WO2015028575A1

WO2015028575A1 - Immunisation method by photochemical internalisation

Info

Publication number: WO2015028575A1
Application number: PCT/EP2014/068314
Authority: WO
Inventors: Anders HØGSET; Pål JOHANSEN
Original assignee: Pci Biotech As
Priority date: 2013-08-28
Filing date: 2014-08-28
Publication date: 2015-03-05

Abstract

The present invention relates to a method of vaccination or immunisation involving the use of a photosensitizing agent, an antigenic molecule, e.g. a vaccine component, and irradiation with light of a wavelength effective to activate the photosensitizing agent. The invention also provides the photosensitizing agent and antigenic molecule for use in such a method. The invention lies in the identification of specific advantageous protocols for achieving enhanced PCI-mediated immunisation e.g. vaccination, in particular in relation to the time at which irradiation is performed after administration of the antigenic molecule and the photosensitizing agent.

Description

IMMUNISATION METHOD BY PHOTOCHEMICAL INTERNALISATION

Vaccination involves administration of antigenic molecules to provoke the immune system to stimulate development of an adaptive immunity to a pathogen. Vaccines can prevent or improve morbidity from infection. Vaccination is the most effective method of preventing infectious diseases, and widespread immunity due to vaccination is largely responsible for the worldwide eradication of smallpox and the restriction of diseases such as polio, measles, and tetanus from much of the world.

The active agent of a vaccine may be intact but inactivated (non-infective) or attenuated (with reduced infectivity) forms of the causative pathogens, or purified components of the pathogen that have been found to be immunogenic (e.g. outer coat proteins of a virus). Toxoids are produced for immunization against toxin- based diseases, such as the modification of tetanospasmin toxin of tetanus to remove its toxic effect but retain its immunogenic effect.

Since most vaccines are taken up by antigen presenting cells through endocytosis and transported via endosomes to lysosomes for antigen digestion and presentation via the MHC class-ll pathway, vaccination primarily activates CD4 T- helper cells and B cells. To combat disorders or diseases such as cancer, as well as intracellular infections, the stimulation of cytotoxic CD8 T-cell responses is important. However, the induction of cytotoxic CD8 T cells usually fails due to the difficulty in delivering antigen to the cytosol and to the MHC class-l pathway of antigen presentation. Photochemical internalisation (PCI) improves delivery of molecules into the cytosol and methods of vaccination which employ PCI are known. PCI is a technique which uses a photosensitizing agent, in combination with an irradiation step to activate that agent, and is known to achieve release of molecules co-administered to a cell into the cell's cytosol. This technique allows molecules that are taken up by the cell into organelles, such as endosomes, to be released from these organelles into the cytosol, following irradiation. PCI provides a mechanism for introducing otherwise membrane-impermeable (or poorly permeable) molecules into the cytosol of a cell in a manner which does not result in widespread cell destruction or cell death.

The basic method of photochemical internalisation (PCI), is described in WO 96/07432 and WO 00/54802, which are incorporated herein by reference. In such methods, the molecule to be internalised (which in the present invention would be the antigenic molecule), and a photosensitizing agent are brought into contact with a cell. The photosensitizing agent and the molecule to be internalised are taken up into a cellular membrane-bound subcompartment within the cell, i.e. they are endocytosed into an intracellular vesicle (e.g. a lysosome or endosome). On exposure of the cell to light of the appropriate wavelength, the photosensitizing agent is activated which directly or indirectly generates reactive species which disrupt the intracellular vesicle's membranes. This allows the internalized molecule to be released into the cytosol.

It was found that in such a method the functionality or the viability of the majority of the cells was not deleteriously affected. Thus, the utility of such a method, termed "photochemical internalisation" was proposed for transporting a variety of different molecules, including therapeutic agents, into the cytosol i.e. into the interior of a cell.

WO 00/54802 utilises such a general method to present or express transfer molecules on a cell surface. Thus, following transport and release of a molecule into the cell cytosol, it (or a part of that molecule) may be transported to the surface of the cell where it may be presented on the outside of the cell i.e. on the cell surface. Such a method has particular utility in the field of vaccination, where vaccine components i.e. antigens or immunogens, may be introduced to a cell for presentation on the surface of that cell, in order to induce, facilitate or augment an immune response.

Whilst vaccination has achieved some noteworthy successes, there remains a need for alternative and improved vaccination methods. The present invention addresses this need.

The present inventors have found that when certain parameters and conditions are used in a PCI-based method of immunisation/vaccination, this results in improved vaccination or an improved immune response. The present inventors have found that the timing of the irradiation with light is an important factor in achieving improved vaccination or an improved immune response. As will be described in more detail in the Examples below, it has been demonstrated that the method of the invention results in improved vaccination or an improved immune response, e.g. production of an increased amount of antigen-specific T cells. For example, Figures 2, 5 and 6 demonstrate that in vivo vaccination of mice using an antigen and a photosensitiser and irradiation with light of a wavelength effective to activate the photosensitiser led to a significantly increased percentage of antigen- specific T cells in the blood and spleen of said mice, when an incubation time of, for example, 18 hours with the antigen and photosensitiser was employed prior to irradiation.

Whilst not wishing to be bound by theory, it is believed that the methods of the invention result in increased antigen presentation on MHC Class I molecules leading to an increased CD8+ T cell responses and hence improved vaccination methods. As discussed below, the present Examples utilise a model system of OT-1 cells, which is used for assessing MHC class I presentation (see e.g.

Delamarre et al. J. Exp. Med. 198: 11 1-122, 2003). In this model system MHC class I presentation of the antigen epitope SIINFEKL leads to activation of the OT-1 T- cells, and the activation can be measured as an increase in proliferation of the antigen-specific T-cells or increased production of IFNy or IL-2. The results with the methods of the present invention show increased numbers of antigen-specific T cells, and increased IL-2 and IFNy production by the T cells, which is correlated with increased or improved antigen presentation.

Thus, in a first aspect the present invention provides an in vivo method of expressing an antigenic molecule or a part thereof on the surface of a cell in a subject, comprising contacting said cell with said antigenic molecule and a photosensitizing agent for 12 to 30 hours before, 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.

In the context of the present invention the "cell" is within a subject or organism.

In such methods said antigenic molecule and said photosensitizing agent, are each taken up into an intracellular vesicle; and when the cell is irradiated the membrane of the intracellular vesicle is disrupted releasing the antigenic molecule into the cytosol of the cell.

The agents may be taken up into the same or a different intracellular vesicle relative to each other. It has been found that active species produced by photosensitizers may extend beyond the vesicle in which they are contained and/or that vesicles may coalesce allowing the contents of a vesicle to be released by coalescing with a disrupted vesicle. As referred to herein "taken up" signifies that the molecule taken up is wholly contained within the vesicle. The intracellular vesicle is bounded by membranes and may be any such vesicle resulting after endocytosis, e.g. an endosome or lysosome.

As used herein, a "disrupted" compartment refers to destruction of the integrity of the membrane of that compartment either permanently or temporarily, sufficient to allow release of the antigenic molecule contained within it.

A "photosensitizing agent" as referred to herein is a compound that is capable of translating the energy of absorbed light into chemical reactions when the agent is activated on illumination at an appropriate wavelength and intensity to generate an activated species. The highly reactive end products of these processes can result in cyto- and vascular toxicity. Conveniently such a photosensitizing agent may be one which localises to intracellular compartments, particularly endosomes or lysosomes.

Photosensitisers may exert their effects by a variety of mechanisms, directly or indirectly. Thus for example, certain photosensitisers become directly toxic when activated by light, whereas others act to generate toxic species, e.g. oxidising agents such as singlet oxygen or other reactive oxygen species, which are extremely destructive to cellular material and biomolecules such as lipids, proteins and nucleic acids.

A range of such photosensitizing agents are known in the art and are described in the literature, including in WO96/07432, which is incorporated herein by reference, and may be used in methods of the invention. There are many known photosensitising agents, including porphyrins, phthalocyanines and chlorins, (Berg et a/., (1997), J. Photochemistry and Photobiology, 65, 403-409). Other photosensitising agents include bacteriochlorins.

Porphyrins are the most extensively studied photosensitising agents. Their molecular structure includes four pyrrole rings linked together via methine bridges. They are natural compounds which are often capable of forming metal-complexes. For example in the case of the oxygen transport protein hemoglobin, an iron atom is introduced into the porphyrin core of heme B.

Chlorins are large heterocyclic aromatic rings consisting, at the core, of three pyrroles and one pyrroline coupled through four methine linkages. Unlike porphyrin, a chlorin is therefore largely aromatic, but not aromatic through the entire circumference of the ring. Particularly preferred are photosensitizing agents which locate to endosomes or lysosomes of cells. Thus, the photosensitizing agent is preferably an agent which is taken up into the internal compartments of lysosomes or endosomes. Preferably the photosensitizing agent is taken up into intracellular compartments by endocytosis. Preferred photosensitizing agents are amphiphilic photosensitizers (e.g. disulphonated photosensitisers) such as amphiphilic phthalocyanines, porphyrins, chlorins, and/or bacteriochlorins, and in particular include sulfonated (preferably disulfonated) meso-tetraphenyl chlorins, porphyrins, phthalocyanines and bacteriochlorins. Particularly preferred are TPPS_2a

(tetraphenylporphine disulfonate), AIPcS_2a (aluminium phthalocyanine disulfonate), TPCS_2a (tetraphenyl chlorin disulfonate) and TPBS_2a (tetraphenyl bacteriochlorin disulfonate), or pharmaceutically acceptable salts thereof. Preferably the photosensitizing agent is TPCS_2a (Disulfonated tetraphenyl chlorin, e.g. Amphinex ®).

