VACCINE

Abstract

The present invention relates to a pharmaceutical combination of compositions for use in the treatment or prevention of a disease having cells bearing a target antigen as a vaccine and to a method for vaccination of a mammal, especially of a human for raising a cellular immune response directed against cells of the mammalian recipient, especially human recipient, which cells express a target antigen. The target antigen can e.g. be an autoantigen like a malignant antigen, i.e. a tumour-specific antigen. The pharmaceutical combination of compositions comprises a first composition and a second composition, wherein the second composition is for administration to recipient subsequent to the administration of the first composition, e.g. 2 to 10 days after the first composition. The pharmaceutical combination of compositions has the advantage of raising an effective antigen-specific T-cell response against cells bearing a target antigen that can be a malignant autoantigen, e.g. for raising an antigen-specific T-cell response against cells bearing a tumour-antigen. A further advantage is that the pharmaceutical combination of compositions can raise an antigen-specific T-cell response within a comparatively short time.

Claims

1. Pharmaceutical combination of compositions for use in medical treatment, the combination comprising a first composition comprising dendritic cells (DC) which are immunologically compatible with a recipient and which are associated with a target antigen and a second composition comprising at least a portion of the target antigen in soluble form and a co-stimulatory antibody effective for activating T-cells and/or the dendritic cells (DC), wherein the second composition is for administration at a time at least 1 day subsequent to administration of the first composition.

2. Pharmaceutical combination according to claim 1, wherein the dendritic cells (DC) are associated with the target antigen by being contacted with the target antigen or by being contacted with a nucleic acid sequence encoding the antigen.

3. Pharmaceutical combination of compositions for use in medical treatment, the combination comprising a first composition comprising an antibody specific for a surface receptor of a dendritic cell (DC) coupled to a target antigen and a second composition comprising at least a portion of the target antigen in soluble form and a co-stimulatory antibody effective for activating T-cells and/or dendritic cells (DC), wherein the second composition is provided for administration at a time at least 1 day subsequent to administration of the first composition.

4. Pharmaceutical combination according to claim 3, wherein the antibody specific for a surface receptor of a dendritic cell (DC) is an anti-DEC205 antibody and/or an anti-DCIR antibody.

5. Pharmaceutical combination according to claim 3, wherein the medical treatment is the treatment of tumour, of viral infections or of infections by intracellular bacteria.

6. Pharmaceutical combination according to claim 3, wherein the second composition further contains a non-specific TLR3 agonist, TLR7 agonist, TLR4 agonist, TLR9 agonist or combinations of at least two of these.

7. Pharmaceutical combination according to claim 3, wherein the co-stimulatory antibody effective for activating professional antigen presenting cells (APC) is selected from the group consisting of anti-CD137 antibody, an anti-CD40 antibody, an anti-OX40 antibody, anti-ICOS antibody, an anti-CD27 antibody, an anti-CD28 antibody, an anti-GITR antibody, specifically anti-human GITR/AITR antibody, an anti-HVEM antibody, an anti-TIM1 antibody, an anti-TIM3 antibody, and mixtures of at least two of these.

8. Pharmaceutical combination according to claim 3, wherein the non-specific TLR3 agonist is Poly(I:C) and/or PolyICLC or a homologue thereof.

9. Pharmaceutical combination according to claim 3, wherein the medical treatment is for raising in a recipient a cellular immune response specifically directed against cells of the recipient bearing the target antigen.

10. Pharmaceutical combination according to claim 3, wherein the first composition is free from an adjuvant.

11. Pharmaceutical combination according to claim 5, wherein the tumour is selected from the group comprising or consisting of hematological malignancies, Hodgkin and non-Hodgkin lymphomas, leukemias, especially acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, monocytic leukemia, myelomas, myeloproliferative diseases, myelodysplastic syndromes and solid cancers, especially originating from brain, head and neck, lung, pleura, heart, liver, kidney, colon, pancreas, stomach, gut, urinary tract, prostate, uterus, ovaries, breast, skin, testes, larynx and sarcoma.

12. Pharmaceutical combination according to claim 5, wherein the tumour antigen is selected from the group consisting of tumour antigens, tumour homogenate or tumour lysate.

