COMPOUND AND METHOD

Abstract

The present invention provides a method of expressing an antigenic molecule or a part thereof on the surface of a cell using a photochemical internalisation method in which a cytokine, preferably GM-CSF, is used to enhance the method. The method may be used to stimulate an immune response and for various therapeutic or prophylactic methods. Pharmaceutical compositions or kits comprising the components for use in the method, cells produced by the method and their use in therapy and prophylaxis also form aspects of the invention.

Claims

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

2. A method as claimed in claim 1, wherein said cytokine is a ligand for a type I or type II cytokine receptor.

3. A method as claimed in claim 1 or 2 wherein said cytokine is a ligand for an IL-2 receptor family member or a GM-CSF receptor family member

4. A method as claimed in claim 1 or 2 wherein said cytokine is an interferon, preferably a type I IFN.

5. A method as claimed in any one of claims 1 to 4 wherein said cytokine is selected from GM-CSF, IL-7, IFN-α, IL-2, IL-15 and IL-21, or a homolog or derivative thereof, wherein preferably the cytokine is GM-CSF.

6. The method as claimed in any one of claims 1 to 5 wherein the antigenic molecule is a molecule capable of stimulating an immune response, preferably a vaccine antigen or vaccine component.

7. The method as claimed in claim 6 wherein the antigenic presentation results in the stimulation of an immune response.

8. The method of any one of claims 1 to 7 wherein the method is performed in vitro or ex vivo.

9. The method of any one of claims 1 to 8 wherein the photosensitising agent is selected from TPCS.sub.2a, AlPcS.sub.2, TPPS.sub.4 and TPBS.sub.2a, preferably TPCS.sub.2a, or a conjugate of a photosensitiser and chitosan as defined in formula (I): ##STR00013## wherein n is an integer greater than or equal to 3; R appears n times in said compound, and in 0.1%-99.9% (preferably 0.5%-99.5%) of said total Rn groups, each R is a group A selected from: ##STR00014## wherein each R.sub.1, which may be the same or different, is selected from H, CH.sub.3 and —(CH.sub.2).sub.b—CH.sub.3; a is 1, 2, 3, 4 or 5; and b is 0, 1, 2, 3, 4 or 5 (in which the counter-ion may be, for example, CI.sup.−; preferably R.sub.1, is CH.sub.3 and b is 1, and ##STR00015## wherein Y is O; S; SO.sub.2; —NCH.sub.3, or —N(CH.sub.2).sub.dCH.sub.3, c=1, 2, 3, 4 or 5; and d=1, 2, 3, 4 or 5, preferably Y is NCH.sub.3 and c is 1, wherein each R group may be the same or different, and in 0.1%-99.9% (preferably 0.5%-99.5%) of said total Rn groups, each R is a group B selected from: ##STR00016## wherein e is 0, 1, 2, 3, 4 or 5; and f is 1, 2, 3, 4 or 5; preferably e and f=1, R.sub.2 is a group selected from: ##STR00017## W is a group selected from O, S, NH or N(CH.sub.3); preferably NH, R.sub.3 is a group selected from: ##STR00018## V is a group selected from CO, SO.sub.2, PO, PO.sub.2H or CH.sub.2; preferably CO, and R.sub.4 is a group (substituted in the o, m or p position), which may be the same or different, selected from H, —OH, —OCH.sub.3, —CH.sub.3, —COCH.sub.3, C(CH.sub.3).sub.4, —NH.sub.2, —NHCH.sub.3, —N(CH.sub.3).sub.2 and —NCOCH.sub.3, preferably H, wherein each R group may be the same or different.

10. The method as claimed in any one of claims 1 to 9 wherein the antigenic molecule is a peptide.

11. The method as claimed in any one of claims 1 to 10 wherein the cell is an antigen presenting cell, preferably a dendritic cell.

12. The method as claimed in any one of claims 1 to 11 wherein said cell is contacted with said antigenic molecule, photosensitising agent and cytokine simultaneously, separately or sequentially.

13. A method of generating an immune response in a subject, comprising administering to said subject an antigenic molecule as defined in claim 1, 6 or 10, a photosensitizing agent as defined in claim 1 or 9, and a cytokine as defined in any one of claims 1 to 5, and irradiating said subject with light of a wavelength effective to activate said photosensitizing agent, wherein an immune response is generated.

