METHOD OF TREATING MELANOMA

Abstract

The present invention relates to a method of treating or preventing melanoma using vaccination or immunisation, wherein said vaccination or immunisation involves the use of a photosensitizing agent, a melanoma antigen (i.e. an antigenic molecule), for example a vaccine component, and irradiation with light of a wavelength effective to activate the photosensitizing agent. The invention also relates to said photosensitizing agent and melanoma antigen for use in such a method, and to cells produced by the method.

Claims

1. A method of expressing a melanoma antigen or a part thereof on the surface of a cell, comprising contacting said cell with said melanoma antigen and a photosensitizing agent and irradiating the cell with light of a wavelength effective to activate the photosensitising agent, wherein said melanoma antigen or a part thereof is released into the cytosol of the cell and subsequently presented on the cell's surface.

2. A method as claimed in claim 1 wherein said method is performed in vivo and said cell is in a subject.

3. A method as claimed in claim 1 wherein said contacting step is performed for 12 to 30 hours, preferably 16-20 hours, for example 18 hours.

4. The method as claimed in claim 1 wherein said photosensitising agent is an amphiphilic porphyrin, chlorin, bacteriochlorin or phthalocyanine, wherein preferably said photosensitising agent is in the form of a conjugate with a chitosan derivative.

5. The method as claimed in claim 4 wherein said photosensitising agent is selected from TPCS.sub.2a, AlPcS.sub.2a, TPPS.sub.2a and TPBS.sub.2a, preferably TPCS.sub.2a. (currently amended) The method as claimed in claim 1 wherein the dose of photosensitizing agent is between 25 and 400 g, preferably between 100 and 300g, for example 250 g.

6. The method as claimed in claim 1 wherein the cell is irradiated for between 1 and 60 minutes, preferably for 3 to 12 minutes, preferably for 6 minutes.

8. The method as claimed in claim 1 wherein the dose of the melanoma antigen is between 1 and 500 g, preferably between 10 and 100 g, preferably 100 g.

9. The method as claimed in claim 1 wherein the antigenic presentation results in the stimulation of an immune response, preferably the stimulation of CD8 T cells.

10. The method as claimed in claim 1 wherein the melanoma antigen is (i) derived from a subject, (ii) derived from one or more melanoma cell lines, or (iii) selected from gp100, MAGE-1, MAGE-3, Melan-A, tyrosinase and tyrosinase-related protein (TRP) 1 or 2 or an antigen comprising a peptide epitope thereof, wherein when said melanoma antigen is derived from a subject or one or more melanoma cell lines said method preferably additionally includes the step of preparing a composition comprising one or more melanoma antigens from one or more subjects or from one or more melanoma cell lines, wherein preferably the melanoma antigen is TRP-2.

11. The method as claimed in claim 1 wherein the cell is an antigen presenting cell, preferably a dendritic cell.

12. The method as claimed in claim 1 wherein said cell is contacted with said melanoma antigen and photosensitising agent simultaneously, separately or sequentially, wherein preferably said contact is achieved by intradermal or intratumoural administration of said melanoma antigen and said photosensitising agent.

13. A cell or population of cells obtainable by a method as claimed in claim 1, wherein preferably the cell is a dendritic cell.

14. (canceled)

15. A method of generating an immune response in a subject, comprising administering to said subject a melanoma antigen and a photosensitizing agent and irradiating said subject with light of a wavelength effective to activate said photosensitizing agent, wherein an immune response is generated, wherein preferably said irradiation is for between 1 and 60 minutes, preferably for 3 to 12 minutes, preferably for 6 minutes, and/or said melanoma antigen and said photosensitizing agent are administered to said subject 12 to 30 hours, preferably 16-20 hours, for example 18 hours, before said irradiation.

16. The method of claim 15 wherein said melanoma antigen and said photosensitising agent are administered to said subject simultaneously, separately or sequentially.

17. A method of generating an immune response in a subject, comprising administering to said subject a cell or population of cells as defined in claim 13.

18. The method as claimed in claim 15 wherein said administration is by intradermal or intratumoural administration.

19. The method as claimed in claim 15 wherein said method is a method of vaccination, preferably therapeutic vaccination.

20. The method as claimed in claim 15 treating or preventing melanoma.

21. The method of claim 2 wherein said subject is a mammal, preferably a cat, dog, horse, donkey, sheep, pig, goat, cow, mouse, rat, rabbit or guinea pig, most preferably the subject is a human.

22-27. (canceled)

Description

[0167] The invention will now be described in more detail in the following non-limiting Examples with reference to the following drawings in which:

[0168] FIG. 1 shows that prophylactic PCI-based immunisation stimulated CD8 T-cell responses and prevented tumour growth in mice. (A-D) Groups of five C57BL/6 mice were immunised intradermally (i.d.) with 10 g OVA protein, with OVA and the photosensitiser TPCS.sub.2a, (OVA-PCI) or left untreated (Untr). 18 h later, all mice were treated with light (4.86 J/cm2). Mice were bled on day 6 and analysed for SIINFEKL-specific CD8+ cells (A), and their CD44 expression (B) and IFN- production (C) by flow cytometry. Survival of mice challenged on day 4 after vaccination with 500,000 OVA-expressing B16 melanoma tumour cells by i.d. injection into one of the flanks (D). Frequency of SIINFEKL-specific CD8.sup.+ and CD44.sup.+ cells (E) and survival after tumour challenge (F) in C57BL/6 and syngeneic MHC class-II-deficient mice immunised with OVA-PCI and challenged with B16 as above. ***, p<0.001; **, p<0.01; n.s. not significant. Shown are means and SEM. The data are representative from 2-4 experiments.

[0169] FIG. 2 shows that therapeutic PCI-based vaccinated prevented tumour growth in mice. (A-D) Groups of five C57BL/6 mice received 500,000 OVA-expressing B16 melanoma tumour cells by i.d. injection into one of the flanks and were immunised 7 days later i.d. with 10 g OVA, OVA-PCI or left untreated as described for FIG. 1. On day 8, all mice were light treated. Tumour growth (A) and survival (B) of mice was monitored until day 15, when mice were euthanized and splenocytes analysed for SIINFEKL-specific CD8+ CD44+ T cells (C) and for intracellular IFN- production (D) by flow cytometry. **, p<0.01; *, p<0.05; n.s. not significant. Shown are means and SEM. The data are representative from 3 experiments.

[0170] FIG. 3 shows that PCI-based vaccination induced CD8 T-cell tumour infiltration and apoptosis. Mice were treated as described in FIG. 2. On day 15, tumours were excised and analysed by immune histochemistry for CD8 (A), CD4 (B) and caspase-3 (D) positive cells as well as stained for haematoxylin and eosin (C) Arrows show apoptotic foci in the H/E staining (C, bottom panel) and caspase-3 positive cells (D, bottom panel). Images are representative of five mice per group.

[0171] FIG. 4 shows that PCI-based vaccination reduced the metastatic potential of melanoma. Groups of four C57BLJ6 mice immunised i.d. with 10 g OVA, OVA-PCI or left untreated as described for FIG. 1. On day 8, the mice received 500,000 OVA-expressing B16 melanoma tumour cells by tail vein injection. On day 19, the mice were killed and lungs isolated for detection of melanoma metastasis.

[0172] FIG. 5 shows that PCI facilitates cytosolic delivery of antigen. Bone marrow DCs from C57BL/6 mice were incubated with TPCS.sub.2a and OVA-Alexa488. (A) After steps of washing, and light activation (3 min LumiSource exposure), the cells were immediately analysed by fluorescence microscopy. (B) DCs were incubated with OVA-Alexa488 as above and light activated. Images of the same microscopic field were made 0, 5, 10, and 15 min after light activation.

[0173] FIG. 6 (A) shows the experimental set up of PCI-mediated immunisation using mice adoptively transferred with OVA-specific CD8 T-cell transgenic OT-1 cells prior to immunisation. (B) After intradermal injection of antigen (OVA) and photosensitiser (TPCS.sub.2a) in the abdominal region, mice were anaesthetised and the site of injection illuminated by placing the mice belly down on a LumiSource light table.

[0174] FIG. 7 shows results with C57BL/6 mice that were spiked with 510.sup.6 OT-I cells and the frequency of SIINFEKL-specific cells were measured in the recipients after 18 hours by MHC I-SIINFEKL pentamer staining and flow cytometry (A). The mice were then immunised with 100 g OVA or with 100 g OVA and 25 g TPCS.sub.2a; control mice were left untreated. After 2 or 18 hours, the TPCS.sub.2a-treated mice were illuminated. On day 6 (B) and 23 (C), mice were bled and stained with MHC I-SIINFEKL pentamer, anti-CD8 and anti-CD44 antibodies and analysed by flow cytometry. Bars show the frequency of triple positive cells relative to the total number of CD8 T cells. (D) shows dot plots of pentamer- and CD44-positive cells from blood analysed by flow cytometry on day 6. Cells were gated on CD8 lymphocytes. (E) shows results on day 14, blood (left panel) and day 23 splenocytes (right panel) that were re-stimulated overnight with SIINFEKL and analysed for CD8, CD44 and IFN- by intracellular staining (ICS). (F) shows results with splenocytes that were re-stimulated with SIINFEKL for analysis of IFN- (left panel) and IL-2 (right panel) by ELISA.

