WO2008149225A2

WO2008149225A2 - Anti-tumour vaccine comprising full-length wt1

Info

Publication number: WO2008149225A2
Application number: PCT/IB2008/001496
Authority: WO
Inventors: Giuseppe Saglio; Enrico Bracco; Daniela Cilloni
Original assignee: Università Degli Studi Di Torino
Priority date: 2007-06-07
Filing date: 2008-06-04
Publication date: 2008-12-11
Also published as: WO2008149225A3; ITTO20070401A1

Abstract

A vaccine for active immunotherapy for the prevention and/or treatment of tumours in humans characterised in that it includes the WTl protein encoded by the Wilms gene.

Description

"Anti-tumour vaccine" ****

Field of the invention The present invention concerns an anti-tumour vaccine, in particular, a vaccine including a tumour- specific antigen allowing performance of an active immunotherapy directed exclusively against tumour cells, provided with low toxicity and efficacious in containing tumour progression.

Technical background of the invention

The Wilms tumour gene [WTl) was first isolated in the homonymous paediatric kidney neoplasia.¹ It encodes a transcription factor (zinc finger) involved in the regulation of cellular proliferation, differentiation and apoptosis .^2"7

This gene, formed by 10 exons located on chromosome llplS¹, has a role that is not yet completely clear. It inhibits the transcription of several growth factors, among which, PDGF⁸, M-CSF⁹, IGF-II³, of growth factor receptors such as IGF-IR¹⁰ and EGFR⁴ and of other genes among which RAR-α⁵, c-myc¹⁶, c- myb¹¹, N-myc¹² and bcl-2⁶, but it can also activate the transcription of the same bcl-213, and of other genes such as Dax-1¹⁴ and RbAp46¹⁵.

WTl is expressed during the period of organogenesis wherein it has a crucial role. WTl gene knockout mice die at the embryonic stage, probably due to cardiac insufficiency and present alterations at the level of the urogenital apparatus¹⁶.

Initially an oncosuppressor role was attributed to the gene since deletions or mutations were found in Wilms tumour, germline mutations in patients presenting a predisposition for developing leukaemias and it also suppresses the growth of Wilms tumour^{17 21}. Unlike other oncosuppressors such as Rb and p53, which are expressed in all tissues, expression of the NTl gene, in addition to having very low values, is limited to the cells of the gonads, uterus, kidney, spleen, of the mesothelia and to the haematopoietic progenitors^22"25. However, a pro-oncogenic role has recently been proposed for the wild-type form of the WTl gene since evidence of high expression levels in various malignant haematological neoplasias and in several solid tumours have been accumulated. The WTl gene is overexpressed in acute myeloid leukaemia, in acute lymphoblastic leukaemia, in chronic myeloid leukaemia, in myelodysplastic syndromes, in Ph negative myeloproliferative disorders, in some non-Hodgkin lymphomas and also in lung tumours, in mesothelioma, in breast tumour, in the squamous cell carcinoma of head and neck, in soft tissue sarcomas, in osteosarcoma, in thyroid tumours, in colon-rectal adenocarcinoma and in glioblastoma. Furthermore, the expression of WTl in acute myeloid leukaemia and in acute lymphoblastic leukaemia is highly correlated with disease progression. In fact, WTl has been shown to be an optimal marker for monitoring residual disease after chemotherapeutic treatment in patients with acute leukaemia. In the myelodysplastic syndromes, WTl expression levels increase with disease progression and are an early indicator of evolution in acute myeloid leukemia^25"39.

Therefore, WTl seems to be involved in tumourigenesis and in maintenance of the neoplastic phenotype .

The anti-tumoural immune response is classically associated with the activity carried out by the CD8+ cytotoxic T lymphocytes: they recognise the endogenous peptides processed on the class I major histocompatibility complex and are able to directly cause the death of the tumour cell⁴⁰.

