PCI method for generating immune respose to antigenic molecule using checkpoint inhibitor and TLR3 ligand

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 a subject an antigenic molecule, a photosensitizing agent, a checkpoint inhibitor and a TLR3 ligand, and irradiating said subject with light of a wavelength effective to activate said photosensitizing agent, wherein an immune response is thereby generated, wherein the antigenic molecule is a peptide, the checkpoint inhibitor is an anti-CTLA4 antibody, an anti-PD-1 antibody, or both an anti-CTLA4 antibody and an anti-PD-1 antibody, and the TLR3 ligand is a double stranded RNA molecule.

2. The method as claimed in claim 1 wherein the checkpoint inhibitor is a monoclonal antibody.

3. The method as claimed in claim 1 wherein the checkpoint inhibitor is (i) an anti-CTLA4 antibody and an anti-PD-1 antibody or (ii) an anti-CTLA4 antibody.

4. The method as claimed in claim 1 wherein said double stranded RNA molecule is Poly(I:C).

5. The method as claimed in claim 1 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.

6. The method as claimed in claim 1 wherein the photosensitising agent is selected from TPCS.sub.2a, AlPcS.sub.2a, TPPS.sub.4 and TPBS.sub.2a, preferably TPCS.sub.2a.

7. The method as claimed in claim 1 wherein the antigenic molecule is a melanoma peptide or Human Papillomavirus (HPV) peptide.

8. The method as claimed in claim 1 wherein said method is a method of vaccination.

9. The method as claimed in claim 1 wherein a disease, disorder or infection is treated or prevented when said immune response is generated, and wherein said disease, disorder or infection is preferably cancer, preferably melanoma or a cancer associated with a papillomavirus.

10. The method as claimed in claim 1 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.

11. The method as claimed in claim 1 wherein said antigenic molecule, photosensitising agent, checkpoint inhibitor and TLR3 ligand are administered to said subject simultaneously, separately or sequentially.

Description

(1) The invention will now be described in more detail in the following non-limiting Examples with reference to the following drawings in which:

(2) 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.

(3) 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.

(4) 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.

(5) 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.

(6) 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.

(7) 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.

(8) 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.

(9) 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.

(10) 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.

(11) 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.

(12) FIG. 11 shows the structure of a lipopolysaccharide.

EXAMPLES

Example 1

(13) 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.

(14) Materials and Methods

(15) Mice

(16) 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.

(17) Tumour Inoculation

(18) 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.

(19) Immunisation Protocol

(20) 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 μg per injection for anti-CTLA4, and 200 μg 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 13E0131 and mAb anti m PD-1, catalog BE0146).

(21) 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

(22) Each immunisation was performed by intradermal administration of a mixture of different combinations of 50 μg HPV long peptide antigen GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR (United Peptides (Herndon, Va.), 25 μg TPCS.sub.2a (Amphinex, PCI Biotech AS) (for the animals receiving PCI treatment) and 5 μg 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.

(23) 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

(24) 18 hours after each immunisation illumination was performed for 6 minutes, using the LumiSource illumination device (PCI Biotech AS).

(25) 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.2×L)/2
where W is the width and L the length diameters of the tumours measured.

(26) 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

(27) 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.).

(28) Intradermal Photosensitisation and Immunisation of Normal Mice.

(29) 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 μg of TRP-2 peptide, 100 μg TPCS.sub.2a and 10 μg high molecular weight poly(IC) as specified below by intradermal injection using 0.3 ml BD Micro-Fine™+insulin syringes with 30G needles (BD, N.J., USA). 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. 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.

(30) Illumination of Immunised Mice

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

(32) Analysis of Immune Responses by Pentamer Staining and Intracellular Staining

(33) 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-γ 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-γ, 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.

(34) The following experimental groups were included:

(35) 1. Untreated Mice were not immunised or illuminated.

(36) 2. TRP-2: Mice were immunised with TRP-2 peptide in all immunisations. They were not illuminated.

(37) 3. TRP-2+poly(IC): Mice were immunised with TRP-2 peptide and 10 μg poly(IC). They were not illuminated.

(38) 4. TRP-2+PCI: Mice were immunised with TRP-2 peptide and 100 μg TPCS.sub.2a and illuminated.

(39) 5. TRP-2+poly(IC)+PCI: Mice were immunised with TRP-2 peptide, 10 μg poly(IC) and 100 μg TPCS.sub.2a and illuminated.

(40) 6. TRP-2+poly(IC)+PCI+anti-CTLA4: Mice were immunised with TRP-2 peptide, 10 μg poly(IC), 100 μg TPCS.sub.2a. Anti-CTLA4 (3 mg/kg, intraperitoneal administration) was administered just before each immunisation. The animals were illuminated.

(41) 7. TRP-2+poly(IC)+anti-CTLA4: Mice were immunised with TRP-2 peptide and 10 μg poly(IC). Anti-CTLA4 (3 mg/kg, intraperitoneal administration) was administered just before each immunisation. The animals were not illuminated.

(42) 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.

(43) 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.

(44) 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

(45) 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.

(46) Materials and Methods

(47) 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.

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

(49) Immunisation Protocol

(50) The treatment schedule is outlined in Table 5.

(51) 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

(52) 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 μg per injection for anti-CTLA4, and 200 μg 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.

(53) The combinations used in the different experimental groups are shown in Table 6.

(54) 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

(55) 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

(56) 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.

(57) Materials and Methods

(58) 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.

(59) Immunisation Protocol

(60) The treatment schedule is outlined in Table 7.

(61) 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

(62) Each immunisation was performed by intradermal administration of a mixture consisting of different combinations of 50 μg HPV long peptide antigen, 25 μg TPCS.sub.2a (for the animals receiving PCI treatment) and 5 μg 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 μg per injection for anti-CTLA4, and 200 μg for anti-PD-1. Tumour sizes were measured as described in Example 1.

(63) The combinations used in the different experimental groups are shown in Table 8.

(64) 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

(65) 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

(66) 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.

(67) Materials and Methods

(68) 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.

(69) Immunisation Protocol

(70) The treatment schedule is outlined in Table 9.

(71) 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

(72) Each immunisation was performed by intradermal administration of a mixture consisting of different combinations of 50 μg HPV long peptide antigen and 25 μg 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 μg 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.

(73) The combinations used in the different experimental groups are shown in Table 10.

(74) 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

(75) 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.