Optionally, the photosensitizing may be attached to or associated with or conjugated to one or more carrier molecules or targeting molecules which can act to facilitate or increase the uptake of the photosensitizing agent.

Thus the photosensitising agent may be linked to a carrier. For example, the photosensitising agent may be provided in the form of a conjugate, e.g. a chitosan- based conjugate, for example a conjugate disclosed in WO2013/189663, which is hereby incorporated by reference. For example, the photosensitising agent may be a conjugate of a photosensitiser and chitosan as defined in Formula (I):

wherein n is an integer greater than or equal to 3,

R appears n times in said compound and

in 0.5%-99.5% of said total Rn groups, each R is a group A selected from:

H,

wherein a is 1 , 2, 3, 4 or 5; and X is Br, CI or OH;

wherein each Ri, which may be the same or different, is selected from H, CH₃ and -(CH₂)_c-CH₃; b is 1 , 2, 3, 4 or 5; and c is 0, 1 , 2, 3, 4 or 5;

wherein Y is O; S; S0_2; -NCH_3; or -N(CH₂)_eCH₃; d=1 , 2, 3, 4 or 5; and e=1 , 2, 3, 4 or 5;

wherein R₂ is -(CH₂)_h-CH₃ or -CO-(CH₂)_h-CH₃; f is 1 , 2, 3, 4 or 5; g is 1 , 2, 3, 4 or 5; and h is 0, 1 , 2, 3, 4 or 5;

wherein R₃ is -(CH₂)_rCH₃, i is an integer from 1 to 200, preferably from 1- 10; j is 0, 1 , 2, 3, 4 or 5; and k is 1 , 2, 3, 4 or 5;

wherein R₃ is -(CH₂)_rCH₃, i is an integer from 1 to 200, preferably from 1- 10; and j is 0, 1 , 2, 3, 4 or 5;

wherein R₃ is -(CH₂)_j-CH₃, i is an integer from 1 to 200, preferably from 1- 10; j is 0, 1 , 2, 3, 4 or 5; and each R₁ , which may be the same or different, is selected from H, CH₃ and -(CH₂)_c-CH₃; and c is 0, 1 , 2, 3, 4 or 5;

wherein R₃= -(CH₂)_j-CH₃, i is an integer from 1 to 200, preferably from 1-10; and j is 0, 1 , 2, 3, 4 or 5;

wherein R₃= -(CH₂)_j-CH₃, i is an integer from 1 to 200, preferably from 1-10; L is 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10; and j is 0, 1 , 2, 3, 4 or 5;

wherein m is 1 , 2, 3, 4 or 5;

wherein each R group may be the same or different; and in 0.5%-99.5% of said total Rn groups, each R is a group B selected from:

wherein

p is 0, 1 , 2, 3, 4 or 5; q is 1 , 2, 3, 4 or 5; and r is 1 , 2, 3, 4 or 5; R₄ is a group selected from:

W is a group selected from O, S, NH or N(CH₃);

R₅ is a group selected from: -(CH₂)_s-CO-; -(CH₂)_s-Z-(CH₂)rCO- and -(CH₂)_S- Z-(CH₂)_rZ-CO-; wherein s is 0, 1 , 2, 3, 4 or 5; t is 0, 1 , 2, 3, 4 or 5;

Z is NH, O, S, or S0_2;

R₆ is a group selected from -CN and CH_3;

and

V is a group selected from CO, S0₂, PO, P0₂H or CH₂; and

R₈ is a group (substituted in the o, m or p position), which may be the same or different, selected from H, -OH, -OCH₃, -CH₃, -COCH₃, C(CH₃)₄, -NH₂, -NHCH₃, -N(CH₃)₂ and -NCOCH_3; wherein each R group may be the same or different. Preferred conjugates are as described in WO2013/189663.

groups are:

preferably each R₁ is CH₃ and b is 1 ; and

wherein preferably Y is -NCH₃ and d is 1. :

and q is 1 ; and

wherein preferably p is 1.

Preferably R₄ is selected from:

; and

TPCc₂

Particularly preferred conjugates are:

TPC_X TPC.

(wherein ό is ₇ and H is R₄ having the preferred form defined above, e.g. TPCai, TPCa₂, TPCci or TPCc₂).

An "antigenic" molecule as referred to herein is a molecule which itself, or a part thereof, is capable of stimulating an immune response, when presented to the immune system or immune cells in an appropriate manner. Advantageously, therefore the antigenic molecule will be a vaccine antigen or vaccine component, such as a polypeptide containing entity.

Many such antigens or antigenic vaccine components are known in the art and include all manner of bacterial or viral antigens or indeed antigens or antigenic components of any pathogenic species including protozoa or higher organisms. Whilst traditionally the antigenic components of vaccines have comprised whole organisms (whether live, dead or attenuated) i.e. whole cell vaccines, in addition sub-unit vaccines, i.e. vaccines based on particular antigenic components of organisms e.g. proteins or peptides, or even carbohydrates, have been widely investigated and reported in the literature. Any such "sub-unit"-based vaccine component may be used as the antigenic molecule of the present invention.

However, the invention finds particular utility in the field of peptide vaccines. Thus, a preferred antigenic molecule according to the invention is a peptide (which is defined herein to include peptides of both shorter and longer lengths i.e.

peptides, oligopeptides or polypeptides, and also protein molecules or fragments thereof e.g. peptides of 5-500 e.g. 10 to 250 such as 15 to 75, or 8 to 25 amino acids).

A vast number of peptide vaccine candidates have been proposed in the literature, for example in the treatment of viral diseases and infections such as AIDS/ HIV infection or influenza, canine parvovirus, bovine leukaemia virus, hepatitis, etc. (see e.g. Phanuphak et al., Asian Pac. J. Allergy. Immunol. 1997, 15(1 ), 41-8; Naruse, Hokkaido Igaku Zasshi 1994, 69(4), 81 1-20; Casal et al., J. Virol., 1995, 69(11 ), 7274-7; Belyakov et al., Proc. Natl. Acad. Sci. USA, 1998, 95(4), 1709-14; Naruse et al., Proc. Natl. Sci. USA, 1994 91 (20), 9588-92; Kabeya et al., Vaccine 1996, 14(12), 1 1 18-22; Itoh et al., Proc. Natl. Acad. Sci. USA, 1986, 83(23) 9174-8. Similarly bacterial peptides may be used, as indeed may peptide antigens derived from other organisms or species.

In addition to antigens derived from pathogenic organisms, peptides have also been proposed for use as vaccines against cancer or other diseases such as multiple sclerosis. For example, mutant oncogene peptides hold great promise as cancer vaccines acting as antigens in the stimulation of cytotoxic T-lymphocytes. (Schirrmacher, Journal of Cancer Research and Clinical Oncology 1995, 121 , 443- 451 ; Curtis Cancer Chemotherapy and Biological Response Modifiers, 1997, 17, 316-327). A synthetic peptide vaccine has also been evaluated for the treatment of metastatic melanoma (Rosenberg et al., Nat. Med. 1998, 4(3), 321-7). A T-cell receptor peptide vaccine for the treatment of multiple sclerosis is described in Wilson et al., J. Neuroimmunol. 1997, 76(1-2), 15-28. Any such peptide vaccine component may be used as the antigenic molecule of the invention, as indeed may any of the peptides described or proposed as peptide vaccines in the literature. The peptide may thus be synthetic or isolated or otherwise derived from an organism.

Once released in the cell cytosol by the photochemical internalisation process, the antigenic molecule may be processed by the antigen-processing machinery of the cell. Thus, the antigenic molecule expressed or presented on the surface of the cell may be a part or fragment of the antigenic molecule which is internalised (endocytosed). A "part" of an antigenic molecule which is presented or expressed preferably comprises a part which is generated by antigen-processing machinery within the cell. Parts may, however, be generated by other means which may be achieved through appropriate antigen design (e.g. pH sensitive bonds) or through other cell processing means. Conveniently such parts are of sufficient size to generate an immune response, e.g. in the case of peptides greater than 5, e.g. greater than 10 or 20 amino acids in size.

As used herein "expressing" or "presenting" refers to the presence of the antigenic molecule or a part thereof on the surface of said cell such that at least a portion of that molecule is exposed and accessible to the environment surrounding that cell, preferably such that an immune response may be generated to the presented molecule or part thereof. Expression on the "surface" may be achieved in which the molecule to be expressed is in contact with the cell membrane and/or components which may be present or caused to be present in that membrane.

According to the present invention, the term "cell" is used herein to describe cells that are within a subject or organism, e.g. an in vivo cell. The term "cell" includes all eukaryotic cells (including insect cells and fungal cells). Representative "cells" thus include all types of mammalian and non-mammalian animal cells, plant cells, insect cells, fungal cells and protozoa. Preferably, however, the cells are mammalian, for example cells from cats, dogs, horses, donkeys, sheep, pigs, goats, cows, mice, rats, rabbits, guinea pigs, but most preferably from humans. The cell which is subjected to the methods, uses etc. of the invention may be any cell which is capable of expressing, or presenting on its surface a molecule which is administered or transported into its cytosol.

The cell is conveniently an immune cell i.e. a cell involved in the immune response. However, other cells may also present antigen to the immune system and these also fall within the scope of the invention. The cells according to the present invention are thus advantageously antigen-presenting cells as described hereinafter. The antigen-presenting cell may be involved in any aspect or "arm" of the immune response as defined herein.

The stimulation of cytotoxic cells requires antigens to be presented to the cell to be stimulated in a particular manner by the antigen-presenting cells, for example MHC Class I presentation (e.g. activation of CD8⁺ cytotoxic T-cells requires MHC-1 antigen presentation). Antibody-producing cells may also be stimulated by presentation of antigen by the antigen-presenting cells.

