METHOD

Abstract

The invention concerns a method of generating an immune response in a subject, comprising administering to the subject an antigenic molecule, a photosensitizing agent, a checkpoint inhibitor, and irradiating said subject with light of a wavelength effective to activate the photosensitizing agent to generate an immune response. Preferably the method is a method of vaccination. The invention also provides related methods, compositions, cells, uses, products and kits.

Claims

1. A method of generating an immune response in a subject, comprising administering to said subject an antigenic molecule, a photosensitizing agent, a checkpoint inhibitor, and irradiating said subject with light of a wavelength effective to activate said photosensitizing agent, wherein an immune response is generated.

2. A method as claimed in claim 1 wherein the checkpoint inhibitor is an antibody, preferably a monoclonal antibody.

3. A method as claimed in claim 1 or 2 wherein the checkpoint inhibitor is selected from anti-CTLA4 and anti-PD-1, preferably (i) anti-CTLA4 and anti-PD-1 or (ii) anti-CTLA4.

4. A method as claimed in any one of claims 1 to 3 wherein the method further comprises contacting said subject with a TLR ligand.

5. A method as claimed in claim 4 wherein said TLR ligand is a TLR3 ligand, preferably a double stranded RNA molecule, preferably Poly(I:C).

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 and preferably comprises more than one antigen.

7. The method as claimed in any one of claims 1 to 6 wherein the photosensitising agent is selected from TPCS.sub.2a, AlPcS.sub.2a, TPPS.sub.4 and TPBS.sub.2a, preferably TPCS.sub.2a.

8. The method as claimed in any one of claims 1 to 7 wherein the antigenic molecule is a peptide, preferably a melanoma peptide or Human Papillomavirus (HPV) peptide.

9. The method as claimed in any one of claims 1 to 8 wherein said method is a method of vaccination.

10. The method as claimed in any one of claims 1 to 9 for treating or preventing a disease, disorder or infection, preferably cancer, preferably melanoma or a cancer associated with a papillomavirus.

11. The method of any one of claims 1 to 10 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.

12. The method of any one of claims 1 to 11 wherein said antigenic molecule, photosensitising agent, checkpoint inhibitor and optionally said TLR ligand are administered to said subject simultaneously, separately or sequentially.

13. A pharmaceutical composition comprising an antigenic molecule as defined in any one of claim 1, 6 or 8, a photosensitizing agent as defined in claim 1 or 7, a checkpoint inhibitor as defined in any one of claims 1 to 3 and optionally a TLR ligand as defined in claim 4 or 5, and one or more pharmaceutically acceptable diluents, carriers or excipients.

14. A method of expressing an antigenic molecule or a part thereof on the surface of a cell, comprising: a) providing a first cell on which said antigenic molecule or part thereof is to be expressed and a second cell to which a checkpoint inhibitor may bind, wherein said first and second cell may be the same cell or different cells, b) contacting at least said first cell with said antigenic molecule, a photosensitizing agent, and optionally a TLR ligand, c) contacting at least the second cell with a checkpoint inhibitor, and d) irradiating at least the first 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 surface of the first cell, wherein said first and second cells are contacted with one another before, during and/or after said irradiation.

15. The method as claimed in claim 14 wherein said antigenic molecule is as defined in claim 6 or 8, said photosensitizing agent is as defined in claim 7, said checkpoint inhibitor is as defined in claim 2 or 3 and/or said TLR ligand is as defined in claim 4 or 5.

16. The method as claimed in claim 15, wherein the antigenic molecule is as defined in claim 6 and the antigenic presentation results in the stimulation of an immune response.

17. The method of any one of claims 14 to 16 wherein the method is performed in vitro or ex vivo.

18. The method as claimed in any one of claims 14 to 17 wherein the first cell is an antigen presenting cell, preferably a dendritic cell or macrophage.

19. The method of any one of claims 14 to 18 wherein said second cell is a T cell.

20. The method of any one of claims 14 to 19 wherein said first and second cells are contacted with one another during the method and said antigenic molecule, photosensitizing agent, checkpoint inhibitor and optionally said TLR ligand are contacted with both cells during said method.

