 7: 1503-14), and thyroxine-binding globulin- binding protein gene promoter (Wang, L., et al, 1997, Proc.Natl.Acad.Sci. USA 94: 11563-11566). The constructs of the invention preferably contain dendritic cell- specific promoters such as the CDl lc promoter (Masood, R., et al. 2001. Int J Mol Med 8:335-343; Somia, N.V., et al. 1995. Proc Acad Sci USA 92:7570-7574), the fascin promoter (Sudowe, S., et al, 2006. J Allergy Clin Immunol. 117: 196-203), the CD83 gene promoter, the CD36 gene promoter or the Dectin-2 promoter {Gene Ther., 2001, 8: 1729-1737).

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 which comprises a polynucleotide or a gene construct of the invention. The person skilled in the art will appreciate that there is no limitation regarding the type of vector which can be used since said vector can be a cloning vector suitable for propagation and for obtaining the suitable polynucleotides or gene constructs or expression vectors in different heterologous organisms suitable for the purification of the conjugates. Thus, suitable vectors according to the present invention include expression vectors in prokaryotes such as pUC18, pUC19, Bluescript and the derivatives thereof, mpl8, mpl9, pBR322, pMB9, CoIEl, pCRl, RP4, phages and shuttle vectors such as pSA3 and pAT28, expression vectors in yeasts such as 2-micron plasmid type vectors, integration plasmids, YEP vectors, centromeric plasmids and the like, expression vectors in insect cells such as the vectors of the pAC series and of the pVL series, expression vectors in plants such as vectors of the pIBI, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pORE series and the like and expression vectors in superior eukaryotic cells either 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 cells. By way of example, the vector in which said DNA sequence is introduced can be a plasmid or a vector which, when it is introduced in a host cell, is integrated in the genome of said cell and replicates together with the chromosome (or chromosomes) in which it has been integrated. Said vector can be obtained by conventional methods known by the persons skilled in the art (Sambrook et al, 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 polynucleotide, a gene construct or a vector of the invention, for which said cell could have been transformed, transfected or infected with a construct or vector provided by this invention. Transformed, transfected or infected cells can be obtained by conventional methods known by the 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 limitation, mammalian, plant, insect, fungal and bacterial cells. Bacterial cells include, without limitation, Gram-positive bacterial cells such as species of the Bacillus, Streptomyces and Staphylococcus genera and Gram-negative bacterial cells such as cells of the Escherichia and Pseudomonas genera. Fungal cells preferably include yeast cells such as Saccharomyces, Pichia pastoris and Hansenula polymorpha. Insect cells include, without limitation, Drosophila cells and Sf9 cells. Plant cells include, among others, crop plant cells such as cereal, medicinal, ornamental or bulbous plants. Mammalian cells suitable for the present invention include epithelial cell lines (porcine, etc.), osteosarcoma cell lines (human, etc.), cell lines of neuroblastoma (human, etc.), epithelial carcinomas (human, etc.), glial cells (murine, etc.), liver cell lines (from monkeys, etc.), CHO (Chinese Hamster Ovary) cells, COS cells, BHK cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, 293 cells or PER.C6 cells, human ECC NTERA-2 cells, D3 cells of the mESC line, human embryonic stem cells such as HS293 and BGV01, SHEF1, SHEF2 and HS181, NIH3T3 cells, 293T cells, REH cells and MCF-7 cells and hMSC cells.

Immunogenic 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 To 11- 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 3 of the present invention shows that the administration of a conjugate according to the present invention with a combination of TLR agonists (imiquimod and poly(LC)) is capable of inducing innate and adaptative responses providing better antitumor effects than the combination of the conjugate with a single TLR agonist. Thus, in another aspect the invention relates to a composition (hereinafter composition one of the invention or first composition of the invention) comprising, together or separately:

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

(ii) at least a TLR ligand. The molar concentrations of the components forming part of the first composition 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 compositions of the invention has been described in detail in the context of the conjugate of the invention. In a preferred particular embodiment, said first component comprises the fibronectin EDA of human origin. In another preferred embodiment, said second component comprises the human TRP-1 protein. In the present invention "TLR receptor ligand" is understood as a molecule which specifically binds to at least one of the TLR (toll-like receptor) receptors and which upon binding is capable of stimulating some of the signals 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.

To 11- 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/to 11- like receptor superfamily. All the members of this family have in common the domain called the Toll-IL-1 receptor (TIL) domain.

It has been estimated that most mammals have between 10 and 15 types of TLRs. Thirteen types of TLRs have been identified up until now in human and mice (Du X. et al., 2000, Eur.Cytokine Netw. 1 1 : 362-71 ; Chuang TH. et al., 2000. Eur. Cytokine Netw. 1 1 : 372-378; Tabeta K, et al.; 2004, Proc. Natl. Acad. Sci. U.S.A. 101 :3516- 3521).

TLR ligands induce several immune responses depending on the cells in which the TLR is expressed as well as depending on the origin of TLR ligand. For example, in the case of microbial ligands, immune cells can produce cytokines which will cause inflammation. In the case of a viral factor, the cells can undergo apoptosis.

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(I:C) 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. 2009 Jul 4 Epub ahead of print). 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, trihydro chloride (Par^Cys) LP which mimics the acetylated amino terminus of a bacterial lipoprotein and OspA LP from Borrelia burgdorferi.

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, a 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 speciesas, lipoarabinomannan from mycobacteria, peptidoglycans from 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, or polyinosinic-polycytidylic acid (Poly IC).

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 descrbed 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 B l ; Hilgers et al, 1986, IntArch. Allergy. Immunol, 79(4):392-6; Hilgers et al, 1987, Immunology, 60(l): 141-6; and EP 0 549 074 B l). Bacterial lipopolysaccharide 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 immuno stimulants 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:

o

i

wherein R4 is ~C-(CH₇)_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. In one embodiment, the TLR agonist is imiquimod (CAS 99011-02-6). Further TLR7 agonists are described in W00285905.

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 R¹ 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:

R¹ 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, or a substituted or unsubstituted O-(alkyl), O-(aryl), O-(heteroaryl), S-(alkyl), -S-(aryl), S-(heteroaryl), aryl, or heteroaryl;

R⁷ is independently H or a substituted or unsubstituted -C(0)(Ci_is alkyl) or -C(0)₂(Ci_ is alkyl), -OC0₂(Ci_i 8 alkyl);

R⁸ is H, -OH ,0-(alkyl), -OC0₂ (CMS alkyl), -OC(O) (CMS alkyl), or a racemic, L- or D-amino acid group -OC(0)CHNH₂R¹;

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, component (i) is a TLR agonist capable of causing a signalling response through TLR- 10. In an alternative embodiment, component (i) is a TLR agonist 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.

In another preferred particular embodiment, the TLR ligand is selected from the group consisting of a TLR3 ligand, a TLR7 ligand and a combination of both. In a more preferred embodiment, the TLR3 ligand is poly(LC) (polyinosinic-polycytidylic acid or polyinosinic-polycytidylic acid sodium salt). In a more preferred manner, the TLR7 ligand is selected from Imiquimod, Resequimod, Gardiquimod, Loxoribine, CL264 or Bropirimine. In another preferred embodiment, the TLR7 ligand is imiquimod.

The authors of the present invention have observed that the conjugates of the invention are capable of improving the antitumor response obtained by means of the use of TLR ligands and of CD40 agonists. Specifically, Figure 6A shows how the administration of said fusion protein with a combination of TLR agonists and CD40 agonist to na^'ive animal models is capable of inducing a higher rate of IFNy production by NK-cells. Moreover, the previously mentioned fusion protein causes in this mice a potent antitumor activity mediated by CD8+ T cells against tumors expressing said protein (Figure C and D). Additionally, as it is shown in example 4 and 5, the administration of the combination of said fusion protein with TLR agonists and CD40 agonist to a melanoma tumor bearing mice induce an innate and adaptative immune response and the tumor growth is completely blocked. Thus, in another aspect the invention relates to a composition (hereinafter composition two of the invention or second composition of the invention) comprising, together or separately: (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.

