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.

Macrophages play a crucial role in the initiation and progression of rheumatoid arthritis (RA). Liposomes can be used to deliver therapeutics to macrophages by exploiting their phagocytic ability. However, since macrophages serve as the immune system’s first responders, it is inadvisable to systemically deplete these cells. By loading the liposomes with the photosensitizer IRDye700DX, we have developed and tested a novel way to perform photodynamic therapy (PDT) on macrophages in inflamed joints. PEGylated liposomes were created using the film method and post-inserted with micelles containing IRDye700DX. For radiolabeling, a chelator was also incorporated. RAW 264.7 cells were incubated with liposomes with or without IRDye700DX and exposed to 689 nm light. Viability was determined using CellTiterGlo. Subsequently, biodistribution and PDT studies were performed on mice with collagen-induced arthritis (CIA). PDT using IRDye700DX-loaded liposomes efficiently induced cell death in vitro, whilst no cell death was observed using the control liposomes. Biodistribution of the two compounds in CIA mice was comparable with excellent correlation of the uptake with macroscopic and microscopic arthritis scores. Treatment with 700DX-loaded liposomes significantly delayed arthritis development. Here we have shown the proof-of-principle of performing PDT in arthritic joints using IRDye700DX-loaded liposomes, allowing locoregional treatment of arthritis.

Systemic sclerosis (SSc) is a rare, severe, auto-immune disease characterized by inflammation, vasculopathy and fibrosis. Activated (myo)fibroblasts are crucial drivers of this fibrosis. By exploiting their expression of fibroblast activation protein (FAP) to perform targeted photodynamic therapy (tPDT), we can locoregionally deplete these pathogenic cells. In this study, we explored the use of FAP-tPDT in primary skin fibroblasts from SSc patients, both in 2D and 3D cultures. Method: The FAP targeting antibody 28H1 was conjugated with the photosensitizer IRDye700DX. Primary skin fibroblasts were obtained from lesional skin biopsies of SSc patients via spontaneous outgrowth and subsequently cultured on plastic or collagen type I. For 2D FAP-tPDT, cells were incubated in buffer with or without the antibody-photosensitizer construct, washed after 4 h and exposed to λ = 689 nm light. Cell viability was measured using CellTiter Glo®®. For 3D FAP-tPDT, cells were seeded in collagen plugs and underwent the same treatment procedure. Contraction of the plugs was followed over time to determine myofibroblast activity. Results: FAP-tPDT resulted in antibody-dose dependent cytotoxicity in primary skin fibroblasts upon light exposure. Cells not exposed to light or incubated with an irrelevant antibody-photosensitizer construct did not show this response. FAP-tPDT fully prevented contraction of collagen plugs seeded with primary SSc fibroblasts. Even incubation with a very low dose of antibody (0.4 nM) inhibited contraction in 2 out of 3 donors. Conclusions: Here we have shown, for the first time, the potential of FAP-tPDT for the treatment of fibrosis in SSc skin.

Background: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.Objectives: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.Methods: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 [111In] 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.Results: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/cm2 radiant exposure, 89.24% ± 3.67% of fibroblasts died in the group incubated with antibody compared to control incubated with buffer only (p<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<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<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.Conclusion:Here we demonstrated the feasibility of ...

BackgroundActivated 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.MethodsThe anti-FAP antibody 28 H1 was conjugated with the photosensitizer IRDye700DX (28 H1–700DX). In vitro 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 ex vivo 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/cm2 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.ResultsThe effect of PDT was optimal at 13.7 J/cm2 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<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.ConclusionsWe 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.Disclosure of InterestNone declared