Antigens may be taken up by antigen-presenting cells by endocytosis and degraded in the endocytic vesicles to peptides. These peptides may bind to MHC class II molecules in the endosomes and be transported to the cell surface where the peptide-MHC class II complex may be recognised by CD4+ T helper cells and induce an immune response. Alternatively, proteins in the cytosol may be degraded, e.g. by proteasomes and transported into endoplasmic reticulum by means of TAP (transporter associated with antigen presentation) where the peptides may bind to MHC class I molecules and be transported to the cell surface (Yewdell and Bennink, 1992, Adv. Immunol. 52: 1-123). If the peptide is of foreign antigen origin, the peptide-MHC class I complex will be recognised by CD8+ cytotoxic T-cells (CTLs). The CTLs will bind to the peptide-MHC (HLA) class I complex and thereby be activated, start to proliferate and form a clone of CTLs. The target cell and other target cells with the same peptide-MHC class I complex on the cells surface may be killed by the CTL clone. Immunity against the foreign antigen may be established if a sufficient amount of the antigen can be introduced into the cytosol (Yewdell and Bennink, 1992, supra; Rock, 1996, Immunology Today 17: 131-137). This is the basis for development of inter alia cancer vaccines. One of the largest practical problems is to introduce sufficient amounts of antigens (or parts of the antigen) into the cytosol. This may be solved according to the present invention.

As mentioned previously, once released in the cell cytosol by the photochemical internalisation process, the antigenic molecule may be processed by the antigen-processing machinery of the cell and presented on the cell surface in an appropriate manner e.g. by Class I MHC. This processing may involve degradation of the antigen, e.g. degradation of a protein or polypeptide antigen into peptides, which peptides are then complexed with molecules of the MHC for presentation. Thus, the antigenic molecule expressed or presented on the surface of the cell according to the present invention may be a part or fragment of the antigenic molecule which is internalised (endocytosed).

A variety of different cell types can present antigen on their surface, including for example, lymphocytes (both T and B cells), dendritic cells,

macrophages etc. Others include for example cancer cells e.g. melanoma cells. These cells are referred to herein as "antigen-presenting cells". "Professional antigen-presenting cells" which are cells of the immune system principally involved in the presentation of antigen to effector cells of the immune system are known in the art and described in the literature and include B lymphocytes, dendritic cells and macrophages. Preferably the cell is a professional antigen-presenting cell.

For antigen presentation by an antigen-presenting cell to a cytotoxic T-cell (CTL) the antigenic molecule needs to enter the cytosol of the antigen-presenting cell (Germain, Cell, 1994, 76, 287-299).

In embodiments of the invention, the cell is a dendritic cell. Dendritic cells are immune cells forming part of the mammalian immune system. Their main function is to process antigenic material and present it on the surface to other cells of the immune system. Once activated, they migrate to the lymph nodes where they interact with T cells and B cells to initiate the adaptive immune response.

Dendritic cells are derived from hematopoietic bone marrow progenitor cells. These progenitor cells initially transform into immature dendritic cells which are characterized by high endocytic activity and low T-cell activation potential. Once they have come into contact with a presentable antigen, they become activated into mature dendritic cells and begin to migrate to the lymph node. Immature dendritic cells phagocytose pathogens and degrade their proteins into small pieces and upon maturation present those fragments at their cell surface using MHC molecules. Dendritic cells arise from monocytes, i.e. white blood cells which circulate in the body and, depending on the right signal, can differentiate into either dendritic cells or macrophages. The monocytes in turn are formed from stem cells in the bone marrow. Monocyte-derived dendritic cells can be generated in vitro from peripheral blood mononuclear cells (PBMCs). Plating of PBMCs in a tissue culture flask permits adherence of monocytes. Treatment of these monocytes with interleukin 4 (IL-4) and granulocyte-macrophage colony stimulating factor (GM-CSF) leads to differentiation to immature dendritic cells (iDCs) in about a week. Subsequent treatment with tumor necrosis factor (TNF) further differentiates the iDCs into mature dendritic cells.

As used herein "contacting" refers to bringing the cells and the

photosensitizing agent and/or the antigenic molecule as defined herein into physical contact with one another under conditions appropriate for internalization into the cells, i.e. at a body temperature of 36-38°C.

The cell may be contacted with the photosensitizing agent and antigenic molecule as defined herein sequentially or simultaneously. Preferably, and conveniently the components are contacted with the cell simultaneously and preferably are applied to the cell together as described in more detail hereinafter. The agents may be taken up by the cell into the same or different intracellular compartments (e.g. they may be co-translocated).

The cells are then exposed to light of suitable wavelengths to activate the photosensitizing compound which in turn leads to the disruption of the intracellular compartment membranes.

"Internalisation" as used herein, refers to the intracellular, e.g. cytosolic, delivery of molecules. In the present case "internalisation" may include the step of release of molecules from intracellular/membrane bound compartments into the cytosol of the cells.

As used herein, "cellular uptake" or "translocation" refers to one of the steps of internalisation in which molecules external to the cell membrane are taken into the cell such that they are found interior to the outer lying cell membrane, e.g. by endocytosis or other appropriate uptake mechanisms, for example into or associated with intracellular membrane-restricted compartments, for example the endoplasmic reticulum, Golgi body, lysosomes, endosomes etc.

The step of contacting the cells with the various agents may be carried out in any convenient or desired way as described herein. In the in vivo methods of the present invention the agents can be administered to the cell or subject via methods as described herein, which results in cell contact.

The comments below discuss the application of the agents to the cells separately. As discussed above however, these agents may be applied to cells together, separately or simultaneously. The agents may be contacted or administered sequentially under some circumstances, as described below. For in vivo methods such as in the present invention, the application may be via direct (i.e. localized) or indirect (i.e. systemic or non-localized) administration as described in more detail hereinbelow.

The photosensitizing agent is brought into contact with the cells at an appropriate concentration for the length of time according to the invention, i.e. 12 to 30 hours, e.g. 16-20 hours, preferably 18 hours, and will depend on such factors as the particular photosensitizing agent used and the target cell type and location. The concentration of the photosensitizing agent is conveniently such that once taken up into the cell, e.g. into, or associated with, one or more of its intracellular

compartments and activated by irradiation, one or more cell structures are disrupted e.g. one or more intracellular compartments are lysed or disrupted. The

photosensitizing agents as described herein may be used in the range 0.05-20 mg/kg body weight when administered systemically. Alternatively, a range of 0.005-20mg/kg body weight may be used for systemic administration. However, the photosensitizing agent is generally administered locally, for example by intradermal, subcutaneous or intratumoural administration, and in that case the dose may be in the region of 1-5000 μg, for example 25-400, or 100-300μg. Preferably the dose is selected from 100μg, 150μg, 200μg and 250μg, or about 250μg. Preferably the dose is 75-125 μg, e.g. 100μg. The doses provided are for a human of average weight (i.e. 70kg). In a preferred embodiment the photosensitiser is administered locally by intradermal administration. For intradermal injection the photosensitiser dose may be dissolved in 100 μΙ-1 ml, i.e. the concentration may be in the range of 1-50000 μg/ml. In smaller animals the concentration range may be different and can be adjusted accordingly though when administered locally, little variation in dosing is necessary for different animals.

The concentration of antigen to be used will depend on the antigen which is to be used. For the in vivo use according to the present invention the protein antigen dose may be in the range 0.5 or 1-500 μg, for example 10-100 μg. For peptide antigens an in vivo dose of 0.1-4000μg, e.g. 0.1-2000μg, 0.1-1000 μg or 0.1-500μg, for example 0.1-100μg, may be employed. In a preferred embodiment the dose is 100μg or about 100μg. Such doses are appropriate for local administration. An appropriate concentration can be determined depending on the efficiency of uptake of the agent in question into the cells in question and the final concentration it is desired to achieve in the cells.

In most cases the photosensitizing agent and the antigenic molecule as defined herein are administered together, but this may be varied. Thus different times or modes or sites of administration (or contact with the cell) are contemplated for the different components, although in a preferred embodiment the antigenic molecule and photosensitizing agent are administered together, preferably via intradermal administration.

Alternatively, the photosensitising agent may be administered separately from the antigen, for example in a separate formulation. In vivo an appropriate method and time of incubation by which the agents are brought into contact with the target cells will be dependent on factors such as the mode of administration and the type of agents which are used. For example, if the agents are injected into a tumour, tissue or organ which is to be treated/irradiated, the cells near the injection point will come into contact with and hence tend to take up the agents more rapidly than the cells located at a greater distance from the injection point, which are likely to come into contact with the agents at a later time point and lower concentration.

According to the present invention the contact between the cell and the photosensitizing agent and antigenic molecule as defined herein is from 12 hours to 30 hours, e.g. 16 hours to 20 hours, preferably 18 hours or about 18 hours.

Preferably the photosensitizing agent and antigenic molecule are contacted with the cell (or administered to the subject) for the same amount of time. However, some variation is possible, e.g. the photosensitizing agent and the antigenic molecule may be applied sequentially, though the time between their administration is small, e.g. from 1 minute to 2 hours or less.

For administration of agents described herein in vivo, any mode of administration common or standard in the art may be used, e.g. injection, infusion, topical administration, transdermal administration, both to internal and external body surfaces etc. The invention can be used in relation to any tissue which contains cells to which the photosensitising agent containing compound or the molecule to be internalized is localized, including body fluid locations, as well as solid tissues. All tissues can be treated as long as the photosensitiser is taken up by the target cells, and the light can be properly delivered. Preferred modes of administration are intradermal, subcutaneous, topical or intratumoural

administration or injection. Preferably administration is by intradermal injection.

