Method of stimulating an immune response

Abstract

The present invention provides a method of stimulating an immune response to an antigenic molecule, by contacting a cell with the antigenic molecule and with a photosensitizing agent, irradiating the cell, and thereby presenting the antigenic molecule, or part thereof, on the surface of said cell and stimulating an immune response.

Claims

1. A method of stimulating a CD8+ cytotoxic T cell immune response to an antigenic molecule in vivo, said method comprising: contacting a cell with said antigenic molecule and with a photosensitizing agent ex vivo, wherein said molecule and said agent are each taken up into an intracellular membrane-restricted compartment of said cell; irradiating said cell ex vivo with light of a wavelength effective to activate the photosensitizing agent, such that the membrane of said intracellular compartment is disrupted, releasing said molecule into the cytosol of the cell, without killing the cell; wherein said released antigenic molecule, or a part thereof of sufficient size to stimulate an immune response, is subsequently presented on the surface of said cell; administering the cell to a mammal after irradiating said cell to thereby stimulate the in vivo immune response to the antigenic molecule; wherein the cell is a cancer cell or an antigen presenting cell selected from the group comprising lymphocytes, dendritic cells and macrophages; and wherein the antigenic molecule stimulates cytotoxic T cells, the photosensitizing agent is selected from the group of a porphyrin, a phthalocyanine and a chlorin, and when said cell is a cancer cell the antigenic molecule stimulates activated cytotoxic T cells.

2. The method of claim 1, wherein the antigenic molecule is a polypeptide.

3. The method of claim 1 wherein the photosensitizing agent is meso tetraphenylporphine with 4 sulfonate groups (TPPS.sub.4), meso-tetraphenylporphine with 2 sulfonate groups on adjacent phenyl rings (TPPS.sub.2a), or aluminum phthalocyanine with 2 sulfonate groups on adjacent phenyl rings (AlPcS.sub.2a).

4. The method of claim 1, wherein the antigenic molecule and/or photosensitizing agent is bound to one or more targeting agents or carrier molecules.

5. The method of claim 1, wherein at least 90% of the cells are not killed.

6. The method of claim 1, wherein at least 95% of the cells are not killed.

7. The method of claim 1, wherein the photosensitizing agent is a sulfonated tetraphenylporphine, a disulfonated aluminum phthalocyanine or a tetrasulfonated aluminum phthalocyanine.

8. A method of stimulating a CD8+ cytotoxic T cell immune response to an antigenic molecule, said method comprising: contacting a cell with an antigenic molecule and with a photosensitizing agent, wherein said antigenic molecule and said agent are each taken up into an intracellular membrane-restricted compartment of said cell; irradiating said cell with light of a wavelength effective to activate the photosensitizing agent, such that the membrane of said intracellular compartment is disrupted, releasing said antigenic molecule into the cytosol of the cell, without killing the cell; wherein said released antigenic molecule, or a part thereof of sufficient size to stimulate an immune response, is subsequently presented on the surface of said cell; wherein presentation of the antigenic molecule, or part thereof, on the surface of said cell results in stimulation of the immune response specific for said antigenic molecule or a part thereof; wherein the antigenic molecule stimulates cytotoxic T cells, and the photosensitizing agent is selected from the group of a porphyrin, phthalocyanine and a chlorin; wherein the cell is a cancer cell or an antigen presenting cell selected from lymphocytes, dendritic cells, or macrophages; and wherein when said method is performed in vitro the cell is contacted with cytotoxic T cells.

9. The method of claim 8, wherein the antigenic molecule is a polypeptide.

10. The method of claim 8, wherein the photosensitizing agent is meso-tetraphenylporphine with 4 sulfonate groups (TPPS.sub.4), meso-tetraphenylporphine with 2 sulfonate groups on adjacent phenyl rings (TPPS.sub.2a), or aluminum phthalocyanine with 2 sulfonate groups on adjacent phenyl rings (AlPcS.sub.2a).