14. The method as claimed in claim 13 wherein said method is a method of vaccination.

15. The method as claimed in claim 13 or 14 for treating or preventing a disease, disorder or infection, preferably cancer.

16. The method of any one of claims 13 to 15 wherein said subject is a non-mammalian animal, preferably a fish, or 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.

17. The method of any one of claims 13 to 16 wherein said antigenic molecule, photosensitising agent and cytokine are administered to said subject simultaneously, separately or sequentially.

18. A pharmaceutical composition comprising an antigenic molecule as defined in any one of claim 1, 6 or 10, a photosensitizing agent as defined in claim 1 or 9, and a cytokine as defined in any one of claims 1 to 5 and one or more pharmaceutically acceptable diluents, carriers or excipients.

19. A cell expressing an antigenic molecule, or a part thereof, on its surface, or a population thereof, which cell is obtainable by a method as defined in any one of claims 1 to 12, wherein preferably the cell is a dendritic cell.

20. A pharmaceutical composition comprising a cell or a population of cells as defined in claim 19 and one or more pharmaceutically acceptable diluents, carriers or excipients.

21. A cell or cell population as defined in claim 19 or a composition as defined in claim 18 or 20 for use in prophylaxis or therapy.

22. A cell or cell population as defined in claim 19 or a composition as defined in claim 18 or 20 for use in stimulating an immune response in a subject, preferably for treating or preventing a disease, disorder or infection in said subject, preferably for vaccination and/or for treating or preventing cancer.

23. Use of a cell population as defined in claim 19 or a composition as defined in claim 18 or 20 for the preparation of a medicament for stimulating an immune response in a subject, preferably for treating or preventing a disease, disorder or infection in said subject, preferably for vaccination and/or for treating or preventing cancer.

24. A use as claimed in claim 23, wherein said stimulation, treatment or prevention comprises administering said medicament to said subject.

25. An antigenic molecule as defined in any one of claim 1, 6 or 10, a photosensitizing agent as defined in claim 1 or 9, and a cytokine as defined in any one of claims 1 to 5 for use in prophylaxis or therapy.

26. An antigenic molecule, photosensitizing agent and cytokine for use as claimed in claim 25 for use in stimulating an immune response in a subject, preferably for treating or preventing a disease, disorder or infection in said subject, preferably for vaccination and/or for treating or preventing cancer, wherein preferably said use comprises a method as defined in any one of claims 1 to 17.

27. The antigenic molecule, photosensitizing agent and cytokine as defined in any one of claims 1 to 5 for use as claimed in claim 25 or 26 wherein said use comprises a method as defined in any one of claims 1 to 12 to prepare a population of cells, wherein preferably the cells are dendritic cells.

28. The antigenic molecule, photosensitizing agent and agent for use as claimed in claim 27, wherein said population of cells are to be administered to said subject.

29. Use of an antigenic molecule as defined in any one of claim 1, 6 or 10 and/or a photosensitizing agent as defined in claim 1 or 9 and/or a cytokine as defined in any one of claims 1 to 5 in the manufacture of a medicament for stimulating an immune response in a subject, preferably for treating or preventing a disease, disorder or infection in said subject, preferably for vaccination and/or for treating or preventing cancer, wherein preferably said immune response is stimulated by a method as claimed in any one of claims 13 to 17.

30. The use of claim 29 wherein said medicament comprises a population of cells expressing an antigenic molecule or a part thereof on the surface of said cells obtainable by a method as defined in any one of claims 1 to 12, for administration to said subject.

31. The use as claimed in claim 30 wherein said antigenic molecule and/or photosensitizing agent and/or cytokine are used in a method as defined in any one of claims 1 to 12 to obtain said population of cells for manufacture of said medicament.

32. A product comprising an antigenic molecule as defined in any one of claim 1, 6 or 10, a photosensitizing agent as defined in claim 1 or 9 and a cytokine as defined in any one of claims 1 to 5 as a combined preparation for simultaneous, separate or sequential use in stimulating an immune response in a subject, preferably for treating or preventing a disease, disorder or infection in said subject, preferably for vaccination and/or for treating or preventing cancer, or for expressing an antigenic molecule or a part thereof on the surface of a cell in a method according to any one of claims 1 to 17.