To achieve the desired outcome, e.g. antigen presentation, generation of an immune response or vaccination, the methods or parts thereof may be repeated. Thus, the method in its entirety may be performed multiple times (e.g. 2, 3 or more times) after an appropriate interval or parts of the method may be repeated, e.g. additional irradiation steps. "Irradiation" to activate the photosensitising agent refers to the

administration of light directly or indirectly as described hereinafter. Thus subjects or cells may be illuminated with a light source for example directly or indirectly, e.g. in vivo when the cells are below the surface of the skin or are in the form of a layer of cells not all of which are directly illuminated, i.e. without the screen of other cells. As discussed above, according to the present invention illumination or irradiation of the cell or subject occurs approximately 12-30 hours after administration of the photosensitizing agent and antigenic molecule as defined herein, preferably 16-20 hours, especially preferably 18 hours (e.g. 17.5 to 18.5 hours) after. In those cases in which sequential administration of the agents is contemplated, the timing of the irradiation is timed such that each of the agents has been administered or contacted with the cell for at least the stated time before irradiation. Thus, for example, the photosensitizing agent may be applied at time 0 and the antigenic molecule 2 hours later and these agents may then be incubated in the subject until 20 hours at which point the subject may be irradiated, i.e. irradiation at 18 hours after the start of incubation of the antigenic molecule and 20 hours after the start of incubation of the photosensitizing agent. In this scenario irradiation is performed within 16-20 hours of the start of incubation of the agents.

The light irradiation step to activate the photosensitising agent may take place according to techniques and procedures well known in the art. The wavelength of light to be used is selected according to the photosensitising agent to be used. Suitable artificial light sources are well known in the art, e.g. using blue (400-475nm) or red (620-750nm) wavelength light. For TPCS_2a, and other disulphonated photosensitisers as described herein, for example a wavelength of between 400 and 500nm, more preferably between 400 and 450nm, e.g. from 430- 440nm, and even more preferably approximately 435nm, or 435nm may be used. Alternatively, chlorins and bacteriochlorins can be activated by red light (e.g. 652nm and 750nm, respectively). Where appropriate the photosensitiser, e.g. a porphyrin or chlorin, may be activated by green light (e.g. around 514nm), for example the KillerRed (Evrogen, Moscow, Russia) photosensitiser may be activated by green light.

Suitable light sources are well known in the art, for example the

LumiSource® lamp of PCI Biotech AS. Alternatively, an LED-based illumination device which has an adjustable output power of up to 60mW and an emission spectra of 430-435nm may be used. For red light, a suitable source of illumination is the PCI Biotech AS 652nm laser system SN576003 diode laser, although any suitable red light source may be used. The time for which the cells are exposed to light in the methods of the present invention may vary. The efficiency of the internalisation of a molecule into the cytosol increases with increased exposure to light to a maximum beyond which cell damage and hence cell death increases.

A preferred length of time for the irradiation step depends on factors such as the target, the photosensitizer, the amount of the photosensitizer accumulated in the target cells or tissue and the overlap between the absorption spectrum of the photosensitizer and the emission spectrum of the light source. Generally, the length of time for the irradiation step is in the order of seconds to minutes or up to several hours, e.g. preferably up to 60 minutes e.g. from 0.25 or 1 to 30 minutes, e.g. from 0.5 to 3 minutes or from 1 to 5 minutes or from 1 to 15 minutes e.g. from 3 to 12 minutes, and preferably approximately 3 minutes, e.g. 2.5 to 3.5 minutes or 6 minutes, e.g. 5.5 to 6.5 minutes, or 12 minutes e.g. 1 1.5 to 12.5 minutes. Shorter irradiation times may also be used, for example 1 to 60 seconds, e.g. 10-50, 20-40 or 25-35 seconds.

Appropriate light doses can be selected by a person skilled in the art and again will depend on the photosensitizer used and the amount of photosensitizer accumulated in the target cells or tissues. The light doses are usually lower when photosensitizers with higher extinction coefficients (e.g. in the red area, or blue area if blue light is used, depending on the photosensitiser used) of the visible spectrum are used. For example, a light dose in the range of 0.24 - 7.2J/cm² at a fluence range of 0.05-20 mW/cm², e.g. 2.0 mW/cm² may be used when an LED-based illumination device which has an adjustable output power of up to 60mW and an emission spectra of 430-435nm is employed. Alternatively, e.g. if the LumiSource® lamp is employed, a light dose in the range of 0.1-6J/cm² at a fluence range of 0.1- 20 (e.g. 13 as provided by Lumisource®) mW/cm² is appropriate. For red light, a light dose of 0.03-1 J/cm², e.g. 0.3J/cm², at a fluence range of 0.1-5 mW/cm², e.g. 0.81 mW/cm², may be used. Furthermore, if cell viability is to be maintained, the generation of excessive levels of toxic species is to be avoided and the relevant parameters may be adjusted accordingly.

The methods of the invention may inevitably give rise to some cell damage by virtue of the photochemical treatment i.e. by photodynamic therapy effects through the generation of toxic species on activation of the photosensitizing agent. Depending on the proposed use, this cell death may not be of consequence and may indeed be advantageous for some applications (e.g. cancer treatment). In most embodiments, however, cell death is avoided to allow the generation of an immune response from the presenting cell. The methods of the invention may be modified such that the fraction or proportion of the surviving cells is regulated by selecting the light dose in relation to the concentration of the photosensitizing agent. Again, such techniques are known in the art.

Preferably, substantially all of the cells, or a significant majority (e.g. at least 75%, more preferably at least 80, 85, 90 or 95% of the cells) are not killed. In making this assessment for the in vivo methods of the invention, cell death (of one or more cell types) within a 1 cm radius of the point of administration (or depth within tissue) may be examined. Cell viability following PCI treatment can be measured by standard techniques known in the art such as by microscopy. As cell death may not occur instantly, the % cell death refers to the percent of cells which remain viable within a few hours of irradiation (e.g. up to 4 hours after irradiation) but preferably refers to the % viable cells 4 or more hours after irradiation.

Thus, in a further aspect the present invention provides a method of generating an immune response in a subject, comprising administering to said subject an antigenic molecule and a photosensitizing agent as defined

hereinbefore, and after 12 to 30 hours, preferably 16-20 hours, e.g. 18 hours, irradiating said subject with light of a wavelength effective to activate said photosensitizing agent, wherein an immune response is generated.

An "immune response" which may be generated may be humoral and cell- mediated immunity, for example the stimulation of antibody production, or the stimulation of cytotoxic or killer cells, which may recognise and destroy (or otherwise eliminate) cells expressing "foreign" antigens on their surface. The term "stimulating an immune response" thus includes all types of immune responses and mechanisms for stimulating them and encompasses stimulating CTLs which forms a preferred aspect of the invention. Preferably the immune response which is stimulated is cytotoxic CD8 T cells. The extent of an immune response may be assessed by markers of an immune response, e.g. secreted molecules such as IL-2 or IFNy or the production of antigen specific T cells (e.g. assessed as described in the Examples).

The stimulation of cytotoxic cells or antibody-producing cells, requires antigens to be presented to the cell to be stimulated in a particular manner by the antigen-presenting cells, for example MHC Class I presentation (e.g. activation of CD8⁺ cytotoxic T-cells requires MHC-I antigen presentation). Preferably the immune response is stimulated via MHC-I presentation.

Preferably the immune response is used to treat or prevent a disease, disorder or infection, e.g. cancer. Preferably the cancer is melanoma and the antigenic molecule is a melanoma antigen (i.e. obtained or derived from a melanoma antigen).

Preferably the method is used for vaccination. As referred to herein, "vaccination" is the use of an antigen (or a molecule containing an antigen) to elicit an immune response which is prophylactic or therapeutic against the development (or further development) of a disease, disorder or infection, wherein that disease, disorder or infection is associated with abnormal expression or presence of that antigen. Preferably the disease is cancer (in which case the vaccination is therapeutic). Prophylactic vaccination may be used to prevent infection.

In a preferred embodiment of the present invention, the subject of the method, e.g. vaccination, is a mammal, preferably a cat, dog, horse, donkey, sheep, pig, goat, cow, mouse, rat, rabbit or guinea pig, but most preferably the subject is a human.

The agents used in the methods of the invention may be administered to the subject separately or sequentially or in some cases simultaneously as described hereinbefore.

Aspects and features discussed above in relation to the method of expressing an antigenic molecule or a part thereof on the surface of a cell of the present invention, where appropriate, are also applicable to the method of generating an immune response above.

The invention also provides a method for introducing an antigenic molecule into the cytosol of a cell in a subject, comprising contacting said cell with the antigenic molecule to be introduced and a photosensitising agent as defined herein, and incubating the cell for 12 to 30 hours and irradiating the cell with light of a wavelength effective to activate the photosensitising agent. Once activated, intracellular compartments within said cell containing said compound release the molecule contained in these compartments into the cytosol.

Compositions comprising the antigenic molecule and/or photosensitizing agents for use in methods of the invention (and products of the invention) may be formulated in any convenient manner according to techniques and procedures known in the pharmaceutical art, e.g. using one or more pharmaceutically acceptable diluents, carriers or excipients. "Pharmaceutically acceptable" as referred to herein refers to ingredients that are compatible with other ingredients of the compositions (or products) as well as physiologically acceptable to the recipient. The nature of the composition and carriers or excipient materials, dosages etc. may be selected in routine manner according to choice and the desired route of administration, purpose of treatment etc. Dosages may likewise be determined in routine manner and may depend upon the nature of the molecule (or components of the composition or product), purpose of treatment, age of patient, mode of administration etc. In connection with the photosensitizing agent, the

potency/ability to disrupt membranes on irradiation, should also be taken into account.