13. Pharmaceutical combination according to claim 2, wherein the dendritic cells (DC) following in vitro contact with the target antigen by being contacted with the target antigen or by being contacted with a nucleic acid sequence encoding the antigen are separated from the medium containing the target antigen or nucleic acid sequence encoding the antigen and are expanded in number by cultivation in cell culture medium.

14. Pharmaceutical combination according to claim 3, wherein the medical treatment comprises the generation of CD8+ T-cells which are specific for the target antigen and/or the generation of CD4+ T-cells which are specific for the target antigen.

15. Pharmaceutical combination according to claim 3, wherein the medical treatment generates activated CD8+ T-cells having specificity for autologous cells comprising the antigen.

16. Pharmaceutical combination according to claim 1, wherein the second composition further contains a non-specific TLR3 agonist, TLR7 agonist, TLR4 agonist, TLR9 agonist or combinations of at least two of these.

17. Pharmaceutical combination according to claim 1, wherein the co-stimulatory antibody effective for activating professional antigen presenting cells (APC) is selected from the group consisting of anti-CD137 antibody, an anti-CD40 antibody, an anti-OX40 antibody, anti-ICOS antibody, an anti-CD27 antibody, an anti-CD28 antibody, an anti-GITR antibody, specifically anti-human GITR/AITR antibody, an anti-HVEM antibody, an anti-TIM1 antibody, an anti-TIM3 antibody, and mixtures of at least two of these.

18. Pharmaceutical combination according to claim 1, wherein the second composition further comprises a non-specific TLR3 agonist that is Poly(I:C) and/or PolyICLC or a homologue thereof.

19. Pharmaceutical combination according to claim 1, wherein the first composition is free from an adjuvant.

Description

[0041] The invention is now described in greater detail by way of mouse experiments with reference to the figures, which show for different first and second compositions administered to experimental animals in

[0042] FIG. 1 flow cytometry results of peripheral blood with staining for CD11a at day 1 prior to administration of the second composition at a), c), e), g), and i) with restimulation with the target antigen (Ndufs1) and at b), d), f), h) and j) without restimulation (Control) with antigen,

[0043] FIG. 2 flow cytometry results of peripheral blood with staining for IFN gamma at day 1 prior to administration of the second composition at a), c), e), g), and i) with restimulation with the target antigen (Ndufs1) and at b), d), f), h) and j) without restimulation (Control) with antigen,

[0044] FIG. 3 a graphical representation of the proportion of malignant antigen-specific CD8+ T-cells from the results of FIG. 2,

[0045] FIG. 4 a)-j) flow cytometry results of peripheral blood with staining for highly CD11a-positive T-cells at day 7 following administration of the second composition,

[0046] FIG. 5 a graphical representation of the proportion of activated, i.e. highly CD11a-positive T-cells from the results of FIG. 4,

[0047] FIG. 6 flow cytometry results of peripheral blood with staining for IFN gamma-positive T-cells at day 7 following administration of the second composition at a), c), e), g), and i) with restimulation with the target antigen (Ndufs1) and at b), d), f), h) and j) without restimulation (Control) with antigen,

[0048] FIG. 7 a graphical representation of the proportion of IFN gamma-positive T-cells in activated T-cells from the results of FIG. 6,

[0049] FIG. 8 a graphical representation of the proportion of antigen-specific T-cells activated by different priming regimens,

[0050] FIG. 9 a) to f) FACS results for one exemplary experimental animal each at different priming regimens,

[0051] FIG. 10 the proportion of CD11a.sup.hi CD8+ T-cells for different second compositions,

[0052] FIG. 11 the proportion of IFN-positive CD8+ T-cells for different second compositions,

[0053] FIG. 12 the in vivo reduction of tumour volume,

[0054] FIG. 13 a graphical representation of the proportion of T-cells in white blood cells (WBC) when stimulated by different compositions,

[0055] FIG. 14 a graphical representation of the proportion of antigen-specific T-cells in white blood cells when stimulated by the compositions used for FIG. 13,

[0056] FIG. 15 a graphical representation of the proportion of antigen-specific T-cells in the T-cell response when stimulated by the compositions used for FIG. 13,

[0057] FIG. 16 a graphical representation of IL-6 contained in CD8+ T-cells raised by the compositions of the invention and by virulent Listerium monocytogenes and by virulent LCM virus,

[0058] FIG. 17 a graphical representation of IFN contained in CD8+ T-cells raised by the compositions of the invention and by virulent Listerium monocytogenes and by virulent LCM virus,

[0059] FIG. 18 a graphical representation of TNF contained in CD8+ T-cells raised by the compositions of the invention and by virulent Listerium monocytogenes and by virulent LCM virus, and in

[0060] FIG. 19 the effect of various TLR agonists in the second composition.