11. The method of claim 8, wherein the antigenic molecule and/or photosensitizing agent is bound to one or more targeting agents or carrier molecules.

12. The method of claim 8, wherein at least 90% of the cells are not killed.

13. The method of claim 8, wherein at least 95% of the cells are not killed.

14. The method of claim 8, wherein the photosensitizing agent is a sulfonated tetraphenylporphine, a disulfonated aluminum phthalocyanine or a tetrasulfonated aluminum phthalocyanine.

15. The method of claim 8 wherein said method is carried out in vitro or in vivo.

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 a schematic representation of how PCI can be utilised to stimulate CTLs. A peptide or protein (P) is applied extracellularly to antigen presenting cells. P is endocytosed and released, into cytosol by PCI. The peptide or protein will thereafter be partly degraded by proteasomes and transported to the cells surface complexed to MHC (HLA) class I where, the complex can be recognised by CTLs.

(3) FIG. 2 shows photochemically induced relocalization of a peptide. BL2-G-E6 cells were incubated with a fluorescein-labelled p21.sup.ras-derived 5-21, Val.sup.12 peptide and AlPcS.sub.2a. The cells were examined for fluorescein-peptide and AlPcS.sub.2a localisation by fluorescence microscopy before (top panels) and 30 minutes after (bottom panels) a 4-min exposure to red light. Bar 20 μm.

(4) FIG. 3 shows the cytotoxicity of a CD8.sup.+ T lymphocyte clone against FM3 melanoma cells after PCI of a MART-1 peptide.

(5) FIG. 4 shows the ability of PCI to deliver HRP into the cytosol. NHIK 3025 cells were treated with 3.2 μg/ml TPPS.sub.2A and 1 mg/ml HRP for 18 hours. The medium was then replaced with drug-free medium before exposure to the indicated light doses. HRP activity was measured in intact cells (•) and in cytosol (o) separated from cytosol-free cell corpses (.Math.) by electropermeabilisation and a density centrifugation technique.

(6) FIG. 5A-5B show photochemically induced expression of GFP. FIG. 5A: expression of GFP in THX cells treated with pEGFP-N1-pLys complex in the absence of AlPcS.sub.2a and light or in the presence of AlPcS.sub.2a followed by exposure to light as indicated on the figure. The cells were analysed by flow cytometry, reckoning the cells on the right side of the drawn line as positive for GFP expression. FIG. 5B: expression of GFP in THX cells treated for 18 hours with a photosensitiser (20 μg/ml AlPcS.sub.2a or 0.25 μg/ml 3-THPP) followed by a 6 hour transfection with pEGF-N1-pLys complex and exposure to light inactivating 50% of the cells. GFP expression was analysed by flow cytometry as described in FIG. 5A.

EXAMPLES

Materials and Methods

(7) Irradiation

(8) Two different light sources were used for treatment of the cells, both consisting of a bank of 4 fluorescent tubes. Cells treated with TPPS.sub.4, TPPS.sub.2a, and 3-THPP (Porphyrin Products, Logan, Utah) were exposed to blue light (model 3026; Appl. Photophysics, London, UK) with a light intensity reaching the cells of 1.5 mW/cm.sup.2 while cells treated with AlPcS.sub.2a (Porphyrin Products, Logan, Utah) were exposed to red light (Philips TL 20W/09) filtered through a Cinemoid 35 filter with a light intensity reaching the cells of 1.35 mW/cm.sup.2.

(9) Fluorescence Microscopy

(10) The cells were analysed by fluorescence microscopy as described in Berg. K., et al., Biochem. Biophys. Acta., 1370: 317-324, 1998. For analysis of fluorescein-labelled molecules the microscope was equipped with a 450-490 nm excitation filter, a 510 nm dichroic beam splitter and a 510-540 nm band pass emission filter.

