Fibrosis in rheumatic connective tissue diseases is marked by CD8+ T cell and pro-fibrotic myofibroblast infiltration, though the role of CD8+ T cells in myofibroblast activity remains unexplored. To address this, we developed a 3D cell culture model of immunity-driven fibrosis by combining the classic mixed lymphocyte reaction and a 3D myofibroblast contractility model. Upon co-culture with myofibroblasts, CD8+ T cells more strongly induced myofibroblast contraction and activation than CD4+ T cells. This was not associated with cytotoxicity but with increased IL-6 production by CD8+ T cells and pSTAT3 and TGFβ signaling in myofibroblasts. Use of the JAK/STAT3-inhibitor tofacitinib or the TGFβ receptor inhibitor SB-505124 inhibited the activated myofibroblast phenotype, and combined use of both inhibitors had a clear additive effect. Our findings reveal a previously underappreciated non-canonical role of CD8+ T cells in fibrosis, providing new light to the mechanisms of the human immune system.

In many autoimmune pathologies, including Rheumatoid Arthritis (RA), only a small percentage of the total B cell population is autoreactive and sustain disease. Yet, current immunotherapy treatments often eliminate the entire B-cell population, leading to immune deficiency. We developed an approach to selectively eliminate autoreactive B cells with targeted photodynamic therapy (tPDT). We designed a construct containing a dimeric peptidic antigen (diCCP4) that selectively binds a patient-derived autoreactive B cell receptor (BCR) and additionally included the photosensitizer IRDye700DX. We tested the construct on a modified Ramos B-cell line (Ramos 3F3), expressing this specific autoreactive BCR sequence. After brief exposure to 689 nm light, the photosensitizer selectively eliminates the modified Ramos cells, while the construct is not cytotoxic to cells lacking the autoreactive BCR. In a 3D coculture of the Ramos autoreactive B cell line with peripheral blood mononuclear cells (PBMCs) we observed only a minimal response of the untargeted cells. These results highlight the potential of tPDT against autoreactive B cells in autoimmune disease.

Background: In rheumatic diseases, like systemic sclerosis and rheumatoid arthritis, myofibroblasts drive fibrosis of tissues (e.g skin, lungs and synovium). This fibrosis is an important cause of morbidity and mortality via tissue stiffening and hardening leading to loss of organ function. In such affected tissues, increased presence of CD8+ T cells has been observed, but these cells have a yet unknown role in myofibroblast formation and activation. Objectives: Our purpose was to examine the role of CD8+ T cells in pro-fibrotic mediated myofibroblast activation and contraction in the developed humanized model of connective tissue disease pathology. Methods: Primary skin fibroblasts and peripheral blood mononuclear cells (PBMCs) or sorted CD8+ T cells were co-cultured in a 3D collagen type 1 hydrogel for up to 96 hours (n=20). Allogeneic mismatching was used as a model to study auto-immune connective tissue biology. T cell activation and cytokine expression was evaluated using flow cytometry. Fibroblast activation was analyzed using immunohistochemistry (IHC) and qPCR and activation-induced contraction was measured macroscopically. Cell death was evaluated using flow cytometry (7AAD/AnnexinV) and IHC (activated caspace-3 and γH2AX). Activation of intracellular signaling pathways in fibroblasts was measured using luciferase reporter cell lines. Results: Co-culture of fibroblasts with PBMCs strongly induced fibroblast contractility and this was further accompanied by an elevated myofibroblast phenotype as measured by increased expression of collagen type 1, fibroblast activation protein (FAP), αSMA and IL-6. Both CD4+ and CD8+ T cells were activated as a result of co-culture as identified by increased CD25 and CD69 expression and elevated IL-2 and IFN-γ production. Notably, enhanced fibroblast cell death was not observed, excluding cytolytic processes as a driving mechanism. Upon co-culture with either sorted CD4+ or CD8+ T cells, CD8+ T cells more strongly induced fibroblast contraction and activation than CD4+ T cells. This was associated with raised IL-6 production by CD8+ T cells and STAT3/TGFβ-induced signaling in fibroblasts. In addition, blocking cytokine release of CD8+ T cells with brefeldin A blocked the CD8+ T cell induced STAT3-signaling in fibroblasts but did not affect their cytolytic capacity. The use of JAK/STAT3-inhibitor tofacitinib or the TGFβ/SMAD2/3 inhibitor SB-505124 blocked the activated fibroblast phenotype. Notably, combined use of both inhibitors illustrated an additive effect and fully blocked myofibroblast activation and contraction. Conclusion: CD8+ T cells drive myofibroblast contraction and activation not via cytotoxicity related programs but via cytokine release. This sheds light on novel mechanisms of immune cell mediated tissue fibrosis. Furthermore, our results suggest that combining JAK/STAT3 inhibition with an anti-fibrotic agent might be promising in mitigating immune cell mediated tissue fibrosis in connective tissue rheumatic disorders and has added value over blocking these pathways individually. This observation is believed to facilitate clinical practice in cases of patients with a fibrotic signature (endotype) that is associated with refractory disease [1]. REFERENCES: [1] Rivellese, F., Surace, A.E.A., Goldmann, K. et al. Rituximab versus tocilizumab in rheumatoid arthritis: synovial biopsy-based biomarker analysis of the phase 4 R4RA randomized trial. Nat Med 28, 1256–1268 (2022). https://doi.org/10.1038/s41591-022-01789-0 Acknowledgements: NIL. Disclosure of Interests: None declared.