[0061] In the following examples and comparative examples, mice were used for representing a human recipient. Mice were divided into groups of 5 mice (strain C57 Bl/6) each. The animals were housed under standard conditions with feed and water ad libitum. Mice were subjected to different prime-boost regimens. Administration of first composition (priming) and of second composition (boosting) was by intravenous (iv) injection. In the figures, the co-stimulatory antibody is designated by its target, e.g. in the figures anti-CD40 antibody is designated as CD40.

[0062] As an example for a malignant antigen, a mouse antigen isolated from HCC tumour, Ndufs1 having amino acid AAVSNMVQKI (SEQ ID NO: 360) was used. Ndufs1 is a model antigen for a homologous tumour antigen. Ndufs1 was prepared by chemical peptide synthesis. The compositions comprised the constituents of the compositions in aqueous medium, preferably in physiological saline.

Example 1

Immunization with Different First Compositions, Followed by Different Second Compositions

[0063] For priming, on day 7 mice received as a first composition either physiological saline (group 1), 100 s soluble Ndufs1 peptide (group 2), 100 g Ndufs1 peptide conjugated to 1 mg PLGA microspheres of 2 m mean diameter (group 3), or 10.sup.6 dendritic cells that were in vitro coated with 10 g Ndufs1 peptide (groups 4 and 5) intravenously. 7 days later (day 0), mice received boosting by intravenous administration of a combination of 100 g Ndufs1 peptide, 100 g of agonistic anti-CD40 antibody (clone 1C10) and 200 g of Poly(I:C) (groups 1 to 4), or again 10.sup.6 dendritic cells that were in vitro coated with 10 g Ndufs1 peptide (group 5) as the second composition. After the administration of the second composition, mice were bled from the mandibular vein on the days indicated below. After red cell lysis, peripheral blood mononuclear cells were stained with the following labelled antibodies: anti-IFN gamma antibody-APC (clone XMG1.2, eBioscience), anti-CD8 antibody-FITC (53-6.7, eBioscience and Becton Dickinson Biosciences), anti-CD11a antibody-PE (M17/4, eBioscience).

[0064] The results of flow cytometry using a model Canto II flow cytometer (Becton Dickinson Biosciences) are shown in FIG. 1.

[0065] The following table summarizes first compositions followed by administration of the second compositions:

TABLE-US-00004 Group (Gr.) results in priming boosting 1 FIG. 1a), 1b) physiological saline Ndufs1 + Poly (I:C) + FIG. 2a), 2b) (no priming) anti-CD40 2 FIG. 1c), d) Ndufs1 only (Ndufs1) Ndufs1 + Poly (I:C) + FIG. 2c), d) anti-CD40 3 FIG. 1e), f) PLGA-Ndufs1 Ndufs1 + Poly (I:C) + FIG. 2e), f) anti-CD40 4 FIG. 1g), h) DC-Ndufs1 Ndufs1 + Poly (I:C) + FIG. 2g), h) anti-CD40 5 FIG. 1i), j) DC-Ndufs1 DC-Ndufs1 FIG. 2i), j)

[0066] The 5 animals of each group were treated identically.

[0067] PLGA-Ndufs1 designates microspheres of poly(lactic-co-glycolic) acid comprising the model antigen Ndufs1. DC-Ndufs1 designates dendritic cells (DCs) isolated from the spleen of a mouse of the same strain without administration of the antigen Ndufs1, which DCs were incubated in RPMI culture medium and loaded with the antigen by adding Ndufs1 to a concentration of approx. 2 g/ml medium.