Components (i) and (ii) of the second composition of the invention have been described in detailed above in the context of the first composition of the invention.

Component (iii) of the second composition of the invention is a "CD40 Agonist". The term "CD40 Agonist", as used herein, refers to a compound that binds to the CD40 receptor and triggers signaling in a manner similar to the endogenous CD40 ligand. Assays adequate for determining whether a compound is capable of acting as a CD40 ligand are those based on the detection of 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. (J.Biol. Chem., 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).

Hereinafter, the term "composition of the invention" will be occasionally mentioned to refer to both to the first composition of the invention and to the second composition of the invention. As a person skilled in the art understands, the compositions of the invention can be formulated as a single component or alternatively presented as separate formulations which can be combined for their subsequent administration. The compositions of the invention can also be presented as parts of a kit, in which each of the components is formulated separately but packaged in a single container.

Medical uses of the conjugates and compositions of the invention

The investigators have observed that the conjugates of the invention are capable of inducing the maturation of dendritic cells, inducing the activation of the antitumor immune response in vivo against the peptide and of eradicating large and well- established melanoma tumors (see Examples 3 to 5 of the invention).

In another aspect, the invention relates to a pharmaceutical composition, or pharmaceutical composition of the invention, comprising a conjugate of the invention, a polynucleotide of the invention, a gene construct of the invention, a vector of the invention, a cell of the invention, or a composition of the invention; and at least one pharmacologically acceptable carrier or adjuvant. "Adjuvant" is understood as any substance intensifying the effectiveness of the pharmaceutical composition of the invention.

The examples of 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 (IFA); mineral gels; block copolymers, Avridine™, SEAM62, adjuvants formed by components of the bacterial cell wall such as adjuvants including lipo saccharides (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, EDIM and mutants of mutant toxins such as CRM- 197, non-toxic mutants of diphtheria toxin; saponins such as ISCOMs (immuno stimulating 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), Tumor 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-( 1 '-2'-dipalmitoyl- s-n-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE) etc; adjuvants derived from the family of CpG molecules, CpG dinucleotides and synthetic oligonucleotides which comprise CpG motifs, C. limosum exoenzyme and synthetic adjuvants such as PCPP, the cholera toxin, Salmonella toxin, alum and the like, aluminum hydroxide, N- acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L- alanyl-D-isoglutamine, MTP-PE and RIBI, containing three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a squalene emulsion at 2%/Tween 80. Other examples of adjuvants include DDA (dimethyl dioctadecyl ammonium bromide), complete and incomplete Freund's adjuvants and QuilA.

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 carriers and the auxiliary substances necessary to manufacture the desired pharmaceutical dosage form of the pharmaceutical composition of the invention will depend, among other factors, on the pharmaceutical dosage form chosen. Said pharmaceutical dosage forms of the pharmaceutical composition will be manufactured according to conventional methods known by the person skilled in the art. A review of different administration methods for active ingredients, excipients which are to be used and processes for producing them can be found in "Tratado de Farmacia Galenica", C. Fauli i Trillo, Luzan 5 , S .A. de Ediciones, 1993. Examples of pharmaceutical compositions include any solid (tablets, pills, capsules, granulates, etc.) or liquid (solutions, suspensions or emulsions) composition for oral, topical or parenteral administration. Furthermore, the pharmaceutical composition can contain, as appropriate, stabilizers, suspensions, preservatives, surfactants and the like.

For use in medicine, the conjugates of the invention can be found in the form of prodrug, salt, solvate or clathrate, either isolated or in combination with additional active agents. The combinations of compounds according to the present invention can be formulated together with an excipient which is acceptable from the pharmaceutical point of view. Preferred excipients for their use in the present invention include sugars, starches, celluloses, gums and proteins. In a particular embodiment, the pharmaceutical composition of the invention will be formulated in a solid (for example tablets, capsules, coated tablets, granulates, suppositories, sterile crystalline or amorphous solids which can be reconstituted to provide liquid forms, etc.), liquid (for example solutions, suspensions, emulsions, elixirs, lotions, unguents etc.) or semisolid (gels, pomades, creams and the like) pharmaceutical dosage form. 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 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.

In the event that nucleic acids (the polynucleotides of the invention, the vectors or the gene constructs) are administered, the invention contemplates pharmaceutical compositions especially prepared for the administration of said nucleic acids. The pharmaceutical compositions can comprise said nucleic acids in naked form, i.e., in the absence of compounds protecting the nucleic acids from their degradation by the nucleases of the organism, involving the advantage of eliminating the toxicity associated to the reagents used for the transfection. Administration routes suitable for the naked compounds include intravascular, intratumoral, intracranial, intraperitoneal, intrasplenic, intramuscular, subretinal, subcutaneous, mucosal, topical and oral route (Templeton, 2002, DNA Cell Biol, 21 :857-867). Alternatively, the nucleic acids can be administered forming part of liposomes, conjugated to cholesterol or conjugated to compounds capable of promoting the translocation through cell membranes such as the Tat peptide derived from HIV-1 TAT protein, the third helix of the homeodomain of the D. melanogaster Antennapedia protein, the herpes simplex virus VP22protein, arginine oligomers and peptides such as those described in WO07069090 (Lindgren, A. et al, 2000, Trends Pharmacol. ScL, 21 :99-103, Schwarze, S.R. et al., 2000, Trends Pharmacol. Sc , 21 :45-48, Lundberg, M et al., 2003, Mol Therapy 8: 143-150 and Snyder, E.L. and Dowdy, S.F., 2004, Pharm. Res. 21 :389-393). Alternatively, the polynucleotide can be administered forming part of a plasmid vector or of a viral vector, preferably vectors based on adenoviruses, on adeno-associated viruses or on retroviruses, such as viruses based on the murine leukemia virus (MLV) or on lentiviruses (HIV, FIV, EIAV). The compositions of the invention can be administered in doses of less than 10 mg per kilogram of body weight, preferably less than 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 or 0.00001 mg per each kg of body weight. The unit dose can be administered by injection, by inhalation or by topical administration.

The dose depends on the severity and response of the condition to be treated and can vary between several days and several months or until observing that the condition remits. The optimum dosage can be determined performing periodic measurements of the concentrations of agent in the organism of the patient. The optimum dose can be determined from the EC50 values obtained by means of previous in vitro or in vivo assays in animal models. The unit dose can be administered once a day or less than once a day, preferably, less than once a day every 2, 4, 8 or 30 days. Alternatively, it is possible to administer an initial dose followed by one or several maintenance doses, generally of a lower amount than the initial dose. The maintenance regimen can involve treating the patient with doses ranging between 0.01 μg and 1.4 mg/kg of body weight per day, for example 10, 1, 0.1 , 0.01 , 0.001 , or 0.00001 mg per kg of body weight per day. The maintenance doses are preferably administered at most once a day every 5, 10 or 30 days. The treatment must be continued during a time which will vary according to the type of disorder that the patient suffers from, its severity and the condition of the patient. After the treatment, the evolution of the patient must be monitored to determine if the dose should be increased in the event that the disease does not respond to the treatment or the dose should be reduced if an improvement of the disease is observed or if undesirable side effects are observed.

The daily dose can be administered in a single dose or in two or more doses according to the particular circumstances. If a repeated administration or frequent administrations are desired the implantation of an administration device such as a pump, a semipermanent catheter (intravenous, intraperitoneal, intracisternal or intracapsular) or a reservoir is recommended. Therefore, in another aspect, the invention relates to a conjugate of the invention, a polynucleotide of the invention, a gene construct of the invention, a vector of the invention, a cell of the invention, a composition of the invention or a pharmaceutical composition of the invention for its use in medicine.