Background: Systemic sclerosis (SSc) is an autoimmune disease characterized by vasculopathy, fibrosis, and immune dysregulation. In SSc pathogenesis, circulating monocytes can be recruited to the skin and differentiate into macrophages. This possibly contributes to the activation and conversion of resident fibroblasts into myofibroblasts, driving fibrosis and skin thickness. However, how these cells exert their functions and via which fibroinflammatory mediator(s) is not fully understood. Objectives: To investigate the role of monocytes in mediating myofibroblast contraction and activation using an innovative 3D collagen hydrogel model. Methods: For our 3D skin model we cocultured human primary dermal fibroblasts with either peripheral blood mononuclear cells (PBMCs) or MACS-sorted CD14+ monocytes in a 3D collagen type 1 hydrogel. Subsequently, monocyte-driven tissue contraction was measured over time. We performed immunostainings for CD68, fibroblast activation protein (FAP), and alpha-smooth muscle actin (α-SMA) to evaluate monocyte/macrophage and fibroblast activation. To investigate the signaling pathways involved in the observed myofibroblast activation, we measured transcription factor-driven luciferase production using reporter constructs in the same dermal fibroblasts. The activity of the following reporter constructs was determined after 24 hours: Sis-Inducible Element (SIE); SMAD-Binding Element (SBE); Nuclear Factor of Activated T-cells 5 Response Element (NFAT-5), and NFκB Response Element (NFκB). Results: After 60 hours of co-culture, hydrogel plugs containing fibroblasts + monocytes displayed a strong spontaneous contraction, seen by a (approx. 80-90%) decrease in the area of the plugs. Hydrogels containing only fibroblasts did not contract. Hydrogels with fibroblasts + monocytes contracted as fast as fibroblasts + PBMCs, but depletion of CD14+ cells from PBMCs slowed down contraction, showing that monocytes strongly activated fibroblasts. The expression of FAP and α-SMA by fibroblasts increased in monocyte-containing hydrogels, compared to fibroblasts cultured without monocytes. Furthermore, CD68 expression also increased in co-culture, indicating enhanced monocyte differentiation/activation into macrophages by fibroblasts. Evaluating which intracellular pathway lead to fibroblast activation, we observed that SIE and NFκB reporter fibroblast constructs were strongly elevated in the presence of monocytes. However, we could not observed enhanced TGF-β activity using the SBE reporter. Together, our results suggest that inflammatory mediators like IL-6, IL-1 and S100A8/9 may contribute to the monocytes-driving myofibroblast contraction. Conclusion: This study highlights the importance of monocytes and the SIE and NFκB intracellular signaling pathways in myofibroblast contraction in a 3D skin model, contributing to understanding the basic mechanisms of these cells in SSc skin fibrosis and thickness. REFERENCES: [1] van Caam, Arjan et al. "Unraveling SSc Pathophysiology; The Myofibroblast." Frontiers in immunology vol. 9 2452. 13 Nov. 2018, doi:10.3389/fimmu.2018.02452. [2] Al-Adwi, Yehya et al. "Macrophages as determinants and regulators of fibrosis in systemic sclerosis." Rheumatology (Oxford) vol. 62,2 (2023): 535-545. Acknowledgements: Grant #2023/04897-7, São Paulo Research Foundation (FAPESP). Disclosure of Interests: None declared.