The invention further provides an antigenic molecule and a photosensitizing agent as defined herein, for use in expressing an antigenic molecule or a part thereof on the surface of a cell, or for use in prophylaxis or therapy or for use in stimulating an immune response, for example for vaccination purposes, e.g. for stimulating CTLs, in a subject, preferably for treating or preventing a disease, disorder or infection in said subject, particularly for treating or preventing cancer, wherein said use comprises a method of the invention as defined herein.

Alternatively defined the present invention provides use of an antigenic molecule and/or a photosensitizing agent, for the preparation of a medicament for use in stimulating an immune response (e.g. for stimulating CTLs) 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 said immune response is stimulated by a method of the invention as defined herein.

Said stimulation, treatment or prevention preferably comprises administering said medicament to said subject.

The invention further provides a product comprising an antigenic molecule, and a photosensitizing agent as defined herein as a combined preparation for simultaneous, separate or sequential use in stimulating an immune response in a subject (or for expressing an antigenic molecule or a part thereof on the surface of a cell or for internalising an antigenic molecule into the cytosol of a cell) in a method as defined herein, preferably to treat or prevent a disease, disorder or infection in a subject.

The present invention also provides 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, for example for use in vaccination or immunisation, or for expressing an antigenic molecule or a part thereof on the surface of a cell or for internalising an antigenic molecule into the cytosol of a cell in a method as defined herein, said kit comprising

a first container containing a photosensitizing agent as defined herein; and a second container containing said antigenic molecule as defined herein.

The products and kits of the invention may be used to achieve cell surface presentation (or therapeutic methods) as defined herein. The antigenic presentation achieved by the claimed invention may advantageously result in the stimulation of an immune response. Preferably an immune response which confers protection against subsequent challenge by an entity comprising or containing said antigenic molecule or part thereof is generated, and consequently the invention finds particular utility as a method of vaccination.

The disease, disorder or infection is any disease, disorder or infection which may be treated or prevented by the generation of an immune response, e.g. by eliminating abnormal or foreign cells which may be identified on the basis of an antigen (or its level of expression) which allows discrimination (and elimination) relative to normal cells. Selection of the antigenic molecule to be used determines the disease, disorder or infection to be treated. Based on the antigenic molecules discussed above, the methods, uses, compositions, products, kits and so forth, described herein may be used to treat or prevent against, for example, infections (e.g. viral or bacterial as mentioned hereinbefore), cancers or multiple sclerosis. Prevention of such diseases, disorders or infection may constitute vaccination.

As defined herein "treatment" refers to reducing, alleviating or eliminating one or more symptoms of the disease, disorder or infection which is being treated, relative to the symptoms prior to treatment. "Prevention" (or prophylaxis) refers to delaying or preventing the onset of the symptoms of the disease, disorder or infection. Prevention may be absolute (such that no disease occurs) or may be effective only in some individuals or for a limited amount of time.

The present invention encompasses all combinations of the preferred aspects described herein. One or more of the preferred features discussed above may be used in combination with any of the other preferred features. By way of example, the incubation time used in methods of the invention, i.e. 12 to 30 hours, preferably 18 hours, can be used with one or more of any of the preferred features discussed above, for example the preferred photosensitizing agent, preferred doses of a photosensitizing agent, particularly a preferred photosensitizing agent, preferred illumination times, preferred dose of antigen, preferred antigens, preferred cell types, preferred light sources, preferred routes of administration and subjects.

For example, methods of the present invention may encompass one or more, for example two, three, four, five or six of the preferred features discussed herein, although additional preferred features can be included.

The incubation time according to methods of the present invention may be used in combination with a preferred photosensitiser at a preferred dose and with a preferred does of antigen, and with a preferred illumination time. Thus for example, in addition to the stated time of irradiation after administration of the agents, the methods of the invention preferably also comprise use of an irradiation time of 3 to 12 minutes (e.g. 6 minutes), use of the photosensitizer TPCS_2a, preferably at 100 to 300μg (e.g. 250μg) and preferably an antigenic molecule dose of 10 to 100μg (preferably 100μg), wherein the agents are applied together in an intradermal injection, preferably to a human. However, this is only one example and other combinations of preferred features may be employed. All combinations of the preferred features are contemplated, particularly as described in the Examples.

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

Figure 1 (A) shows the experimental set up of PCI-mediated immunisation using mice adoptively transferred with OVA-specific CD8 T-cell transgenic OT-1 cells prior to immunisation. (B) After intradermal injection of antigen (OVA) and photosensitiser (TPCS_2a) in the abdominal region, mice were anaesthetised and the site of injection illuminated by placing the mice belly down on a LumiSource light table.

Figure 2 shows results with C57BL/6 mice that were spiked with 5x 10⁶ OT-I cells and the frequency of SIINFEKL-specific cells were measured in the recipients after 18 hours by MHC l-SIINFEKL pentamer staining and flow cytometry (A). The mice were then immunised with 100 μg OVA or with 100 μg OVA and 25 μg TPCS_2a; control mice were left untreated. After 2 or 18 hours, the TPCS_2a -treated mice were illuminated. On day 6 (B) and 23 (C), mice were bled and stained with MHC I- SIINFEKL pentamer, anti-CD8 and anti-CD44 antibodies and analysed by flow cytometry. Bars show the frequency of triple positive cells relative to the total number of CD8 T cells. (D) shows dot plots of pentamer- and CD44-positive cells from blood analysed by flow cytometry on day 6. Cells were gated on CD8 lymphocytes. (E) shows results on day 14, blood (left panel) and day 23

splenocytes (right panel) that were re-stimulated overnight with SIINFEKL and analysed for CD8, CD44 and IFN-γ by intracellular staining (ICS). (F) shows results with splenocytes that were re-stimulated with SIINFEKL for analysis of IFN-γ (left panel) and IL-2 (right panel) by ELISA.

Figure 3 shows results with C57BL/6 mice that were spiked with 1.6x10⁶ OT-I cells. After eight hours, the mice were immunised with 10 μg OVA, with 10 μg OVA and 25 μg TPCS_2a, or with 10 μg OVA and 250 μg TPCS_2a. On day 8 the mice were bled and analysed for (A) MHC l-SIINFEKL pentamer, CD44 and CD8 staining. On day 1 1 the mice were euthanized and their splenocytes analysed for (B) CD8 and CD44 and intracellular IFN-γ, as well as secretion of IL-2 (C) and IFN-γ (D)

measured by ELISA. Bars show the frequency of triple positive cells relative to the total number of CD8 T cells.

Figure 4 shows (A) J774 cells that were incubated overnight with 25 μg/ml OVA- Alexa488 (left panel) or with OVA-Alexa488 and 0.05 μ9 πτιΙ TPCS_2a (right panel). After washing and 90 minutes incubation in fresh medium, the cells were illuminated, and the cellular uptake and distribution of OVA-Alexa488 was analysed by fluorescence microcopy. (B) J774 cells were incubated with 1.0 μg/ml TPCS_2a and 25 μg/ml OVA-Alexa488 as above and analysed for cellular uptake, distribution and co-localisation of OVA-Alexa488 and TPCS_2a by fluorescence microcopy. Co- localisation of the two compounds causes emission of yellow fluorescence.

Figure 5 shows results with C57BL/6 mice that were spiked with 1.6*10⁶ OT-I cells. After eight hours, the mice were immunised with 100μg OVA, or with 100μg OVA and 25μg TPCS_2a; control mice were left untreated. After 2, 6 or 18 hours, the TPCS_2a -treated mice were illuminated. On day 0 and day 7 mice were bled and stained with MHC l-SIINFEKL pentamer and anti-CD8 antibodies and analysed by flow cytometry (A). On days 0, 7, 14 blood cells and day 23 splenocytes were stained with anti-CD8 antibodies and pentamer and analysed by flow cytometry (B). Each circle represents the results for a different animal.

Figure 6 shows a similar study to that shown in Figure 5 but timepoints of 18 hours and 42 hours after illumination were assayed. On day 0 and day 7 mice were bled and stained with MHC l-SIINFEKL pentamer and anti-CD8 antibodies and analysed by flow cytometry (A). On days 0, 7 blood cells and day 14 splenocytes were stained with anti-CD8 antibodies and pentamer and analysed by flow cytometry (B).

(C) shows splenocytes that were re-stimulated overnight with SIINFEKL and analysed for IFN-γ by ELISA. IFN-y was also analysed on day 14 by flow cytometry

(D) .

Figure 7 shows a similar study to that shown in Figure 5 but the illumination time was varied between 3, 6 and 12 minutes (incubation time was 18 hours). On days 0, and 9 the mice were bled and the cells analysed for MHC l-SIINFEKL pentamer and CD8 staining by flow cytometry (A). On day 0 and day 9 mice were bled and stained with MHC l-SIINFEKL pentamer and anti-CD8 antibodies and assessed by flow cytometry (B). (C) shows splenocytes from day 14 that were re-stimulated overnight with SIINFEKL and analysed for IL-2 and IFN-γ by ELISA.

Figure 8 shows a similar study to that shown in Figure 5 but the photosensitiser dose was varied between 25, 50 and 100 μg TPCS_2a- An illumination time of 6 minutes and incubation time of 18 hours was used. On day 7 the mice were bled and cells stained with pentamer and anti-CD8 antibodies and assessed by flow cytometry (A). On day 7 blood cells were stained with anti-CD8 antibodies and analysed by flow cytometry (B). On day 12 splenocytes were analysed for IFN-γ, CD8 and CD44 staining (left panel) and MHC l-SIINFEKL pentamer and CD8 staining, (right panel) by flow cytometry (C).