33. A kit for use in stimulating an immune response in a subject, preferably for treating or preventing a disease, disorder or infection in said subject, preferably for vaccination and/or for treating or preventing cancer, or for expressing an antigenic molecule or a part thereof on the surface of a cell in a method according to any one of claims 1 to 17, said kit comprising a first container containing a photosensitizing agent as defined in claim 1 or 9; a second container containing said antigenic molecule as defined in any one of claim 1, 6 or 10; and a third container containing a cytokine as defined in any one of claims 1 to 5.

34. A method of generating an immune response in a subject, preferably to treat or prevent a disease, disorder or infection in said subject, preferably for vaccination and/or for treating or preventing cancer, comprising preparing a population of cells according to the method of any one of claims 1 to 12, and subsequently administering said cells to said subject.

Description

[0158] The invention will now be described in more detail in the following non-limiting Examples with reference to the following drawings in which:

[0159] FIG. 1A shows Scheme 1: synthetic route for synthesis of compound 5. Reagents and conditions: (a) propionic acid, reflux, 1 h (20%); (b) NaNO.sub.2 (1.8 eq), TFA, rt, 3 min. 67%); (c) SnCl.sub.2.2H.sub.2O, conc. HCI, 60° C., 1 h (88%); (d) Bromoacetyl bromide, Et.sub.3N, CH.sub.2Cl.sub.2, rt, 1 h (64%) (e) Piperazine, CH.sub.2Cl.sub.2, rt, 1 h (94%).

[0160] FIG. 1B shows Scheme 2. Synthesis of N-modified Chitosan derivatives (TPP-CS-TMA & TPP-CS-MP). Here A-represents 1.sup.st batch compounds and B-presents 2.sup.nd batch compounds. Reagents and conditions: (a) MeSO.sub.3H/H.sub.2O, 10° C.-rt, 1 h, (90%); (b) TBDMSCl, imidazole, DMSO, rt, 24 h (96%); (c) Bromoacetyl bromide, Et.sub.3N, CH.sub.2Cl.sub.2, −20° C., 1 h (92%); (d) compound 5 i.e. TPP—NH-Pip (0.1 or 0.25 eq), Et.sub.3N, CHCl.sub.3, rt, 2 h (92-90%) (e) NMe.sub.3or 1-methyl piperazine, CHCl.sub.3, rt, 24 h (f) TBAF, NMP, 55° C., 24 h or conc. HCl/MeOH, rt, 24 h.

[0161] FIG. 1C shows Scheme 3 —Synthesis scheme for compounds 1, 3 20 and 21. Reactions and conditions: ((a) Propionic acid, reflux, 1 h, (20%); (b) NaNO.sub.2 (1.8 eq.), TFA, rt, 3 min.; (c) SnCl.sub.2.2H.sub.2O, conc. HCl, 60° C., 1 h, (54%); (d.sub.1) p-Toluenesulfonylhydrazide, K.sub.2CO.sub.3, pyridine, reflux, 24 h; (d.sub.2) o-Chloranil, CH.sub.2Cl.sub.2, rt, (80%); (e) Chloroacetyl chloride, Et.sub.3N, CH.sub.2Cl.sub.2, rt, 2 h, in situ-(f) Piperazine, CH.sub.2Cl.sub.2, rt, 12 h, (61%). All derivatives of compound 20 and 21 will contain the TPCa.sub.1 and the TPCa.sub.2 isomer. However only the TPCa.sub.1 structure is shown in schemes and in the structure drawings.

[0162] FIG. 1D shows Scheme 4-synthesis scheme for compounds 22-28. Reactions and conditions: (a) Acetyl chloride, MeOH, reflux, 24 h, (87%); (b) BF.sub.3.Et.sub.2O, CHCl.sub.3, rt, p-chloranil, 48 h, (14%); (c) 2N KOH (in MeOH), THF:Pyridine (10:1), reflux, 24 h (71%); (d.sub.1) p-Toluenesulfonylhydrazide, K.sub.2O0.sub.3, Pyridine, reflux, 24h; (d.sub.2) o-chloranil, CH.sub.2Cl.sub.2: MeOH (75:25), rt, (70%); (e) EDCI.HCl, HOBT, Et.sub.3N, N-Boc-piperazine 5, DMF, rt, 24 h (54%) (f) TFA, CH.sub.2Cl.sub.2, rt, 1 h (89%). All derivatives of compound 26-28 will contain the TPCc.sub.1 and the TPCc.sub.2 isomer. However, only the TPCc.sub.1 structure is shown in schemes and in the structure drawings.