21. The method as claimed in any one of claims 14 to 20 wherein said first and second cells are contacted with said antigenic molecule, photosensitising agent and checkpoint inhibitor, and optionally said TLR ligand, simultaneously, separately or sequentially.

22. 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 14 to 21, wherein preferably the cell is a dendritic cell.

23. A pharmaceutical composition comprising a cell or a population of cells as defined in claim 22 and one or more pharmaceutically acceptable diluents, carriers or excipients.

24. A cell or cell population as defined in claim 22 or a composition as defined in claim 13 or 23 for use in prophylaxis or therapy.

25. A cell or cell population as defined in claim 22 or a composition as defined in claim 13 or 23 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.

26. Use of a cell population as defined in claim 22 or a composition as defined in claim 13 or 23 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.

27. A use as claimed in claim 26, wherein said stimulation, treatment or prevention comprises administering said medicament to said subject.

28. An antigenic molecule as defined in any one of claim 1, 6 or 8, a photosensitizing agent as defined in claim 1 or 7, a checkpoint inhibitor as defined in any one of claims 1 to 3, and optionally a TLR ligand as defined in claim 4 or 5, for use in prophylaxis or therapy.

29. An antigenic molecule, photosensitizing agent, checkpoint inhibitor and optionally a TLR ligand for use as claimed in claim 28 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 12 or 14 to 21.

30. The antigenic molecule, photosensitizing agent, checkpoint inhibitor and optional TLR ligand as defined in any one of claim 1 to 6, 7 or 8 for use as claimed in claim 28 or 29 wherein said use comprises a method as defined in any one of claims 14 to 21 to prepare a population of cells expressing an antigenic molecule or a part thereof on its surface, wherein preferably the cells are dendritic cells.

31. The antigenic molecule, photosensitizing agent, checkpoint inhibitor and optional TLR ligand for use as claimed in claim 30, wherein said population of cells are to be administered to said subject.

32. Use of an antigenic molecule as defined in any one of claim 1, 6 or 8 and/or a photosensitizing agent as defined in claim 1 or 7 and/or a checkpoint inhibitor as defined in any one of claims 1 to 3, and optionally a TLR ligand as defined in claim 4 or 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 1 to 12.

33. The use of claim 32 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 14 to 21, for administration to said subject.

34. The use as claimed in claim 33 wherein said antigenic molecule and/or photosensitizing agent and/or checkpoint inhibitor and optionally said TLR ligand, are used in a method as defined in any one of claims 14 to 21 to obtain said population of cells for manufacture of said medicament.

35. A product comprising an antigenic molecule as defined in any one of claim 1, 6 or 8, a photosensitizing agent as defined in claim 1 or 7, a checkpoint inhibitor as defined in any one of claims 1 to 3, and optionally a TLR ligand as defined in claim 4 or 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 12 or 14 to 21.

36. 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 12 or 14 to 21, said kit comprising a first container containing a photosensitizing agent as defined in claim 1 or 7; a second container containing said antigenic molecule as defined in any one of claim 1, 6 or 8; and a third container containing a checkpoint inhibitor as defined in any one of claims 1 to 3; and optionally a fourth container containing a TLR ligand as defined in claim 4 or 5.

37. 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 14 to 21, and subsequently administering said cells to said subject.

Description

[0221] The invention will now be described in more detail in the following non-limiting Examples with reference to the following drawings in which:

[0222] FIG. 1 shows the effect of PCI with the checkpoint inhibitors anti-CTLA4 and anti-PD-1 in the TC-1 mouse model for HPV-induced cancer. The results show average tumour volume as a % of volume at the vaccination time point.

[0223] FIG. 2 shows the median values (% antigen-specific, CD44+ cells of the total CD8+ cells) for TRP-2 pentamer staining after re-stimulation of spleen cells (isolated from mice after in vivo treatment as indicated) with the TRP-2 peptide.

[0224] FIG. 3 shows the results from interferon-gamma (IFN-gamma) intracellular staining after re-stimulation of spleen cells (isolated from mice after in vivo treatment as indicated) with the TRP-2 peptide.