There are clear clinical evidences of the efficacy of the anti-tumoural immune response. This can be inferred from the ability of the graft-vs-leukaemia contested upon allogenic transplant of haematopoietic stem cells for the treatment of leukaemias to achieve patient recover⁴¹: in fact, the donor immune system cells are able to recognise and eliminate the neoplasia in a reasonable percentage of cases.

However, this immune reaction is not directed only against the tumour cells, but also affects healthy tissues causing damage to various organs (graft-vs-host disease) ; this is difficult to predict and control once established, even with immunosuppressant drugs.

In order to better exploit the anti-tumoural activity of the immune system, already from the beginning of the 90s, donor lymphocyte infusion was started as treatment for relapse after allogenic transplant, even if, in face of a potential benefit of this procedure, the possibility of developing severe organ damage (graft-vs-host disease) was further increased.⁶⁰

Instead, vaccination with a tumour-specific antigen has the object of selectively directing the activity of the patients immune system against the neoplastic cells expressing the tumour-specific antigen, drastically reducing toxicity against healthy tissues . The WTl protein has a high immunogenic potential. In fact, in humans the spontaneous formation of CD8+ T lymphocytes against the WTl antigen has been observed in patients affected by acute myeloid leukaemia and myelodysplastic syndrome, but also in patients with lung and breast tumours^44'45. The WTl protein is able to stimulate both a CD8+ T cytotoxic response and a humoral response already physiologically in vivo. A vaccination approach could amplify this immune reaction so to make it efficacious in destroying tumour cells. Therefore, WTl protein has two characteristics that make it an ideal antigen for an active onco- haematological immunotherapy approach: high immunogenic potential and a high degree of tumour-specific association. Starting from these observations, evaluation was begun of the possible role of WTl as an antigen against which the body could direct its own immune system in an attempt to eliminate tumour cells.

Experimentation employing peptides derived from the amino acid sequence of the WTl protein are currently ongoing. These peptides that bind to the class I major histocompatibility complex (MHC I) are characterised by common sequences of 8-9 amino acids and can bind to specific MHC I. In a preclinical study, inoculation of mice with these peptides determines the formation of CD8+ cytotoxic T lymphocytes with specificity against the WTl antigen: in vitro such lymphocytes are able to selectively kill tumour cells overexpressing WTl. Mice immunized with these peptides reject inoculated tumour cells overexpressing WTl and survive for a long time. Furthermore, none of the mice vaccinated with these peptides show signs of organ toxicity from CD8+ cytotoxic T lymphocytes specific for WTl antigen⁴². Therefore, the immune response directed against WTl is able to impact only the tumour cells containing high levels of this protein and to spare healthy tissues.

In another study the peptides were designed so to be able to bind also to the class II major histocompatibility complex (MHC II). In this case attempting to exploit also the humoral side of the immune response, which seems to be responsible for the long-term maintenance of the anti-tumour response. Vaccination of mice with these peptides induces the formation of both CD8+ cytotoxic T lymphocytes and antibodies specific against the WTl antigen. Also in this case no sign of toxicity at the histopathologic level is registered, confirming the absence of auto aggression against healthy tissues by the cell-mediated and humoral immune systems of the organism, stimulated against the WTl antigen⁴³.

In particular, various peptides able to evoke a cytotoxic immune response against the WTl antigen have been identified in human, but they must bind selectively to specific human MHC I. The peptides 9-mer

(a. a. 235-243, of sequence CYTWNQMNL) and Dbl26 (a. a.

126-134, of sequence RMFPNAPYL), were derived from the most represented histocompatibility complexes in the