[0163] FIGS. 1E-1F show Scheme 5A and Scheme 5B, respectively. Reagents and conditions (6A): (a) compound 21 i.e. TPC—NH-Pip (0.1 eq), Et.sub.3N, CHCl.sub.3, rt, 2 h (78%) (b) NMe.sub.3 or 1-methyl piperazine, CHCl.sub.3, rt, 24 h. Reagents and conditions (6b): a) compound 28 i.e. TPC-CO-Pip (0.1 eq), Et.sub.3N, NMP, 75° C., 12 h (89%) (b) NMe.sub.3 or 1-methyl piperazine, CHCl.sub.3, rt, 24 h.

[0164] FIGS. 2A-2B show the effect of the adjuvant GM-CSF. Mice were immunised with 10 μg OVA, with 10 μg OVA and 10 μg GM-CSF, with 10 μg OVA and 150 μg TPCS.sub.2a, with 10 μg OVA, 10 μg GM-CSF, and 150 μg TPCS.sub.2a or left untreated. Mice receiving TPCS.sub.2a were illuminated. On day 7 the mice were bled, and the frequency of OVA-specific CD8 T-cells was analyzed by flow cytometry. (FIG. 2A) shows representative dot plots from the flow cytometry analysis. The cells were first gated on CD8 expression, and then the CD8.sup.+ population was analysed for SIINFEKL pentamer binding (y-axis) and CD44 expression (x-axis). The population within the ellipses thus represents the CD8.sup.+, pentamer.sup.+, CD44.sup.+ cells, representing the antigen-specific (pentamer binding), activated (CD44 expression) CD8.sup.+ cells. (FIG. 2B) shows the average values (% antigen-specific, CD44.sup.+ cells of the total CD8+ cells) for the experimental groups (5 animals in each group, error bars: standard error of the mean).

[0165] FIG. 3 shows the effect of GM-CSF on vaccination of mice with a HPV 16 E7 peptide antigen. Mice were immunised with HPV alone or HPV with GM-CSF, with or without PCI. The figure shows the % of total CD8 cells expressing the HPV pentamer.

[0166] FIG. 4 shows the effect of GM-CSF, optionally with Poly(IC) on vaccination of mice with a HPV 16 E7 peptide antigen. Mice were immunised with HPV, poly(IC) and/or GM-CSF with or without PCI, as indicated in the figure. 2 immunisations were used in the results shown in the last two bars in the figure. The figure shows the % of total CD8 cells expressing the HPV pentamer.

EXAMPLES

Example 1

Effect of Cytokines on In Vivo Vaccination with OVA

Materials and Methods

Mice

[0167] C57BL/6 mice are purchased from Harlan (Horst, The Netherlands). OT-I mice transgenic for the T-cell receptor that recognises the MHC class-I restricted epitope OVA.sub.257-264 from ovalbumin (OVA) are bred in facilities at the University of Zurich (originally purchased from Taconic Europe (Ry, Denmark)). All mice are kept under specified pathogen-free (SPF) conditions, and the procedures performed are approved by Swiss Veterinary authorities. In the OT-1 mice, the gene for the T-cell receptor has been engineered in such a way that nearly all of the CD8+ T-cells in these mice (called OT-1 cells) will specifically recognize the specific peptide epitope (SIINFEKL) from the ovalbumin (OVA) antigen.