Patients with pancreatic ductal adenocarcinoma (PDAC) have a dismal 5 year survival of 9%. One important limiting factor for treatment efficacy is the dense tumor-supporting stroma. The cancer-associated fibroblasts in this stroma deposit excessive amounts of extracellular matrix components and anti-inflammatory mediators, which hampers the efficacy of chemo- and immunotherapies. Systemic depletion of all activated fibroblasts is, however, not feasible nor desirable and therefore a local approach should be pursued. Here, we provide a proof-of-principle of using fibroblast activation protein (FAP)-targeted photodynamic therapy (tPDT) to treat PDAC. FAP-targeting antibody 28H1 and irrelevant control antibody DP47GS were conjugated to the photosensitizer IRDye700DX (700DX) and the chelator diethylenetriaminepentaacetic acid. In vitro binding and cytotoxicity were evaluated using the fibroblast cell-line NIH-3T3 stably transfected with FAP. Biodistribution of 111In-labeled antibody-700DX constructs was determined in mice carrying syngeneic tumors of the murine PDAC cell line PDAC299, and in a genetically engineered PDAC mouse model (CKP). Then, tPDT was performed by exposing the subcutaneous or the spontaneous PDAC tumors to 690 nm light. Induction of apoptosis after treatment was assessed using automated analyses of immunohistochemistry for cleaved caspase-3. 28H1-700DX effectively bound to 3T3-FAP cells and induced cytotoxicity upon exposure to 690 nm light, whereas no binding or cytotoxic effects were observed for DP47GS-700DX. Although both 28H1-700DX and DP47GS-700DX accumulated in subcutaneous PDAC299 tumors, autoradiography demonstrated that only 28H1-700DX reached the tumor core. On the contrary, control antibody DP47GS-700DX was only present at the tumor rim. In CKP mice, both antibodies accumulated in the tumor, but tumor-to-blood ratios of 28H1-700DX were higher than that of the control. Notably, in vivo FAP-tPDT caused upregulation of cleaved caspase-3 staining in both subcutaneous and in spontaneous tumors. In conclusion, we have shown that tPDT is a feasible approach for local depletion of FAP-expressing stromal cells in murine models for PDAC.