Japanese population (HLA-A*2402) and Caucasian population (HLA-A*0201) respectively, capable of stimulating the greatest immune response in the respective populations. Mononuclear cells, taken from the peripheral blood of healthy donors with compatible HLA and pulsed in vitro with the peptides derived from WTl, develop specific CD8+ cytotoxic T lymphocytes. The latter are able to selectively kill WTl overexpressing leukaemia cells^46"49, without inhibiting the formation of colonies from normal bone marrow cells: this indicates that the CD8+ cytotoxic T lymphocytes evoked through exposure to the WTl antigen do not attack the normal haematopoietic progenitors which express extremely low levels of ΗT1^A1' ^50~51. This observation, together with the studies performed in mice, confirms that also in human, stimulation of the immune system against the WTl antigen would not cause damage to those tissues expressing WTl physiologically, probably as a consequence of the different WTl expression levels in dysplastic or neoplastic cells with respect to healthy cells.^22"25 Based on these results the first phase I and II clinical studies were initiated in patients affected by acute leukaemia, myelodysplastic syndrome, lung tumour and breast tumour. Preliminary results show that the vaccination is very safe and without toxicity thus confirming what had already been demonstrated by the preclinical studies; in fact, no self-immunity phenomenon is registered, even against tissues expressing WTl physiologically. Furthermore, it is observed that the vaccination shows a good activity against onco-haematological tumours: two patients affected by myelodysplastic syndrome show a decrease in the number of blasts and in bone marrow WTl expression levels, one patient with relapsing acute myeloid leukaemia achieved complete remission, while in other patients with acute myeloid leukaemia in clinical remission a reduction in the minimal residual disease is observed, both in blast number and in WTl expression. The peptide vaccination appears to be active also against solid tumours: clinical responses are detected in two patients affected by lung tumours and in another two affected by metastatic breast tumours^52"55.

Recently, the hypothesis that two other neoplasia might respond to vaccination with antigens contained in WTl has been evaluated: the non Hodgkin lymphomas (NHL) and multiple myeloma. In NHL this is justified by increased immunohistochemical expression levels of WTl in the neoplastic cells, while in multiple myeloma this is justified by the observation in vitro that CD8+ cytotoxic T lymphocytes specific against the WTl antigen lyse multiple myeloma cells, even though in these cells an increase in NTl expression level has not been demonstrated^{5 ~57}.

Preliminary data from phase II studies in human confirm a positive effect of peptide vaccination in patients affected by both onco-haematological neoplasias and by solid tumours⁴²' ^52~55> ^58,59 _{^ τhe} positive effect of such preliminary results is even more appreciable in light of the fact that patients selected for phase II studies have very often been treated with multiple cycles of various chemotherapeutics and are in very advanced stages of the disease, with a very depressed immune system and therefore normally little susceptible to vaccine stimulation.

Since to date, vaccination through antigens contained in WTl has been shown to be very safe and without toxic affects, in the future, patients can be selected that have less advanced disease, and therefore a more efficient immune system in which the true activity and efficacy of this immunotherapeutic approach can be evaluated.

The anti-tumour vaccines containing WTl-derived peptides currently under study, in spite of the positive responses in terms of antibody response, present some very important disadvantages: 1) they cause an HLA-dependent response and therefore can not be used in all patients, 2) they only generate a CD8+ type of lymphocyte response against only one epitope of the entire antigen, and 3) they do not allow the generation of a humoral immune response. ^■ On the contrary, the approach described herein, providing for the use of the entire WTl protein, causes both a cell- mediated type and a humoral-type of immune response. They are both critical in mediating the short term and long term anti-tumour response⁶¹. Furthermore, vaccination with the entire protein causes an immune response directed against various epitopes of the WTl antigen and this positively influences the efficacy of the anti-tumour response. Finally, this type of vaccination is HLA independent and this represents an extremely advantageous condition for the development of a vaccine-therapy clinical protocol.

General description of the invention

Object of the present invention is the identification of a tumour-specific antigen allowing the performance of an active immunotherapy directed exclusively against tumour cells and overcoming the inconveniences associated with the use of peptides.

According to the present invention, such object is achieved by means of the solution specifically recalled in the claims that follow. The claims form an integral part of the technical teaching provided herein relative to the invention.

The invention concerns the use of the full-length WTl protein, in particular the human WTl protein, as a vaccine for active immunotherapy directed exclusively against tumour cells. The sequence of the human WTl protein is accessible from the Genbank amino acid sequence database with the access number NP_077744.