[0175] FIG. 8 shows results with C57BL/6 mice that were spiked with 1.610.sup.6 OT-I cells. After eight hours, the mice were immunised with 10 g OVA, with 10 g OVA and 25 g TPCS.sub.2a, or with 10 g OVA and 250 g TPCS.sub.2a. On day 8 the mice were bled and analysed for (A) MHC I-SIINFEKL pentamer, CD44 and CD8 staining. On day 11 the mice were euthanized and their splenocytes analysed for (B) CD8 and CD44 and intracellular IFN-, as well as secretion of IL-2 (C) and IFN- (D) measured by ELISA. Bars show the frequency of triple positive cells relative to the total number of CD8 T cells.

[0176] FIG. 9 shows (A) J774 cells that were incubated overnight with 25 g/ml OVA-Alexa488 (left panel) or with OVA-Alexa488 and 0.05 g/ml TPCS.sub.2a (right panel). After washing and 90 minutes incubation in fresh medium, the cells were illuminated, and the cellular uptake and distribution of OVA-Alexa488 was analysed by fluorescence microscopy. (B) J774 cells were incubated with 1.0 g/ml TPCS.sub.2a and 25 g/ml OVA-Alexa488 as above and analysed for cellular uptake, distribution and co-localisation of OVA-Alexa488 and TPCS.sub.2a by fluorescence microscopy. Co-localisation of the two compounds causes emission of yellow fluorescence.

[0177] FIG. 10 shows results with C57BL/6 mice that were spiked with 1.610.sup.6 OT-I cells. After eight hours, the mice were immunised with 100 g OVA, or with 100 g OVA and 25 g TPCS.sub.2a; control mice were left untreated. After 2, 6 or 18 hours, the TPCS.sub.2a -treated mice were illuminated. On day 0 and day 7 mice were bled and stained with MHC I-SIINFEKL pentamer and anti-CD8 antibodies and analysed by flow cytometry (A). On days 0, 7, 14 blood cells and day 23 splenocytes were stained with anti-CD8 antibodies and pentamer and analysed by flow cytometry (B). Each circle represents the results for a different animal.

[0178] FIG. 11 shows a similar study to that shown in FIG. 10 but time points of 18 hours and 42 hours after illumination were assayed. On day 0 and day 7 mice were bled and stained with MHC I-SIINFEKL pentamer and anti-CD8 antibodies and analysed by flow cytometry (A). On days 0, 7 blood cells and day 14 splenocytes were stained with anti-CD8 antibodies and pentamer and analysed by flow cytometry (B). (C) shows splenocytes that were re-stimulated overnight with SIINFEKL and analysed for IFN- by ELISA. IFN- was also analysed on day 14 by flow cytometry (D).

[0179] FIG. 12 shows a similar study to that shown in FIG. 10 but the illumination time was varied between 3, 6 and 12 minutes (incubation time was 18 hours). On days 0, and 9 the mice were bled and the cells analysed for MHC I-SIINFEKL pentamer and CD8 staining by flow cytometry (A). On day 0 and day 9 mice were bled and stained with MHC I-SIINFEKL pentamer and anti-CD8 antibodies and assessed by flow cytometry (B). (C) shows splenocytes from day 14 that were re-stimulated overnight with SIINFEKL and analysed for IL-2 and IFN- by ELISA.

[0180] FIG. 13 shows a similar study to that shown in FIG. 10 but the photosensitiser dose was varied between 25, 50 and 100 g TPCS.sub.2a. An illumination time of 6 minutes and incubation time of 18 hours was used. On day 7 the mice were bled and cells stained with pentamer and anti-CD8 antibodies and assessed by flow cytometry (A). On day 7 blood cells were stained with anti-CD8 antibodies and analysed by flow cytometry (B). On day 12 splenocytes were analysed for IFN-, CD8 and CD44 staining (left panel) and MHC I-SIINFEKL pentamer and CD8 staining, (right panel) by flow cytometry (C).

[0181] FIG. 14 shows results with C57BL/6 mice that were spiked with 210.sup.6 OT-I cells. One day later, the mice were immunised with 20 g OVA, with 20 pg OVA and 200 g TPCS.sub.2a, or left untreated. On day 54, the mice were euthanized and the splenocytes analysed by flow cytometry for (A) MHC I-SIINFEKL pentamer and CD8 staining, or (B) intracellular IFN- and CD8 and CD44 staining. Bars show the frequency triple positive cells relative to the total number of CD8 T cells. (C) Secretion of IFN- into 96-hours splenocyte cultures was measured by ELISA.

[0182] FIG. 15 shows the effect of PCI-based vaccination on tumour growth. C57BL/6 mice were spiked with 110.sup.4 OT-I cells. One day later, the mice were immunised with 20 g OVA, with 20 g OVA and 200 pg TPCS.sub.2a, or left untreated. The abdomen was shaved before vaccination. The abdominal region was illuminated for six minutes 18 hours after vaccination. On day 4 after immunisation, the mice received an intradermal injection of 510.sup.5 SIINFEKL-expressing B16 mouse melanoma cells. Two weeks thereafter, the tumour volume was measured (A) and the tumour photographed (B). n.s.: not significant; *: p<0.05 as analysed by Kruskal-Wallis test.

[0183] FIG. 16 shows the effect of PCI-based vaccination on tumour growth. Methods similar to those in FIG. 1 were used, but using 150 g TPCS.sub.2a and/or 10 g OVA and 2.510.sup.5 OVA-expressing B16 mouse melanoma cells. The frequency of OVA-specific CD8 T-cells was analyzed by flow cytometry (A). The tumour growth was monitored from day 13 after vaccination (B) until the volume of the tumours reached the endpoint, 1000 mm.sup.3. On day 36 the experiment was ended. (C) shows the average results for tumour growth.

[0184] FIG. 17 shows the effect of therapeutic vaccination on tumour size. Methods similar to those in FIG. 2 were used but using 150 g TPCS.sub.2a and/or 10 g OVA. On days 7 and 14 after vaccination the animals were bled (by tail bleeding), and the frequency of OVA-specific CD8 T-cells was analyzed by flow cytometry (A). The tumour growth was monitored from day ten after injection of tumour cells until the volume of the tumours reached the endpoint, 1000 mm.sup.3. On day 35 the experiment was terminated. (B) shows the average results for tumour growth.

[0185] FIG. 18 shows results of a further prophylactic vaccination with male mice. Methods similar to those in FIG. 1 were used except that 150 g TPCS.sub.2a and/or 10 g OVA were used. frequency of OVA-specific CD8 T-cells was analysed by flow cytometry.

[0186] FIG. 19 shows the effect of the adjuvants poly(IC) and CpG. Mice were immunised with 10 g of OVA, with 100 g OVA, with 10 g OVA and 150 g TPCS.sub.2a, with 10 g OVA and 50 g ODN2935 CpG oligonucleotide, with 10 g OVA, 50 g ODN2935 CpG oligonucleotide and 150 g TPCS.sub.2a, with 10 g OVA and 50 g Poly(IC), with 10 g OVA, 50 g Poly(IC) and 150 g TPCS.sub.2a or left untreated. Mice receiving TPCS.sub.2a were illuminated. Mice were bled on day 7 and the frequency of OVA-specific CD8 T-cells was analyzed by flow cytometry. On day 14 spleen cells were obtained and restimulated by SIINFEKL peptide and analysed by Interferon-gamma ELISA. (A) shows the average values (% antigen-specific, CD44.sup.+ cells of the total CD8.sup.+ cells) in blood at day 7 for the experimental groups (5 animals in each group, error bars: standard error of the mean). (B) shows results from interferon-gamma (IFN-gamma) ELISA after restimulation of day 14 spleen cells with SIINFEKL peptide.

[0187] FIG. 20 shows the results of a study in which normal mice were immunised at day 0 and at day 14 with 50 g of TRP-2 peptide, 100 g TPCS.sub.2a and 10 g poly(IC) as shown. On day 7 after immunisation mice were bled by tail bleeding and erythrocytes were removed by lysis. The frequency of antigen specific CD8 T-cells in the blood was monitored by flow cytometry after staining the cells with anti-CD8 and anti-CD44 antibodies and TRP-2 pentamers. The activation status of the cells was analysed by testing the expression of CD44 by flow cytometry. FIG. 20 shows the average values (% antigen-specific, CD44+cells of the total CD8+ cells) for the TRP-2 pentamer stained experimental groups after the second immunisation.

[0188] FIG. 21 shows the results of a study in which normal mice were immunised at day 0 and at day 14 and at day 35 with 200 g of TRP-2 peptide, 100 g TPCS.sub.2a and 10 g poly(IC) as shown. On day 7 after immunisation mice were bled by tail bleeding and erythrocytes were removed by lysis. The frequency of antigen specific CD8 T-cells in the blood was monitored by flow cytometry after staining the cells with anti-CD8 and anti-CD44 antibodies and TRP-2 pentamers. The activation status of the cells was analysed by testing the expression of CD44 by flow cytometry. FIG. 21 shows the average values (% antigen-specific, CD44+ cells of the total CD8+ cells) for the TRP-2 pentamer stained experimental groups after the second immunisation.

[0189] FIG. 22 shows results from the same study as FIG. 21. Interferon-gamma (IFN-gamma) intracellular staining after re-stimulation of spleen cells with the TRP-2 peptide is shown.

