SAT0052 PHOTODYNAMIC THERAPY TARGETING ACTIVATED FIBROBLASTS INDUCES SYNOVIAL CELL DEATH IN EXPERIMENTAL ARTHRITIS
Authors: Daphne Dorst, Mark Rijpkema, Mijke Buitinga, Peter Laverman, Marti Boss, Christian Klein, Anne Freimoser-Grundschober, Birgitte Walgreen, Peter van der Kraan, Martin Gotthardt, Marije Koenders
Journal: Annals of the Rheumatic Diseases (2019)

<h3>Background:</h3> Activated synovial fibroblasts (SF) contribute to rheumatoid arthritis (RA) by producing a multitude of cytokines, chemokines and proteases thus aggravating disease. Activated SF can be distinguished from quiescent fibroblasts by their expression of fibroblast activation protein (FAP). Selective depletion of FAP+ SF in inflamed joints could decrease their contribution to the arthritis process and thus constitute a viable treatment option. Further focussing of the treatment to only those areas affected by the disease can be accomplished by applying targeted photodynamic therapy (tPDT). In tPDT a light sensitive molecule, a photosensitizer (PS), is conjugated to a targeting moiety. Upon activation by light this construct produces reactive oxygen species, killing the targeted cells. <h3>Objectives:</h3> To this end we developed and tested a therapy that selectively depletes activated SF by targeting FAP on these cells with an antibody, 28H1, to which the PS, IRDye700DX, for tPDT is attached. Here we investigated the feasibility of using FAP-tPDT to induce cell death in murine arthritic synovium ex vivo. <h3>Methods:</h3> After conjugation of the IDRye700DX to 28H1 (28H1-700DX), binding and specificity of the conjugate was determined. Subsequently, tPDT efficiency in vitro was established using a 3T3 fibroblast cell line stably transfected with FAP. Biodistribution using an [<sup>111</sup>In] In-DTPA-28H1 conjugate with and without IRDye700DX was performed in healthy C57BL/6N mice as well as in C57BL/6N mice with antigen induced arthritis (AIA). Finally, the potential of FAP-tPDT to induce targeted cell death in the synovial lining was determined by treating knee joints from mice with AIA ex vivo. <h3>Results:</h3> Conjugation of IRDye700DX to the antibody did not negatively influence the immunoreactive fraction or binding capacity of the conjugate (94.7% for 28H1-700DX). 28H1-700DX was able to efficiently induce FAP-specific cell death in vitro. At 17.6 J/cm<sup>2</sup> radiant exposure, 89.24% ± 3.67% of fibroblasts died in the group incubated with antibody compared to control incubated with buffer only (p&lt;0.001). Biodistribution of the compound with the PS showed increased accumulation in the liver compared to the antibody without PS (31.46 ± 5.49% injected dose per gram tissue (%ID/g) versus 5.32 ± 1.17 %ID/g for the antibody with or without PS, respectively (p&lt;0.001)). However, despite this increased clearance to the liver, accumulation in the inflamed joints was increased in the group injected with the antibody-PS construct (1.61 ± 0.08 %ID versus 1.13 ±0.06 %ID for the antibody with or without PS (p&lt;0.001)). Interestingly, ex vivo FAP-tPDT of knee joints of arthritic mice caused significant photo-bleaching of the PS (19.69 ± 2.02% fluorescent signal remaining versus 96.00 ± 25.98% compared to the unexposed control at baseline, p=0.047). Furthermore FAP-tPDT induced marked apoptosis as was indicated by an increased staining of the markers caspase-3 and yH2AX evident in the synovium of treated knee joints. <h3>Conclusion:</h3> Here we demonstrated the feasibility of conjugating a PS to an antibody targeting FAP on activated SF without negatively impacting the binding capacity thereof. Furthermore we showed that this construct can then be used to deliver cell specific cytotoxicity through tPDT both in vitro and ex vivo in a mouse model of arthritis. This approach may have therapeutic potential in the treatment of RA. <h3>Disclosure of Interests:</h3> Daphne Dorst: None declared, Mark Rijpkema: None declared, Mijke Buitinga: None declared, Peter Laverman: None declared, Marti Boss: None declared, Christian Klein Employee of: Roche pharmaceutics, Anne Freimoser-Grundschober Employee of: Roche pharmaceutics, Birgitte Walgreen: None declared, Peter van der Kraan: None declared, Martin Gotthardt: None declared, Marije Koenders: None declared