SAT0061 Targeting activated synovial fibroblasts using photodynamic therapy in rheumatoid arthritis
Authors: D.N. Dorst, M. Rijpkema, M. Buitinga, M. Brom, D.L. Bos, A. Freimoser, C. Klein, B. Walgreen, P. van der Kraan, M. Gotthardt, M.I. Koenders
Journal: Annals of the Rheumatic Diseases (2018)

<h3>Background</h3> Activated synovial fibroblasts (SF) play an important role in the pathogenesis of rheumatoid arthritis (RA). They contribute to the pro-inflammatory environment in the joint as well as to the degradation of cartilage. Depleting SF could ameliorate both the symptoms of joint inflammation and degradation in RA. SF are characterised by the expression of Fibroblast Activation Protein (FAP). Here, we investigated the potential of photodynamic therapy (PDT) targeting FAP to selectively induce cell death in these cells as well as in synovial tissue from RA patients. In PDT, a light-sensitive molecule is delivered to a target cell and activated with light of a specific wavelength. This causes cell death through the production of reactive oxygen species. <h3>Methods</h3> The anti-FAP antibody 28 H1 was conjugated with the photosensitizer IRDye700DX (28 H1–700DX). <i>In vitro</i> PDT assays were performed with 3 T3 fibroblasts stably transfected with FAP. 3T3-FAP cells were incubated with 28 H1–700DX or a control conjugate for 4 hours, and exposed to varying 690 nm light exposures. Subsequently, cell viability was measured using the CellTiter-Glo assay. For <i>ex vivo</i> evaluation of PDT efficiency, human RA synovial tissue obtained after joint replacement surgery was processed into standardised 6 mm biopsies and used for FAP-based PDT. The biopsies were incubated with 28 H1–700DX for 4 hours, subjected to 52 J/cm<sup>2</sup> light exposure and fixed in formalin after 1 hour. Tissue was then embedded in paraffin and stained for the presence of gH2AX and caspase 3 as indicators of DNA double-strand breaks and early apoptosis on sequential slides. The presence of FAP was also determined on subsequent slides. <h3>Results</h3> The effect of PDT was optimal at 13.7 J/cm<sup>2</sup> light exposure to 3T3-FAP cells incubated with 6.67 pM 28 H1–700DX, which dramatically reduced cell viability with 89.27%±2.48 compared to control (p&lt;0.001). No cell death was observed with the control 700DX-conjugate (p=0.16). In the PDT experiment on human RA synovial biopsies, the groups incubated with 28 H1–700DX and exposed to light showed apparent cell death in the synovial tissue as evidenced by the positive staining of both the gH2AX and caspase 3 markers (figure J and K). Staining of these markers co-localised with areas of high FAP staining (figure L). This was not the case in the control samples that were not exposed to either 28 H1–700DX and/or light (figure A – I). All biopsies did show FAP staining indicating that the cell death was only achieved when the biopsies were exposed to both the antibody and the light. <h3>Conclusions</h3> We have demonstrated fibroblast-specific cell death by targeted PDT using 700DX-conjugated 28 H1. Furthermore, we demonstrated that PDT also induces cell death of FAP-positive cells in synovial tissue from RA patients, suggesting FAP-targeted PDT as a promising new tool in treating RA. <h3>Disclosure of Interest</h3> None declared