[0190] FIG. 23 shows results from the same study as FIG. 21. TNF-alpha intracellular staining after re-stimulation of spleen cells with the TRP-2 peptide is shown.

[0191] FIG. 24 shows the results of a study in which mice were immunised at day 0 and at day 14 with 110.sup.6 irradiated B16-F10 melanoma cells, 150 g TPCS.sub.2a and 10 g poly(IC) as shown. At day 21 510.sup.5 B16-F10 cells were injected intradermally, and the size of the tumours was measured at least 2 times per week. FIG. 24 shows the average tumour volume with day 0 being the day the cells were injected.

EXAMPLES

Example 1

Materials and Methods

[0192] Animals C57BL/J6 mice were purchased from Harlan (Horst, The Netherlands). CD8 T-cell receptor transgenic OT-I mice (B6.129S6-Rag2tm1Fwa Tg(TcraTcrb)1100Mjb) from Taconic Europe (Ry, Denmark) and MHC class II-deficient mice (B6.129S2-H2dlAb1-Ea/J) from Jackson Laboratories (Bar Harbor, Maine) and bred in our own SPF facilities at the University of Zurich; the OT-I CD8 T cells recognise the H-2K.sup.b-restricted epitope SIINFEKL from ovalbumin (OVA, aa257-264). All mice were kept under SPF conditions, and the procedures performed were approved by Swiss Veterinary authorities (licence 69/2012).

Materials and Cells

[0193] Chicken OVA was purchased from Sigma-Aldrich (Buchs, Switzerland) and the SIINFEKL peptide from EMC microcollections (Tuebingen, Germany). The photosensitiser tetraphenyl chlorin disulfonate (TPCS.sub.2a) was from PCI Biotech (Lysaker, Norway). OVA and TPCS.sub.2a were mixed in PBS, kept light protected, and administered to mice within 60 minutes of preparation. TPCS.sub.2a was activated by illumination with LumiSource (PCI Biotech). B16.F10 melanoma cells (ATCC CRL-6322), originally from C57BL/6 mice, were used to make a stable transfectant that expressed the whole OVA antigen.

Intradermal Photosensitisation and Immunisation of Mice

[0194] C57BL/6 mice were immunised at 6-10 weeks of age. One day prior to immunisation, 10,000 OT-I spleen and lymph node cells were administered by intravenous injection into the recipient C57BL/6 mice (see Example 2). The next day, the fur was shaven off the abdominal region and 100 l of the vaccine preparations were injected intradermally. The doses of OVA and TPCS.sub.2a were 10 g and 100 g, respectively. After 18 hours, the anaesthetised mice were placed on the light source for six minutes illumination (4.86 J/cm2) for activation of TPCS.sub.2a.

Analysis of Immune Responses by Flow Cytometry and ELISA

[0195] The frequency of antigen-specific CD8 T cells was monitored in blood and spleen by flow cytometry using H-2K.sup.b/SIINFEKL Pro5 pentamer (Proimmune, Oxford, UK). Cell-surface expression of CD4, CD8, and CD44 and intracellular production of IFN- was analysed by flow cytometry after Fc-receptor blocking with anti-CD16/32. The intracellular staining was after overnight incubation at 37 C. with 0.1 g SIINFEKL. Brefeldin A (2.5 g/ml) was added during the last 4 hours. The cells were fixed with 4% formaldehyde for 10 minutes, permeabilised in 0.1% NP40 for 3 minutes, and stained with anti-IFN- for 35 minutes. All staining were performed at 4 C. and all steps followed by washing in PBS/FCS 2%. FACS antibodies were from eBioscience (Vienna, Austria) or BD Pharmingen (Basel, Switzerland). Acquisition was performed on FACSCanto (BD Biosciences, San Jose, USA) and data analysed with FlowJo 8.5.2 (Tree Star, Inc., Ashland, Oreg.). For analysis of cytokine secretion by ELISA, 210.sup.5 splenocytes were re-stimulated in round-bottom 96-well plates with 0.1 g SIINFEKL. Supernatants were collected after 24-72 hours and analysed using cytokine ELISA kits (eBioscience).

Fluorescence Microscopy of Cytosolic Antigen Release

[0196] Cells (J774.1 (ATCC no. TIB-67 mouse monocyte macrophage cell line) or bone marrow DCs from C57BL/6 mice) were incubated with 0.05 or 1.0 g/ml TPCS.sub.2a and 25 g/ml OVA-Alexa488 for 18 hours and washed three times in drug-free culture medium prior to incubation for four hours before light activation (3 min LumiSource exposure). The cells were subsequently washed in ice-cold PBS with Ca.sup.2+ and Mg.sup.2+ prior to microscopy. Images of cellular localization and PCI-induced cytosolic release of OVA were obtained by epi-fluorescence microscopy using a Plan-Apochromat 63/1.40 Oil differential interference contrast (DIC) objective or 40/0.95 Plan-Apochromat phase contrast (Korr Ph3 M27) objective with a Zeiss Axioimager Z.1 microscope (Carl Zeiss, Oberkochen, Germany). Fluorescence of Alexa488-labelled OVA was obtained by using a 470/40 nm band pass (BP) excitation filter with a beam splitter at 495 nm and a 525/50 nm BP emission filter. TPCS.sub.2a fluorescence was obtained by using a 395-440 nm BP excitation filter with a beam splitter at 460 nm, and a 620 nm long pass filter. Micrographs were recorded with a digital AxioCam MRm camera and processed and analysed by use of the Axiovision Software (Carl Zeiss).

Vaccination Against Intradermal Melanoma and Monitoring of Tumour Growth

[0197] If not otherwise stated, C57BL/6 mice were vaccinated as described above 4-5 days prior to, or 7-8 days after, the tumour challenge with 500,000 OVA-expressing B16-melanoma cells injected intradermally into one of the mouse flanks; 10,000 OT-I cells were transferred intravenously to the recipient one day before prophylactic vaccination, or one day before B16 injection in the therapeutic vaccination model. In the therapeutic vaccination model, the 7-8 days after B16 injection represents the time required for the tumour to develop to a palpable size. The tumour growth was monitored by measuring the size of the neoplasm with a calliper. The tumour volume was calculated using of the modified ellipsoid formula: (lengthwidth.sup.2)/2.

[0198] Histological Analysis Of Tumour and Tumour Infiltrates

[0199] C57BLJ6 mice received 500,000 OVA-expressing B16-melanoma cells intradermally as described above. Six days later, the mice were vaccinated with 10 g OVA 100 g TPCS.sub.2a as described above. On day 7, the mice were treated with 4.86 J/cm2 light. On day 15, the mice were euthanized and the tumours excised and cut in two pieces, one snap frozen in liquid nitrogen and one fixed in formalin (2 days) and 60% ethanol before embedded in paraffin. The frozen tissues were sectioned and stained with anti-mouse CD8, CD4, and caspase-3 antibodies for immune histochemistry. Paraffin sections were stained with haematoxylin and eosin.

Monitoring of Metastatic Potential of Melanoma

[0200] C57BLJ6 mice were vaccinated as above with OVA with or without TPSC.sub.2a and challenged four days later with 500,000 OVA-expressing B16 cells given by intravenous injection. On day 19, mice were euthanized and spleens and lungs were harvested. The lungs were analysed by counting melanoma spots as a measure for metastasis. Spleen cells were analysed by flow cytometry for CD8 T-cell activation as described above.

Results

Photosensitisation Enables MHC-class I Antigen Presentation of Protein Vaccines

[0201] Mice were immunised intradermally with OVA protein with or without photosensitiser and the immune responses analysed by measuring the frequency of pentamer (H2K.sup.d-SIINFEKL)-binding CD8 T cells in blood. No or little so-called cross priming was observed in mice immunised with OVA alone. In contrast, concomitant photosensitisation with TPCS.sub.2a and illumination of the skin 18 hours later, resulted in photochemical internalisation (PCI) and MHC class-I antigen presentation of the endogenous protein with strong activation of antigen-specific CD8 T cells (FIG. 1A). The CD8 T cells had an activated CD44 phenotype (FIG. 1B) and were IFN- producers (FIG. 10).

[0202] When splenocytes from immunised mice were re-stimulated in vitro with the MHC class-I-binding peptide SIINFEKL, cells from mice immunised with OVA-PCI secreted significantly more IL-2, TNF- and IFN-, as measured by ELISA of culture supernatants, than cells from mice immunised with OVA alone (data not shown).

[0203] PCI-adjuvated immunisation also prevented growth of melanoma when B16 cells were given intradermally five days after immunisation. While no untreated mice survived the tumour challenge longer than 26 days, nine out of ten OVA-PCI immunised mice did not develop tumours (FIG. 1D). Fifty percent of the mice immunised with OVA without concomitant photosensitisation developed tumours and succumbed by days 13 (n=2), 18 (n=2) and 32 (n=1). The protection against tumour growth was also long lived, as mice challenged six weeks after immunisation still showed effective protection (data not shown). In general, tumour protection after immunisation and photosensitisation (OVA-PCI) reflected the stronger activation of SIINFEKL-specific CD8 T cells as analysed by the correlation of the two parameters from several experiments (p<0.01 by Spearman's p, n=63).