In another aspect, the invention relates to the use of a conjugate of the invention, a polynucleotide of the invention, a gene construct of the invention, a vector of the invention, a cell of the invention, a composition of the invention or a pharmaceutical composition of the invention for the manufacture of a vaccine. In other words, the invention relates to a conjugate of the invention, a polynucleotide of the invention, a gene construct of the invention, a vector of the invention, a cell of the invention, a composition of the invention or pharmaceutical composition of the invention for its use as vaccine. Alternatively, the invention relates to a method for the vaccination of a subject which comprises the administration to said subject of a conjugate of the invention, a polynucleotide of the invention, a gene construct of the invention, a vector of the invention, a cell of the invention, a composition of the invention or a pharmaceutical composition of the invention.

As used herein, the term "vaccine" refers to a formulation which contains a conjugate or a composition according to the present invention and which is in a form that is capable of being administered to a vertebrate and which induces a protective immune response sufficient to induce immunity 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 conjugate or composition of the invention. As it will be understood, the immune response generated by the vaccine may be a humoral or a cellular immune response.

The expression "humoral 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 "cellular immune response", is used herein to describe an immune response against foreign antigen(s) that is mediated by antibodies produced by B-cells. The vaccine is systemically or locally administered. The vaccine can be administered by means of a single administration, or with a boost by means of multiple administrations as has been previously described for the administration of the compositions of the invention.

In another aspect, the invention relates to the use of a conjugate of the invention, a polynucleotide of the invention, a gene construct of the invention, a vector of the invention, a cell of the invention, a composition of the invention or a pharmaceutical composition of the invention for the manufacture of a medicament for the prevention and/or the treatment of a melanoma. Alternatively, the invention relates to a conjugate of the invention, a polynucleotide of the invention, a gene construct of the invention, a vector of the invention, a cell of the invention, a composition of the invention or a pharmaceutical composition of the invention for the prevention and/or the treatment of a melanoma. Alternatively, the invention relates to a method for the prevention and/or the treatment of melanoma which comprises administering to a subject in need thereof a conjugate of the invention, a polynucleotide of the invention, a gene construct of the invention, a vector of the invention, a cell of the invention, a composition of the invention or a pharmaceutical composition of the invention.

The term "melanoma" includes, but is not limited to, melanomas, metastatic melanomas, melanomas derived from either melanocytes or melanocyte related nevus cells, melanocarcinomas, melanoepitheliomas, melanosarcomas, melanoma in situ, superficial spreading melanoma, modular melanoma, lentigo malignant melanoma, acral lentiginous melanoma, invasive melanoma and familial atypical mole and melanoma (FAM-M) syndrome.

Moreover, the term "melanoma" refers not only to primary melanomas but also to "melanoma metastasis" which, as used herein, refers to the spread of melanoma cells to regional lymph nodes and/or distant organs. This event is frequent given that melanomas contain multiple cell populations characterized by diverse growth rates, karyotypes, cell-surface properties, antigenicity, immunogenicity, invasion, metastasis, and sensitivity to cytotoxic drugs or biologic agents. Melanoma shows frequent metastasis to brain, lungs, lymph nodes, and skin. Thus, the conjugates of the present invention are also adequate for the treatment of melanoma metastasis. Methods of vaccination with dendritic cells of the invention

Another cancer therapy approach is to take advantage of the normal dendritic cell role as an immune educator. As has been previously mentioned, dendritic cells capture virus antigens among others and present them to T cells to recruit their help in an initial T cell immune response. This works well against foreign cells entering the body, but cancer cells frequently evade the "self '/"foreign" substance detection system. The investigators, by modifying the dendritic cells, are capable of activating a special type of autoimmune response which includes an attack of T cells against cancer cells. Due to the fact that a tumor agent alone is not enough to generate an immune response, it is possible to contact an immature dendritic cell with a conjugate of the invention, a polynucleotide of the invention, a cell of the invention, a composition of the invention or a pharmaceutical composition of the invention, which results in the activation of the dendritic cells, the capture of the melanome-associated antigen or antigens and the presentation thereof in the surface associated to the major histocompatibility antigen. These cells thus activated can be administered to the patient, such that the presentation of the tumor antigens to the immune system of the patient occurs, which eventually results in the generation of an immune response mediated by T cells on the cancer cells of the patient. Thus, in another aspect, the invention relates to an in vitro method for obtaining mature dendritic cells presenting at least one melanoma-associated antigen, comprising:

(i) contacting dendritic cells with a conjugate of the invention, a polynucleotide of the invention, a gene construct of the invention, a vector of the invention, a cell of the invention, a composition of the invention or a pharmaceutical composition of the invention in conditions suitable for the maturation of the dendritic cells to take place, and

(ii) recovering the mature dendritic cells. Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) and have a unique capacity to interact with the non-activated T lymphocytes (naive T lymphocytes) and initiate the primary immune response, activating the CD4+ helper T lymphocytes and the CD8+ cytotoxic T lymphocytes.

In the absence of inflammation and of immune response, dendritic cells patrol through the blood, peripheral tissues, lymph and secondary lymphoid organs. In the peripheral tissues, dendritic cells capture self and foreign antigens. The antigens captured are processed giving rise to fragments thereof which pass to class I and II MHC molecules (for the activation of CD8+ or CD4+ T lymphocytes, respectively). This process of antigen capture, degradation and load is called antigen presentation. However, in the absence of stimulation, the peripheral dendritic cells present the antigens inefficiently. The exogenous signal or signals coming from the pathogens or the endogenous signal or signals induce the dendritic cells so that they initiate a development process called maturation, which transforms the dendritic cells into APCs and into T lymphocyte activators.

There are different types of dendritic cells which can be used in the invention. On one hand there are myeloid dendritic cells (mDCs) which are similar to the monocytes and which in turn are divided into two subtypes: mDC-1, which are the most common and which are the main stimulators of the cells T and mDC-2, which are rarer and the main function of which is to fight against wound infections. On the other hand, there are plasmacytoid dendritic cells (pDCs), which are similar to plasma cells but which have certain features typical of myeloid dendritic cells.

Immature dendritic cells are derived from bone marrow hematopoietic stem cells. These stem cells differentiate into immature cells having a high endocytic capacity and low capacity to activate T cells. These cells have in their membrane different membrane receptors such as TLRs. The bacterial and viral products, as well as inflammatory cytokines and other typical molecules, induce the maturation of the dendritic cells by means of direct interaction with the surface receptors of innate dendritic cells. T lymphocytes, through CD40- dependent and independent pathways, and endothelial cells contribute to the final maturation of dendritic cells by means of direct cell-to-cell contact and by means of cytokine secretion. Soon after a danger signal arises, the efficiency of the antigen capture, the intracellular transport and the degradation, and the intracellular MHC molecule traffic are modified. The peptide load, as well as the half-life and the transfer to the cell surface of the molecules MHC are increased. The expression in the surface of the T cell co-stimulating molecules also increases. Dendritic cells thus become the most potent APCs, and the only ones capable of activating the non-activated T lymphocytes and initiating the immune response. Together with the modification of the capacities thereof in antigen presentation, the maturation also induces the massive migration of dendritic cells out of the peripheral tissues. The modifications in the expression of chemokine receptors and adhesion molecules, as well as the important changes in the cytoskeleton organization, contribute to the migration of dendritic cells through the lymph to the secondary lymph organs.

Dendritic cells respond to two types of signals: to the direct recognition of the pathogens (by means of receptors with a specific recognition pattern) and to the indirect recognition of the infection (by means of inflammatory cytokines, internal cell compounds and specific immune responses). In response to these signals, dendritic cells are activated and initiate their maturation process, which transforms them into efficient T cell stimulators. One of the most efficient signals for the maturation of DCs is mediated by the interactions of the to 11- like receptors, TLRs, (TLR1-9) with their respective ligands (reviewed by Kaisho and Akira, Biochimica et Biophysica Acta, 2002, 1589: 1-13).

The immature dendritic cells used in the present invention can be primary culture cells. Thus, the dendritic cells used in the method of the invention can be autologous or heterologous. As used herein, the term "autologous" means that the cells are from the same individual.

As used herein, the term "heterologous" means that the cells are from a different individual.