(11) Preparation of Plasmid-pLys Complexes and Treatment of Cells

(12) Plasmid-pLys complexes (charge ratio, 1.7) were prepared by gently mixing 5 μg plasmid (pEGFP-N1; Clontech Laboratories, Inc., Palo Alto, Calif.) in 75 μl of HBS with 5.3 μg pLys (mW 20700; Sigma, St. Louis, Mo.) in 75 μl of HBS. The solutions were incubated for 30 min at room temperature, diluted with culture medium and added to the cells. THX cells were incubated with 20 μg/ml AlPcS.sub.2a for 18 hours at 37° C., washed and incubated in sensitizer-free medium for 3 hours before incubation with plasmid-pLys complexes for 2 hours. The pEGFP-N1/pLys treated THX cells were washed once and incubated for 2 hours in culture medium without additions before exposure to light. The cells were incubated at 37° C. for 2 days, subcultured and further incubated for an additional 5 days before analysis of GFP expression by flow cytometry.

(13) HCT-116 cells were incubated with 20 μg/ml AlPcS.sub.2a for 18 hours, washed and transfected with plasmid-pLys complexes for 6 hours before light exposure in plasmid-free medium. After 40 hours incubation at 37° C. the GFP expression was studied by microscopy.

(14) Flow Cytometry Analysis

(15) The cells were trypsinized, centrifuged, resuspended in 400 μl of culture medium and filtered through a 50 μm mesh nylon filter. The cells were then analysed in a FACStar plus flow cytometer (Becton Dickinson). Green Fluorescent Protein (GFP) was measured through a 510-530 nm filter after excitation with an argon laser (200 mW) tuned on 488 nm. AlPcS.sub.2a was measured through a 650 nm longpass filter after excitation with a krypton laser (50 mW) tuned on 351-356 nm. Cell doublets were discriminated from single cells by gating on the pulse width of the GFP fluorescence signal. The data were analysed with PC Lysys II software (Becton Dickinson).

(16) Preparation of Fluorescein-Peptide and Treatment of Cells

(17) The fluorescein-labelled Val.sup.12-p21.sup.ras-peptide (residues 5-21) were synthesised and provided by Alan Cuthbertson, Nycomed Amersham).

(18) BL2-G-E6 cells were incubated with 30 μg/ml of the fluorescein-labelled p21.sup.ras-derived peptide for 18 hours followed by 20 μg/ml AlPcS.sub.2a for 18 hours and 1 hour in drug-free medium before exposure to red light.

Example 1

(19) Photochemical Internalisation (PCI) can be Used to Enable Peptides to Enter the Cytosol of Cells

(20) To evaluate PCI for cytosolic delivery of cancer-specific peptides, a fluorescein-labelled p21.sup.ras peptide encompassing residues 5-21 and containing a Val.sup.12 mutation (G12V) was used (Gjertsen, M. K., et al., Int. J. Cancer, 72: 784-790, 1997). In BL2-6-E6 mouse fibroblasts, the ras peptide colocalised well with AlPcS.sub.2a, indicating endocytic uptake of the peptide (FIG. 2). After a 4-min exposure to light, the fluorescein-labelled ras peptide and AlPcS.sub.2a were found to be located diffusely in the cytoplasm. Similar effects were not Observed in cells exposed to the fluorescein-labelled ram peptide and light only (data not shown).