PCI-Based Stimulation of CD8 T cells is MHC Class II Independent

[0204] The hypothesised mechanism of PCI-adjuvated stimulation of CD8 T-cell responses is the light-activated release of antigen from endosomes, and thereby prevention of the default MHC class-II pathway of antigen presentation. However, as activation of

[0205] CD8 T-cell responses is mostly dependent on CD4 help and MHC class II, it was tested whether the stimulation of CD8 T-cell responses with protein and photosensitiser was dependent on MHC class II. Mice were immunised with OVA, photosensitiser and light exposure as described above. The frequencies of pentamer (H2K.sup.d-SIINFEKL)-binding CD8 T cells in blood were not significantly different in MHC class II wild type and deficient mice (FIG. 1E). Indeed, there was actually a slightly higher frequency in the MHC class II-deficient mice. Moreover, when mice were subsequently challenged with the B16 melanoma, wild type mice had no survival benefit as compared to MHC class II-deficient mice (FIG. 1F).

Therapeutic Vaccination with PCI-Based Vaccines Improves Survival in Melanoma-Bearing Mice

[0206] To study if PCI-based immunisation would also reduce the growth of already established neoplasms, mice were immunised after appearance of tumours under the skin. The tumours grew readily in non-immunised mice and in mice immunised with antigen only (FIG. 2A). In contrast, no growth was observed in mice that received the photosensitive vaccine OVA-PCI. Correspondingly, the survival, as measured by time to reach a tumour volume of 50 mm.sup.3, was significantly improved in OVA-PCI-immunised as compared to OVA-immunised mice (FIG. 2B; p=0.018 by log rank Mantel-Cox test). When splenocytes were re-stimulated in vitro with SIINFEKL and analysed by flow cytometry, therapeutic PCI-based vaccination also stimulated proliferation of antigen-specific CD8 T cells (FIG. 2C) that produced IFN- (FIG. 2D).

Photosensitive Cancer Vaccines Stimulate TILS to Melanoma and Apoptosis in Tumours

[0207] Tumours were excised and analysed 10 days after therapeutic vaccination of tumour bearing mice. As illustrated in FIG. 3A, photosensitisation of skin caused a heavy infiltration of CD8 T cells into the intradermal tumour, whereas vaccination with antigen only represented no benefit compared to no treatment. PCI-adjuvated tumour vaccination had no effect on the infiltration of CD4-positive TILs (FIG. 3B). The hematoxylin and eosin (H/E) staining of tumour sections indicated apoptotic cells and foci in melanomas from mice vaccinated with OVA-PCI, but not in mice vaccinated with protein alone or in untreated mice (FIG. 3C). In part, this apoptosis was mediated by caspase-3, as caspase-3 positive cells were observed in OVA-PCI-treated mice, but not in OVA-treated or untreated mice (FIG. 3D).

[0208] Photosensitive vaccines decrease the metastatic potential of melanoma in mice To study the effect of PCI-based vaccination on metastasis, mice were immunised with OVA alone or with OVA-PCI as described above and challenged one week later with B16 melanoma cells given intravenously; the dissemination of cells to the lung with growth of tumours is an established method for the study of B16 metastatic potential in mice. Nineteen days later, the mice were euthanized and the lungs excised. The lungs of non-immunised mice had innumerable (>100) tumour spots on the surface (FIG. 4, top panel). OVA-immunised mice had on average approx. 50 melanoma spots (FIG. 4, middle panel), while mice immunised with OVA and the photosensitiser TPCS.sub.2a had an average of 10 lung metastases (FIG. 4, bottom panel).

Antigen and Photosensitiser Co-Localise in Endosomes of Dendritic Cells and are Released into Cytosol Upon Light Activation

[0209] The hypothesised effect of photosensitisation on the stimulation of CD8 T cells immunisation assumes that antigen presenting cells such as DCs have affinity for the photosensitiser and that the photosensitiser translocates to the endosome upon endocytosis of extracellular protein vaccine. Moreover, the effect also assumes that the antigen and the photosensitiser are contained in the same endosomes. To test this, bone-marrow derived DCs were incubated in vitro with the photosensitiser TPCS.sub.2a and with Alexa488-labelled OVA protein. After washing of the cells, they were analysed by fluorescence microscopy, which showed co-localisation of antigen and photosensitiser in DC endosomes (FIG. 5A). When the cells were activated by light, release of the antigen into the cytosol could be observed over 15 minutes (FIG. 5B). At this time point, endosomal compartments were no longer visible, and the fluorescent antigen had diffused throughout the cytosol with direct access to the MHC class-I machinery of antigen presentation.

Conclusion

[0210] In the current study, it was tested whether PCI could be utilised as adjuvant for induction of anti-tumour CD8 T-cell responses in mice after intradermal administration. Eighteen hours after the vaccination with protein antigen and the photosensitiser TPCS.sub.2a, a time period that allowed uptake of vaccine into dermal antigen-presenting cells, the injection site was illuminated with light to activate the photosensitiser contained in endosomes. Light activation triggered the release of antigen to cytosol where it was degraded and MHC class-I-presented to CD8 T cells. This method of vaccination stimulated strong immune responses with proliferation and cytokine secretion of CD8 T cells but not of CD4 T cells. The immune responses protected against tumour development in a prophylactic model of mouse melanoma. More importantly, therapeutic vaccination of melanoma-bearing mice prevented further growth of the tumours, while control vaccination without photosensitiser had no effect on tumour growth and mouse survival.

[0211] The PCI-based tumour vaccine worked exclusively through stimulation and recruitment of CD8-positive tumour-infiltrating lymphocytes (TILs), cells known to be of vital importance in the immunological fight against tumours. We observed that tumours from PCI-treated mice were heavily infiltrated by CD8-positive T cells, while control vaccination without photosensitiser caused no such infiltration. In contrast, PCI had no effect on the recruitment of CD4-positive TILs. Tumour vaccination with the PCI-based vaccine induced apoptosis in the skin melanoma, which correlated with caspase-3 expression and IFN- secretion, a cytokine that inhibits tumour cell growth by inducing apoptosis and by reducing their capability to enter cell cycling.

[0212] By fluorescence microscopy, it was verified that antigen and photosensitiser were taken up into endosomes of DCs and that the endosomes were disrupted upon light exposure. Indeed, the light-induced disruption of endosomes and release of antigen into the cytosol was so effective that it may suggest a total turn-off of MHC class-II antigen presentation. Although the purpose of PCI-adjuvated vaccination is to trigger MHC class-I-restricted CD8 T-cell responses, the generation of primary CD8 T-cell responses to non-inflammatory antigens typically requires MHC class II-restricted CD4 T-helper cells. PCI-based vaccination in wild type and MHC class II-deficient mice was therefore compared. Surprisingly, the stimulation of CD8 T-cell responses and the fitness of the CD8 T cells to control tumour growth were not impaired in MHC class II-deficient mice. T-helper-cell-independent PCI-based vaccination is considered important as many tumour patients are treated with CD4 T-cell-sensitive immune suppressive agents, which could impair the efficacy of vaccination.

[0213] Altogether, the results presented here show that photochemical internalisation enables high amounts of exogenous protein vaccine to access the cytosol were it is degraded for MHC class-I-restricted antigen presentation. The subsequent strong stimulation of CD8 T-cell responses prevented tumour growth in of murine model of melanoma.

Example 2

Materials and Methods

Mice

[0214] Female C57BL/6 mice (used at 6-10 weeks of age) and Rag2 deficient OT-I mice were as described in Example 1 and bred in the facilities at the University of Zurich as described in Example 1.

Materials

[0215] The antigen chicken ovalbumin (OVA; Grade V) was purchased from Sigma-Aldrich (Buchs, Switzerland) and dissolved in PBS. The octapeptide OVA aa257-264 (SIINFEKL) and photosensitiser TPCS.sub.2a were obtained as described in Example 1. TPCS.sub.2a was at a concentration of 30 mg/ml in polysorbate 80, mannitol and 50 mM Tris pH 8.5. TPCS.sub.2a was protected from light and kept at 4 C. Prior to vaccination OVA and TPCS.sub.2a were mixed together in PBS and kept protected from light. The light used for activation of the photosensitiser was LumiSource (PCI Biotech), which contains four 18 W Osram L18/67 standard light tubes with a fluence rate of 13.5 mW/cm.sup.2 and emits light at 435 nm.

Intradermal Photosensitisation and Immunisation of Mice

[0216] One day prior to the immunisation, spleens and lymph nodes were isolated from female OT-1 mice, and erythrocytes were removed by lysis (RBC Lysing Buffer Hybri-Max from Sigma-Aldrich) from the homogenised cell suspensions. The remaining cells were washed in PBS, filtered through 70 micron nylon strainers, and 210.sup.6 OT-1 cells were administered by intravenous injection into recipient female C57BLJ6 mice; the adoptive transfer of SIINFEKL-specific CD8 T cells facilitates monitoring of the immune response by flow cytometry. One day or 8 hours later, mice were bled by tail bleeding, and the blood was collected in heparin-containing tubes for analysis of the baseline frequency of OVA-specific CD8 T cells.

[0217] Then, the mice were shaved on the abdominal area, and the vaccines, consisting of OVA or of a mixture of OVA and TPCS.sub.2a, were injected intradermally using syringes with 29G needles. The vaccines were kept light protected and used within 60 minutes of preparation. The vaccines were given in two injections of 50 l each, on the left and right side of the abdominal mid line. OVA was tested at 10 to 100 g per dose. The TPCS.sub.2a dose was 7.5 to 250 g.