Classically, the CD8+ T-type lymphocyte response is the one most implicated in suppression of neoplastic cell growth, but there is evidence that the humoral response is the one that maintains the immune response against the tumour cells over time⁴³. It is known that peptide vaccines mainly evoke a CD8+ T cytotoxic mediated type of immune response. Furthermore, it is know that peptide vaccines have the limitation of being administrable only in patents with specific MHC I. On the contrary, the vaccine according to the present invention is able to induce a good CD8+ T lymphocyte cytotoxic response and to invoke a humoral response, since it allows the exploitation of the entire reactivity potential of the immune system against different epitopes of the same antigen. Furthermore, the activity of such vaccine is HLA- independent, therefore it can be used for the vaccination of subjects having diverse HLA. This vaccine is free of toxicity for healthy tissues physiologically expressing WTl, such as the haematopoietic tissues, kidney, spleen, gonads and lymph nodes, while it shows a surprising activity in reducing tumour growth.

Brief description of the Figures

Figure 1 : SDS-PAGE of the bovine albumin serum (BSA) curve at the concentrations of 1 μg, 3 μg, 5 μg, 10 μg and 15 μg. - Figure 2 : SDS-PAGE of the bacterial lysate at time 0 after induction (1), after 3 hours (2) and after 6 hours (3) and GST-WTl purification product (4) .

Figure 3 : Histology of the lymph node, spleen, ovary, and kidney of GST+AD (1, 3, 5 and 9) and of WT1+AD (2, 4, 6 and 10) stained with haematoxylin eosin, and bone marrow smears from GST+AD (7) and of WT1+AD (8) stained with giemsa and Wright stain. - Figure 4 : Dot blot of increasing dilution of serum from mice vaccinated with GST+AD [1/500 (1), 1/2000 (2), 1/3000 (3) and blank (4)] and of serum from mice vaccinated with WTl protein associated with the adjuvant [1/500 (5), 1/2000 (6), 1/3000 (7) and blank (8)] .

Figure 5 : luminescence values at various decreasing dilutions of serum observed with the dot blot method corresponding to the presence of anti WTl Ig in the group of mice vaccinated with GST+AD.

Figure 6 : luminescence values at various decreasing dilutions of serum observed with the dot blot method corresponding to the presence of anti WTl

Ig in the group of mice inoculated with WTl protein associated with the adjuvant.

Figure 7 : dot blot of sera of mice inoculated with the WTl protein associated with the adjuvant (1, 2, 3, 7, 8 and 9), with PBS only (4 and 10) and with GST associated with the adjuvant (5, 6, 11 and 12).

Figure 8 : luminescence values observed with the dot blot method corresponding to the presence of anti WTl Ig in the group of mice injected with the WTl protein associated with the adjuvant (WT1+AD) , with the WTl protein only (WTl), with PBS only (PBS) and with GST associated with the adjuvant (GST+AD) .

Figure 9 : mean cytotoxicity in the groups of treated mice. - Figure 10 : evaluation of the tumour surface (A) and mass (B) in the groups of mice vaccinated (WT1+AD) and in the controls (CTRL) treated with GST+AD and PBS.

Detailed description of the invention The invention will now be described in detail, by way of non-limiting example only. Preparation of the GST-WTl fusion protein

The complete sequence encoding murine WTl (NM_144783), 1339 nucleotide bases, was amplified by PCR from a classical mammalian expression vector containing the WTl cDNA driven by a strong promoter such as CMV kindly provided by Dr. Hastie, using the following primers: forward primer: 5'-CGGAATTCATGGGTTCCGACGTGCGGGAC-S' (SEQ ID No. : 1) reverse primer: 5'- CCGCTCGAGTCAAAGCGCCACGTGGAGTTTGG-3' (SEQ ID No . : 2 ) .