Dendritic cells can be generated in vitro from peripheral blood mononuclear cells (PBMCs) using a protocol which would basically consist of seeding PBMCs in a culture bottle such that the adhesion of said cells is allowed. After that the cells would be treated with interleukin 4 (IL4) and granulocyte-macrophage colony- stimulating factor (GM-CSF) leading to the differentiation of the cells into immature dendritic cells (iDCs) in approximately one week. Optionally, the cells can be maturated treating them with tumor necrosis factor alpha (TNFa).

Dendritic cells can be obtained using standard methods from suitable sources. These tissues suitable for the isolation of dendritic cells include the peripheral blood, spinal cord, tumor-infiltrating cells, peritumor tissue-infiltrating cells, biopsies of lymph nodes, thymus, spleen, skin, umbilical cord blood, monocytes obtained from peripheral blood, CD34- or CD14-positive cells obtained from peripheral blood, as well as any other suitable tissue or fluid.

Optionally, stable cell cultures can be used. Document WO9630030 describes methods for obtaining dendritic- like cell/tumor cell hybridomas and pluralities of dendritic- like cell/tumor cell hybrids. These hybrids and hybridomas are generated for the fusion of tumor cells with dendritic- like cells. For example, immortal tumor cells from an autologous tumor cell line can be fused with autologous HLA-matched allogeneic dendritic- like cells. The autologous tumor cell lines can be obtained from primary tumors and from their metastases. Alternatively, immortal dendritic- like cells of an autologous or allogeneic HLA-matched dendritic- like cell line can be fused with autologous tumor cells. Document WO/2002/048167 also describes methods for generating stable dendritic cell lines. Another cell line that can be used is CB1 (Paglia P. et al. 1993. Journal of Experimental Medicine, Vol 178, 1893-1901). A first step of the method of the invention consists of contacting dendritic cells with a conjugate of the invention, a polynucleotide of the invention, a vector of the invention, a gene construct of the invention, a cell of the invention, a composition of the invention or a pharmaceutical composition of the invention in conditions suitable for the maturation of the dendritic cells to take place.

The invention contemplates any possible way of contacting the dendritic cells with a conjugate of the invention, a polynucleotide of the invention, a vector of the invention, a gene construct of the invention, a cell of the invention, a composition of the invention or a pharmaceutical composition of the invention. The person skilled in the art will appreciate that, depending on the type of compound, the contacting is carried out differently. Thus, in the event that it is the conjugate of the invention or a peptide of the invention, these can be directly added to the culture medium in which the cells are located or can be bound to a surface of plastic, glass, etc. which will be exposed to the dendritic cells. Forms to bind the components of the conjugate of the invention as well as the peptide of the invention to solid surfaces are known by a person skilled in the art.

In the event that it is a gene construct of the invention or a vector of the invention, the techniques used to introduce said components in a cell (cell of the invention) have been previously described in the section of gene constructs of the invention. In a particular embodiment, the cells of the invention have in their membrane the components of the conjugate of the invention such that they are accessible to the dendritic cells.

"Conditions suitable for the maturation to take place", is understood as all those culture conditions (oxygen, temperature, humidity, nutrients etc) which allow activating the dendritic cells, after being contacted with a conjugate of the invention, a polynucleotide of the invention, a vector of the invention, a cell of the invention, a composition of the invention or a pharmaceutical composition of the invention, such that at least one melanoma-associated antigen has been presented. This activation occurs when the immature dendritic cells have phagocytized some of the presented antigens and have degraded said antigens into small pieces, presenting said parts in their surface by using histocompatibility system molecules (MCH). The mature dendritic cells simultaneously upregulate membrane receptors acting as co-receptors in the activation of the T cells, such as CD80 (B7.1), CD86 (B7.2), and CD40, such that their capacity to activate said T cells is thus increased. The mature dendritic cells also upregulate the expression of CCR7, a receptor which induces the travel of the dendritic cells throughout the blood stream to the spleen and from there their passage to the lymph system. The mature dendritic cells are capable of activating helper T cells, killer T cells and B lymphocytes presenting the antigens which they have processed.

Thus, mature dendritic cell presenting at least one melanoma-associated antigen is understood as that dendritic cell which, after capturing a melanoma-associated antigen, is capable of presenting said antigen in the surface of its membranes bound to the major histocompatibility complex (MHC) after having processed it. Additionally, the mature dendritic cells can present the above indicated upexpressed membrane receptors. In another step, the cells are maintained under conditions suitable for the internalization, processing and presentation of one or more peptides of derivatives of the conjugate of the invention. The conditions suitable for the internalization, processing and presentation of the at least one antigenic peptide derived from the conjugate of the invention can be determined using standard assays for determining the activation of dendritic cells.

The maturation of the DCs can be followed using a number of molecular markers and of phenotypic alterations of the cell surface. These changes can be analyzed, for example, using flow cytometry techniques. The maturation markers are typically marked using specific antibodies and the DCs expressing a marker or a group of markers can be separated from the total of DCs using, for example, FACS cell sorting. The DC maturation markers include genes which appear expressed at high levels in mature DCs compared with immature DCs. These markers include, but are not limited to MHC class II antigens of the cell surface (in particular HLA-DR), co-stimulating molecules such as CD40, CD80, CD86, CD83, cell traffic molecules such as CD45, CD1 lc and CD18, etc. Furthermore, the maturation of DCs can be determined measuring the expression of certain Notch ligands such as the Delta-like ligand (DLL4), Jaggedl and Jagged2 which are associated with the induction of the Thl response. On the other hand, mature dendritic cells can be identified using their ability to stimulate the proliferation of allogeneic T cells in a mixed lymphocyte reaction (MLR). Furthermore, it has been generally seen that while immature DCs are very efficient in the internalization of antigens but have a low antigen presentation, mature DC cells have a low internalization of antigens but are very efficient in presenting antigens.

The antigen-presenting function of the dendritic cells can be measured using MHC- limited, antigen-dependent T cell activation assays as well as other assays which are well known for the persons skilled in the art such as the capacity for in vitro stimulation in peripheral blood lymphocytes, for example, determining the amount of IFN-γ produced by CD8+ lymphocytes in the presence of DCs. This determination can be carried out using the technique called ELISPOT. The activation of T cells can additionally be determined measuring for example the induction of cytokine production by the stimulated dendritic cells. The stimulation of the cytokine production can be determined using a large variety of standard techniques, such as ELISA, which are well- known by a person skilled in the art.

Other cytotoxicity assays such as the binding of target cells with tritiated thymidine ([³H]-TdR) can be used. 3H-TdR is incorporated in the nucleus of the cells. The release of ([³H]-TdR) is a measure of cell death due to DNA fragmentation.

In a second step of the method of the invention the mature dendritic cells obtained in step (i) are recovered.

Different strategies can be used to analyze the mature cells obtained in step a) of the method of the invention. For example, the membrane markers expressed by mature cells and which have been previously described, such as for example CD80, can be used. The expression of cell surface markers can be determined, for example, by means of flow cytometry using conventional methods and apparatuses. For example, the Becton Dickinson Calibur FACS (fluorescent-activated cell sorting) system using commercially available antibodies and usual protocols known in the art can be used. Thus, the cells presenting a signal for a specific cell surface marker in the flow cytometry above the background signal can be selected. The background signal is defined as the signal intensity given by a non-specific antibody of the same isotype as the specific antibody used to detect each surface marker in the conventional FACS analysis. In order for a marker to be considered positive, the observed specific signal has to be more than 20%, preferably, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 500%, 1000%, 5000%, 10000% or above, intense in relation to the intensity of the background signal using conventional methods and apparatuses (for example, a Becton Dickinson Calibur FACS system used with commercially available antibodies and usual protocols known in the art).

The dendritic cells obtained by means of the method according to the invention have proved to be useful for the treatment of diseases which respond to the generation of an immune response against melanoma-associated antigens. Using said method, mature cells presenting at least one antigen from the conjugate of the invention are obtained. Thus in another aspect, the invention relates to antigen-presenting dendritic cells presenting at least one antigen from the conjugate of the invention and which are CD40- positive obtained by means of the method of the invention or dendritic cells of the invention.