[0225] FIG. 4 shows the results from TNF-alpha intracellular staining after re-stimulation of spleen cells (isolated from mice after in vivo treatment as indicated) with the TRP-2 peptide.

[0226] FIG. 5 shows the effect of PCI with the checkpoint inhibitors anti-CTLA4 and anti-PD-1 (used together) in the TC-1 mouse model for HPV-induced cancer. The results show median tumour volume after tumour inoculation.

[0227] FIG. 6 shows the effect of PCI with the checkpoint inhibitors anti-CTLA4 and anti-PD-1 (used together) in the TC-1 mouse model for HPV-induced cancer. The results show animal survival after tumour inoculation.

[0228] FIG. 7 shows the effect of PCI with the checkpoint inhibitors anti-CTLA4 and anti-PD-1 (used together) when used in conjunction with poly(IC) in the TC-1 mouse model for HPV-induced cancer. The results show median tumour volume after tumour inoculation.

[0229] FIG. 8 shows the effect of PCI with the checkpoint inhibitors anti-CTLA4 and anti-PD-1 (used together) when used in conjunction with poly(IC) in the TC-1 mouse model for HPV-induced cancer. The results show animal survival after tumour inoculation.

[0230] FIG. 9 shows the effect of PCI with the checkpoint inhibitor anti-PD-1 in the TC-1 mouse model for HPV-induced cancer. The results show median tumour volume after tumour inoculation.

[0231] FIG. 10 shows the effect of PCI with the checkpoint inhibitor anti-PD-1 in the TC-1 mouse model for HPV-induced cancer. The results show animal survival after tumour inoculation.

EXAMPLES

Example 1

[0232] The study was performed to investigate the effect of PCI in combination with the checkpoint inhibitors anti-CTLA4 and anti-PD-1 in the TC-1 mouse model for HPV-induced cancer.

Materials and Methods

Mice

[0233] C57BL/6 mice were purchased from Harlan (Horst, The Netherlands). All mice were kept under specified pathogen-free (SPF) conditions, and the procedures performed were approved by Swiss Veterinary authorities.

Tumour Inoculation

[0234] Mice were inoculated subcutaneously on their right flank with 200,000 TC-1 tumour cells (licensed from The Johns Hopkins University, 3400 N. Charles St., Baltimore, Md. 21218-2695) on day 0.

Immunisation Protocol

[0235] The further treatment schedule is outlined in Table 3. The checkpoint inhibitors anti-CTLA4 and anti-PD-1 were administered by intraperitoneal injection at the time points shown in Table 3. The doses of the checkpoint inhibitors were 100 g per injection for anti-CTLA4, and 200 g for anti-PD-1. Both checkpoint inhibitors were obtained from Bio X Cell, 10 Technology Drive, Suite 2B, West Lebanon, NH03784-1671, USA (mAb anti m CTLA-4, catalog BE0131 and mAb anti m PD-1, catalog BE0146).

TABLE-US-00005 TABLE 3 Checkpoint inhibitor Day administration (i.p.) 0 Tumour inoculation 5 1.sup.st immunisation x 6 1.sup.st illumination 10 x 13 x 17 x 20 2.sup.nd immunisation x 21 2.sup.nd illumination 24 x 27 x 31

[0236] Each immunisation was performed by intradermal administration of a mixture of different combinations of 50 g HPV long peptide antigen GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR (United Peptides (Herndon, Va.), 25 g TPCS.sub.2a (Amphinex, PCI Biotech AS) (for the animals receiving PCI treatment) and 5 g high molecular weight Polyinosinic-polycytidylic acid (Poly(IC)) (InvivoGen (San Diego, USA)). The combinations in the different experimental groups are shown in Table 4, below.

TABLE-US-00006 TABLE 4 Group no. Treatment No. of animals 1 Untreated control 5 2 anti-PD-1 5 3 HPV peptide + poly(IC) 5 4 HPV peptide + PCI 5 5 HPV peptide + poly(IC) + PCI 5 6 HPV peptide + anti-PD-1 5 7 HPV peptide + poly(IC) + anti-PD-1 5 8 HPV peptide + PCI + anti-PD-1 5 9 HPV peptide + poly(IC) + PCI + anti-PD-1 5 10 anti-CTLA4 5 11 HPV peptide + poly(IC) + PCI + anti-CTLA4 5 12 HPV peptide + anti-CTLA4 2

[0237] 18 hours after each immunisation illumination was performed for 6 minutes, using the LumiSource illumination device (PCI Biotech AS).

