Objective In RA, synovial fibroblasts become activated. These cells express fibroblast activation protein (FAP) and contribute to the pathogenesis by producing cytokines, chemokines and proteases. Selective depletion in inflamed joints could therefore constitute a viable treatment option. To this end, we developed and tested a new therapeutic strategy based on the selective destruction of FAP-positive cells by targeted photodynamic therapy (tPDT) using the anti-FAP antibody 28H1 coupled to the photosensitizer IRDye700DX. Methods After conjugation of IRDye700DX to 28H1, the immunoreactive binding and specificity of the conjugate were determined. Subsequently, tPDT efficiency was established in vitro using a 3T3 cell line stably transfected with FAP. The biodistribution of [111In]In-DTPA-28H1 with and without IRDye700DX was assessed in healthy C57BL/6N mice and in C57BL/6N mice with antigen-induced arthritis. The potential of FAP-tPDT to induce targeted damage was determined ex vivo by treating knee joints from C57BL/6N mice with antigen-induced arthritis 24 h after injection of the conjugate. Finally, the effect of FAP-tPDT on arthritis development was determined in mice with collagen-induced arthritis. Results 28H1-700DX was able to efficiently induce FAP-specific cell death in vitro. Accumulation of the anti-FAP antibody in arthritic knee joints was not affected by conjugation with the photosensitizer. Arthritis development was moderately delayed in mice with collagen-induced arthritis after FAP-tPDT. Conclusion Here we demonstrate the feasibility of tPDT to selectively target and kill FAP-positive fibroblasts in vitro and modulate arthritis in vivo using a mouse model of RA. This approach may have therapeutic potential in (refractory) arthritis.

Objective Activated synovial fibroblasts are key effector cells in rheumatoid arthritis (RA). Selectively depleting these based upon their expression of fibroblast activation protein (FAP) is an attractive therapeutic approach. Here we introduce FAP imaging of inflamed joints using [68Ga]Ga-FAPI-04 in an RA patient, and aim to assess feasibility of anti-FAP targeted photodynamic therapy (FAP-tPDT) ex vivo using 28H1-IRDye700DX on RA synovial explants. Methods Remnant synovial tissue from RA patients was processed into 6 mm biopsies and, from several patients, into primary fibroblast cell cultures. Both were treated using FAP-tPDT. Cell viability was measured in fibroblast cultures and biopsies were evaluated for histological markers of cell damage. Selectivity of the effect of FAP-tPDT was assessed using flowcytometry on primary fibroblasts and co-cultured macrophages. Additionally, one RA patient intravenously received [68Ga]Ga-FAPI-04 and was scanned using PET/CT imaging. Results In the RA patient,FAPI-04 PET imaging showed high accumulation of the tracer in arthritic joints with very low background signal. In vitro, FAP-tPDT induced cell death in primary RA synovial fibroblasts in a light dose dependent manner. An upregulation of cell damage markers was observed in the synovial biopsies after FAP-tPDT. No significant effects of FAP-tPDT were noted on macrophages after FAP-tPDT of neighbouring fibroblasts. Conclusion In this study the feasibility of selective FAP-tPDT in synovium of rheumatoid arthritis patients ex vivo is demonstrated. Furthermore, this study provides the first indication that FAP-targeted PET/CT can be used to image arthritic joints, an important step towards application of FAP-tPDT as a targeted locoregional therapy for RA.

Lipopolysaccharides or endotoxins elicit an excessive host inflammatory response and lead to life-threatening conditions such as endotoxemia and septic shock. Lipopolysaccharides trigger mobilization and stimulation of leukocytes and exaggerated production of pro-inflammatory molecules including cytokines and proteolytic enzymes. Matrix metalloproteinase-9 (MMP-9) or gelatinase B, a protease stored in the tertiary granules of polymorphonuclear leukocytes, has been implicated in such inflammatory reactions. Moreover, several studies even pinpointed MMP-9 as a potential target molecule to counter excessive inflammation in endotoxemia. Whereas the early effect of lipopolysaccharide-induced inflammation in vivo on the expression of MMP-9 in various peripheral organs has been described, the effects on the bone marrow and during late stage endotoxemia remain elusive. We demonstrate that TIMP-free MMP-9 is a major factor in bone marrow physiology and pathology. By using a mouse model for late-stage endotoxemia, we show that lipopolysaccharides elicited a depletion of neutrophil MMP-9 in the bone marrow and a shift of MMP-9 and MMP-9-containing cells towards peripheral organs, a pattern which was primarily associated with a relocation of CD11bhighGr-1high cells. In contrast, analysis of the tissue inhibitors of metalloproteinases was in line with a natural, systematic upregulation of TIMP-1, the main tissue inhibitor of TIMP-free MMP-9, and a general shift toward control of matrix metalloproteinase activity by tissue inhibitors of metalloproteinases.

Systemic sclerosis (SSc) is a rare autoimmune disease with limited treatment options that is characterized by fibrosis in various organs. To screen the effectiveness of new therapies, there is an urgent need for reliable in vitro models. Key is that diseased cells' characteristics are maintained, which is challenging in currently used setups. In this study, an in vitro 3D culture system is described using the biocompatible polyisocyanide (PIC‐RGD) hydrogel and SSc patient‐derived fibroblasts from affected (lesional cells) and from healthy‐skin (healthy cells). In contrast to the standard collagen‐coated 2D cultures, the cells in the 3D PIC‐RGD gels maintain the native phenotype and functionality of the primary cells. The functionality of the model is studied in the presence of the fibrosis stimulator transforming growth factor β1 (TGFβ1) and the suppressor tumor necrosis factor (TNFα). In this study, it is observed that lesional cells have a stronger fibrotic character with increased contraction, proliferation, and expression of collagen, and myofibroblast markers α‐smooth muscle actin and fibroblast activation protein. The high tunability of the hydrogel, which can maintain the native functionality of fibroblasts in in vitro cultures, delivers a crucial step in developing these materials into an effective tool for personalized medicine approaches of SSc patients.