[0218] On day 0, prior to vaccination and on various days thereafter (e.g. day 6, 7, 8, 9, 14, 23, as indicated) mice were bled by tail bleeding and erythrocytes were removed by lysis, before analysis of antigen-specific CD8 T cells by flow cytometry. At the end of the experiment (typically 11, 12, 14 or 23 days), the mice were euthanized and the splenocytes analysed ex vivo. At various time points after the TPCS.sub.2a injection (0-48 hours), the mice were anaesthetised by intraperitoneal injection of a mixture of ketamine (25 mg/kg body weight) and xylazin (4 mg/kg) and placed on a light source (for illumination and activation of the photosensitiser TPCS.sub.2a). The light dose was 6 minutes, if not otherwise stated. The whole procedure is illustrated in the scheme of FIG. 6A. The illumination of mice using LumiSource is imaged in FIG. 6B.

Analysis of Immune Responses

[0219] The frequency of OVA-specific CD8 T-cells in blood was monitored by staining the cells with anti-CD8 antibody and H-2K.sup.b/SIINFEKL ProS pentamer (Proimmune, Oxford, UK) for analysis by flow cytometry. The activation status of the cells was further analysed by testing the expression of CD44 and CD69 by flow cytometry. Intracellular staining for IFN- was done after overnight stimulation of splenocytes in 24-well plates with the CD8 epitope OVA.sub.257-264 (SIINFEKL) at 37 C. Brefeldin A was added during the last 4 hours. The cells were then washed and fixed with 4% formaldehyde in PBS for 10 min on ice. Anti-CD16/32 was added to block unspecific binding to Fc receptors. The cells were then permeabilised with 0.1% NP40 in PBS for 3 min and washed before staining with anti-IFN-, anti-CD8 and ant-CD44 antibodies (eBioscience or BD Pharmingen). The cells were acquired using FACSCanto (BD Biosciences, San Jose, USA) and analysed using FlowJo 8.5.2 software (Tree Star, Inc., Ashland, Oreg.).

[0220] Alternatively, 210.sup.5 splenocytes were re-stimulated in 96-well plates with OVA protein or the SIINFEKL. After 24 and 72 hours, supernatants were collected and analysed for IL-2 or IFN- by ELISA (eBioscienceperformed according to the manufacturer's instructions).

Live Cell Fluorescence Microscopy

[0221] Fifty thousand J774.1 cells (ATCC no. TIB-67 mouse monocyte macrophage cell line) were seeded out on no. 1.5 glass coverslips (Glasswarenfabrik Karl Hecht KG, Sondheim, Germany) in 4-well plates overnight. The cells were incubated with 0.05 or 1,0 g/ml TPCS.sub.2a for 18 hours and washed three times in drug-free culture medium prior to incubation with 25 g/ml OVA-Alexa488 for four hours. Cells were subsequently washed in ice-cold PBS with Ca.sup.2+ and Mg.sup.2+ prior to microscopy. Images of cellular localization and PCI-induced cytosolic release of OVA was obtained by epi-fluorescence microscopy using a Plan-Apochromat 63/1.40 Oil differential interference contrast (DIC) objective or 40/0.95 Plan-Apochromat phase contrast (Korr Ph3 M27) objective with a Zeiss Axioimager Z.1 microscope (Carl Zeiss, Oberkochen, Germany). Fluorescence of Alexa488-labelled OVA was obtained by using a 470/40 nm band pass (BP) excitation filter with a beam splitter at 495 nm and a 525/50 nm BP emission filter. TPCS.sub.2a fluorescence was obtained by using a 395-440 nm BP excitation filter with a beam splitter at 460 nm, and a 620 nm long pass filter. Micrographs were recorded with a digital AxioCam MRm camera and processed and analysed by use of the Axiovision Software (Carl Zeiss).

Vaccination and Effect on Tumour Growth

[0222] Animals were immunised intradermally as described above with 10 g OVA with or without 200 g TPCS.sub.2a. The abdominal region was illuminated for six minutes 18 hours after vaccination. One day prior to vaccination, the mice received 10,000 OT-I cells intravenously. On day four after vaccination, the mice received 5105 SIINFEKL-expressing B16 mouse melanoma cells by intradermal injection into one of the flanks. The B16 melanoma cell line is of spontaneous origin in C57BL/6 mice, and the SIINFEKL-expressing line was kindly provided by Emmanuel Contassot (University of Zurich). The growth of the solid tumour was monitored by measuring the tumour size by calliper 14 days after tumour injection, the endpoint of the investigation. The tumour volume was calculated using use of the modified ellipsoid formula: (lengthwidth.sup.2)/2.

Results

[0223] Analysis of the Effect of the Length of Immunisation Before Illumination on the PCI-Mediated Generation of an Immune Response.

[0224] To facilitate analysis of MHC-class I antigen presentation, we used the class-I binding octapeptide SIINFEKL from OVA (aa257-264) in combination with SIINFEKLspecific CD8 T cells from T-cell receptor transgenic OT-I mice. OT-I lymphocytes were purified from OT-I mice, and 210.sup.6 cells were adoptively transferred to syngeneic and sex-matched wild type C57BL/6 mice. One day after the transfer approximately 1.4% of all CD8-positive T cells in peripheral blood was SIINFEKL-specific (FIG. 7A); the frequency of SIINFEKL-specific CD8 T cells in C57BL/6 mice, which did not receive an adoptive transfer of OT-I cells was less than 0.05% (data not shown).

[0225] The mice were then typically immunised with 10-100 g OVA protein or with a mixture of OVA and 7.5-250 g of the photosensitiser TPCS.sub.2a by intradermal administration in the abdominal region. At different time point thereafter, the mice were anaesthetised and placed belly-down onto the light source, and the site of vaccination was illuminated for six minutes. By day six after vaccination, the frequency of SIINFEKL-specific CD8 T cells in the peripheral blood of mice vaccinated 100 OVA g had increased to approximately 3.5% (FIG. 7B). A similar frequency was measured in mice that also received 25 g TPCS.sub.2a and were illuminated two hours after vaccination (FIG. 7B). However, when mice were illuminated 18 hours post-vaccination, a significant increase in the number of SIINFEKL-specific CD8 T cells was measured in blood (FIG. 7C; P=0.0286 by Mann Whitney). Typically, a retraction of the number of SIINFEKL-specific CD8 T cells in blood was observed 10-15 days after vaccination. By day 23 post-vaccination, the numbers of antigen-specific CD8 T cells had retracted to baseline levels in mice immunised with OVA alone or OVA plus TPCS.sub.2a and illuminated two hours after administration (FIG. 7C). Also, mice immunised with OVA and TPCS.sub.2a and illuminated at 18 hours after immunization showed reduced frequencies after 23 days, but still significantly higher than baseline (P=0.0294 by Mann Whitney). While the SIINFEKL-specific cells in blood had a non-activated phenotype with lack of activation markers such as CD44 (FIG. 7D), CD25 and CD69 (not shown), both immunisation with OVA and OVA-PCI caused strong up-regulation of these markers by day six.

[0226] On day 14, the mice were bled and the PBMCs cells re-stimulated with SIINFEKL overnight. After staining for surface CD8 and CD44 and intracellular IFN-, the cells were acquired by flow cytometry and the frequency of triple-positive cells within all CD8-positive cells was calculated. OVA-immunised mice had a 4-fold increased frequency as compared to control mice that had received OT-I transfer only (FIG. 7E, left panel). The increase in IFN--producing CD44-positive cells after PCI treatment was 6-fold (illumination at 2 hours) and 15-fold (18 hours).

[0227] On day 23, mice were euthanized and splenocytes cultured overnight with SIINFEKL. The cells were then analysed for intracellular IFN- by flow cytometry (FIG. 7E, right panel) or for the secretion of IL-2 (24 hours) and IFN- (72 hours) by ELISA (FIG. 7F). The intracellular IFN-y staining showed barely detectable frequencies of CD44-positive IFN-y producing cells in splenocytes from OVA-immunised mice that did not receive parallel PCI treatment (FIG. 7E, right panel). Clearly higher frequencies of IFN- producing cells were detected in splenocytes from mice that received PCI-treatment. Again, 18 hours interval between immunisation and illumination was most beneficial. Splenocytes from all OVA-immunised mice showed significant production of both IL-2 and IFN- when compared to non-immunised OT-I recipients. Although not statistically significant, there was a clear tendency for increased cytokine secretion in splenocytes from mice that were also PCI-treated.

[0228] Since immunisation with PCI did not produce good responders in all animals tested (typically 3-4 out of 5), we further tested the effect of the time interval between TPCS.sub.2a administration and illumination on the stimulated immune response. Intervals of 6-8 hours or of 42 hours did not suggest an adjuvant effect for PCI (data not shown). Repeatedly, an interval of approximately 18 hours was required to gain an adjuvant effect of PCI. This was observed without exceptions in four independent experiments.

Analysis of the Effect of the Ddose of Photosensitizer on the PCI-Mediated Generation of an Immune Response.