The resulting DNA fragment was cut at the 5' and 3' ends with the restriction enzymes Eco RI and Xho I respectively and cloned into the same restriction sites of the pGEX-4T-l vector (Amersham Biosciences) containing the glutathione-S-transferase protein (GST) under the control of an isopropyl-b-D-thiogalactaside (IPTG, VWR) inducible promoter. The construct was transformed into E. CoIi (JM109) to produce the GST-WTl fusion protein. The E. CoIi transformed with the construct were incubated at 37° in Luria Bertani (LB) culture media with ampicillin [100 μg/ ml] starting from an 0.D 600 > 0.8 and in the presence of 1 mM IPTG for 6 hours. The bacteria are then washed twice with 10 ml of cold IX PBS and re-suspended in 3 ml of lysis buffer (3 ml of IX PBS; PMSF [100 μg/ml] , aprotinin [1 μg/ml] and DTT [1 mM] ) . The cells were "sonicated" 3 times for 45' ' with pauses of 5' , in addition, the third time 1% X-IOO Triton is added (VWR) . The lysate was centrifuged for 15' at 13,000 RPM and the supernatant was transferred to clean test-tubes. The lysis product was incubated with glutathione-conjugated beads [100 μl of beads for each 100 ml of bacterial culture] (Amersham Biosciences) for 30' at 4°C with agitation and the beads were then washed 3 times with 1 ml of RIPA buffer (Tris HCl pH 8 [50 mM] , NaCl [150 mM] , NP40 [1%], DOC [0.5%] and SDS [0.1%]) and a fourth time with IX PBS. The GST-WTl protein was eluted from the beads by boiling at 100⁰C for 5' . The product of the purification was checked and quantified by SDS- PAGE. SDS-PAGB The total lysate or the purification product was dissolved in 3X SDS sample buffer (Bio-Labs), boiled for 5' at 95°C and loaded on a 10% polyacrylamide gel.

The gel is run at constant voltage (100V), then stained for 30' with coomassie blue and finally destained overnight in a solution of ethanol (5%) and acetic acid (7%). A bovine serum albumin (BSA) curve is also constructed for the purpose of quantifying the purification product, as is shown in figures 1 and 2. Dot Blot to detect the presence of antibodies directed against the WTl antigen

The dot blot instrument was prepared with two layers of absorbent paper (M3 Whatman) on which the re- hydrated nitrocellulose membrane (Amersham Biosciences) was positioned. The instrument is started and into each well 0.5 μg of GST-WTl protein (7.15 μg of protein per cm² of nitrocellulose membrane) are spotted.

The membrane is left for 30' in a humid chamber and then saturated with 5% bovine serum albumin (BSA, Sigma) . Three washes are performed with 0.1% TBS-Tween (Sigma) and then the membrane is repositioned in the dot blot instrument where 20 μl of serum, diluted 1:500 with water, are spotted.

The membrane is then left again for 30' in a humid chamber. Three 15' washes are performed with 0.1% TBS- Tween. At this point the membrane is incubated for 60' with peroxidase conjugated anti-mouse (Santa Cruz Biotechnology) and it is then washed 4 times for 10' with 0.1% TBS-Tween. The product of the reaction is detected with the ECL solution (Amersham Biosciences) and read with a luminometer (Viktor, Perkin Elmer) . Groups of mice used in the study

The mice used in this study were 54 8-week old females of the C57BL/6J strain divided into 3 groups as is shown in table 1. Table 1 .

WT1+AD = WTl protein associated with the adjuvant GST+AD = GST associated with the adjuvant ) PBS = only PBS

Administration scheme of the WTl protein vaccine

The various groups of mice listed above were vaccinated subcutaneously with the vaccination protocol reported in table 2 for a duration of 4 weeks. Table 2.

(*⁾ WTl+AD = WTl protein associated with the adjuvant GST+AD = GST associated with the adjuvant I PBS = only PBS

The murine cell lines used

The cell line TRAMP-C2 (ATCC, CRL-2731, USA) is maintained in Dulbecco's Modified Eagle's Medium (DMEM, Cambrex) culturing media with 10% Fetal Bovine Serum (FBS, Invitrogen) and 4 mM L-glutamine. TRAMP-C2 are murine prostate adenocarcinoma cells derived from C57BL/6J mice and expressing high levels of WTl⁴³. The C1498 cell line is maintained in Dulbecco's Modified Eagle's Medium (DMEM) culture medium with 10% Fetal Bovine Serum (FBS) and 4 mM L-glutamine. The C1498 are murine acute leukaemia cells derived from C57BL/6J mice . Cytotoxicity or chrome release test

The mice were sacrificed by cervical dislocation and lymphocytes were obtained through spleen homogenization.