[0068] FIGS. 1-3 refer to analyses at day 1, i.e. 6 days following administration of the respective first composition and 1 day prior to administration of the respective second composition.

[0069] FIG. 1 shows the results of analysis for CD11a, indicating the activated T-cells of the total T-cells (CD8), with addition of antigen Ndufs1 (Figs. a), c), e), g) and i)) in one sample of each animal and without added antigen (Control) for each sample of each animal (Figs. b), d), f), h) and j)).

[0070] The analysis for target antigen-specific T-cells was by measuring IFN gamma following re-stimulation with antigen Ndufs1 (FIG. 2 a), c), e), g) and i)) in a sample of each animal in comparison to the samples from the same animals without added antigen (FIG. 2 b), d), f), h) and j)). For measurement of IFN gamma produced by each T-cell (CD8), secretion of IFN gamma was hindered by addition of brefeldin A (GolgiPlug, available from Becton Dickinson), followed by staining using a labelled anti-IFN gamma antibody and measurement by flow cytometry.

[0071] The flow cytometry analyses after administration of the first compositions only (P, priming) are summarized in FIG. 3, showing that a first composition consisting of DCs in vitro loaded the model tumour antigen Ndufs in groups 4 and 5 (DC-Ndufs1) resulted in approx. 0.02% specific CD8+ T-cells in peripheral blood lymphocytes (PBL), whereas priming with PLGA microspheres with the antigen, group 3 (PLGA-Ndufs1) or antigen alone in group 2 (Ndufs1) resulted in numbers of antigen-specific CD8+ T-cells in PBL close to background obtained without antigen (Gr. 1, -).

[0072] Following administration of the second compositions at day 0 to the same animals, samples were taken 7 days later (Day 7).

[0073] The flow cytometry results from FIG. 4 for staining with anti-CD11a antibody (CD11a) and anti-CD8 antibody (CD8), using the encircled areas, are summarized in FIG. 5, showing that CD11a high positive T-cells (+++CD11a), which are activated T-cells, are generated to approx. 27-28% by the first compositions of PLGA microspheres comprising the antigen or DC loaded with antigen, followed by administration of the second composition comprising the antigen, the TLR3 agonist Poly(I:C) and the co-stimulating antibody anti-CD40 antibody. In contrast, the priming with antigen-primed DC followed by boosting with antigen-primed DC gave the lowest number of activated CD8+ T-cells.

[0074] FIG. 6 shows the flow cytometry results for staining with anti-IFN gamma (IFN gamma) and anti-CD8+(CD8), following re-stimulation with antigen Ndufs1 (FIG. 6 a), c), e), g) and i)) and without added antigen (FIG. 6 b), d), f), h) and j)). It is clearly seen that the priming with a first composition comprising DC loaded with antigen followed by boosting with a second composition comprising the antigen in combination with the TLR3 agonist and with the co-stimulating antibody according to the invention yields the most effective generation of a proportion of antigen-specific CD8+ T-cells (insets in FIG. 6g)) in activated T-cells, which are highly CD11a positive CD8+ T-cells (+++CD11a CD8 T-cells), approx. 16% (Group 4). This proportion is significantly higher than that obtained for both priming and boosting by antigen-loaded DC (Group 5).

[0075] Interestingly, FIG. 6 e) shows that priming by a first composition of PLGA microspheres coated with the Ndufs1 antigen does not yield a detectable level of antigen-specific CD8+ T-cells upon restimulation with Ndufs1 plus TLR3 agonist and the co-stimulatory antibody anti-CD40 (CD40). Currently, this absence of a boosting effect by the second composition is assumed to be caused by the antigen being a tumour-specific antigen and/or when the first composition contains the antigen bound to PLGA microspheres.

[0076] The analytical data for the different first and second compositions for the experimental animals of each group are summarized in FIG. 7. The data show that the combination of compositions according to the invention, priming by antigen-loaded DC followed by boosting with the antigen in combination with an co-stimulatory antibody and, optionally a TLR agonist, results in the highest activation of antigen-specific CD8+ T-cells (Gr. 4), whereas priming with antigen-coated PLGA carrier did not result in a relevant antigen-specific CD8+ T-cell generation when using the same boost (Gr. 3).