[0238] Tumour sizes were measured two or three times per week by measuring two perpendicular diameters with a digital caliper. Tumour volumes were calculated using the following formula:

V=(W.sup.2L)/2

where W is the width and L the length diameters of the tumours measured.

[0239] The results are shown in FIG. 1, which shows a reduction in tumour volume in groups in which a checkpoint inhibitor was administered with Poly(IC), HPV and PCI.

Example 2

Materials and Methods

[0240] C57BL/6 mice, TPCS.sub.2a and Poly(IC) were as described in Example 1. The TRP-2 peptide (sequence SVYDFFVWL) was obtained from United Peptides (Herndon, Va.).

Intradermal Photosensitisation and Immunisation of Normal Mice.

[0241] 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 high molecular weight 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. In some experimental groups (see below) anti-CTLA4 (3 mg/kg, intraperitoneal administration) was administered just before each immunisation.

Illumination of Immunised Mice

[0242] 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

[0243] 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 peptides and subsequently analysed with intracellular staining for interferon-gamma (IFN-gamma) and tumour necrosis factor alpha (TNF-alpha). Intracellular staining for IFN- was performed after overnight stimulation of splenocytes in 24-well plates with the TRP-2 peptides 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.). Intracellular staining for tumour necrosis factor alpha (TNF-alpha) was performed as described for IFN-gamma using anti-TNF-alpha antibodies.

[0244] The following experimental groups were included:

1. Untreated Mice were not immunised or illuminated.
2. TRP-2: Mice were immunised with TRP-2 peptide in all immunisations. They were not illuminated.
3. TRP-2+poly(IC): Mice were immunised with TRP-2 peptide and 10 g poly(IC). They were not illuminated.
4. TRP-2+PCI: Mice were immunised with TRP-2 peptide and 100 g TPCS.sub.2a and illuminated.
5. TRP-2+poly(IC)+PCI: Mice were immunised with TRP-2 peptide, 10 g poly(IC) and 100 g TPCS.sub.2a and illuminated.
6. TRP-2+poly(IC)+PCI+anti-CTLA4: Mice were immunised with TRP-2 peptide, 10 g poly(IC), 100 g TPCS.sub.2a. Anti-CTLA4 (3 mg/kg, intraperitoneal administration) was administered just before each immunisation. The animals were illuminated.
7. TRP-2+poly(IC)+anti-CTLA4: Mice were immunised with TRP-2 peptide and 10 g poly(IC). Anti-CTLA4 (3 mg/kg, intraperitoneal administration) was administered just before each immunisation. The animals were not illuminated.

[0245] FIG. 2 shows the median values (% antigen-specific, CD44+ cells of the total CD8+ cells) for TRP-2 pentamer 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 significant, but small increase in antigen-specific cells were observed over what was seen with antigen alone (group 2). The addition of anti-CTLA4 to the TRP-2 peptide+poly(IC) combination (group 7) did not seem to increase the response over what was seen with TRP-2 peptide+poly(IC). Combining TRP-2 peptide+poly(IC) with PCI clearly enhanced the immunological response (group 5), and adding anti-CTLA4 to this combination increased the response more than two times further, showing a synergistic effect of PCI and anti-CTLA4 on the proliferation of antigen specific CD8+, CD44+ T-cells in this experimental system.

[0246] FIG. 3 shows the results from interferon-gamma (IFN-gamma) intracellular staining after re-stimulation of spleen cells with the TRP-2 peptide. For this marker the combination of anti-CTLA4 with PCI and poly(IC) did not seem increase the marker expression over what was seen with the PCI+poly(IC) combination.