[0229] We then reduced the OVA immunisation dose in order to titrate out the effect of OVA and increasing doses of TPCS.sub.2a was titrated into the vaccine. Immunisation with 10 g OVA alone produced no measurable effect on SIINFEKL-specific CD8 T cells in blood as compared to untreated animals (data not shown). Several experiments with TPCS.sub.2a at 10, 25, 50, 100 and 250 g showed that increasing TPCS.sub.2a doses also increased the measured OVA-specific immune response (data not shown). Representatively, PCI with 25 g TPCS.sub.2a caused 40% good responders, 40% week responders and 20% non-responders as measured for SIINFEKL-specific CD8 T cells in blood on day 8, while PCI with 250 g TPCS.sub.2a produced 100% good responders (FIG. 8A). On day 11 the splenocytes were tested by flow cytometry for IFN-y production. Immunisation with OVA alone showed weak responders in all mice tested, whereas immunisation with OVA and PCI caused better responders in nine out of ten (90%) mice tested (FIG. 8B). Again, PCI with 250 g TPCS.sub.2a showed 100% responders and the highest frequency of IFN- producing cells. Whereas intracellular staining and flow cytometry qualitatively measures whether cells can produce cytokines, ELISA measures how much cytokine the cell can produce. We therefore re-stimulated the day 11 splenocytes with SIINFEKL in vitro and analysed IL-2 (FIG. 8C) and IFN- (FIG. 8D) after 24 and 72 hours, respectively. Immunisation with OVA alone produced weak but clearly measurable IL-2, but not IFN- secretion. Immunisation with OVA and PCI at 25 g TPCS.sub.2a did not cause an increase in IL-2, but a strong increase in IFN- secretion as compared to immunisation with OVA alone. At 250 g TPCS.sub.2a, strong secretion of both IL-2 and IFN- was detected. Finally, while PCI with TPCS.sub.2a had a dose-dependent adjuvant effect with regards to the immune response measured, higher TPCS.sub.2a doses also caused more local inflammation with transient erythema on days 1-3 after illumination (data not shown).

[0230] To study the mechanism by which PCI mediates the adjuvant effect, murine J774 cells, an antigen-presenting macrophage cell line, were incubated with Alexa488-labelled OVA with or without parallel PCI treatment. As shown in the fluorescence micrograph of FIG. 9A, in cells treated with OVA alone, antigen uptake was observed and the antigen was located close to the cell surface in concise spherical shaped bodies, suggesting that the antigen was contained in vesicles, e.g. endosomes. After parallel PCI treatment of the cells, cytosol and in some cases also the nucleus have diffuse green fluorescence suggesting that the antigen is freely floating in the cytosol, hence, released from the endosomes. Since the photosensitiser TPCS.sub.2a is auto-fluorescent, it enabled the study of the relative localisation of antigen and TPCS.sub.2a after incubation of J774 cells with Alexa488-labelled OVA (green) and the photosensitiser (red). Again, after light activation of sensitised cells, the antigen showed a diffuse distribution throughout the cytosol and the nucleus (FIG. 9B). The TPCS.sub.2a photosensitiser showed a similar distribution and the merge of the two images demonstrates that antigen and photosensitiser are co-localised.

[0231] Further analysis of the effect of the length of immunisation before illumination on the PCI-mediated generation of an immune response. To further examine the effect of the incubation time prior to illumination, a further study was conducted as generally described above but using 25 g TPCS.sub.2a, 100 g OVA, 6 minutes illumination time, and 2, 6 or 18 hours incubation time. FIG. 10 shows results with C57BL/6 mice that were spiked with 510.sup.6 OT-I cells. After 18 hours, the mice were immunised with 100 g OVA, or with 100 g OVA and 25g TPCS.sub.2a; control mice were left untreated. After 2, 6 or 18 hours, the TPCS.sub.2-treated mice were illuminated. On day 0 and day 7 mice were bled and the cells stained with anti-CD8 antibodies and MHC I-SIINFEKL pentamer and assessed by flow cytometry analysis (A). On days 0, 7, 14 blood cells and day 23 splenocytes were stained with anti-CD8 antibodies and MHC I-SIINFEKL pentamer and assessed by flow cytometry (B). Individual circles in this and other figures show the results for individual animals. It can be seen that 18 hours incubation time produced an increase in antigen-specific CTLs.

Further Analysis of the Effect of the Length of Immunisation Before Illumination on the PCI-Mediated Generation of an Immune Response.

[0232] A similar study to the above study was carried out to further test different times of incubation prior to illumination. Time points of 18 hours and 42 hours after illumination were assayed (FIG. 11). On day 0 and day 7 mice were bled and stained with MHC I-SIINFEKL pentamer and anti-CD8 antibodies and assessed by flow cytometry (A) On days 0 and 7 blood cells and day 14 splenocytes cells were stained with anti-CD8 antibodies and pentamer and analysed by flow cytometry (B). (C) shows splenocytes that were re-stimulated overnight with SIINFEKL and analysed for IFN- by ELISA. IFN- was also analysed on day 14 by flow cytometry (D).

Analysis of the Effect of the Length of Illumination on the PCI-Mediated Generation of an Immune Response.

[0233] A similar study to the above study was carried out to test the illumination time, which was varied between 3, 6 and 12 minutes (incubation time was 18 hours) (FIG. 12). On days 0 and 9 blood cells and day 14 splenocytes were analysed for MHC I-SIINFEKL pentamer and CD8 staining by flow cytometry (A). On day 0 and day 9 mice were bled and stained with MHC I-SIINFEKL pentamer and anti-CD8 antibodies and analyzed by flow cytometry (B). (C) shows splenocytes (day 14) that were re-stimulated overnight with SIINFEKL and analysed for IL-2 and IFN- by ELISA.

Analysis of the Effect of the Photosensitizer Dose on the PCI-Mediated Generation of an Immune Response.

[0234] A similar study was carried out to test the photosensitiser dose, which was varied between 25, 50 and 100 g TPCS.sub.2a (FIG. 13). An illumination time of 6 minutes and incubation time of 18 hours was used. On day 7 the mice were bled and blood cells stained with MHC I-SIINFEKL pentamer and anti-CD8 antibodies and assessed by flow cytometry (A). On day 7 blood cells were stained with anti-CD8 antibodies and pentamer analysed by flow cytometry (B). On day 12 splenocytes were analysed for IFN-, CD8 and CD44 staining (left panel) and MHC I-SIINFEKL pentamer and CD8 staining, (right panel) by flow cytometry (C).

Analysis of the Length of the Adjuvant Effect of PCI

[0235] The longevity of the memory of the observed CD8-positive immune responses was tested in mice immunised as described above using 20 g OVA with or without 200 g TPCS.sub.2a. The abdominal region was illuminated for six minutes 18 hours after vaccination. After 54 days, the mice were euthanized and the splenocytes analysed directly for the frequency and function of SIINFEKL-specific CD8 T cells. As shown in FIG. 14A, the frequencies of measurable SIINFEKL-specific CD8 T cells in mice treated with OVA or with OVA and PCI were not different from untreated mice. However, re-stimulation with SIINFEKL overnight revealed that PCI-treatment enabled stimulation of antigen-specific CD8 memory cells, which reacted by secretion of the effector cytokine IFN-. This was observed both by intracellular staining and flow cytometry (FIG. 14B) and by ELISA (FIG. 14C). By both assay, a statistically significant difference was observed between OVA alone and OVA-PCI treated mice (P<0.01).

Effect of Vaccination on Tumour Growth

[0236] Mice received SINFEKL-expressing mouse melanoma B16 cells four days after vaccination and the tumour growth was measured on day 14 post-injection of the melanoma cells. The results (FIG. 15) showed that PCI-based vaccination can prevent subsequent tumour growth. In non-vaccinated mice, the transfer of 210.sup.6 OT-I cells totally prevented B16 growth (data not shown). Therefore, the number of transferred cells was reduced to 110.sup.4 OT-I cells. When compared to untreated controls, a significantly reduced B16 tumour growth was observed in mice that received PCI-based vaccination with OVA (P<0.05 by Kruskal-Wallis test) but not after vaccination with OVA alone (FIG. 15A). When all data were transformed to binary data (0=no growth); 1=growth) and analysed by the Chi-square test, PCI-based vaccination had a significantly stronger suppressing effect on tumour growth than vaccination with OVA alone (P=0.048). FIG. 15B shows representative micrographs of tumours on day 14 from differently treated mice.

Example 3

[0237] Similar methods to those described in Example 1 were carried out with minor variation in the doses and protocols.

Effect of Prophylactic Vaccination on Tumour Growth

[0238] Female mice were given 1.010.sup.4OT-I cells i.v. one day prior to intradermal immunization with 150 g TPCS.sub.2a and/or 10 g OVA. The vaccine was given in two injections, each of 50 l to the left and right of the abdominal mid line. The abdomen was shaved before vaccination. The abdominal region was illuminated for six minutes 18 hours after vaccination. On day 4 after immunization, the mice received 2.510.sup.5 OVA-expressing B16 mouse melanoma cells by intradermal injection into the right flank. Two days later the mice were bled (by tail bleeding), and the frequency of OVA-specific CD8 T-cells was analyzed by flow cytometry (FIG. 16A). The tumour growth was monitored (B) from day 13 after vaccination until the volume of the tumours reached the endpoint, 1000 mm.sup.3. On day 36 the experiment was ended. (C) shows the average tumour growth.