In another aspect, the invention relates to dendritic cells of the invention, for their use in medicine. Said dendritic cells of the invention can be used to cause an immune response in a patient using the latter as a DC vaccination, i.e. by means of the administration to said patient of said cells. Thus in another aspect, the invention relates to a dendritic cell of the invention for the generation of an immune response in a patient. In other words, the invention relates to a method for causing an immune reaction in a subject which comprises the administration to a subject of the antigen-presenting cell.

The vaccination with DC is carried out by administering the antigen-presenting DC to a subject (for example a human patient) in whom an immune response is induced. The immune response typically includes a CTL response against target cells which are marked with the antigenic peptides (for example the components of the conjugate of the invention). These target cells are typically cancer cells. When the modified DCs are to be administered to a patient, these cells are preferably isolated from stem cells of the same patient (i.e., DCs are administered to an autologous patient). However, the cells can be administered to allogeneic patients who are compatible with respect to the HLA or to allogeneic patients where there is no coincidence. In this latter case immunosuppressive drugs must be administered to the patient receiving the cells.

The cells can be administered in any suitable form, preferably with a carrier (for example saline solution). The administration will normally be intravenous, but intra- articular, intramuscular, intradermal, intraperitoneal or subcutaneous administrations are also acceptable. The administration or immunization can be repeated at different time intervals.

The DC injection can be combined with the administration of cytokines which act such that the number of DCs and their activity is maintained such as GM-CSF, IL-12.

The dose administered to a patient must be efficient to induce an immune response which can be detected using assays which measure the proliferation of T cells, the cytotoxicity of T lymphocytes and/or the beneficial therapeutic effect of response of the patients over time. 106 to 109 DC cells are typically injected, provided that they are available. The vaccines can be administered one or more times to a patient to achieve beneficial results. The time between the first and the successive dose or doses of the vaccine depend on a variety of factors, which include but are not limited to, the health of the patient, age, weight, etc. The vaccine can be administered at any suitable time interval, for example including but without being limited to, once a week, once a day every two weeks, once a day every three weeks, once a month. In a particular embodiment, the vaccine can be administered indefinitely. In another particular embodiment, the vaccine is administered three times in intervals of two weeks. The doses of the vaccine also depend on a variety of factors, which include but are not limited to the health of the patient, stability, age, weight, etc. Once a sufficient immunity level involving a clinical benefit has been achieved, booster doses can be used, which are generally administered with a lower frequency (for example monthly or half-yearly doses).

The DCs used in the method for causing an immune response are preferably formulated so that they can be used as an off-the-shelf drug or ready for their use in the event that there is histocompatibility between the cells of the preparation and those of the treated patient. The lack of compatibility between the subject object of the therapy and the dendritic cells can result in a reduction of the effect of the vaccine, either because there is a premature elimination of the cells (especially after multiple administrations) or due to the generation of a strong anti-allotypic response distracting the immune system from the intended target. In this context, the use of vaccines in which at least some of the HLA Class I alleles in the dendritic cells of the invention (especially in the locus A and more particularly in the A2 allele) are shared with the patient is advantageous. Thus at least some of the tumor antigens will be presented in autologous class I molecules, whereby the antitumor response will be increased and the anti-allotypic response will be reduced.

A partial coincidence can be obtained using a vaccine with DCs made with cells having two or more of the most common HLA- A allotypes (HLA-A2, Al, A19, A3, A9, and A24). A total coincidence for most of the patients can be achieved by providing the physician with a set of different DCs from where different possibilities having only an allotype in the locus HLA-A can be selected. The treatment will involve the identification of one or more HLA allotypes in the patient using standard tissue-taping methods, and the treatment of the patients with the DCs having the HLA allotype or allotypes which coincide with those of the patient. For example a patient who is HLA- A2 and HLA-A19 can be treated either with cells homozygous for HLA-A2 or for HLA-A19 or with a mixture of both.

Potential negative effects of the lack of coincidence of HLA can also be treated generating immunotolerance against the foreign allotypes. During the preparation of the vaccine the DC cells are divided into two groups: a group for generating tolerogenic immature cells and the other group for generating mature DCs for the antigen presentation. The tolerogenic cells are designed such that they generate an acceptance of the mature cells. The tolerogenic cells will be administered one or more times to the patient so that a sufficient degree of absence of immune response (measured for example in a mixed lymphocyte reaction) is generated. Once the patient is tolerant (after one week or one month) the mature antigen-presenting cells are administered to the subject in the amount and in the frequency which is necessary for an immune response against the target tumor antigen.

In a particular embodiment, the antigen-presenting dendritic cells of the invention are autologous to the subject to be treated. The composition of the vaccine can include an adjuvant. The adjuvant can be any available adjuvant or a combination thereof. Examples of adjuvants have been mentioned in the section of medical uses of the conjugates and compositions of the invention. In another aspect the invention relates to the use of a cell of the invention for the manufacture of a medicament for the prevention and/or treatment of a melanoma or of a metastasis thereof. Thus in another aspect, the invention relates to a dendritic cell of the invention for its use in the prevention and/or treatment of a melanoma or of a metastasis thereof. In other words the invention relates to a method for the prevention and/or treatment of a melanoma or of a metastasis thereof in a subject which comprises the administration to said subject of the dendritic cell of the invention.

The terms "melanoma" and "metastasis" have been described in detail above and are used in the same sense in the therapeutic methods using the dendritic cells according to the invention.

The invention is described below by means of the following examples which must be considered as merely illustrative and not as limitations thereof. EXAMPLES

MATERIALS AND METHODS Reagents

Peptide OVA(257-264) was synthesized by the solid-phase method of Merrifield using a manual multiple solid-phase peptide synthesizer. OVA protein was purchased from Sigma Aldrich (Madrid, Spain). EDA and EDA-OVA fusion protein were produced as described (Lasarte et al., J Immunol 2007;178:748-56). Imiquimod was used as Aldara™ cream (Meda Pharma; Madrid, Spain). Poly(I :C) was obtained from Amersham (Barcelona, Spain) (Code Ner. 27-4732) and agonistic anti-CD40 antibody was purified by ammonium sulfate precipitation from ascytic fluid of nude mice injected with the FGK45.5 hybridoma cells (LLopiz et al., Cancer Immunol Immunother 2008;57(1): 19-29).

Production and purification of the recombinant protein EDA-TRP2(59-257) (also named M-EDA-Linker-TRP2(59-257)-Linker-6xHist, SEQ ID NO:351)

Plasmid pET20b-EDA, expressing the extra domain A from fibronectin, (Lasarte et al., J Immunol 2007;178(2):748-56) was used for the construction of the expression plasmid pET20b-EDA-TRP2 (59-257), to express a fusion protein containing a fragment of 198 amino acids from TRP2 antigen (aminoacids 59-257) linked to the C terminus of EDA and carrying six histidines at the carboxy terminus as described (Mansilla et al, J Hepatol 2009;51(3):520-7). EDA-TRP2(59-257) was purified from inclusion bodies (8M urea-20mM HEPES) by a semi-preparative isoelectrofocussing (Rotofor, Biorad) followed by affinity chromatography using Nickel Sepharose 6 Fast flow resin (GE Healtcare Biosciences, Uppsala, Sweden). The resulting protein was refolded in a sepharose G25 column using a urea gradient size-exclusion chromatography and purified from endotoxins by using Endotrap columns (Profos Ag, Regensburg, Germany). Purified protein was analyzed by SDS-PAGE and stained with Coomassie blue (Bio-Safe Coomassie reagent, Bio-Rad, Hercules, CA) for confirmation of size and purity. Mice

Six to eight weeks-old female C57BL/6 mice were obtained from Harlan (Barcelona, Spain) and were maintained in pathogen-free conditions and treated according to guidelines of our institution, after study approval by the review committee.