Background Rheumatoid arthritis (RA) is one of the most prevalent and debilitating joint diseases worldwide. RA is characterized by synovial inflammation (synovitis), which is linked to the development of joint destruction. Magnetic resonance imaging and ultrasonography are widely being used to detect the presence and extent of synovitis. However, these techniques do not reveal the activation status of inflammatory cells such as macrophages that play a crucial role in synovitis and express CD64 (Fc gamma receptor (FcγR)I) which is considered as macrophage activation marker. Objectives We aimed to investigate CD64 expression and its correlation with pro-inflammatory cytokines and pro-damaging factors in human-derived RA synovium. Furthermore, we aimed to set up a molecular imaging modality using a radiolabeled CD64-specific antibody as a novel imaging tracer that could be used to determine the extent and phenotype of synovitis using optical and nuclear imaging. Methods First, we investigated CD64 expression in synovium of early- and late-stage RA patients and studied its correlation with the expression of pro-inflammatory and tissue-damaging factors. Next, we conjugated an anti-CD64 antibody with IRDye 800CW and diethylenetriamine penta-acetic acid (DTPA; used for 111 In labeling) and tested its binding on cultured THP1 cells, ex vivo RA synovium explants and its imaging potential in SCID mice implanted with human RA synovium explants obtained from RA patients who underwent total joint replacement. Results We showed that CD64 is expressed in synovium of early and late-stage RA patients and that FCGR1A /CD64 expression is strongly correlated with factors known to be involved in RA progression. Combined, this makes CD64 a useful marker for imaging the extent and phenotype of synovitis. We reported higher binding of the [ 111 In]In-DTPA-IRDye 800CW anti-CD64 antibody to in vitro cultured THP1 monocytes and ex vivo RA synovium compared to isotype control. In human RA synovial explants implanted in SCID mice, the ratio of uptake of the antibody in synovium over blood was significantly higher when injected with anti-CD64 compared to isotype and injecting an excess of unlabeled antibody significantly reduced the antibody-binding associated signal, both indicating specific receptor binding. Conclusion Taken together, we successfully developed an optical and nuclear imaging modality to detect CD64 in human RA synovium in vivo.

Novel photosensitizers with inherently "drug-like" structures are essential for the development of new therapies for challenging diseases. LightOx has developed a novel class of donor-acceptor diarylacetylene [1] that elicits the generation of radical oxygen species (ROS) in cells when activated by 360-430 nm light. We sought to apply these photosensitizers to PDT of fibrotic skin diseases, but initially found that light of this wavelength, alone, already causes a cytotoxic effect in the cultured 3T3 fibroblast cell line as well as in primary dermal fibroblast cells, whereas tissue explants did not show these effects. Herein we report an investigation into an experimental approach that enables the in vitro study of PDT on fibroblasts using blue light. 3T3 cells stably transfected with FAP (3T3-FAP, Roche, PETR4906) and primary dermal fibroblasts were incubated with two different diarylacetylene photosensitizers (compound 1 and 2, LightOx Ltd., UK), counterstained with Didye (DID, Thermofisher) and uptake of the PS was visualized using confocal microscopy. For PDT the cells were exposed to light of 405 or 430 nm wavelength after PS incubation. Cell viability was subsequently measured using the XTT assay. To adjust the ROS/ antioxidant balance, either Vitamin C or uric acid were added prior to light exposure. In addition to cultured fibroblasts, skin from mice with bleomycin-induced skin fibrosis was also exposed to 405 nm light. The paraffin-embedded and formalin-fixed tissue was subsequently stained for apoptosis using an antibody targeting cleaved caspase-3 (9661S, Cell signalling technologies). Both fibroblast types showed comparable PS uptake, with pronounced accumulation of the photosensitizer in the cytoplasm of the cells. For compound 1, dark-toxicity was observed at the highest PS dose (50 µM), but lower doses showed healthy cells with bright cytoplasmic PS fluorescence. By comparison, the signal from compound 2 was less bright, but did not show dark-toxicity. As described previously, light in the 405 nm range was toxic to fibroblasts [2]. To overcome this in vitro problem, antioxidants were added to the medium right before light exposure. Vitamin C addition did not improve cell survival, but the addition of uric acid did prevent light-induced toxicity (102±8.1% live cells versus 14,2±1.6% for cells in medium with and without uric acid, followed by 2.5 J/cm2 405 nm light exposure, respectively). By pre-incubating these cells with the PS we could induce PS specific cytotoxicity (8.3±0.9% live cells remaining after light exposure), paving the way for the in vitro evaluation of these types of PS for PDT of UV sensitive cells. Importantly, no increase in apoptosis was observed in fibrotic skin explants after 405 nm light exposure.

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.