The tumour cells expressing NTl (TRAMP-C2) were previously treated with mitomycin C (Sigma) for 30 minutes to block their growth and then put into contact with the lymphocytes taken from the mice in a 1:20 ratio in 10 ml of RPMI 10% FBS, in the presence of interleukin-2 [1 ng/ml] (Listarfish) . The lymphocytes were incubated with the tumour cells for 6 days, after which they were reacted with tumour cells labelled with ⁵¹Cr - 50 μCi - (Perkin Elmer) in 96-well plates. The plates are left at 37°C for 4 hours and then centrifuged: the supernatant is transferred into LumaPlate plates (Perkin Elmer) for reading in a scintillation counter.

Infection of marine acute leukaemia, cells (C1498) with the lentiviral vector expressing WTl The lentiviral vector expressing WTl henceforth referred to as LV-WTl was constructed by cloning the complete coding sequence of WTl between the Bam HI and Sal I sites of the pCCL vector (United States Patent 6924144) . Aliquots containing a high lentiviral titre were produced in HEK-293-T cells (ATCC, CRL-1573, USA) following co-transfection with vectors expressing the genes encoding the proteins required for viral packaging, with the calcium phosphate technique. The supernatants of these cells were collected 36 hours after transfection, filtered (0.22 μm pore diameter) and used directly to infect exponentially growing C1498 cells . Statistical analysis For statistical analysis the Student's t test for independent data and its non-parametric analog: the Mann Whitney test were used.

EXAMPLES Example 1

Evaluation of organ toxicity in vaccinated mice

The group of C57BL/6J mice injected with the WTl protein in association with the adjuvant (WT1+AD) , the mice treated with only GST and adjuvant (GST+AD) and those treated with only PBS (PBS) were sacrificed and evaluated both through histological analysis and blood cell counts to exclude a possible organ toxicity.

In particular, the histological analysis performed on tissue sections from kidney, lymph node, spleen, gonad and on transversal sections of the femoral bone did not reveal pathological alterations (figure 3). Bone marrow smears did not reveal significant qualitative or quantitative anomalies at the level of haematopoietic progenitors.

The blood cell counting performed in vaccinated mice, as is shown in table 3, does not show statistically significant differences in the values of Hb (p> 0.05), white blood cells (p> 0.05), formula and platelets (p> 0.05) with respect to the control groups.

Table 3.

Mean Hb WBC Platelets

WT1+AD(* ) 13. 975 5135 1128000

GST +AD ( ** ) 16. 4 6150 1463000

PBS(***) 14. 275 7357. 5 1020000

(*) WT1+AD = WTl protein associated with the adjuvant (**) GST+AD = GST associated with the adjuvant (***) PBS = only PBS

Example 2

Evaluation of the development of anti-WTl antibodies after vaccination with the protein.

In the group of C57BL/6J mice vaccinated with the WTl protein in association with the adjuvant, mice treated with only GST and adjuvant and with only PBS the presence of total antibodies directed against WTl was evaluated through the dot blot technique, see figures 4, 5, 6 and 7.

The mice treated with only PBS or GST and adjuvant do not develop antibodies. The mice inoculated with the entire WTl protein with adjuvant develop significant levels of antibodies, as is shown in figure 8.

Example 3 Evaluation of cytotoxic activity in vaccinated mice

On the same groups of mice described above, the activity of cytotoxic T lymphocytes was evaluated with the ⁵¹Cr release test. The test demonstrated that the vaccinated mice (group WT1+AD) develop a mean cytotoxicity level of approximately 30%, which instead reaches a value of only approximately 5% in the GST+AD group and only approximately 2% in the PBS group as is shown in figure 9.