Immunisation Protocol

[0168] On day 0 female C57BL/6 mice are injected with 1.5×10.sup.6 splenocytes from Rag2/OT-1 mice intravenously in the tail vein. In this way the mice that are vaccinated have a “background” of CD8 T-cells that can respond to the SIINFEKL-epitope from OVA if, and only if, this is properly presented on MHC class I on antigen presenting cells. Thus, the transfer of OT-1 cells “amplifies” the detection system in the vaccinated mice making it possible to easily assay for the effect of in vivo vaccination by measuring antigen specific CD8+ T-cells and IFN-y and IL-2 production. 4 hours later the animals are vaccinated by intradermal injection at the abdomen (2×50 μl of solutions containing the ingredients specified below). 14 groups of 4 animals receive total doses of: [0169] Group 1: 250 μg TPCS.sub.2a (Amphinex)+10 μg ovalbumin (OVA, Grade V, Sigma-Aldrich). [0170] Group 2: 250 μg TPCS.sub.2a+10 μg ovalbumin+10 μg GM-CSF. [0171] Group 3: 250 μg TPCS.sub.2a+10 μg ovalbumin+500 000 IU IL-2. [0172] Group 4: 250 μg TPCS.sub.2a+10 μg ovalbumin+10 μg IL-7. [0173] Group 5: 250 μg TPCS.sub.2a+10 μg ovalbumin+10 μg IL-15. [0174] Group 6: 250 μg TPCS.sub.2a+10 μg ovalbumin+10 μg IL-21. [0175] Group 7: 250 μg TPCS.sub.2a+10 μg ovalbumin+3,000,000IU IFNα. [0176] Group 8: 10 μg ovalbumin. [0177] Group 9: 10 μg ovalbumin+25 μg GM-CSF. [0178] Group 10: 10 μg ovalbumin+500,000IU IL-2. [0179] Group 11: 10 μg ovalbumin+10 μg IL-7. [0180] Group 12: 10 μg ovalbumin+10 μg IL-15. [0181] Group 13: 10 μg ovalbumin+10 μg IL-21. [0182] Group 14: 10 μg ovalbumin+3,000,000IU IFNα.

[0183] On day 1 the animals of groups 1-7 are anaesthetized and illuminated for 6 minutes with blue light using a LumiSource lamp (PCI Biotech AS). The animals are illuminated about 18 h after injection of the antigen solution, the fluence rate of the illumination is about 13 mW/cm.sup.2. On day 7 the mice are bled from the tail vein and the blood cells are stained with SIINFEKL pentamer (Prolmmune), and CD8 and CD44 antibodies for flow cytometry analysis (see protocols below). On day 14 the mice are euthanized and the spleens are collected. One aliquot of the splenocytes is restimulated with the SIINFEKL peptide (EMC microcollections, Tuebingen, Germany), stained for intracellular IFN-γ expression and analysed by Flow cytometry analysis (see below). Another aliquot of the splenocytes is resuspended in cell culture medium, kept in this medium overnight (purely for practical reasons) without restimulation stained by SIINFEKL-pentamer as described above and analysed by flow cytometry (see protocol below).

[0184] SIINFEKL-Pentamer-Staining of Spleen Cells

[0185] SIINFEKL-pentamer staining and flow cytometry on spleen cells is performed on cells that have been resuspended in cell medium and kept in this medium overnight (purely for practical reasons) without restimulation.

[0186] SIINFEKL-Pentamer Staining and Flow Cytometry

[0187] 5-10 drops of whole tail blood are collected and 0.5 ml of Red Cell Lyse solution (Sigma) is added. After 5-6 minutes, cells are spun down and washed twice with 0.5 ml PBS. The cell pellet is resuspended in FACS buffer (2% FCS/PBS with 0.01% Na-azide), transferred to a U-formed 96 well plate and incubated with FcR-blocking antibodies (1.0 μl Anti-CD16/CD32 from Pharmingen) for 10 min on ice, (1 μl+49 μl FACS buffer). Without washing, the SIINFEKL-pentamer-PE (Prolmmune; 5 μl per sample) is added, mixed and incubated at 37° C. for 15 min. Without washing, a fluorescence-labeled CD8 or CD44 is added to a final concentration of 1:100, and incubated on ice for 25-45 min. Cells are washed in 100 μl FACS buffer and suspended in 100 μl FACS buffer. Cells are analysed with FACSCanto.