Example 2

(21) Use of PCI to Induce Antigen Presentation and CD8.sup.+ T Lymphocyte Mediated Cell Killing

(22) FM3 melanoma cells (2×10.sup.5/well in 6 well plates), grown in RPMI 1640 medium with 10% foetal calf serum (FCS), not expressing MART-1 peptide were treated with 10 μg/ml of the photosensitizing agent AlPcS.sub.2a for 18 hours. The cells were then released from the substratum with EDTA (0.1 M) in Dulbecco's phosphate-buffered saline (PBS) and kept in solution during loading of the cells with .sup.51Cr (60 μCi/ml Na.sub.2CrO.sub.4) for 1 hour in 100% FCS followed by 5 hours incubation with 5 μg/ml MART-1 peptide in RPMI 1646 in 10% FCS, while the cells were still kept in solution. The sequence of the MART-1 peptide was: TAEEAAGIGILTVILG. The cells were then washed twice in RPMI 1640 medium containing 10% FCS and seeded out in 96-well plates (2000/well in 100 μl medium (RPMI 1640/10% FCS). The cells were then exposed to light for the times as indicated in FIG. 3 ((Philips TL 20W/09) filtered through a Cinemoid 35 filter with a light intensity reaching the cells of 1.35 mW/cm.sup.2 (Rodal et al., 1998, J. Photochem. Photobiol. B: Biol. 45: 150-9)). 18 hours after light exposure the medium was removed and medium containing MART-1/HLA-A2 specific cytotoxic T lymphocytes (CTLs—40,000/well added in 100 μl) were added. After 4 hours of incubation the medium was separated from FM3 cells and the .sup.51Cr released to the medium (as an indicator of lysed cells) was counted as well as the spontaneous and maximum release as previously described (Possum et al., 1995, Cancer Immunol. Immunother. 40: 165-172). The percentage specific chromium release was calculated by the formula: (experimental release−spontaneous release)/(maximum release−spontaneous release)×100. It can be seen from the results shown in FIG. 3 that FM3 cells after PCI of a MART-1 peptide as outlined above show light dependent susceptibility to CD8.sup.+ T lymphocyte cytotoxicity.

Example 3

(23) PCI Induces the Release of a Large Fraction of the Endocytosed Molecule

(24) This was shown by PCI induced internalisation/endocytosis of Horseradish Peroxidase (HRP).

(25) By using HRP, it is demonstrated (see FIG. 4) that PCI induces the release of a large fraction (>60%) of endocytosed HRP into the cytosol.

(26) In this experiment NHIK 3025 cells (carcinoma cells in situ from human cervix) were treated with the photosensitizing agent TPPS.sub.2, (3.2 μg/ml) and 1 mg/ml HRP for 18 hours. The medium was then replaced with drug free medium before exposure to the light doses as indicated in FIG. 4, HRP activity was measured according to the procedure described in Steinman et al., J. Cell. Biol., 68: 665-687, 1976. Cytosol was separated from cytosol-free cell corpses by electropermeabilisation and a density centrifugation technique (Berg et al., Int. J. Cancer 59: 814-822, 1994).

Example 4

(27) PCI can be Used to Enhance the Delivery of Functional Genes

(28) To demonstrate this, THX cells were transfected with a pLys-complex of a plasmid (pEGFP-N1) coding for green fluorescent protein (GFP). The expression of GFP was analysed by flow cytometry (FIG. 5, a and b) and fluorescence microscopy (data not shown). As can be seen from FIG. 5a, treatment with, AlPcS.sub.2a and light led to a strong increase in the percentage of the cells expressing GFP. The fraction of the cells that was positive for this reporter molecule increased from 1% at no light treatment to 50% after a 5-min light exposure. GFP expression was not enhanced by light in cells treated with pEGFP-pLys in the absence of a photosensitizer. A complex of an irrelevant plasmid (encoding hems oxygenase) and pLys did not induce green fluorescence when combined with AlPcS.sub.2a and light (data not shown). Consequently, in a light-directed manner, PCI can substantially increase the efficiency of transfection of a functional gene to THX cells. Similar results were obtained using TPPS.sub.2a as a photosensitizer and BHK-21 and HCT-116 as target cells (data not shown). The essentially non-lysosomally located sensitizer 3-THPP induced only a minor increase in GFP expression (FIG. 5b). PCI of pEGFP-N1 not complexed with pLys did not induce the expression of GFP (data not shown).