Cell lines

Parental EL-4 thymoma cells (H-2b), OVA-transfected E.G7-OVA cells and the NK- sensitive cell line YAC-1 were purchased from American Type Culture Collection (Manassas, VA) and were grown as described (Llopiz et al., Int J Cancer 2009;125:2614-2623). B16-OVA (Brown et al, Immunology 2001;102:486-497) and B16.F10 tumor cells (Fidler IJ. Cancer Res 1975;35:218-224), obtained from Dr. G Kroemer, Paris were grown in DMEM containing 10% fetal calf serum and antibiotics.

Immunization of mice

Na^'ive C57BL/6 mice received combinations of Imiquimod cream (topical application; 2.5 mg/mouse) plus subcutaneous administration of poly(LC) (50 μg/mouse), anti- CD40 (50 μg/mouse), OVA protein (100-500 μg/mouse), EDA (25 μg/mouse), EDA- OVA protein (125 μg/mouse) or EDA-TRP2(59-257) protein (2 nmoles/mouse). Six days later animals were sacrificed and splenocytes were obtained for immunological analysis.

Tumor treatment experiments

B16 melanoma is a transplantable murine tumor cell widely studied in models of human melanoma. B16 cell lines (Fidler et al., Cancer Res 1975;35:218-24) are poorly immunogenic tumors which have many characteristics of tumors found in patients, making it a good model to develop immunotherapeutic strategies with potential clinical applications.

C57BL/6 mice were injected intradermally with 10⁵ B16-OVA cells and when the tumor diameter reached 4-5 mm, treatment protocols containing combinations of the following adjuvants and antigens were applied: Imiquimod cream was topically applied (2.5 mg/mouse) to shaved skin at the tumor site, whereas poly(LC) (50 μg/mouse), anti- CD40 (50 μ§/ηκηΐ8ε), OVA protein (500

and EDA-OVA protein (125 μ /ηιου8ε) were administered intratumor (i.t.). Untreated mice challenged with tumor cells were used as positive controls of tumor growth. Tumor volume was calculated according to the formula: V= (length x width2)/2. Mice were killed when tumor diameter reached 17 mm.

Analysis by real-time PCR of OVA mRNA expression

Total R A extraction from tumor cell lines and real-time PCR were performed as described (larrea 2007), using primers TATTCGTTCAGCCTTGCCAG (sense SEQ ID NO:352) and CTTTCTCCCACAGTCCTTTG (antisense SEQ ID NO: 353) for OVA, and CGCGTCCACCCGCGAG (sense SEQ ID NO: 354) and CCTGGTGCCTAGGGCG (antisense SEQ ID NO:355) for β-actin. Results were normalized according to β-actin. The amount of each transcript was expressed by the formula: 2ACt [ACt = Ct(P-actin)-Ct(gene)].

ELISPOT

Cells producing IFN-γ were enumerated by ELISPOT assays using a kit from BD- Biosciences (San Diego, CA) as described (Llopiz et al, Int J Cancer 2009;125:2614- 2623). For T-cell responses, splenocytes were stimulated with peptides OVA(257-264) (1 μ^ιηΐ), TRP-2(180-188) (10 μ^ιηΐ), OVA protein (10 μ^πιΐ) or 4 x 10⁴ irradiated (20000 rads) tumor cells. When measuring NK cell derived production of IFN-γ, splenocytes were incubated with 4 x 10⁴ mitomycin C treated- Y AC- 1 cells.

Flow cytometry

Expression of MHC Kb class I molecules in tumor cells was analyzed using anti Kb- FITC labeled antibodies. To analyze NK cells and DC, spleens were treated with collagenase and DNAse for 15 minutes and homogenized. Then, cells were first incubated for 10 min with Fc Block™ (BD-Biosciences) and stained with specific antibodies. For NK cell analysis, cells were stained with anti-CD69-FITC, anti-CD3-PE and anti-NKl .l-APC-labeled antibodies. DC were analyzed using anti-CD 1 lc-APC and anti-CD86-FITC-labeled antibodies. T-cell activation was analyzed after stimulation of splenocytes with 1 ng/ml of OVA(257-264) in the presence of GolgiStop and GolgiPlug (BD-Biosciences) with or without anti-CD 107-FITC antibodies for 4 hours. Then cells were labeled with anti-CD8-APC and fixed, permeabilized and stained with anti-IFN-γ- PE. For triple cytokine analysis, after surface staining with anti-CD8-FITC antibodies, cells were stained with anti-IFN-γ-ΡΕ, anti-TNF-a-PE-Cy7 and anti-IL-2-APC labeled antibodies . All antibodies were from BD-Biosciences, except anti-NKl . l (e- Bioscience). Expression of the different markers was analyzed with a FACSCalibur flow cytometer (Becton Dickinson) and Flowjo software (Tree Star Inc; Ashland, OR).

Measurement of cytokines by ELISA

IL-12 and TNF-a content in the serum obtained from the retroorbital plexus of mice, and IFN-γ from 48 h culture supernatants of splenocytes stimulated with different concentrations of OVA(257-264) were measured using OptEIA Sets from BD- Biosciences. 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

Immunotherapy based on adjuvant administration has a poor effect on B16-OVA tumor bearing mice, inducing innate but not adaptive immunity.

The ability of a single prophylactic administration of adjuvant molecules poly(I:C) and agonistic anti-CD40 antibodies plus OVA protein to induce potent T-cell responses which protect 100% of mice from the growth of subcutaneous EG7.0VA tumors has been described (LLopiz et ah, Cancer Immunol Immunother 2008;57(1): 19-29). However, the same therapy applied to the B16-OVA melanoma model results in no difference in tumor growth and survival between vaccinated and control mice (Figures 1A-B), demonstrating the poor immunogenicity and recognition of B16-OVA cells. Indeed, OVA expression of B16-OVA cells was very low, as compared with EG.70VA cells, and the same low expression was obtained for MHC class I K^b molecules (Figures 1C-D). Thus, we considered B16-OVA cells as a good tumor model to develop our immunotherapeutic strategies.

Since repeated intra-tumor administration of poly(I:C) and anti-CD40 rejected 35 % of established EG.70VA tumors (LLopiz et al., Cancer Immunol Immunother 2008;57(1): 19-29), the effect of this repeated adjuvant administration was tested on the B16-OVA model. For these experiments, the combination of topical application of Aldara™ cream (containing the TLR7 ligand imiquimod) and intra-tumor injection of anti-CD40 antibodies was used. Mice bearing seven-day intradermal tumors (5 mm) received repeated adjuvant administrations in a twenty-day interval. As shown in Figure 2A, a slight delay (p < 0.05) was observed in tumor growth in the group of treated mice, but all mice died by day 40.

Rejection of EG7.0VA tumors after administration of poly(I:C) plus anti-CD40 was associated to the induction of innate and adaptive immunity (LLopiz et al., Cancer Immunol Immunother 2008;57(1): 19-29). Therefore, these parameters were analysed when using imiquimod and anti-CD40 as adjuvants. Na^'ive mice received a single administration of imiquimod plus anti-CD40 and one and two days later, innate immunity was evaluated in the spleen. NK cell activity was analyzed by measuring CD69 upregulation on CD3^"NK1.1⁺ cells and IFN-γ production against the NK- sensitive YAC-1 cell line. A high activity in both functions was found at 24 h (Figure 2B) after adjuvant administration. Regarding DC, a clear upregulation of CD86 was observed (Figure 2C). Finally, analysis of serum cytokines showed a peak for IL-12 at 6 h (226 pg/ml), whereas TNF-a levels grew until 48 h (625 pg/ml). Analysis of innate immunity in tumor bearing mice showed that these animals already had activated responses, which was not further enhanced by adjuvant administration (Figure 3). Analysis of anti-tumor adaptive immune responses was carried out in tumor-bearing mice, untreated or treated with imiquimod plus anti-CD40. None of these groups displayed IFN-γ production against the CD8 T-cell epitope OVA(257-264) or OVA protein in ELISPOT assays (Figure 2D). These results suggest that administration of these adjuvants to B16-OVA tumor-bearing mice induces innate but not adaptive immunity.

EXAMPLE 2

Combination of adjuvants imiquimod and anti-CD40 with the tumor antigen OVA induces adaptive immunity with better antitumor effects.