[0188] Splenocyte Restimulation Ex Vivo

[0189] Splenocytes are isolated and prepared for intracellular staining by crushing the spleen and separating cells in 2% FCS/PBS, by agitation in lysis buffer (Sigma) for 1-2 minutes and washing in 2% FCS/PBS. 1 ml of the cell suspension in complete medium is added per well of a 24-well plate (500,000 cells/ml) and 5 μg/ml SIINFEKL is added to each well and incubated overnight at 37° C. Brefeldin A (1-2 μg/ml) is added to each well and incubated for 4 hours at 37° C. Cells are transferred to U-formed 96 well plates, washed in 2% FCS/PBS and resuspended in 50 μl FACS buffer with FcR-blocking antibodies (1.0 μl anti-CD16/CD32 from Pharmingen), and incubated on ice for 10 minutes. Without washing, cells are incubated with surface antibodies CD8 or CD44 for 20-45 min on ice (dark), washed in FACS buffer and fixed by resuspending in 100 μl paraformaldehyde (PFA) (1% in PBS) for 10-20 minutes on ice. Cells are washed in FACS buffer, resuspended in 100 μl NP40 (0.1% in PBS) and incubated for 3 minutes on ice. After washing in FACS buffer, a fluorescence-labelled interferon-gamma antibody is added and incubated for 35 min on ice in the dark. After washing and suspension in FACS buffer, the cells are analysed with FACSCanto using FlowJo 8.5.2 software (Tree Star, Inc., Ashland, Oreg.).

[0190] Flow Cytometry

[0191] The frequency of OVA-specific T-cells is determined by flow cytometry (FACSCanto from BD Biosciences, San Jose, USA). Before the flow cytometry run a compensation is performed using beads stained with each antibody separately. Before antibody staining, the red blood cells are lysed using Red Cell Lyse solution (Sigma). 10 000 CD8.sup.+ events are recorded for each sample, and the percentage of SIINFEKL-pentamer positive cells is calculated using FlowJo 8.5.2 software from Tree Star, Inc. (Ashland,Oreg.) http://www.flowjo.com/.

[0192] ELISA

[0193] ELISA is performed using the Ready-set Go! kit (eBioscience) for the relevant molecules according to the manufacturer's instructions.

[0194] Mice are vaccinated in vivo by the immunisation protocol described above. Blood is isolated after 7 days and spleen after 14 days. Blood is analysed for antigen-specific CD8+ T cells and spleen cells are either analysed directly for antigen-specific CD8+ T-cells or for IFN-γ or IL-2 production after restimulation in vitro.

[0195] Level of Antigen-Specific T-cells in Blood and Spleen

[0196] The level of antigen-specific T-cells is measured by flow cytometry, using a fluorescently labelled antigen-specific “pentamer” that binds specifically to the antigen-specific T-cells. The number of antigen specific CD8+ T-cells in % of the total CD8+ T-cells in the animal is determined (see the staining and flow cytometry analysis described in the immunisation protocol and details of SIINFEKL staining).

[0197] The endogenous T-cells serve as an internal control for the antigen-specificity of the effect, since a general stimulation effect on T-cells will affect also the endogenous T-cells not leading to an increase in the % of the antigen-specific cells. Typically the % of OT-1 cells is measured before vaccination and at time point(s) after vaccination. The effect of the antigen alone (“conventional vaccination”) is compared to the effect of antigen+PCI.

[0198] Level of IFN-γ Production in Spleen Cells after Ex Vivo Stimulation with Antigen (Flow Cytometry)

[0199] Spleens removed on day 14 of vaccination are subject to splenocyte isolation and restimulation with SIINFEKL antigen peptide and intracellular staining for IFN-γ production for analysis of CD8+ T cells by flow cytometry as described in the protocols above.

[0200] Level of IFN-γ and IL-2 Production in Spleen Cells after Ex Vivo Stimulation with Antigen (ELISA)

[0201] Spleens removed on day 14 of vaccination are subject to splenocyte isolation and restimulation with SIINFEKL antigen peptide and IFN-γ and IL-2 production analysis by ELISA as described in the protocols above.

Example 2

Effect of GM-CSF on In Vivo Vaccination with OVA

[0202] Materials and Methods

[0203] Animals

[0204] C57BL/6 mice were purchased from Harlan (Horst, The Netherlands). CD8 T-cell receptor transgenic OT-I mice (B6.129S6-Rag2tm1Fwa Tg(TcraTcrb)1100 Mjb) were purchased from Taconic Europe (Ry, Denmark) or from Jackson Laboratories (Bar Harbor, Maine). 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 the veterinary authorities in Switzerland and Norway.

[0205] Materials and Cells

[0206] Chicken OVA was purchased from Sigma-Aldrich (Buchs, Switzerland), the SIINFEKL peptide from EMC microcollections (Tuebingen, Germany), and GM-CSF from Preprotech (Wien).The photosensitiser tetraphenyl chlorine disulfonate (TPCS.sub.2a) was from PCI Biotech (Lysaker, Norway). OVA, TPCS.sub.2a and, when relevant, GM-CSF 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).