The cancer associated fibroblast (CAF) orchestrates an aberrant tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC), contributing to poor patient survival. Fibroblast activation protein (FAP)-targeted depletion of the CAF in PDAC could modulate the tumor microenvironment and make it more susceptible to immune response or systemic therapies. Here, we developed a FAP-targeting minibody conjugated to the photosensitizer IRDye700DX for ablation of the CAF with photodynamic therapy. We characterized efficiency of the minibody-IRDye700X in vitro and in vivo in a murine subcutaneous PDAC model.

Fibroblast activation protein (FAP), expressed on cancer-associated fibroblasts, is a target for diagnosis and therapy in multiple tumour types. Strategies to systemically deplete FAP-expressing cells show efficacy; however, these induce toxicities, as FAP-expressing cells are found in normal tissues. FAP-targeted photodynamic therapy offers a solution, as it acts only locally and upon activation. Here, a FAP-binding minibody was conjugated to the chelator diethylenetriaminepentaacetic acid (DTPA) and the photosensitizer IRDye700DX (DTPA-700DX-MB). DTPA-700DX-MB showed efficient binding to FAP-overexpressing 3T3 murine fibroblasts (3T3-FAP) and induced the protein’s dose-dependent cytotoxicity upon light exposure. Biodistribution of DTPA-700DX-MB in mice carrying either subcutaneous or orthotopic tumours of murine pancreatic ductal adenocarcinoma cells (PDAC299) showed maximal tumour uptake of 111In-labelled DTPA-700DX-MB at 24 h post injection. Co-injection with an excess DTPA-700DX-MB reduced uptake, and autoradiography correlated with FAP expression in the stromal tumour region. Finally, in vivo therapeutic efficacy was determined in two simultaneous subcutaneous PDAC299 tumours; only one was treated with 690 nm light. Upregulation of an apoptosis marker was only observed in the treated tumours. In conclusion, DTPA-700DX-MB binds to FAP-expressing cells and targets PDAC299 tumours in mice with good signal-to-background ratios. Furthermore, the induced apoptosis indicates the feasibility of targeted depletion of FAP-expressing cells with photodynamic therapy.

Abstract Objective Activated synovial fibroblasts are key effector cells in 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-FAPI-04 in a 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 flow cytometry on primary fibroblasts and co-cultured macrophages. Additionally, one RA patient intravenously received 68Ga-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.

Background Systemic sclerosis (SSc) is a rare, severe auto-immune disease characterized by inflammation, vasculopathy and fibrosis. Activated (myo)fibroblasts are crucial drivers of fibrosis. By exploiting their expression of fibroblast activation protein (FAP) to perform targeted photodynamic therapy (tPDT), we can locoregionally deplete these pathogenic cells. Objectives We explored the use of FAP-tPDT to selectively target primary skin fibroblasts from SSc patients, both in 2D and 3D cultures, as well as in biopsies from fibrotic skin lesions in the murine bleomycin-induced skin fibrosis model. Methods The FAP targeting monoclonal antibody (clone 28H1) was conjugated with the photosensitizer IRDye700DX. Primary skin fibroblasts were obtained from lesional skin biopsies of SSc patients via spontaneous out-growth and subsequently cultured on plastic or collagen type I. For 2D FAP-tPDT, cells were incubated in buffer with or without the antibody-photosensitizer (Ab-PS) construct, washed after 4 h and exposed to λ = 689 nm light (50 J/cm 2 at 280 mW/cm 2 ). 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. Skin fibrosis was induced in mice by 3x/week injections of 1.5 IU bleomycin intradermally on the back of the mouse. After 5 weeks the mice were sacrificed and biopsies were taken from affected and not affected skin. These were incubated with or without the FAP targeting Ab-PS construct or a control Ab-PS. After washing, the biopsies were exposed to light, incubated for 1h and subsequently formalin fixed and paraffin embedded. Sections were immuno stained for the presence of cleaved caspase-3. Results FAP-tPDT resulted in antibody-dose dependent cytotoxicity in primary skin fibroblasts upon light exposure. Cells not exposed to light or incubated with a control Ab-PS 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. In fibrotic skin biopsies from mice with bleomycin-induced skin fibrosis, an upregulation of apoptosis, as evidenced by increased caspase-3 staining (Figure 1), was observed in response to FAP-tPDT. The same treatment on biopsies of control skin did not increase caspase-3 staining, nor did incubation with a control Ab-PS construct or light alone. Figure 1. Cleaved caspase-3 staining of biopsies from the skin of mice with bleomycin-induced dermal fibrosis upon treatment with FAP-tPDT. The red arrows indicate the presence of positive fibroblasts. Conclusion Here we have shown, for the first time, the potential of FAP-tPDT for the selective targeting of pathogenic fibroblasts and treatment of fibrosis in SSc skin both in relevant patient-derived culture models as well as an in vivo model of fibrosis. Disclosure of Interests Daphne Dorst: None declared, Arjan van Caam: None declared, Elly Vitters: None declared, Birgitte Walgreen: None declared, Monique Helsen: None declared, Christian Klein Employee of: CK is an employee of Roche pharmaceutics, Shreya Gudi: None declared, Tirza Wubs: None declared, Jyoti Kumari: None declared, Przemysław Błyszczuk: None declared, Madelon Vonk: None declared, Peter van der Kraan: None declared, Marije Koenders: None declared.