The lack of adaptive immunity in B16-OVA-bearing mice treated with adjuvants alone led us to hypothesize that inclusion of an antigen together with adjuvants would be able to induce T-cell responses. Indeed, it has been shown that repeated administration of OVA together with poly(I:C) and anti-CD40 enhanced EG7.0VA tumor rejection from 35% to 100% (LLopiz et ah, Cancer Immunol Immunother 2008;57(1): 19-29)). Immunization of na^'ive mice with OVA plus the adjuvant combination Imiquimod and anti-CD40 had a strong effect on the induction of T-cell responses (Figure 4A), as observed when combining OVA plus poly(I:C) and anti-CD40 (LLopiz et ah, Cancer Immunol Immunother 2008;57(1): 19-29)). Thus tumor-bearing mice were treated with OVA plus Imiquimod and anti-CD40. In experiments similar to that of Figure 2, mice received repeated administrations of OVA plus Imiquimod and anti-CD40 during a twenty-day period. In this case, tumor volume was kept below 500 mm³ during the treatment period, as compared to untreated animals, which at day 20 had a mean tumor volume above 2000 mm³ (p < 0.05) (Figure 4B). Moreover, only 33 % of untreated mice were alive at the end of treatment (day 20), whereas 100% of treated mice survived (p < 0.05) (Figure 4C). Also, as compared to mice treated only with adjuvants (Figure 2A), once treatment finished, animals treated with OVA plus imiquimod and anti-CD40 had slow-growing tumors until day 30, when they started to grow and finally all mice died at day 40. The effect of OVA plus the combination of poly(I:C) and anti- CD40 was also tested since a similar strategy was known to induce rejection of EG7.0VA tumors. As when using OVA plus imiquimod and anti-CD40, a significant delay in tumor growth was observed (p<0.01), but without tumor rejections (Figure 5). The lack of tumor rejection in treated animals could not be attributed to the absence of T-cell responses, because tumor-bearing mice treated with as few as two immunizations showed clear responses against tumor antigens (a representative example of mice treated with OVA plus Imiquimod and anti-CD40 is shown in Figure 4D). Thus, a protocol which combines the tumor antigen with double-adjuvant mixtures induces T- cell responses, delays tumor growth during treatment period and some days beyond, but does not induce tumor rejection.

EXAMPLE 3

A multiple adjuvant combination and antigen targeting strategy enhances innate immunity and induces polyfunctional high avidity T-cell responses.

The poor but significant effect that antigen plus adjuvant administration had on tumor growth suggested that new strategies inducing enhanced T-cell responses would be more suitable to attain tumor rejection. Thus, looking for a higher synergistic effect between adjuvants, all three molecules (anti-CD40, poly(LC) and Imiquimod) were included in a multiple adjuvant combination (MAC). Moreover, to enhance not only innate immunity, but also to target antigen to APC, OVA was coupled to the extra domain A of fibronectin (EDA-OVA), a TLR4 ligand which enhances innate and adaptive immunity, and when coupled to antigens, targets them to TLR4-expressing antigen presenting cells (Lasarte et ah, J Immunol 2007;178:748-56). It was first characterized the potency of this mixture on the activation of innate immunity. In naive mice, analysis of CD69 upregulation on NK cells did not show differences between groups receiving a single administration of the different adjuvant combinations (data not shown). However, EDA-OVA+MAC induced the strongest NK-cell derived IFN-γ production of the four combinations tested (Figure 6A), mainly due to the additional effect of poly(LC) (p<0.01; EDA-OVA+MAC vs. EDA-OVA+Imiquimod+anti-CD40, or OVA+MAC vs. OVA+Imiquimod+anti-CD40). Analysis of DC maturation showed that all groups reached similar levels of CD86, both in the percentage of CD86⁺ cells and in the fluorescence intensity (data not shown). Analysis of adaptive immunity in na^'ive mice immunized with the different combinations showed a synergistic effect for EDA-OVA+MAC in ELISPOT assays measuring the number of IFN-y-producing cells, against OVA(257-264) peptide and OVA protein, as compared with immunization with OVA plus triple or double adjuvant combinations (Figure 6B) (p<0.01 ; EDA-OVA+MAC vs. remaining groups). Antigen targeting to EDA was necessary to obtain these high responses, since immunization with MAC plus EDA and free OVA induced lower responses, mainly for those mediated by CD8 T-cells (Figure 7). Besides quantitative parameters of T-cell activation, the quality of the CD8 T-cell responses was analyzed by stimulating the splenocytes with decreasing OVA(257-264) concentrations. Interestingly, splenocytes from mice immunized with EDA-OVA+MAC and stimulated with concentrations as low as 0.1-1 ng/ml of OVA(257-264) still produced high amounts of IFN-γ (Figure 6C), as compared to the other groups. Finally, other qualitative aspects were analyzed at the single cell level by studying polyfunctional CD8 T-cells (cells producing several cytokines and displaying different effector functions). These analyses were carried out on high avidity T-cells, by stimulating splenocytes with 1 ng/ml of OVA(257-264). First, a higher number of CD8 T-cells with lytic activity against OVA(257-264), determined by the expression of CD 107, was found in splenocytes from mice immunized with EDA-OVA+MAC; and second, these animals had the highest number of CD8 T-cells simultaneously producing IFN-γ, TNF-a and IL-2 (Figure 6D). These results suggest that immunization with EDA-OVA+MAC induces stronger responses with a higher avidity for the antigen and able to display several effector functions.

EXAMPLE 4

Therapeutic effects of EDA-OVA+MAC administration in B16-OVA tumor-bearing mice.

EDA-OVA+MAC, characterized as the best combination to induce innate and adaptive immune responses, was next used to treat B16-OVA tumor-bearing mice. When mice with seven-day tumors were treated with this strategy, tumor growth was completely blocked until day 30, whereas all untreated mice had already died by day 28. Moreover, beyond this day, tumor growth was slower in the treated group, and half of mice rejected their tumor and still survived at day 80 (Figures 8A-B). These results agree with the ability of splenocytes from mice immunized with EDA-OVA+MAC to recognize in vitro B16-OVA tumor cells (Figure 8C), as opposed to the lack of recognition found in splenocytes from OVA plus Imiquimod and anti-CD40 immunized mice which, as shown in Figures 4B-C, did not reject their tumors.

EXAMPLE 5 Administration of EDA-OVA+MAC to tumor bearing mice induces T-cell responses against different tumor antigens.

To analyze the role of the different tumor antigens recognized by T-cells, at day 80 (60 days after finishing treatment), re-challenge experiments were carried out in surviving animals. None of cured animals after treatment with EDA-OVA+MAC developed tumors when re-challenged with B16-OVA cells, whereas a quick tumor growth was observed in control untreated mice. Moreover, when equivalent mice cured after treatment with EDA-OVA+MAC were re-challenged with B16.F10 tumor cells, which do not express OVA, 80 % of mice remained tumor free (Figure 9A). Analysis in cured animals of responses against tumor antigens expressed by B16-OVA tumor cells showed that they not only had responses against OVA, but also against the melanoma antigen TRP-2 (Figure 9B), suggesting that this treatment not only induces responses against the antigen administered, but also against other antigens expressed by tumor cells.

EXAMPLE 6

Biological activity of the EDA-TRP2(59-257) protein was assessed with a bioassay using human THP-1 monocytic cells.

Treatment of mice bearing subcutaneous B 16. OVA tumors with EDA-OVA + MAC showed that this therapy was able to reject established tumors. Since the effect of this therapy was dependent on CD8 T cells, therapeutic experiments were repeated using a true melanoma tumor antigen, such as TRP2. For these experiments, parental untransduced B16.F10 tumor cell were used. First, a fragment of TRP2, encompassing amino acids 59 to 257 was cloned, fused to EDA and expressed in bacteria (see methods of the invention).