[0077] Further, the proportion of IFN-positive cells in CD8+ T-cells was determined for different first and second compositions administered: no priming (), tumour antigen only (Ndufs), PLGA coated with antigen (PLGA-Ndufs), DC coated with tumour antigen (DC Ndufs) Ndufs1 plus Poly(I:C) plus anti-CD40 antibody (COAT Ndufs), followed as indicated in FIG. 8 by the second composition Ndufs1 plus Poly(I:C) plus anti-CD40 antibody (Ndufs+ PolyI:C+CD40+COAT). As a further comparison, both the first and second composition were DC coated with Ndufs1 as the antigen (DC-Ndufs). The data of FIG. 8 show that only the combination of the first and second compositions according to the invention result in an effective generation of antigen-specific IFN-positive CD8+ T-cells. Priming by PLGA coated with antigen (PLGA-Ndufs) gave a highly significant lower proportional response by IFN positive CD8+ T-cells than priming with DC coated with the same tumour antigen (DC-Ndufs), when both were followed by a boost of the tumour antigen Ndufs1, TLR agonist Poly(I:C) and co-stimulatory antibody anti-CD440 (Ndufs+Poly I:C+CD40) (COAT).

[0078] FIG. 9 (all axis same scale, Y-axis IFN, X-axis CD8+) shows FACS results for individual experimental mice analysed in FIG. 8 which have a mean immune response in the respective group as indicated by the inset number showing the relative proportion of IFN positive CD8+ T-cells, e.g. for the compositions of the invention in FIG. 9 d) a value of 11.8% for the first composition of DC-Ndufs and the second composition of Ndufs1, stimulatory antibody and TLR agonist (COAT). The box indicates tumour-antigen specific CD8+ T-cells. The negative control in FIG. 9a) consisting of priming by antigen Ndufs1 (Ndufs) and boosting by COAT gave a marginal response of IFN positive CD8+ T-cells (0.56), FIG. 9b) for priming by antigen alone (Ndufs) and boosting by COAT gave a response of 0.64, and PLGA coated with antigen gave an even lower specific response. The comparative priming and boosting by DC coated with antigen (DC-Ndufs) of FIG. 9f) gave a response a little higher than the other comparative compositions.

[0079] Also the results of FIG. 9 show that essentially only the combination of compositions according to the invention results in an effective generation of tumour-antigen specific CD8+ T-cells.

Example 2

Generation of Antigen-Specific CD8+ T-Cell Response Against Tumour-Antigen

[0080] Following administration of the first composition at day 7, consisting of 10.sup.6 dendritic cells that were in vitro coated with 10 g Ndufs1 peptide in a vehicle, mice were administered with second compositions of the antigen 100 g Ndufs1 and varying amounts of co-stimulatory antibody, exemplified by anti-CD40, and varying amounts of TLR agonist poly(LC). The results are shown in FIG. 10 for no boost by a second composition (left hand col., -Ndufs, -Poly I:C, -antibody (CD40)), and with the amounts indicated.

[0081] The results show that the co-stimulatory antibody of the second composition has a significant effect on the generation of the T-cell response, whereas the TLR agonist supports the effect the second composition, e.g. a comparison of 10 g anti-CD40 with 20 g or 200 g Poly(I:C) shows raising similar proportions of CD11a.sup.hi CD8+ T-cells in total CD8+ T-cells; the same can be seen for 100 g anti-CD40, drastically raising the proportion of CD11a.sup.hi CD8+ T-cells compared to 10 g anti-CD40, whereas 20 g or 200 g Poly(I:C) have a less important impact.

[0082] The results of the analysis of IFN-positive CD8+ T-cells in relation to total CD8+ T-cells are shown in FIG. 11. The data show that the combination of a co-stimulatory antibody and a TLR agonist in the second composition improves the proportion of activated tumour-specific CD8+ T-cells.

Example 3

In Vivo Treatment of Tumour

[0083] As an example for a tumour, mice were subcutaneously injected with 10.sup.7 CMT 64 cells (mouse lung carcinoma) to generate solid subcutaneous tumours seven days prior to the beginning of the immunisations.