BackgroundSystemic sclerosis (SSc) is a rare, severe auto-immune disease characterized by inflammation, vasculopathy and fibrosis. Activated (myo)fibroblasts are crucial drivers of fibrosis. By exploiting their expression of fibroblast activation protein (FAP) to perform targeted photodynamic therapy (tPDT), we can locoregionally deplete these pathogenic cells.ObjectivesWe explored the use of FAP-tPDT to selectively target primary skin fibroblasts from SSc patients, both in 2D and 3D cultures, as well as in biopsies from fibrotic skin lesions in the murine bleomycin-induced skin fibrosis model.MethodsThe FAP targeting monoclonal antibody (clone 28H1) was conjugated with the photosensitizer IRDye700DX. Primary skin fibroblasts were obtained from lesional skin biopsies of SSc patients via spontaneous out-growth and subsequently cultured on plastic or collagen type I. For 2D FAP-tPDT, cells were incubated in buffer with or without the antibody-photosensitizer (Ab-PS) construct, washed after 4 h and exposed to λ = 689 nm light (50 J/cm2 at 280 mW/cm2). 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. Skin fibrosis was induced in mice by 3x/week injections of 1.5 IU bleomycin intradermally on the back of the mouse. After 5 weeks the mice were sacrificed and biopsies were taken from affected and not affected skin. These were incubated with or without the FAP targeting Ab-PS construct or a control Ab-PS. After washing, the biopsies were exposed to light, incubated for 1h and subsequently formalin fixed and paraffin embedded. Sections were immuno stained for the presence of cleaved caspase-3.ResultsFAP-tPDT resulted in antibody-dose dependent cytotoxicity in primary skin fibroblasts upon light exposure. Cells not exposed to light or incubated with a control Ab-PS 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. In fibrotic skin biopsies from mice with bleomycin-induced skin fibrosis, an upregulation of apoptosis, as evidenced by increased caspase-3 staining (Figure 1), was observed in response to FAP-tPDT. The same treatment on biopsies of control skin did not increase caspase-3 staining, nor did incubation with a control Ab-PS construct or light alone.Figure 1.Cleaved caspase-3 staining of biopsies from the skin of mice with bleomycin-induced dermal fibrosis upon treatment with FAP-tPDT. The red arrows indicate the presence of positive fibroblasts.ConclusionHere we have shown, for the first time, the potential of FAP-tPDT for the selective targeting of pathogenic fibroblasts and treatment of fibrosis in SSc skin both in relevant patient-derived culture models as well as an in vivo model of fibrosis.Disclosure of InterestsDaphne Dorst: None declared, Arjan van Caam: None declared, Elly Vitters: None declared, Birgitte Walgreen: None declared, Monique Helsen: None declared, Christian Klein Employee of: CK is an employee of Roche pharmaceutics, Shreya Gudi: None declared, Tirza Wubs: None declared, Jyoti Kumari: None declared, Przemysław Błyszczuk: None declared, Madelon Vonk: None declared, Peter van der Kraan: None declared, Marije Koenders: None declared.