The activity of EDA-TRP2(59-257) was tested in vitro using THP-1 human monocyte cell line. THP-1 cells were plated at 2* 10⁵cells/well in a 96 well plate and cultured with EDA-TRP2(59-257) (1 μΜ), EDA-TRP2(59-257) previously digested with proteinase- K (using agarose-proteinase K beads according to manufacturer's instructions (Sigma, St Louis)), LPS (0.1 μg/ml) or culture medium for 15 hours. Human TNF-a released to the medium by THP-1 cells in response to the stimuli was measured by ELISA (BD Biosciences) according to manufacturer^'s instructions. As shown in Figure 10, EDA-TRP2(59-257) protein activated THP-1 cells (measured as the production of TNF-a), and this activity is due to the protein fraction and not to potential non-protein components, such as LPS, since treatment with proteinase K abolishes the effect. EXAMPLE 7

Immunization with EDA-TRP2(59-257) + MAC

In next experiments, the ability of EDA-TRP2(59-257) + MAC to induce a cellular immune response was analyzed in immunization experiments. Mice were immunized with this combination and immune responses against CD8 epitope TRP2(180-188) and against TRP2 protein were studied.

C57BL/6 na^'ive mice (n=3) were immunized with EDA-TRP2(59-257) + MAC (subcutaneous administration of poly(LC) (50 μg/mouse), anti-CD40 (50 μg/mouse), EDA-TRP2(59-257) protein (2 nanomoles/mouse) as well as topical application of Imiquimod cream (2.5 mg/mouse)). Six days later animals were sacrificed and splenocytes were obtained for immunological analysis. T-cells producing IFN-γ were enumerated by ELISPOT assays using a kit from BD-Bio sciences (San Diego, CA) according to manufacturer instructions. Briefly, plates (Multiscreen HTS; Millipore, Bedford, MA) were coated overnight with anti-IFN-γ antibody, washed with PBS and blocked for 2 h with RPMI containing 10% fetal calf serum. Then, 4 x 10⁵ splenocytes were cultured in triplicate in the absence or in the presence of peptide TRP-2(180-188) (10 μg/ml), proteins EDA-TRP2(59-257), EDA-OVA or EDA (all at 2 μΜ). One day later plates were washed with PBS and incubated with biotinylated anti-IFN-γ antibody. After 2 hours, plates were washed and incubated with a 1/100 dilution of streptavidin- peroxidase. One hour later plates were washed and developed with freshly prepared 3- amino-9-ethylcarbazole solution. The reaction was stopped with distilled water and spots were counted using an automated ELISPOT reader (CTL; Aalen, Germany).

These experiments showed that a single immunization with EDA-TRP2(59-257) + MAC could induce CD8 T cell responses against peptide TRP2(180-188) as well as against the TRP2 fragment (figure 11).

EXAMPLE 8 Tumor treatment experiments

Once the ability of EDA-TRP2(59-257) to activate innate and adaptive immunity was characterized, this construct was used in therapeutic experiments in mice with established B16.F10 tumors.

Mice (n=10 per group) were injected intradermally with 10⁵ B16.F10 cells and when the tumor diameter reached 4-5 mm, mice were treated three weeks twice per week with: poly(LC) (50 μg/mouse), anti-CD40 (50 μg/mouse) and EDA-TRP2(59-257) (2 nanomoles/mouse) all administered intratumor, as well as with Imiquimod cream (daily topical application to shaved skin at the tumor site; 2.5 mg/mouse). Untreated mice challenged with tumor cells were used as positive controls of tumor growth. Tumor volume was calculated according to the formula: V= (length x width²)/2. Mice were killed when tumor diameter reached 17 mm.

These experiments showed that, while tumors grew quickly in untreated mice, mice treated with EDA-TRP2(59-257) + MAC did not show any tumor growth for 10 days, and after that, they had slow-growing tumors (figure 12A). Moreover, all untreated mice died by day 13, whereas in the group of mice treated with EDA-TRP2(59-257) + MAC, 50% of animals still survived 37 days after starting treatment (figure 12B).

Claims

A conjugate comprising:

(i) the fibronectin extracellular domain A (EDA) or a functionally equivalent variant thereof and

(ii) at least one melanoma-associated antigenic protein or peptide or an antigenic fragment of said protein or peptide,

wherein components (i) and (ii) are covalently coupled.

The conjugate according to claim 1 , wherein the fibronectin EDA is of human origin.

The conjugate according to claims 1 or 2, wherein component (ii) comprises an antigenic peptide selected form tables I, II, III and IV.

The conjugate according to any of claims 1 to 3, wherein component (ii) is selected from the group consisting of TRPl/gp75, TRP2, Tyrosinase, gplOO (Pmell7), Melan-A/MART-1, OAl, RAB38/NY-MEL-1, a Melanoma Antigen Gene (MAGE) family member, a B Melanoma Antigen (BAGE) family member, a GAGE family member, a LAGE-l/NY-ESO-1 family member, GnTV, CDK4 and catenin.

The conjugate according to claims 1 or 2, wherein component (ii) comprises the sequence SEQ ID NO: 356.

The conjugate according to claim 1 to 5, wherein components (i) and (ii) form a single polypeptide chain.

The conjugate according to claim 6 which comprises the sequence SEQ ID NO:350.

The conjugate according to claim 6 which comprises the sequence SEQ ID NO:351.

9. A polynucleotide or gene construct encoding a conjugate according to any of claims 5 to 8.

10. A vector comprising a polynucleotide or gene construct according to claim 9.

11. A cell containing a polynucleotide or a gene construct according to claim 9 or a vector according to claim 10.

12. A composition comprising, together or separately:

(i) a conjugate according to any of claims 1 to 8, a polynucleotide or gene construct according to claim 9, a vector according to claim 10, a host cell according to claim 11 and

(ii) at least a TLR ligand.

13. A composition comprising, together or separately:

(i) a conjugate according to any of claims 1 to 8, a polynucleotide or gene construct according to claim 9, a vector according to claim 10, a host cell according to claim 11 ,

(ii) at least a TLR ligand and

(iii) a CD40 agonist.

14. The composition according to claims 12 or 13 wherein component (ii) is selected from the group of a TLR3 ligand, a TLR7 ligand or a combination thereof.

15. The composition according to claim 14 wherein the TLR3 ligand is poly(I:C).

16. The composition according to claims 14 or 15 wherein the TLR7 ligand is imiquimod.

17. The composition according to any of claims 13 to 16, wherein the CD40 agonist (iii) is an anti-CD40 antibody.

18. A pharmaceutical composition comprising a conjugate according to claims 1 to 8, a polynucleotide or gene construct according to claim 9, a vector according to claim 10, a cell according to claim 11 or a composition according to any of claims

12 to 17 and at least one pharmacologically acceptable carrier or adjuvant.

19. A conjugate according to any of claims 1 to 8, a polynucleotide or gene construct according to claim 9, a vector according to claim 10, a cell according to claim 11 or a composition according to any of claims 12 to 18 for its use in medicine.

20. Use of a conjugate according to any of claims 1 to 8, a polynucleotide or gene construct according to claim 8, a vector according to claim 10, a cell according to claim 11 or a composition according to any of claims 12 to 18 for the manufacture of a vaccine.

21. Use of a conjugate according to any of claims 1 to 8, a polynucleotide or gene construct according to claim 9, a vector according to claim 10, a cell according to claim 11 or a composition according to any of claims 12 to 18 for the manufacture of a medicament for the prevention or the treatment of a melanoma or of a metastasis thereof.

22. An in vitro method for obtaining mature dendritic cells presenting at least one melanoma-associated antigen, comprising:

(i) contacting dendritic cells with a conjugate according to any of claims

1 to 8, a polynucleotide or gene construct according to claim 9, a vector according to claim 10, a cell according to claim 11 or a composition according to any of claims 12 to 18 in conditions suitable for the maturation of the dendritic cells to take place and

(ii) recovering the mature dendritic cells.

23. A dendritic cell which is obtained by a method according to claim 22.

24. The dendritic cell according to claim 23 for its use in medicine.

25. Use of a dendritic cell according to claim 23 for the manufacture of a medicament for the prevention or the treatment of a melanoma or of a metastasis thereof.