[0084] Mice were administered with 10.sup.6 dendritic cells that were in vitro coated with 10 g Ndufs1 peptide intravenously on day 7. 7 days later (day 0), mice received the same composition again (DC-DC Ndufs),

or according to the invention with 10.sup.6 dendritic cells that were in vitro coated with 10 g Ndufs1 peptide intravenously on day 7. 7 days later (day 0), mice received boosting by intravenous administration of a combination of 100 g Ndufs1 peptide, 100 g of agonistic anti-CD40 antibody (clone 1C10) and 200 g of Poly(I:C) (DC-COAT Ndufs), or mice were left without treatment as a negative control (Untreated).

[0085] The results are shown in FIG. 12, demonstrating that the treatment by DC coated with antigen followed by the boosting second composition comprising the tumour antigen, a co-stimulatory antibody and a TLR agonist resulted in a significantly reduced growth of the tumour (CMT 64), whereas the administration of DC coated with the antigen as both the first and the second composition did not result in a significant difference of tumour growth, possibly in added tumour growth.

Example 4

Immunization with First Compositions Containing DC Primed with SIINFEKL, Followed by Different Second Compositions

[0086] For priming, on day 7 mice received as a first composition 10.sup.6 dendritic cells that were in vitro coated with 10 g SIINFEKL peptide, the antigenic epitope of hen ovalbumin (OVA), intravenously and at day 0 were challenged with a second composition by intravenous administration of a combination of 100 g SIINFEKL peptide, 100 g of agonistic antibody, and 200 g of Poly(I:C) for the co-stimulatory antibodies indicated in FIGS. 8 to 10: anti-CD40 (CD40) (clone 1C10), anti-CD134 (CD134) (also termed OX40), anti-CD137 2EI (CD137 2EI), anti-CD137 3H3 (CD137 3H3), anti-CD278 (CD278), corresponding to ICOS.

[0087] As a negative control, the co-stimulatory antibody was replaced by rat IgG2 (RatIgG2) in the second composition. As a positive control, mice on day 7 received Listerium monocytogenes expressing ovalbumin (LM-OVA) followed again by LM-OVA at day 0 as the second composition. As a further negative control, mice were not treated at day 7 nor at day 0 (naiv).

[0088] 7 days after the administration of the second composition, mice were bled from the mandibular vein. After red cell lysis, peripheral blood mononuclear cells were stained with the following labelled antibodies: anti-CD8 antibody-FITC (53-6.7, eBioscience and Becton Dickinson Biosciences), anti-CD ha a antibody-PE (M17/4, eBioscience), TET+ was detected by SIINFEKL-specific tetramers in order to identify antigen-specific activated T-cells.

[0089] The proportion of antigen-specific activated T-cells (CD8+ and CD11a+++ T-cells) of white blood cells (WBC) is shown in FIG. 13 at day 7 (7 Days after 2.sup.nd challenge) following the administration of the second composition shows high proportions of activated T-cells for the agonistic antibody anti-CD40 (CD40), and for anti-CD137 3H3, which are higher than the proportion obtained by the positive control LM-OVA.

[0090] FIG. 14 shows that the antigen-specific CD8 T-cell response to the heterologous antigen SIINFEKL in relation to total white blood cells (WBC) was most intense for boosting with anti-CD40 or anti-CD137 3H3 as the co-stimulatory antibody, and also higher than the positive control LM-OVA.

[0091] FIG. 15 shows that proportion of the antigen-specific CD8 T-cell response in total activated CD8+ T-cells for the co-stimulatory antibodies anti-CD40 (CD40), anti-CD134 (CD134), anti-CD137 2EI (CD137 2EI), anti-CD137 3H3 (CD137 3H3) and anti-CD278 (CD278) (ICOS) was higher than the negative control (naiv) and negative control RatIgG2 for all of the co-stimulatory antibodies tested. Accordingly, these results show that a co-stimulatory antibody which according to the invention is directed against a surface receptor of T-cells and/or of DCs, in the second composition raises the antigen-specific CD8+ T-cell response.

[0092] In comparison to the negative control RatIgG, these data show that the presence of a co-stimulatory antibody has a high influence on the generation of the antigen-specific CD8+ T-cell response.