Example 4

Antitumour effect of the vaccine

Two weeks after the last immunisation, 75 x 10³ C1498 cells (expressing high levels of WTl) were inoculated subcutaneously in the groups of mice treated with WT1+AD, GST+AD and PBS. Two weeks after the inoculation, a palpable tumour mass is observed. At 8 weeks after the inoculation the tumour mass reaches considerable size and at this point the mice were sacrificed and the masses analysed. The group of mice immunised with WT1+AD show tumour masses and surfaces of significantly reduced size with respect to the control groups GST+AD and PBS (p< 0.03 and p<0.01 respectively) as is shown in figure 10.

Naturally, the details and the embodiments may vary- appreciably with respect to what has been described and illustrated without departing from the scope of the present invention, as defined by the annexed claims. SEQUENCE LISTING

<110> Umversita degli Studi di Torino

<120> Anti-tumor vaccine

<130> BWO9922-CF <150> TO2007A000401

<151> 2007-06-07

<160> 2 <170> Patentln version 3.3

<210> 1

<211> 29

<212> DNA <213> artificial

<220>

<223> forward primer for cloning WTl antigen <400> 1 cggaattcat gggttccgac gtgcgggac 29

<210> 2 <211> 32

<212> DNA

<213> artificial

<220> <223> reverse primer for cloning WTl antigen

<400> 2 ccgctcgagt caaagcgcca cgtggagttt gg 32

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Claims

1. A vaccine for active immunotherapy for the prevention and/or treatment of tumours in humans characterised in that it includes the full-length WTl protein encoded by the Wilms gene.

2. A vaccine according to claim 1, wherein the WTl protein is of human origin.

3. A vaccine according to claim 1 or claim 2, wherein the WTl protein is associated with an adjuvant.

4. A vaccine according to any of the previous claims, wherein the WTl protein is produced with recombinant DNA technology.

5. A vaccine according to any of the previous claims, wherein the WTl protein is a fusion protein.

6. A vaccine according to any of the previous claims, wherein the tumour is selected from among acute myeloid leukaemia, acute lymphoblastic leukaemia, chronic myeloid leukaemia, myelodysplastic syndromes, Ph negative myeloproliferative disorders, non-Hodgkin lymphoma, multiple myeloma, lung tumour, mesothelioma, breast tumour, squamous carcinoma of head and neck, soft tissues sarcoma, osteosarcoma, thyroid tumour, colorectal adenocarcinoma and glioblastoma.

7. A vaccine according to any of the previous claims, wherein the vaccine is suitable for subcutaneous, intradermal, intervenous, intramuscular aaddmmiinniissttrraattiioonn,, oorr ffoorr vveennoouuss iinnffuussiioonn.

8. The use of the full-length WTl protein encoded by the Wilms tumour gene for the production of a vaccine for the prevention and/or immunotherapy of tumours in humans.

9. Use according to claim 8, wherein the WTl protein is of human origin.

10. Use according to claim 8 or claim 9, wherein the WTl protein is associated with an adjuvant.

11. Use according to any of the claims 8 to 10, wherein the WTl protein is produced with recombinant DNA technology.

12. Use according to any of the claims 8 to 11, wherein the WTl protein is a fusion protein.

13. Use according to any of the claims 8 to 12, wherein the tumour is selected from among acute myeloid leukaemia, acute lymphoblastic leukaemia, chronic myeloid leukaemia, myelodysplastic syndromes, Ph negative myeloproliferative disorders, non-Hodgkin lymphoma, multiple myeloma, lung tumour, mesothelioma, breast tumour, squamous carcinoma of head and neck, soft tissue sarcoma, osteosarcoma, thyroid tumour, colorectal adenocarcinoma and glioblastoma.

14. Use according to any of the claims 8 to 13, wherein the vaccine is suitable for subcutaneous, intradermal, intervenous, intramuscular administration, or for venous infusion.