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.

Abstract 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.

Background Many experiments to study inflammation, hyperplasia, and fibrosis in the synovium have been performed in animal models of RA and OA. However, the predictive value of these models for the screening of potential drugs in RA is variable and for OA, none were sufficiently effective in clinical trials. Translational arthritis research with human cells is often performed in monolayer culture where the absence of extracellular matrix and other cell types results in alterations of cell functions and loss of phenotype. Objectives To improve the predictive value of preclinical arthritis research by developing and optimizing innovative translational models to study human synovial pathology in vitro and in vivo. Methods Synovial biopsies from RA patients were obtained during joint replacement surgery and processed for either (1) explant cultures, (2) 3D-synovial micromasses, (3) RA-SCID transplantation studies, and/or (4) a biobank for corresponding mRNA and IHC profiling. For explant culture, 3mm biopsies were cultured for 24hr w/o various inhibitors, and cytokine production was analyzed by Luminex. 3D-micromasses were generated from primary RA FLS and CD14+ PBMCs, stimulated for 3 weeks with 10 ng/ml TNFα or TGFβ and analyzed by histology, IHC and QPCR. For target validation and preclinical imaging, 6mm biopsies were engrafted SC into SCID mice. Radionuclide- and fluorescently-labelled anti-CD64 antibodies were injected IV, and targeting was determined by biodistribution, µSPECT/CT and IVIS imaging analysis. Results Our first assay with RA synovium explants demonstrated to be highly suitable to test the therapeutic efficacy of inhibitors for TNFα, TLR4, p38 and the JAK-pathway, resulting in significantly reduced production of proinflammatory mediators after 24hr of culture. In contrast to the explant cultures, our 3D synovial micromasses could be followed for weeks. In this second translational model, lining formation was observed at day 7 and the micromasses could be stimulated to mimic RA- or OA-like features of synovial hyperplasia or fibrosis respectively. Long-term exposure to the RA-related cytokine TNFα lead to hyperplasia of the lining and an altered macrophage phenotype characterized by reduced CD163 expression. Conversely, the repair-related growth factor TGFβ induced fibrosis-like changes in the micromass lining, a hallmark of OA. This was accompanied by an increased expression of PLOD2, COL1A1 and αSMA. Our third preclinical model, the RA synovium SCID mouse, was previously validated using adalimumab, secukinumab, and rituximab, and was now used to study CD64 as a potential marker to image synovitis. Gene expression of FCGRI (CD64) in synovial explants from RA patients was shown to correlate positively to gene expression of pro-inflammatory factors IL1B, TNFA, IL8, S100A8, MCP1, and with damage-associated genes MMP2 and MMP13. Interestingly, dual-labelled 111In-DTPA-IrDye800CW-anti-CD64 antibody showed high uptake in the synovial transplants of the SCID mice, and specifically visualized the subcutaneous synovial grafts by both µSPECT/CT and IVIS imaging. Conclusion The development of these translational models allows us to bridge the gap between preclinical and clinical drug development research. Whereas our synovial explant assay is ideal for short-term interventions, and the RA-SCID mouse a great translational model for in vivo preclinical studies, donor variability and access to sufficient tissue may be challenging. In such cases, the 3D synovial micromass model may be an excellent alternative. Especially in combined setting, these 3 translational approaches will improve target validation and preclinical development of novel anti-rheumatic drugs. Reference [1] Abdollahi-Roodsaz, J Clin Invest2008; Koenders, Arthritis Rheum 2012; Broeren, ALTEX 2019 Disclosure of Interests None declared

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 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. Disclosure of Interests: 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