Effect of Therapeutic Vaccination on Tumour Size Female mice were given 1.010.sup.4 OT-I cells i.v. one day prior to intradermal injection of 5.010.sup.5 OVA-expressing B16 mouse melanoma cells into the right flank. One week later the mice were vaccinated intradermally with 150 g TPCS.sub.2a and/or 10 g OVA in the abdominal region. The vaccine was given in two injections, each of 50 l to the left and right of the abdominal mid line. The abdomen was shaved before vaccination. The abdominal region was illuminated for six minutes 18 hours after vaccination. On days 7 and 14 after vaccination the animals were bled (by tail bleeding), and the frequency of OVA-specific CD8 T-cells was analyzed by flow cytometry (FIG. 17A). The tumour growth was monitored from day ten after injection of tumour cells until the volume of the tumours reached the endpoint, 1000 mm.sup.3. On day 35 the experiment was terminated. (B) shows the average tumour growth.

[0239] In a further study of prophylactic vaccination male mice were given 1.010.sup.4 OT-I cells i.v. 6 hours before intradermal immunization with 150 g TPCS.sub.2a and/or 10 g OVA. The vaccine was given in two injections, each of 50 l to the left and right of the abdominal mid line. The abdomen was shaved before vaccination. The abdominal region was illuminated for six minutes 18 hours after vaccination. On day 4 after immunization, the mice received 5.010.sup.5 OVA-expressing B16 mouse melanoma cells by intradermal injection into the right flank. Two days later the mice were bled (by tail bleeding), and the frequency of OVA-specific CD8 T-cells analyzed by flow cytometry (FIG. 18). The tumour growth will be monitored from day 18 after vaccination.

Example 4

Materials and Methods

Animals

[0240] C57BLJ6 mice were as described in Example 1. OT-I mice were as described in Example 1 and were purchased from Taconic Europe (Ry, Denmark) or from Jackson Laboratories (Bar Harbor, Maine). All mice were kept under SPF conditions, and the procedures performed were approved by the veterinary authorities in Switzerland and Norway.

Materials and Cells

[0241] OVA and TPCS.sub.2a were as for Example 1 and TPCS.sub.2a was illuminated as described in Example 1. In addition, Poly(IC) (high MW) and CpG oligonucleotide ODN 2395 were from InvivoGen (San Diego, USA). ODN 2395 is a type C CpG ODN which has the sequence

TABLE-US-00002 5-tcgtcgttttcggcgc:gcgccg-3
(palindrome underlined). OVA, TPCS.sub.2a and when relevant Poly(IC) were mixed in PBS, kept light protected, and administered to mice within 60 minutes of preparation.

Intradermal Photosensitisation and Immunisation Of Mice

[0242] The preparation and administration of OT-I cells to recipient female C57BL/6 mice and baseline analysis was as described in Example 2.

[0243] Then, the mice were shaved on the abdominal area, and the vaccines, consisting of OVA or of different mixtures of OVA, TPCS.sub.2a, Poly(IC) (50 g) or CpG oligonucleotide (50 g) were injected intradermally using syringes with 29G needles. The vaccines were kept light protected and used within 60 minutes of preparation. The vaccines were given in two injections of 50 l each, on the left and right side of the abdominal mid line. OVA was used at a dose of 10 or 100 g, and the TPCS.sub.2a dose was 150 g. 18 hours after the vaccine injection, the mice were anaesthetised by intraperitoneal injection of a mixture of ketamine (25 mg/kg body weight) and xylazin (4 mg/kg) and placed on the LumiSource light source (for illumination and activation of the photosensitiser TPCS.sub.2a). The illumination time was 6 minutes.

[0244] On days 7 and 14 thereafter mice were bled by tail bleeding and erythrocytes were removed by lysis, before analysis of antigen-specific CD8 T cells by flow cytometry. At the end of the experiment (day 14), the mice were euthanized and the splenocytes analysed ex vivo.

Analysis of Immune Responses

[0245] The frequency of OVA-specific CD8 T-cells in blood was monitored by staining the cells with anti-CD8 antibody and H-2K.sup.b/SIINFEKL ProS pentamer (Proimmune, Oxford, UK) for analysis by flow cytometry. The activation status of the cells was further analysed by testing the expression of CD44 by flow cytometry. The cells were analysed using FACSCanto (BD Biosciences, San Jose, USA) and analysed using FlowJo 8.5.2 software (Tree Star, Inc., Ashland, Oreg.).

[0246] For ELISA analysis 2x10.sup.5 splenocytes were re-stimulated in 96-well plates with 0.005 g/ml of the SIINFEKL peptide. After 72 hours, supernatants were collected and analysed for IFN- by ELISA (eBioscienceperformed according to the manufacturer's instructions).

Poly(IC) and CpG Experiment.

[0247] The experiment was performed as described under Materials and Methods, and mouse blood samples from day 7 after vaccination were analysed by flow cytometry as described. Spleen cells from day 14 were restimulated by SIINFEKL peptide and analysed by Interferon-gamma ELISA as described. All mice received OT-1 cells as described. [0248] The following experimental groups were included: [0249] 1. Untreated: Mice received OT-1 cells, but were not vaccinated or illuminated. [0250] 2. OVA: Mice were vaccinated with 10 g of OVA. They were not illuminated. [0251] 3. OVA 100 g: Mice were vaccinated with a mixture of 100 g OVA. They were not illuminated. [0252] 4. OVA 10 g PCI: Mice were vaccinated with a mixture of 10 g OVA+150 g TPCS.sub.2a. Illuminated as described. [0253] 5. CpG OVA: Mice were vaccinated with a mixture of 10 g OVA+50 g ODN2935 CpG oligonucleotide. They were not illuminated. [0254] 6. CpG OVA/PCI: Mice were vaccinated with a mixture of 10 g OVA+50 g ODN2935 CpG oligonucleotide+150 g TPCS.sub.2a. Illuminated as described. [0255] 7. Poly(IC) OVA: Mice were vaccinated with a mixture of 10 g OVA+50 g Poly(IC). They were not illuminated. [0256] 8. Poly(IC) OVA/PCI: Mice were vaccinated with a mixture of 10 g OVA+50 g Poly(IC)+150 g TPCS.sub.2a. Illuminated as described.

[0257] FIG. 19A shows the average values (% antigen-specific, CD44.sup.+ cells of the total CD8.sup.+ cells) for the experimental groups. It can be seen that the CpG and Poly(IC) adjuvants when used alone had only a very modest (for CpG) or no significant (for Poly(IC) effect, and that PCI used alone was substantially more potent than either of these adjuvants. However, a clear synergistic effect was seen when PCI was used in combination with CpG or Poly(IC), and was most prominent for the combination PCI+Poly(IC).

[0258] FIG. 19B shows the results from interferon-gamma (IFN-gamma) ELISA after restimulation of spleen cells with SIINFEKL peptide. Firstly it can be seen that the IFN-gamma production was totally dependent on restimulation (bars from unstimulated cells are barely visible), showing that the production was strictly antigen specific. It can also be seen that while there was virtually no effect with the cells from the CpG or Poly(IC) groups (nor with OVA alone), in all the PCI-treated groups a strong effect of restimulation could be observed, again with a synergistic effect in the PCI+CpG and the PCI+Poly(IC) groups, with the latter representing the better combination.

Example 5

Materials and Methods

[0259] C57BLJ6 mice, TPCS.sub.2a and OVA peptide were as described in Example 1. The TRP-2 peptide (sequence SVYDFFVWL) and gp100 (sequence KVPRNQDWL) was obtained from United Peptides (Herndon, Va.) and Poly(IC) (high molecular weight, average size of 1.5-8 kb) from InvivoGen (San Diego, USA).

Intradermal Photosensitisation and Immunisation of Normal Mice.

[0260] Preparation of the mice for immunization was performed as described in Example 2. The mice were shaved on the abdominal area and immunised at day 0 and at day 14 with a mixture of TRP-2 peptide and gp-100 peptide (50 g of each), 100 g TPCS.sub.2a and 10 g poly(IC) as specified below by intradermal injection using syringes with 29G needles. The vaccines were kept light protected and used within 60 minutes of preparation. The vaccines were given in two injections of 50 l each, on the left and right side of the abdominal mid line. At a specified time point after vaccine injection the mice were anaesthetised by intraperitoneal injection of a mixture of ketamine (25 mg/kg body weight) and xylazin (4 mg/kg) and illuminated where relevant.

Illumination of Immunised Mice.

[0261] Illumination with LumiSource was performed for 6 min, 18 hours after immunisation.

Analysis of Immune Responses by Pentamer Staining

[0262] On day 7 after immunisation mice were bled by tail bleeding and erythrocytes were removed by lysis. The frequency of antigen specific CD8 T-cells in the blood was monitored by flow cytometry after staining the cells with anti-CD8 and anti-CD44 antibodies and TRP-2 pentamers. The activation status of the cells was analysed by testing the expression of CD44 by flow cytometry. The cells were analysed using FACSCanto (BD Biosciences, San Jose, USA) and analysed using FlowJo 8.5.2 software (Tree Star, Inc., Ashland, Oreg.).