Figure 9 shows results with C57BL/6 mice that were spiked with 2x 10⁶ OT-I cells. One day later, the mice were immunised with 20 μg OVA, with 20 μg OVA and 200 μg TPCS_2a, or left untreated. On day 54, the mice were euthanized and the splenocytes analysed by flow cytometry for (A) MHC l-SIINFEKL pentamer and CD8 staining, or (B) intracellular IFN-γ and CD8 and CD44 staining. Bars show the frequency triple positive cells relative to the total number of CD8 T cells. (C)

Secretion of IFN-γ into 96-hours splenocyte cultures was measured by ELISA.

Figure 10 shows the effect of PCI-based vaccination on tumour growth. C57BL/6 mice were spiked with 1 x 10⁴ OT-I cells. One day later, the mice were immunised with 20 μg OVA, with 20 μg OVA and 200 μg TPCS_2a, or left untreated. The abdomen was shaved before vaccination. The abdominal region was illuminated for six minutes 18 hours after vaccination. On day 4 after immunisation, the mice received an intradermal injection of 5^10⁵ SIINFEKL-expressing B16 mouse melanoma cells. Two weeks thereafter, the tumour volume was measured (A) and the tumour photographed (B). n.s.: not significant; *: p<0.05 as analysed by Kruskal-Wallis test.

Figure 11 shows the effect of different light doses with the LED-based blue light illumination device (average of 2 animals per group). Figure 12 shows(% antigen-specific, CD44+ cells of the total CD8+ cells) for the experimental groups (average of 5 animals per group; error bars are standard-error- of-the-mean).

EXAMPLES

Materials and methods

Mice

For immunisation, female C57BL/6 mice were purchased from Harlan (Horst, The Netherlands) and used at 6-10 weeks of age. Rag2 deficient OT-I mice transgenic for the T-cell receptor that recognises the MHC class-l restricted epitope OVA₂₅7-264 (SIINFEKL) from ovalbumin (OVA) were originally purchased from Taconic Europe (Ry, Denmark) and bred in the facilities at the University of Zurich. All mice were kept under specified pathogen-free (SPF) conditions, and the procedures performed were approved by Swiss Veterinary authorities (licence 69/2012).

Materials

The antigen chicken ovalbumin (OVA; Grade V) was purchased from Sigma-Aldrich (Buchs, Switzerland) and dissolved in PBS. The octapeptide OVA aa257-264 (SIINFEKL) was purchased from EMC microcollections (Tuebingen, Germany). The photosensitiser TPCS_2a (tetraphenyl chlorin disulfonate or Amphinex®) was provided by PCI Biotech (Lysaker, Norway) at a concentration of 30 mg/ml in polysorbate 80, mannitol and 50 mM Tris pH 8.5. TPCS_2a was protected from light and kept at 4°C. Prior to vaccination OVA and TPCS_2a were mixed together in PBS and kept protected from light. The light used for activation of the photosensitiser was LumiSource™ (PCI Biotech), which contains four 18 W Osram L18/67 standard light tubes with a fluence rate of 13.5 mW/cm² and emits light at 435 nm.

Intradermal photosensitisation and immunisation of mice

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 2x10⁶ 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.

Then, the mice were shaved on the abdominal area, and the vaccines, consisting of OVA or of a mixture of OVA and TPCS_2a, were injected intradermal^ using syringes with 29G needles. The vaccines were kept light protected and used within 60 minutes of preparation. The vaccines were given in two injections of 50 μΙ each, on the left and right side of the abdominal mid line. OVA was tested at 10 to 100 μg per dose. The TPCS_2a dose was 7.5 to 250 μg.

On day 0, prior to vaccination and on various days thereafter (e.g. day 6, 7, 8, 9, 14, 23, as indicated) mice were bled by tail bleeding and erythrocytes were removed by lysis, before analysis of antigen-specific CD8 T cells by flow cytometry. At the end of the experiment (typically 1 1 , 12, 14 or 23 days), the mice were euthanized and the splenocytes analysed ex vivo. At various time points after the TPCS_2a injection (0-48 hours), the mice were anaesthetised by intraperitoneal injection of a mixture of ketamine (25 mg/kg body weight) and xylazin (4 mg/kg) and placed on a light source (for illumination and activation of the photosensitiser TPCS_2a). The light dose was 6 minutes, if not otherwise stated. The whole procedure is illustrated in the scheme of Figure 1A. The illumination of mice using LumiSource™ is imaged in Figure 1 B.

Analysis of immune responses

The frequency of OVA-specific CD8 T-cells in blood was monitored by staining the cells with anti-CD8 antibody and H-2K^b/SIINFEKL Pro5 pentamer (Proimmune, Oxford, UK) for analysis by flow cytometry. The activation status of the cells was further analysed by testing the expression of CD44 and CD69 by flow cytometry. Intracellular staining for IFN-γ was done after overnight stimulation of splenocytes in 24-well plates with the CD8 epitope OVA₂₅₇-₂64 (SIINFEKL) at 37 °C. Brefeldin A was added during the last 4 hours. The cells were then washed and fixed with 4% formaldehyde in PBS for 10 min on ice. Anti-CD16/32 was added to block unspecific binding to Fc receptors. The cells were then permeabilised with 0.1 % NP40 in PBS for 3 min and washed before staining with anti-IFN-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, OR). Alternatively, 2x 10⁵ splenocytes were re-stimulated in 96-well plates with OVA protein or the SIINFEKL. After 24 and 72 hours, supernatants were collected and analysed for IL-2 or IFN-γ by ELISA (eBioscience - performed according to the manufacturer's instructions).

Live cell fluorescence microscopy

Fifty thousand J774.1 cells (ATCC no. TIB-67 mouse monocyte macrophage cell line) were seeded out on no. 1.5 glass coverslips (Glasswarenfabrik Karl Hecht KG, Sondheim, Germany) in 4-well plates overnight. The cells were incubated with 0.05 or 1 ,0 μg/ml TPCS_2a for 18 hours and washed three times in drug-free culture medium prior to incubation with 25 μg/ml OVA-Alexa488 for four hours. Cells were subsequently washed in ice-cold PBS with Ca²⁺ and Mg²⁺ prior to microscopy. Images of cellular localization and PCI-induced cytosolic release of OVA was obtained by epi-fluorescence microscopy using a Plan-Apochromat 63x/1.40 Oil differential interference contrast (DIC) objective or 40x/0.95 Plan-Apochromat phase contrast (Korr Ph3 M27) objective with a Zeiss Axioimager Z.1 microscope (Carl Zeiss, Oberkochen, Germany). Fluorescence of Alexa488-labeled OVA was obtained by using a 470/40 nm band pass (BP) excitation filter with a beam splitter at 495 nm and a 525/50 nm BP emission filter. TPCS_2a fluorescence was obtained by using a 395-440 nm BP excitation filter with a beam splitter at 460 nm, and a 620 nm long pass filter. Micrographs were recorded with a digital AxioCam MRm camera and processed and analysed by use of the Axiovision Software (Carl Zeiss).

Vaccination and effect on tumour growth

Animals were immunised intradermally as described in the Materials and Methods section with 10 μg OVA with or without 200 μg TPCS_2a- The abdominal region was illuminated for six minutes 18 hours after vaccination. One day prior to vaccination, the mice received 10,000 OT-I cells intravenously. On day four after vaccination, the mice received 5x 105 SIINFEKL-expressing B16 mouse melanoma cells by intradermal injection into one of the flanks. The B16 melanoma cell line is of spontaneous origin in C57BL/6 mice, and the SIINFEKL-expressing line was kindly provided by Emmanuel Contassot (University of Zurich). The growth of the solid tumour was monitored by measuring the tumour size by calliper 14 days after tumour injection, the endpoint of the investigation. The tumour volume was calculated using use of the modified ellipsoid formula: (length ^χ width²)/2. Example 1 : Analysis of the effect of the length of immunisation before illumination on the PCI-mediated generation of an immune response.

To facilitate analysis of MHC-class I antigen presentation, we used the class-l binding octapeptide SIINFEKL from OVA (aa257-264) in combination with

SIINFEKL- specific CD8 T cells from T-cell receptor transgenic OT-I mice. OT-I lymphocytes were purified from OT-I mice, and 2x10⁶ cells were adoptively transferred to syngeneic and sex-matched wild type C57BL/6 mice. One day after the transfer approximately 1.4% of all CD8-positive T cells in peripheral blood was SIINFEKL-specific (Fig. 2A); the frequency of SIINFEKL-specific CD8 T cells in C57BL/6 mice, which did not receive an adoptive transfer of OT-I cells was less than 0.05% (data not shown).