[0207] Intradermal Photosensitisation and Immunisation of Mice

[0208] 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 2×10.sup.6 OT-1 cells were administered by intravenous injection into recipient female C57BL/6 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.

[0209] Then, the mice were shaved on the abdominal area, and the vaccines, consisting of OVA or of different mixtures of OVA, TPCS.sub.2a and GM-CSF (10 μg) were injected intradermally using syringes with 29 G needles. The vaccines were kept light protected and used within 60 minutes of preparation. The vaccines were given in two injections of 50 μl each, on the left and right side of the abdominal mid line. OVA was used at a dose of 10 or 100 μg, and the TPCS.sub.2a dose was 150 μg. 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.

[0210] On day 7 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, the mice were euthanized.

[0211] Analysis of Immune Responses

[0212] 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.).

[0213] GM-CSF Experiment.

[0214] 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. All mice received OT-1 cells as described. The following experimental groups were included:

[0215] 1. Untreated: Mice received OT-1 cells, but were not vaccinated or illuminated.

[0216] 2. OVA: Mice were vaccinated with 10 μg of OVA. They were not illuminated.

[0217] 3. OVA+GM-CSF: Mice were vaccinated with a mixture of 10 μg OVA+10 μg GM-CSF. They were not illuminated.

[0218] 4. OVA PCI: Mice were vaccinated with a mixture of 10 μg OVA+150 μg TPCS.sub.2a. Illuminated as described.

[0219] 5. OVA+gm-CSF PCI: Mice were vaccinated with a mixture of 10 μg OVA+10 μg gm-CSF+150 μg TPCS.sub.2a. Illuminated as described.

[0220] FIG. 2A shows representative dot plots from the flow cytometry analysis. The population within the ellipses thus represents the CD8.sup.+, pentamer.sup.+, CD44.sup.+ cells, representing the antigen-specific (pentamer binding), activated (CD44 expression) CD8.sup.+ cells. It can be seen that the number of cells in this population is increased (as compared to the OVA group) in the OVA+GM-CSF and the OVA PCI groups), and that the effect is further significantly increased in the OVA+GM-CSF PCI group.

[0221] FIG. 2B. shows the average values (% antigen-specific, CD44.sup.+ cells of the total CD8+ cells) for the experimental groups, again showing a substantial increase in the OVA+GM-CSF PCI group over all the other groups.

Example 3

Effect of GM-CSF on In Vivo Vaccination with HPV

[0222] Materials and Methods

[0223] Animals

[0224] C57BL/6 mice were purchased from Harlan (Horst, The Netherlands). All mice were kept under SPF conditions, and the procedures performed were approved by the veterinary authorities in Norway.

[0225] Materials and Cells

[0226] The HPV 16 E7 peptide antigen (sequence QAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR, the CD8 epitope is underlined) was obtained from United Peptides (Herndon, Va.). High MW Poly(IC) was from InvivoGen (San Diego, USA). GM-CSF (recombinant murine) was purchased from PeproTech Inc., Rocky Hill, USA (Catalog# 315-03). The photosensitiser tetraphenyl chlorin disulfonate (TPCS.sub.2a) was from PCI Biotech (Lysaker, Norway), and HPV pentamer were from Proimmune (Oxford, UK), (Proimmune peptide codes 502H).

[0227] Intradermal Photosensitisation and Immunisation of Mice.

[0228] The mice were shaved on the abdominal area, and the vaccines, consisting of 50 pg HPV long peptide antigen, 100 μg TPCS.sub.2a and 10 μg GM-CSF and/or poly(IC) (as specified below) were injected intradermally using syringes with 29 G needles. The vaccines were kept light protected and used within 60 minutes of preparation. The vaccines were given in two injections of 50 μl each, on the left and right side of the abdominal mid line. 18 hours after immunisation the mice were anaesthetised by intraperitoneal injection of a mixture of ketamine (25 mg/kg body weight) and xylazin (4 mg/kg) and illuminated as described below.

[0229] On day 7 after each immunisation mice were bled by tail bleeding and erythrocytes were removed by lysis, before analysis of antigen-specific CD8 T cells by flow cytometry.

[0230] Illumination of Immunised Mice.

[0231] TPCS.sub.2a was activated by illumination with LumiSource™ (PCI Biotech). Illumination with LumiSource was performed for 6 min, 18 hours after immunisation.