[0263] The following experimental groups were included: [0264] 1. Untreated TRP-2: Mice were not immunised or illuminated, blood samples were stained with TRP-2 pentamer. [0265] 2. TRP-2/poly(IC): Mice were immunised with a mixture of TRP-2 peptide and gp-100 peptide (50 g of each), and 10 g poly(IC). They were not illuminated. Blood samples were stained with TRP-2 pentamer. [0266] 3. TRP-2/PCI: Mice were immunised with a mixture of TRP-2 peptide and gp-100 peptide (50 g of each) and 100 g TPCS.sub.2a and illuminated. Blood samples were stained with TRP-2 pentamer. [0267] 4. TRP-2/poly(IC)/PCI: Mice were immunised with a mixture of TRP-2 peptide and gp-100 peptide (50 g of each), 100 g TPCS.sub.2a and 10 g poly(IC) and illuminated. Blood samples were stained with TRP-2 pentamer.

[0268] FIG. 20 shows the average values (% antigen-specific, CD44+cells of the total CD8+cells) for the TRP-2 pentamer stained experimental groups after the second immunisation. It can be seen that when the TRP-2 antigen was used with poly(IC) alone (group 2) or with PCI alone (group 3) no significant increase in antigen-specific cells were observed over what was seen in untreated animals. In comparison, the combination of poly(IC) and PCI (group 4) gave a clear synergistic effect leading to a significant increase in the number of antigen-specific CD8+T-cells.

Example 6

Materials.

[0269] C57BLJ6 mice (Harlan Laboratories, Netherlands) and TPCS.sub.2a (PCI Biotech, Norway) were as described in Example 1. The TRP-2 peptide and Poly(IC) were as described in Example 5.

Intradermal Photosensitisation and Immunisation of Normal Mice.

[0270] Preparation of the mice for immunization was performed as described in Example 2. The mice were shaved on the abdominal area (3-4 cm.sup.2) and immunised at day 0, day 14 and day 35 with 200 g of TRP-2 peptide, 100 g TPCS.sub.2a and 10 g poly(IC) as specified below by intradermal injection using 0.3 ml BD Micro-Fine+insulin syringes with 30G needles (BD, NJ, USA). The vaccines were kept light protected and used within 60 minutes of preparation. The vaccines were given in two injections of 50 l each, on the left and right side of the abdominal mid line. At a specified time point after vaccine injection the mice were anaesthetised by subcutaneous injection of a mixture of Zoletil (10 mg/kg body weight, Virbac, Norway) and illuminated where relevant.

Illumination of Immunised Mice.

[0271] Illumination of the vaccination site with LumiSource (PCI Biotech) was performed for 6 min, 18 hours after immunisation.

Analysis of Immune Responses by Pentamer Staining and Intracellular Staining.

[0272] On day 7 after each immunisation mice were bled by tail bleeding and erythrocytes were removed by lysis. The frequency of antigen specific CD8 T-cells in the blood was monitored by flow cytometry after staining the cells with anti-CD8 and anti-CD44 antibodies and TRP-2 pentamers. The activation status of the cells was analysed by testing the expression of CD44 by flow cytometry. The cells were analysed by using the BD LSRII flow cytometer with the FACSDiva software (BD Biosciences, San Jose, USA) and further analysed and processed using the FlowJo 8.5.2 software (Tree Star, Inc., Ashland, Oreg.).

[0273] On day 60 after the first immunisation the animals were sacrificed, the spleens were removed and the spleen cells were re-stimulated with the TRP-2 peptide and subsequently analysed with intracellular staining for interferon-gamma (IFN-gamma) as described in Example 2 and intracellular staining for tumour necrosis factor alpha (TNF-alpha) was performed as described for IFN-gamma using anti-TNF-alpha antibodies. (Antibodies against both TNF-alpha and IFN-gamma (carrying different fluorophores) were included in the same sample.)

[0274] The following experimental groups were included: [0275] 1. Untreated Mice were not immunised or illuminated. [0276] 2. TRP-2: Mice were immunised with 200 g TRP-2 peptide in all immunisations. They were not illuminated. [0277] 3. TRP-2+poly(IC): Mice were immunised with 200 g TRP-2 peptide and 10 g poly(IC). They were not illuminated. [0278] 4. TRP-2+PCI: Mice were immunised with 200 g TRP-2 peptide and 100 g TPCS.sub.2a and illuminated. [0279] 5. TRP-2+poly(IC) +PCI: Mice were immunised with 200 g TRP-2 peptide, 10 g poly(IC) and 100 g TPCS.sub.2a and illuminated.

[0280] FIG. 21 shows the average values (% antigen-specific, CD44+cells of the total CD8+cells) for the TRP-2 pentamer stained blood samples after the third immunisation. It can be seen that when the TRP-2 antigen was used with poly(IC) alone (group 3) or with PCI alone (group 4) a significant, but small increase in antigen-specific cells were observed over what was seen with antigen alone (group 2). In comparison, the combination of TRP-2, poly(IC) and PCI (group 5) gave a clear synergistic effect leading to a substantial (about 5 times) increase over what was seen with the individual treatments alone.

[0281] FIG. 22 shows the results from interferon-gamma (IFN-gamma) intracellular staining after re-stimulation of spleen cells with the TRP-2 peptide. It can be seen that when the TRP-2 antigen was used with poly(IC) alone (group 3) or with PCI alone (group 4) a small increase in the percentage of IFN-gamma producing cells were observed (over what was achieved with the TRP-2 peptide alone). Again, the combination of TRP-2, poly(IC) and PCI (group 5) gave a clear synergistic effect leading to a substantial (about 8 times) increase over what was seen with the best of the individual treatments alone (TRP-2 +poly(IC), group 3).

[0282] FIG. 23 shows the results from TNF-alpha intracellular staining after re-stimulation of spleen cells with the TRP-2 peptide. It can be seen that when the TRP-2 antigen was used with poly(IC) alone (group 3) or with PCI alone (group 4) a small increase in the percentage of TNF-alpha producing cells were observed (over what was achieved with the TRP-2 peptide alone). Again, the combination of TRP-2, poly(IC) and PCI (group 5) gave a clear synergistic effect leading to a substantial (about 6 times) increase over what was seen with the best of the individual treatments alone (TRP-2 +poly(IC), group 3).

Example 7 PCI-Mediated Prophylactic Vaccination with Melanoma Cell Extracts.

[0283] Materials and methods were as described in Example 6, where appropriate.

Preparation of Melanoma Cell Extract

[0284] B16-F10 mouse melanoma cells (as described in Example 1 but without OVA transfection/expression) were harvested when in the logarithmic growth phase i.e. cell culture flasks were 50% confluent. The medium was aspirated and the flask rinsed briefly with 2 ml trypsin/EDTA (0.25% (w/v) Trypsin-0.53 mM EDTA) and aspirated again. 2 ml trypsin/EDTA were added, tilting the flask to ensure that all cells were covered. The side of the flask was tapped periodically until cells detached and slid down the culturing surface. 12 ml cold CM (Dulbecco's Modified Eagle's Medium with 10% FBS) was added to neutralize the trypsin and the suspension was pipetted vigorously to obtain a single-cell suspension. The suspension was transferred to a 15-ml conical centrifuge tube and the cells were pelleted by centrifugation for 10 min at 528g/1500 rpm (Rotina 380R, Hettich, Germany) at 4 C. The supernatant was decanted and the cell concentration was adjusted to 110.sup.7/ml in ice-cold Hanks Balanced Salt Solution (5.4 mM KCl, 0.3 mM Na.sub.2HPO.sub.4.7H.sub.2O, 0.4 mM KH.sub.2PO.sub.4, 4.2 mM NaHCO.sub.3, 1.3 mM CaCl.sub.2, 0.5 mM MgCl.sub.2.6H.sub.2O, 0.6 mM MgSO.sub.4.7H.sub.2O, 137 mM NaCl, 5.6 mM D-glucose, 0.02% phenol red; pH adjusted to 7.4 with 1 M HCl or 1 M NaOH).

[0285] Irradiation of B16-F10 cells was conducted with an X-ray generator (Faxitron CP160, 160kV, 6.3 mA, Arizona, USA) with a total dose of 50 Gy.

[0286] For mouse vaccination the irradiated cells were kept on ice before vaccination of 6- to 12-week-old female C57BL/6 mice.

Experimental Groups:

[0287] There were 5 mice in each experimental group. The mice were vaccinated twice (days 0 and 14) with a total of 110.sup.6 cells B16-F10 cells per mouse per vaccination, divided in two 50 L injections per mouse. The experimental groups were as follows: [0288] Group 1: no treatment, no illumination. [0289] Group 2: Irradiated B16-F10 cells, no illumination. [0290] Group 3: Irradiated B16-F10 cells+10 g Poly (IC), no illumination [0291] Group 4: Irradiated B16-F10 cells+150 g TPCS.sub.2a+illumination. [0292] Group 5: Irradiated B16-F10 cells+10 g poly(IC)+150 g TPCS.sub.2a+illumination.

[0293] At day 21 510.sup.5 B16-F10 cells were injected intradermally, and the size of the tumours was measured at least 2 times per week. The results are shown with day 0 as the day the tumour cells were administered.

[0294] From FIG. 24 it can be seen that the tumours in the groups receiving PCI-mediated vaccination (groups 4 and 5) grew substantially slower than in the groups receiving the vaccine alone (group 2) or the vaccine with the poly(IC) adjuvant without PCI (group 3), both of which did not differ from the untreated animals.