The mice were then typically immunised with 10 - 100 μg OVA protein or with a mixture of OVA and 7.5 - 250 μg of the photosensitiser TPCS_2a by intradermal administration in the abdominal region. At different time point thereafter, the mice were anaesthetised and placed belly-down onto the light source, and the site of vaccination was illuminated for six minutes. By day six after vaccination, the frequency of SIINFEKL-specific CD8 T cells in the peripheral blood of mice vaccinated 100 OVA μg had increased to approximately 3.5% (Fig. 2B). A similar frequency was measured in mice that also received 25 μg TPCS_2a and were illuminated two hours after vaccination (Fig. 2B). However, when mice were illuminated 18 hours post-vaccination, a significant increase in the number of SIINFEKL-specific CD8 T cells was measured in blood (Fig. 2C; P=0.0286 by Mann Whitney). Typically, a retraction of the number of SIINFEKL-specific CD8 T cells in blood was observed 10-15 days after vaccination. By day 23 post-vaccination, the numbers of antigen-specific CD8 T cells had retracted to baseline levels in mice immunised with OVA alone or OVA plus TPCS_2a and illuminated two hours after administration (Fig. 2C). Also, mice immunised with OVA and TPCS_2a and illuminated at 18 hours after immunization showed reduced frequencies after 23 days, but still significantly higher than baseline (P=0.0294 by Mann Whitney). While the SIINFEKL-specific cells in blood had a non-activated phenotype with lack of activation markers such as CD44 (Fig. 2D), CD25 and CD69 (not shown), both immunisation with OVA and OVA-PCI caused strong up-regulation of these markers by day six. On day 14, the mice were bled and the PBMCs cells re-stimulated with SIINFEKL overnight. After staining for surface CD8 and CD44 and intracellular IFN-γ, the cells were acquired by flow cytometry and the frequency of triple-positive cells within all CD8-positive cells was calculated. OVA-immunised mice had a 4-fold increased frequency as compared to control mice that had received OT-I transfer only (Fig. 2E, left panel). The increase in IFN-y-producing CD44-positive cells after PCI treatment was 6-fold (illumination at 2 hours) and 15-fold (18 hours).

On day 23, mice were euthanized and splenocytes cultured overnight with

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

Since immunisation with PCI did not produce good responders in all animals tested (typically 3-4 out of 5), we further tested the effect of the time interval between TPCS_2a administration and illumination on the stimulated immune response.

Intervals of 6-8 hours or of 42 hours did not suggest an adjuvant effect for PCI (data not shown). Repeatedly, an interval of approximately 18 hours was required to gain an adjuvant effect of PCI. This was observed without exceptions in four independent experiments.

Example 2: Analysis of the effect of the dose of photosensitizer on the PCI- mediated generation of an immune response.

We then reduced the OVA immunisation dose in order to titrate out the effect of OVA and increasing doses of TPCS_2a was titrated into the vaccine. Immunisation with 1 C^g OVA alone produced no measurable effect on SIINFEKL-specific CD8 T cells in blood as compared to untreated animals (data not shown). Several experiments with TPCS_2a at 10, 25, 50, 100 and 250 μg showed that increasing TPCS_2a doses also increased the measured OVA-specific immune response (data not shown). Representatively, PCI with 25 μg TPCS_2a caused 40% good responders, 40% week responders and 20% non-responders as measured for SIINFEKL-specific CD8 T cells in blood on day 8, while PCI with 250 μg TPCS_2a produced 100% good responders (Fig. 3A). On day 1 1 the splenocytes were tested by flow cytometry for IFN-γ production. Immunisation with OVA alone showed weak responders in all mice tested, whereas immunisation with OVA and PCI caused better responders in nine out of ten (90%) mice tested (Fig. 3B). Again, PCI with 250 μg TPCS_2a showed 100% responders and the highest frequency of IFN-γ producing cells. Whereas intracellular staining and flow cytometry qualitatively measures whether cells can produce cytokines, ELISA measures how much cytokine the cell can produce. We therefore re-stimulated the day 1 1 splenocytes with SIINFEKL in vitro and analysed IL-2 (Fig. 3C) and IFN-γ (Fig. 3D) after 24 and 72 hours, respectively. Immunisation with OVA alone produced weak but clearly measurable IL-2, but not IFN-γ secretion. Immunisation with OVA and PCI at 25 μg TPCS_2a did not cause an increase in IL-2, but a strong increase in IFN-γ secretion as compared to immunisation with OVA alone. At 250 μg TPCS_2a, strong secretion of both IL-2 and IFN-y was detected. Finally, while PCI with TPCS_2a had a dose- dependent adjuvant effect with regards to the immune response measured, higher TPCS_2a doses also caused more local inflammation with transient erythema on days 1-3 after illumination (data not shown).

To study the mechanism by which PCI mediates the adjuvant effect, murine J774 cells, an antigen-presenting macrophage cell line, were incubated with Alexa488- labelled OVA with or without parallel PCI treatment. As shown in the fluorescence micrograph of Figure 4A, in cells treated with OVA alone, antigen uptake was observed and the antigen was located close to the cell surface in concise spherical shaped bodies, suggesting that the antigen was contained in vesicles, e.g.

endosomes. After parallel PCI treatment of the cells, cytosol and in some cases also the nucleus have diffuse green fluorescence suggesting that the antigen is freely floating in the cytosol, hence, released from the endosomes. Since the photosensitiser TPCS_2a is auto-fluorescent, it enabled the study of the relative localisation of antigen and TPCS_2a after incubation of J774 cells with Alexa488- labelled OVA (green) and the photosensitiser (red). Again, after light activation of sensitised cells, the antigen showed a diffuse distribution throughout the cytosol and the nucleus (Fig. 4B). The TPCS_2a photosensitiser showed a similar distribution and the merge of the two images demonstrates that antigen and photosensitiser are co-localised.

Example 3: Further analysis of the effect of the length of immunisation before illumination on the PCI-mediated generation of an immune response.

To further examine the effect of the incubation time prior to illumination, a further study was conducted as generally described above but using 25μg TPCS_2a, ^ 00μg OVA, 6 minutes illumination time, and 2, 6 or 18 hours incubation time. Figure 5 shows results with C57BL/6 mice that were spiked with 5x10⁶ OT-I cells. After 18 hours, the mice were immunised with 10C^g OVA, or with 10C^g OVA and 25μg TPCS_2a; control mice were left untreated. After 2, 6 or 18 hours, the TPCS_2a - treated mice were illuminated. On day 0 and day 7 mice were bled and the cells stained with anti-CD8 antibodies and MHC l-SIINFEKL pentamer and assessed by flow cytometry analysis (A). On days 0, 7, 14 blood cells and day 23 splenocytes were stained with anti-CD8 antibodies and MHC l-SIINFEKL pentamer and assessed by flow cytometry (B). Individual circles in this and other figures show the results for individual animals. It can be seen that 18 hours incubation time produced an increase in antigen-specific CTLs.

Example 4: Further analysis of the effect of the length of immunisation before illumination on the PCI-mediated generation of an immune response.

A similar study to Example 3 was carried out to further test different times of incubation prior to illumination. Timepoints of 18 hours and 42 hours after illumination were assayed (Figure 6). On day 0 and day 7 mice were bled and stained with MHC l-SIINFEKL pentamer and anti-CD8 antibodies and assessed by flow cytometry (A) On days 0 and 7 blood cells and day 14 splenocytes cells were stained with anti-CD8 antibodies and pentamer and analysed by flow cytometry (B).

(C) shows splenocytes that were re-stimulated overnight with SIINFEKL and analysed for IFN-γ by ELISA. I FN-y was also analysed on day 14 by flow cytometry

(D) . Example 5: Analysis of the effect of the length of illumination on the PCI- mediated generation of an immune response.

A similar study to Example 3 was carried out to test the illumination time, which was varied between 3, 6 and 12 minutes (incubation time was 18 hours) (Figure 7). On days 0 and 9 blood cells and day 14 splenocytes were analysed for MHC I- SIINFEKL pentamer and CD8 staining by flow cytometry (A). On day 0 and day 9 mice were bled and stained with MHC l-SIINFEKL pentamer and anti-CD8 antibodies and analyzed by flow cytometry (B). (C) shows splenocytes (day 14) that were re-stimulated overnight with SIINFEKL and analysed for IL-2 and IFN-γ by ELISA.

Example 6: Analysis of the effect of the photosensitizer dose on the PCI- mediated generation of an immune response.

A similar study was carried out to test the photosensitiser dose, which was varied between 25, 50 and 100 μg TPCS_2a (Figure 8). An illumination time of 6 minutes and incubation time of 18 hours was used. On day 7 the mice were bled and blood cells stained with MHC l-SIINFEKL pentamer and anti-CD8 antibodies and assessed by flow cytometry (A). On day 7 blood cells were stained with anti-CD8 antibodies and pentamer analysed by flow cytometry (B). On day 12 splenocytes were analysed for IFN-γ, CD8 and CD44 staining (left panel) and MHC l-SIINFEKL pentamer and CD8 staining, (right panel) by flow cytometry (C).

Example 7: Analysis of the length of the adjuvant effect of PCI

The longevity of the memory of the observed CD8-positive immune responses was tested in mice immunised as described in the Materials and Methods section using 20 μg OVA with or without 200 μg TPCS_2a- The abdominal region was illuminated for six minutes 18 hours after vaccination. After 54 days, the mice were euthanized and the splenocytes analysed directly for the frequency and function of SIINFEKL- specific CD8 T cells. As shown in Figure 9A, the frequencies of measurable SIINFEKL-specific CD8 T cells in mice treated iwith OVA or with OVA and PCI were not different from untreated mice. However, re-stimulation with SIINFEKL overnight revealed that PCI-treatment enabled stimulation of antigen-specific CD8 memory cells, which reacted by secretion of the effector cytokine IFN-γ. This was observed both by intracellular staining and flow cytometry (Fig. 9B) and by ELISA (Fig. 9C). By both assay, a statistically significant difference was observed between OVA alone and OVA-PCI treated mice (P<0.01 ).

Example 8: Effect of vaccination on tumour growth

Mice received SIINFEKL-expressing mouse melanoma B16 cells four days after vaccination and the tumour growth was measured on day 14 post injection of the melanoma cells.

The results (Figure 10) showed that PCI-based vaccination can prevent subsequent tumour growth. In non-vaccinated mice, the transfer of 2x 10⁶ OT-1 cells totally prevented B16 growth (data not shown). Therefore, the number of transferred cells was reduced to 1 x 10⁴ OT-I cells. When compared to untreated controls, a significantly reduced B16 tumour growth was observed in mice that received PCI-based vaccination with OVA (P<0.05 by Kruskal-Wallis test) but not after vaccination with OVA alone (Fig. 10A). When all data were transformed to binary data (0=no growth); 1 = growth) and analysed by the Chi-square test, PCI- based vaccination had a significantly stronger suppressing effect on tumour growth than vaccination with OVA alone (P=0.048). Figure 10B shows representative micrographs of tumours on day 14 from differently treated mice.