Example 5

Activity of CD8+ T-Cell Immune Response

[0093] For comparing the effect of the combination of the first and second composition for activity against cells expressing a malignant antigen, SIINFEKL was used as the antigen according to the invention (DC COAT) in comparison to virulent Listerium monocytogenes, representing an intracellular bacterial antigen, and in comparison to LCM virus representing an intracellular viral antigen.

[0094] For priming according to the invention, on day 7 mice received as a first composition 10.sup.6 dendritic cells that were in vitro coated with 10 g SIINFEKL peptide intravenously and at day 0 were challenged with a second composition by intravenous administration of a combination of 100 g SIINFEKL peptide, 100 g of anti-CD40 (CD40) (clone 1C10) as the co-stimulatory antibody, and 200 g of Poly(I:C). This combination is designated as DC COAT in FIGS. 16-18.

[0095] Virulent Listerium monocytogenes (Virulent LM) was administered at a dose of 510.sup.4 du/mouse at day 7 at day 0.

[0096] LCM virus (LCMV, Armstrong wild-type strain) was administered at a concentration of 210.sup.5 at day 0.

[0097] As a negative control, mice were left without treatment or challenge.

[0098] For analysis, at day 7 following day 0, cytokines IL-6, IFN and TNF were analysed from spleen lysate. The increased production of these cytokines as measured in spleen lysate indicates expansion of CD8+ T-cells and CD8+ T-cell activation.

[0099] Results are shown in FIGS. 16 to 18. The results show that the administration of the first and second compositions according to the invention (DC COAT) gave rise to CD8+ T-cell expansion as indicated by producing the significantly highest production of TNF, indicating anti-tumour activity, in comparison to both intracellular pathogens represented by virulent LM and virulent LCM virus.

[0100] The results show that production of TNF was best induced by the combination according to the invention when compared to the virulent bacterium or virus, demonstrating the high efficacy of the combination of the first and second compositions according to the invention for generating an antigen-specific CD8+ T-cell response.

[0101] The production of IFN by the CD8+ T-cells raised by DC COAT was not significantly higher than in the negative control and not significantly lower than that raised by virulent LM (FIG. 16), but significantly lower than that raised by LCM virus. This level of IL-6 shows the systemic inflammation induced by the boost (second composition) as a further proof of functional CD8+ T-cells induced by the vaccine combination of first and second compositions of the invention.

[0102] The production of IFN in CD8+ T-cells raised by the compositions of the invention are very significantly higher than that in the negative control and at a level comparable to that raised by LM or LCM virus. This level of IFN shows the active secretion of interferon by the CD8+ T-cells that were induced by the vaccine combination of first and second compositions. This active secretion of IFN is in contrast to anergic T-cells, which are IFN-secretion defective.

[0103] These results show a good anti-tumour function of the CD8+ T-cells raised by the compositions of the invention.

Example 6

TLR Agonists in Second Composition

[0104] In order to assess the effect of a TLR agonist in the second composition, on day 7 mice received as a first composition 10.sup.6 dendritic cells that were in vitro coated with 10 g SIINFEKL peptide intravenously and at day 0 were challenged with a second composition by intravenous administration of a combination of 100 g SIINFEKL peptide, 100 g of anti-CD40 (CD40) (clone 1C10) as the co-stimulatory antibody, and a TLR agonist. The results are shown in FIG. 19, showing that the highest proportion of antigen-specific CD8+ T-cells was obtained for the TLR agonist being 200 g Poly(I:C) (Poly I:C), with the negative control (No TLR agonist) resulting in comparatively low proportions of antigen-specific CD8+ T-cells and the TLR7 agonist (Imiquimod), the TLR9 agonist CpG oligodesoxynucleotide (CpG ODN) or lipopolysaccharide of E. coli (LPS) giving significantly higher proportions of antigen-specific CD8+ T-cells.

[0105] These data show that the invention also in the absence of a TLR agonist in the second composition raises antigen-specific CD8+ T-cells, and that presence of a TLR agonist in the second composition is preferred, especially Poly(I:C) is the preferred TLR agonist.

[0106] Generally, it is preferred that the first composition is free from a co-stimulatory antibody and/or free from a TLR agonist.