UNCORRECTED PROOF 1 Intradermal photosensitisation facilitates stimulation of MHC class-I 2 restricted CD8 T-cell responses of co-administered antigen 3 Monika Q1 Håkerud a,b,c , Ying Waeckerle-Men a , Pål Kristian Selbo b,c , Thomas M. Kündig a , 4 Anders Høgset c , Pål Johansen a, ⁎ 5 a Department of Dermatology, University Hospital Zurich, Gloriastrasse 31, 8091 Zurich, Switzerland 6 b Department of Radiation Biology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway 7 c PCI Biotech AS, Strandveien 55, 1366 Lysaker, Norway 8 9 abstract article info 10 Article history: 11 Received 15 August 2013 12 Accepted 17 November 2013 13 Available online xxxx 14 15 16 17 Keywords: 18 Q3 Antigen delivery 19 Cytosolic delivery 20 Endosomes 21 Photochemical internalization 22 Cytotoxicity 23 T cells 24 Melanoma 25 The protection or treatment of several immunological disorders is dependent on the antigen-specific and cyto- 26 toxic CD8 T cells. However, vaccines aimed at stimulating CD8 T-cell responses are typically ineffective because 27 vaccine antigens are primarily processed by the MHC class-II and not the MHC class-I pathway of antigen presen- 28 tation: the latter requires cytosolic delivery of antigen. In order to facilitate targeting of antigen to cytosol, the an- 29 tigen was combined with the photosensitiser TPCS2a (disulfonated tetraphenyl chlorin) and administered 30 intradermally to mice. The photosensitiser was activated by illumination of the injection site. This photochemical 31 internalization (PCI) strongly increased the stimulation of CD8 T-cell responses as measured by antigen-specific 32 proliferation and secretion of pro-inflammatory cytokines. Fluorescence microscopy showed that delivery to cy- 33 tosol was TPCS 2a dependent and occurred by light-induced disruption of TPCS 2a - and antigen-containing 34 endosomes. PCI-based vaccination prevented growth of malignant B16 cells as compared with vaccination with- 35 out PCI. In conclusion, PCI represents a potent tool for delivery of antigens to cytosol for stimulation of cytotoxic 36 CD8 T-cell responses. This study demonstrated a first proof-of-principle for PCI-mediated immunisation with 37 potential application in cancer immunotherapy. 38 © 2013 Published by Elsevier B.V. 39 40 41 42 43 1. Introduction 44 Vaccines most probably represent the single medical intervention 45 with the greatest impact on the global human health: childhood vac- 46 cines against bacterial toxins, measles, poliomyelitis and more prevent 47 millions of fatalities each year. Vaccines primarily protect by means of 48 neutralising antibodies [1] which are a result of antigen presentation 49 via the MHC class-II pathway, the default pathway of exogenous anti- 50 gens. When current vaccines fail to work, e.g. in tuberculosis, Hansens' 51 disease (leprosy), most parasitic infections, some viral infections such 52 as herpes and human immunodeficiency viruses, and tumours, it is 53 mainly because protection or treatment is dependent on T cells, espe- 54 cially cytotoxic CD8 T cells. The signals for CD8 T-cell stimulation is me- 55 diated via peptide antigens loaded on MHC class-I molecules, and this 56 generally occurs when the peptide is generated intracellularly; the 57 source of such peptides is typically worn-out endogenous proteins or 58 proteins from infecting virus. A major challenge in vaccine research 59 and development is therefore to find ways to access the MHC class I 60 pathway of antigen presentation with exogenous antigens. 61 Phagocytosis or endocytosis normally leads to phago-lysosome fu- 62 sion and MHC class II antigen presentation [2]. Several technologies 63 have been proposed for stimulation of CD8 T-cells, e.g. recombinant 64 but non-replicating viruses that are able to infect antigen-presenting 65 cells by other ways than phagocytosis [3–5]. We recently demonstrated 66 that endocytosed antigens can be redirected to the MHC class I pathway 67 of antigen presentation by using the drug-delivery technology photo- 68 chemical internalisation (PCI) [6]. Firstly, the protein antigen ovalbumin 69 (OVA) and the photosensitiser disulfonated tetraphenyl chlorine 70 (TPCS 2a ) [7], were co-incubated with bone-marrow-derived dendritic 71 cells in vitro, and the cells illuminated with defined light doses and 72 energies. Secondly, the dendritic cells were used to stimulate antigen- 73 specific CD8 T-cell responses in vitro and in vivo. The strength of 74 the stimulated CD8 T-cell responses was strongly dependent on 75 the photosensitisation of dendritic cells. This current report is the 76 first study to show direct immunisation with a vaccine and using 77 photosensitiser and light as adjuvants. The intradermal application of 78 photosensitiser and light strongly facilitated MHC class-I presentation 79 and CD8 T-cell stimulation of the co-delivered protein antigen in mice. 80 This PCI method could open new avenues in the vaccination or Journal of Controlled Release xxx (2013) xxx–xxx ⁎ Corresponding author at: Department of Dermatology, University Hospital Zurich, Gloriastrasse 31, 8091 Zurich, Switzerland. Tel.: +41 44 255 8616; fax: +41 44 255 4418. E-mail address: pal.johansen@usz.ch (P. Johansen). COREL-06964; No of Pages 8 0168-3659/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.jconrel.2013.11.017 Contents lists available at ScienceDirect Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel Please cite this article as: M. Håkerud, et al., Intradermal photosensitisation facilitates stimulation of MHC class-I restricted CD8 T-cell responses of co-administered antigen, J. Control. Release (2013), http://dx.doi.org/10.1016/j.jconrel.2013.11.017