Similar results were observed in therapeutic vaccination in which vaccination as described above was performed on test mice with melanomas developed from intradermally injected melanoma cells. Tumour growth was significantly reduced by vaccination (data not shown).

For Examples 9 and 10 the following Materials and Methods were employed: Animals

C57BL/6 mice were purchased from Harlan (Horst, The Netherlands). CD8 T-cell receptor transgenic OT-I mice (B6.129S6-Rag2tm1 Fwa Tg(TcraTcrb)1 100Mjb) from Taconic Europe (Ry, Denmark) or from Jackson Laboratories (Bar Harbor, Maine). The OT-I CD8 T cells recognise the H-2K^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.

Materials and cells

Chicken OVA was purchased from Sigma-Aldrich (Buchs, Switzerland), and the SIINFEKL peptide from EMC microcollections (Tuebingen, Germany). The photosensitiser tetraphenyl chlorin disulfonate (TPCS_2a) was from PCI Biotech (Lysaker, Norway). The chitosan-TPC (tetraphenylchlorin) conjugate was from PCI Biotech (Lysaker, Norway).

SIINFEKL pentamers were from Proimmune (Oxford, UK), (Proimmune peptide code 093).

Intradermal photosensitisation and immunisation of mice with adoptively transferred OT-1 cells.

One day prior to the immunisation, spleens and lymph nodes were isolated from female OT-1 mice, and erythrocytes were removed by lysis ( BC 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 2x10⁶ 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.

For intradermal immunisation the mice were shaved on the abdominal area, and the vaccines, consisting of OVA or of mixtures of OVA and TPCS_2a or chitosan conjugate were injected intradermal^ using syringes with 29G needles. The vaccines were kept light protected and used within 60 minutes of preparation. The vaccines were given in two injections of 50 μΙ each, on the left and right side of the abdominal mid line. 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 illuminated as described below according to the individual experiments.

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

Analysis of immune responses by pentamer staining.

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 pentamer corresponding to the 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, OR).

Example 9: Effect of illumination dose with a LED-based blue light

illumination device

The experiment was performed as described above for vaccination of mice with OT- 1 cells. The animals were immunised at day 0 with a mixture of 10 μg OVA protein and 150 μg TPCS_2a as specified below. Illumination with different light doses was performed with the LED-based blue light illumination device (LED-based illumination device emitting blue light was used as described herein (PCI Biotech AS)) at a fluence rate of 2 mW/cm². Blood samples from day 7 after immunisation were stained by SIINFEKL pentamer, CD8 and CD44 antibodies, and analysed by flow cytometry as described. The following experimental groups were included:

1. Untreated: Mice received OT-1 cells, but were not immunised or illuminated.

2. OVA without TPCS_2a: Mice were immunised with 10 μg of OVA. They were not illuminated.

3. OVA with PCI. Mice were immunised with 10 μg of OVA and 150 μg TPCS_2a, and were illuminated with light doses of 0.24 to 7.2 J/cm² as indicated in Figure 1 1.

Figure 1 1 shows the effect of different light doses with the LED-based blue light illumination device (average of 2 animals per group). It can be seen that an immune response can be induced by light doses in the range of 0.24-7.2 J/cm², with an apparent peak level between 0.48 and 3.6 J/cm². Example 10: Effect of using a chitosan-photosensitiser conjugate as the photosensitiser

The experiment was performed as described above for vaccination of mice with OT- 1 cells. The animals were immunised at day 0 with a mixture of 10 μg OVA protein and 25 μg of chitosan-TPC (tetraphenylchlorin) conjugate of the structure shown below. Illumination for 6 min was performed with the LumiSource (LumiSource™ (PCI Biotech)) illumination device, 18 hours after immunisation. Blood samples from day 7 were stained by SIINFEKL pentamer, CD8 and CD44 antibodies, and analysed by flow cytometry as described. The following experimental groups were included:

1. Untreated: Mice received OT-1 cells, but were not immunised or illuminated.

2. OVA 10 μg: Mice were immunised with 10 μg of OVA. They were not illuminated.

3. OVA 100 μg: Mice were immunised with 100 μg of OVA. They were not illuminated.

4. Chitosan conjugate: Mice were immunised with 10 μg of OVA and 25 μg of chitosan-TPC conjugate and illuminated.

Figure 12 shows (% antigen-specific, CD44+ cells of the total CD8+cells) for the experimental groups (average of 5 animals per group; error bars are standard-error- of-the-mean). The results show that the chitosan-TPC conjugate (labelled as 044A in Figure 12) can be used as a photosensitiser for PCI-based intradermal immunisation (group 4), and that the effect that can be achieved with this conjugate with 10 μg of protein (OVA) antigen is significantly better that what is achieved with a 10 times higher amount of antigen (group 3) without the PCI treatment.

Claims

1. An in vivo method of expressing an antigenic molecule or a part thereof on the surface of a cell in a subject, comprising contacting said cell with said antigenic molecule and a photosensitizing agent for 12 to 30 hours 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. The method as claimed in claim 1 , wherein the cell is contacted with said antigenic molecule and photosensitizing agent for 16-20 hours.

3. The method as claimed in claim 2 wherein the cell is contacted with said antigenic molecule and photosensitizing agent for 18 hours.

4. The method as claimed in any one of claims 1 to 3 wherein said photosensitising agent is an amphiphilic porphyrin, chlorin, bacteriochlorin or phthalocyanine.

5. The method as claimed in claim 4 wherein said photosensitising agent is selected from TPCS_2a, AIPcS_2a, TPPS_2a and TPBS_2a.

6. The method as claimed in claim 5 wherein said photosensitising agent is TPCS_2a.

7. The method as claimed in any one of claims 1 to 6 wherein the dose of photosensitizing agent is between 25 and 400μg, preferably between 100 and 300μg.

8. The method as claimed in claim 7 wherein the dose of photosensitizing agent is 250μg.

9. The method as claimed in any one of claims 1 to 8 wherein the cell is irradiated for between 1 and 60 minutes.

10. The method as claimed in claim 9 wherein the cell is irradiated for 3 to 12 minutes, preferably for 6 minutes.

1 1 . The method as claimed in any one of claims 1 to 10 wherein the dose of the antigenic molecule is between 1 and 50C^g.

12. The method as claimed in claim 1 1 wherein the dose of the antigenic molecule is between 10 and 10C^g, preferably 10C^g.

13. The method as claimed in any one of claims 1 to 12 wherein the antigenic molecule is a molecule capable of stimulating an immune response, preferably a vaccine antigen or vaccine component.

14. The method as claimed in claim 13 wherein the antigenic presentation results in the stimulation of an immune response.

15. The method as claimed in any one of claims 1 to 14 wherein the antigenic molecule is a peptide.

16. The method as claimed in any one of claims 1 to 15 wherein the cell is an antigen presenting cell, preferably a dendritic cell.

17. The method as claimed in any one of claims 1 to 16 wherein said cell is contacted with said antigenic molecule and photosensitising agent simultaneously, separately or sequentially, wherein preferably said contact is achieved by intradermal administration of said antigenic molecule and said photosensitising agent.

18. A method of generating an immune response in a subject, comprising administering to said subject an antigenic molecule as defined in claim 1 , 1 1 , 12, 13 or 15, a photosensitizing agent as defined in claim 1 or 4 to 8 and after 12 to 30 hours, preferably 16-20 hours, irradiating said subject with light of a wavelength effective to activate said photosensitizing agent, wherein an immune response is generated, wherein preferably said irradiation is performed as defined in claim 9 or 10.

19. The method as claimed in claim 18 wherein said administration is by intradermal administration.

20. The method as claimed in claim 18 or 19 wherein said method is a method of vaccination.

21. The method as claimed in any one of claim 18 to 20 for treating or preventing a disease, disorder or infection, preferably cancer.

22. The method of any one of claims 18 to 21 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.

23. The method of any one of claims 18 to 22 wherein said antigenic molecule and said photosensitising agent are administered to said subject simultaneously, separately or sequentially.

24. An antigenic molecule as defined in any one of claims 1 , 1 1 , 12, 13 or 15, and a photosensitizing agent as defined in claim 1 or 4 to 8 for use in expressing said antigenic molecule or a part thereof on the surface of a cell or for use in prophylaxis or therapy wherein said use comprises a method as defined in any one of claims 1 to 23.

25. An antigenic molecule and photosensitizing agent for use as claimed in claim 24 to stimulate an immune response or for stimulating CTLs 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.

26. Use of an antigenic molecule as defined in any one of claims 1 , 1 1 , 12, 13 or 15 and/or a photosensitizing agent as defined in claim 1 or 4 to 8 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 said immune response is stimulated by a method as defined in any one of claims 18 to 23.

27. A product comprising an antigenic molecule as defined in any one of claims 1 , 11 , 12, 13 or 15, and a photosensitizing agent as defined in any one of claims 1 or 4 to 8 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, wherein said immune response is stimulated or said antigenic molecule or part thereof expressed by a method as defined in any one of claims 1 to 23.

28. 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, wherein said immune response is stimulated or said antigenic molecule or part thereof expressed by a method as defined in any one of claims 1 to 23, said kit comprising

a first container containing a photosensitizing agent as defined in any one of claims 1 or 4 to 8; and

a second container containing said antigenic molecule as defined in any one of claims 1 , 1 1 , 12, 13 or 15.