[0247] FIG. 4 shows the results from TNF-alpha intracellular staining after re-stimulation of spleen cells with the TRP-2 peptide. In accordance with the results shown in FIG. 2 it can be seen that the addition of anti-CTLA4 to the TRP-2 peptide+poly(IC) combination (group 7) did not seem to increase the response over what was seen with TRP-2 peptide+poly(IC) (group 3). However adding anti-CTLA4 to the TRP-2 peptide+poly(IC)+PCI combination significantly increased the TNF-alpha expression response, showing a synergistic effect of PCI and anti-CTLA4 on antigen induced TNF-alpha expression in CD8+, CD44+ T-cells in this experimental system.

Example 3

[0248] The study was performed to investigate the effect of PCI vaccination in combination with the checkpoint inhibitors anti-CTLA4 and anti-PD-1 (used together) in the TC-1 mouse model for HPV-induced cancer.

Materials and Methods

[0249] Mice were as described in Example 1. Mice were inoculated subcutaneously on their right flank with 100,000 TC-1 tumour cells (licensed from The Johns Hopkins University, 3400 N. Charles St., Baltimore, Md. 21218-2695) on day 0.

[0250] Checkpoint inhibitors anti-CTLA4 and anti-PD-1, photosensitizing agent TPCS.sub.2a, and HPV long peptide antigen were as described in Example 1.

Immunisation Protocol

[0251] The treatment schedule is outlined in Table 5.

TABLE-US-00007 TABLE 5 Treatment schedule Checkpoint inhibitor Day administration (i.p.) 0 Tumour inoculation 4 x 7 1.sup.st immunisation x 8 1.sup.st illumination 11 x 14 2.sup.nd immunisation x 15 2.sup.nd illumination 18 x 21 3.sup.rd immunisation x 22 3.sup.rd illumination 25 x

[0252] The PCI treated animals were illuminated 18 hours after each immunization. Illumination was performed for 6 min, using the LumiSource illumination device (PCI Biotech AS). The checkpoint inhibitors anti-CTLA4 and anti-PD-1 were administered together by intraperitoneal injection at the time points shown in Table 5. The doses of the checkpoint inhibitors were 100 g per injection for anti-CTLA4, and 200 g for anti-PD-1. Tumour sizes were measured two or three times per week by measuring two perpendicular diameters with a digital caliper. Tumour volumes were calculated as described in Example 1.

[0253] The combinations used in the different experimental groups are shown in Table 6.

TABLE-US-00008 TABLE 6 Experimental Groups Group No. of no. Treatment animals 1 Untreated 5 2 anti-PD-1/antiCTLA4 (i.p.) 5 3 HPV peptide (i.d.) + anti-PD-1/antiCTLA4 (i.p.) 5 4 HPV peptide + PCI (i.d.) anti-PD-1/antiCTLA4 (i.p.) 5

[0254] The results are shown in FIG. 5 from which it can be seen that administering the combination of the checkpoint inhibitors anti-CTLA4 and anti-PD-1 had little effect on tumour growth, even if these inhibitors were combined with the HPV long peptide antigen, expressed by the tumour. However, if PCI was added to the treatment regimen with the checkpoint inhibitors a significant inhibition of tumour growth was observed. Thus, a clear tumour shrinkage was induced, with an onset about one week after the initial PCI treatment, indicating a PCI-induced immunologically mediated anti-tumour effect. The effect of PCI also translated into an improved survival of the animals, as can be seen from FIG. 6.

Example 4

[0255] The study was performed to investigate the effect of PCI vaccination in combination with the checkpoint inhibitors anti-CTLA4 and anti-PD-1 (used together) and TLR ligand poly(IC) in the TC-1 mouse model for HPV-induced cancer.

Materials and Methods

[0256] Mice were inoculated with TC-1 tumour cells as described in Example 3. Checkpoint inhibitors anti-CTLA4 and anti-PD-1, photosensitizing agent TPCS.sub.2a, HPV long peptide antigen and Poly(IC) were as described in Example 1.

Immunisation Protocol

[0257] The treatment schedule is outlined in Table 7.