[0232] Analysis of Immune Responses by Pentamer Staining.

[0233] The frequency of antigen specific CD8 T-cells in blood was monitored by flow cytometry after staining the cells with anti-CD8 and anti-CD44 antibodies and a pentamer corresponding to the HPV antigen used. 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.).

Example 3

Effect of PCI with HPV Long Peptide Antigen and GM-CSF

[0234] The experiment was performed as described under Materials and Methods. The animals were immunised at day 0 and at day 14 with vaccine mixtures as specified below. Illumination for 6 min was performed with the LumiSource illumination device, 18 hours after immunisation. Blood samples from day 6 after each immunisation were stained with HPV pentamer, CD8 and CD44 antibodies, and analysed by flow cytometry as described. The following experimental groups were included:

[0235] 2×HPV: Mice were immunised 2 times with 50 μg HPV long peptide. The mice were not illuminated.

[0236] 2×HPV+GM-CSF: Mice were immunised 2 times with 50 μg HPV long peptide and 10 pg GM-CSF. The mice were not illuminated.

[0237] 2×HPV+GM-CSF+PCI: Mice were immunised 2 times with 50 μg HPV long peptide, 100 μg TPCS.sub.2a and 10 μg-GM-CSF. The mice were illuminated at both immunisations.

[0238] Results

[0239] As can be seen in FIG. 3 two immunisations with antigen+GM-CSF did not improve the immunological CD8-cell response over what was observed with antigen alone. However, combining GM-CSF with PCI substantially increased the CD8-cell response.

Example 3b

Effect of PCI with HPV Long Peptide Antigen, GM-CSF and Poly(IC)

[0240] The experiment was performed as described under Materials and Methods. The animals were immunised at day 0 and at day 14 with vaccine mixtures as specified below. Illumination for 6 min was performed with the LumiSource illumination device, 18 hours after immunisation. Blood samples from day 6 after each immunisation were stained with HPV pentamer, CD8 and CD44 antibodies, and analysed by flow cytometry as described. The following experimental groups were included:

[0241] 2×HPV: Mice were immunised 2 times with 50 μg HPV long peptide. The mice were not illuminated.

[0242] 2×HPV+poly(IC): Mice were immunised 2 times with 50 μg HPV long peptide and 10 μg poly(IC). The mice were not illuminated.

[0243] 1.sup.st: HPV+p(IC)+PCI. 2.sup.nd: HPV+PCI: Mice were immunised with a mixture of 50 μg HPV long peptide, 10 μg poly(IC) and 100 μg TPCS.sub.2a (1.sup.st immunisation), and 50 μg HPV long peptide and 100 μg TPCS.sub.2a (2.sup.nd immunisation). The mice were illuminated at both immunisations.

[0244] 1.sup.st: HPV+p(IC)+GM-CSF+PCI. 2.sup.nd: HPV+GM-CSF+PCI: Mice were immunised with a mixture of 50 μg HPV long peptide, 10 μg poly(IC), 10 μg GM-CSF and 100 μg TPCS.sub.2a (1.sup.st immunisation), and 50 μg HPV long peptide, 10 μg GM-CSF and 100 μg TPCS.sub.2a (2.sup.nd immunisation). The mice were illuminated at both immunisations.

[0245] Results

[0246] In this experiment, the first immunisation was performed with the HPV antigen alone, with antigen+poly(IC) or with combinations of the antigen+poly(IC)+PCI, or the antigen+poly(IC)+GM-CSF+PCI. The second immunisations were performed with the same combinations, but, for samples treated with PCI, without poly(IC). It can be seen from FIG. 4 that while the treatment regimen with PCI+poly(IC) alone had a positive effect on the immunological response, adding GM-CSF to the same treatment regimen substantially enhanced the response. Immunisation with antigen+GM-CSF+PCI improved the immunological CD8-cell response significantly over what was observed with antigen alone (FIG. 3), but the magnitude of the response was substantially smaller than what was observed for the combination of GM-CSF and poly(IC) and PCI (FIG. 4). Taken together with the observation that without PCI the use of poly(IC) did not improve the immunological response over what was achieved with antigen alone, the experiments indicate that when used with PCI GM-CSF and poly(IC) PCI can act synergistically to enhance the CD8 response to a peptide antigen.