TABLE-US-00009 TABLE 7 Treatment schedule Checkpoint inhibitor Day administration (i.p.) 0 Tumour inoculation 4 x 7 1.sup.st immunisation x 8 1.sup.st illumination 11 x 14 2.sup.nd immunisation x 15 2.sup.nd illumination 18 x 21 3.sup.rd immunisation x 22 3.sup.rd illumination 25 x

[0258] Each immunisation was performed by intradermal administration of a mixture consisting of different combinations of 50 g HPV long peptide antigen, 25 g TPCS.sub.2a (for the animals receiving PCI treatment) and 5 g poly(IC). The PCI treated animals were illuminated 18 hours after each immunization. Illumination was performed for 6 min, using the LumiSource illumination device (PCI Biotech AS). The checkpoint inhibitors anti-CTLA4 and anti-PD-1 were administered together by intraperitoneal injection at the time points shown in Table 7. The doses of the checkpoint inhibitors were 100 g per injection for anti-CTLA4, and 200 g for anti-PD-1. Tumour sizes were measured as described in Example 1.

[0259] The combinations used in the different experimental groups are shown in Table 8.

TABLE-US-00010 TABLE 8 Experimental Groups Group No. of no. Treatment animals 1 Untreated 5 2 anti-PD-1/antiCTLA4 (i.p.) 5 3 HPV peptide + poly(IC) (i.d.) + anti-PD-1/antiCTLA4 5 (i.p.) 4 HPV peptide + poly(IC) + PCI (i.d.) + anti-PD-1/ 5 antiCTLA4 (i.p.) 5 HPV peptide + poly(IC) + PCI (i.d.) 5

[0260] The results are shown in FIG. 7 from which it can be seen that administering the TLR3 ligand poly(IC) in addition to the combination of the check point inhibitors anti-CTLA4 and anti-PD-1 had a significant inhibitory effect on the tumour growth. When PCI was added to this treatment a strongly increased anti-tumour effect was observed, with the median tumour volume shrinking to a size below the size at the start of the experiment, and the shrinkage lasting for at least two weeks. The effect of PCI also translated into a strongly improved survival of the animals as can be seen from FIG. 8.

Example 5

[0261] The study was performed to investigate the effect of PCI vaccination in combination with the checkpoint inhibitor anti-PD-1 in the TC-1 mouse model for HPV-induced cancer.

Materials and Methods

[0262] Mice were inoculated with TC-1 tumour cells as described in Example 3. Checkpoint inhibitor anti-PD-1, photosensitizing agent TPCS.sub.2a and HPV long peptide antigen were as described in Example 1.

Immunisation Protocol

[0263] The treatment schedule is outlined in Table 9.

TABLE-US-00011 TABLE 9 Treatment Schedule Anti-PD-1 administration Day (i.p.) 0 Tumour inoculation 4 x 7 1.sup.st immunisation x 8 1.sup.st illumination 11 x 14 2.sup.nd immunisation x 15 2.sup.nd illumination 18 x 21 3.sup.rd immunisation x 22 3.sup.rd illumination 25 x 28 4.sup.th immunisation x 29 4.sup.th illumination 32 x 36 x

[0264] Each immunisation was performed by intradermal administration of a mixture consisting of different combinations of 50 g HPV long peptide antigen and 25 g TPCS.sub.2a (for the animals receiving PCI treatment). The PCI treated animals were illuminated 18 hours after each immunization. Illumination was performed for 6 min, using the LumiSource illumination device (PCI Biotech AS). 200 g of the checkpoint inhibitor anti-PD-1 was administered by intraperitoneal injection at the time points shown in Table 9. Tumour sizes were measured as described in Example 1.

[0265] The combinations used in the different experimental groups are shown in Table 10.

TABLE-US-00012 TABLE 10 Experimental Groups Group no. Treatment No. of animals 1 Untreated 5 2 HPV (i.d.) + anti-PD-1 (i.p.) 8 3 HPV (i.d.) + anti-PD-1 (i.p.) + PCI 8

[0266] The results are shown in FIG. 9 from which it can be seen that administering the checkpoint inhibitor anti-PD-1 together with the HPV peptide antigen had a small inhibitory effect on tumour growth. However, if PCI was added to this treatment regimen a significant increase in the inhibition of tumour growth was achieved. Thus, a clear tumour shrinkage was observed, something that was not seen with the checkpoint inhibitor (+antigen) alone. The effect of PCI also translated into an improved survival of the animals, as can be seen from FIG. 10.