Our previous study demonstrated that heat shock protein 90 (Hsp90) is overexpressed in the involved skin of patients with systemic sclerosis (SSc) and in experimental dermal fibrosis. Pharmacological inhibition of Hsp90 prevented the stimulatory effects of transforming growth factor-beta on collagen synthesis and the development of dermal fibrosis in three preclinical models of SSc. In the next step of the preclinical analysis, herein, we aimed to evaluate the efficacy of an Hsp90 inhibitor, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), in the treatment of established experimental dermal fibrosis induced by bleomycin. Treatment with 17-DMAG demonstrated potent antifibrotic and anti-inflammatory properties: it decreased dermal thickening, collagen content, myofibroblast count, expression of transforming growth factor beta receptors, and pSmad3-positive cell counts, as well as leukocyte infiltration and systemic levels of crucial cytokines/chemokines involved in the pathogenesis of SSc, compared to vehicle-treated mice. 17-DMAG effectively prevented further progression and may induce regression of established bleomycin-induced dermal fibrosis to an extent comparable to nintedanib. These findings provide further evidence of the vital role of Hsp90 in the pathophysiology of SSc and characterize it as a potential target for the treatment of fibrosis with translational implications due to the availability of several Hsp90 inhibitors in clinical trials for other indications.

Background:AX-202 is a monoclonal antibody that inhibits the bioactivity of S100A4. S100A4 is an alarm signal that is released from cells in response to stress or injury and functions as an amplifying mechanism of inflammation and fibrosis in the diseased tissue microenvironment. Previous in vitro studies have found that S100A4 induces fibroblast activation, sensitizes fibroblasts to the effects of TGFβ, drives epithelial-mesenchymal transition, and stimulates monocyte cytokine release (1-3). Moreover, S100A4-/- mice are protected from fibrosis in several animal models (1). In patients with systemic sclerosis (SSc), S100A4 is elevated both in lesional tissue and systemically and correlates with skin involvement, disease activity, and pulmonary function.Objectives:The aim of this study was to assess the antifibrotic effects of murine AX-202 in two pre-clinical models of SSs reflecting both inflammation-mediated and inflammation non-mediated fibrosis and confirm the in vivo activity of humanized AX-202.Methods:We first evaluated the effects of murine AX-202 in the bleomycin-induced skin fibrosis model and the tight-skin 1 (Tsk-1) model. In the bleomycin (BLM) model, fibrosis was induced by 3 weeks of BLM s.c. injections followed by 3 weeks of AX-202 treatment in parallel with continued BLM s.c. injections. The control groups included NaCl s.c. injections for 6 weeks, BLM s.c. injections for 6 weeks, or BLM s.c. injections for 3 weeks, followed by NaCl s.c. injections for 3 weeks. Three dosing regimens of AX-202 were tested: 3.75, 7.5, or 12.5 mg/kg i.p. every 3rd day. In the Tsk-1 model, treatment with 7.5 mg/kg i.p. every 3rd day was administered from week 5 until week 10. The control groups included pa mice, Tsk-1 mice, and Tsk-1 mice treated i.p. with isotype IgG. We subsequently evaluated the effects of humanized AX-202 in the model of BLM-induced skin fibrosis in a similar design as used for the murine AX-202 study. Three dosing regimens were tested: 8 mg/kg and 16 mg/kg i.p. every 3rd day and 24 mg/kg i.v. once weekly.Results:In the BLM model, murine AX-202 (7.5 mg/kg) was effective both in the prevention of progression of pre-established skin fibrosis and in the induction of regression of fibrosis as assessed by the dermal thickness (-55%, p<0.0001 vs BLM for 6 weeks, and -23%, p<0.0001 vs BLM for 3 weeks), myofibroblast count and hydroxyproline content. Murine AX-202 also ameliorated fibrosis in the Tsk-1 model as assessed by the hypodermal thickness (-24%, p=0.01 vs Tsk-1 isotype control), myofibroblast count, and hydroxyproline content. In both models, the antifibrotic effects were associated with a reduction in pSMAD3 expression. Humanized AX-202 was effective in the prevention of progression of pre-established skin fibrosis in all doses tested across all endpoints (dermal thickness, myofibroblast counts, hydroxyproline content). In the two groups treated with 16 mg/kg i.p. and 24 mg/kg i.v., humanized AX-202 also induced regression of fibrosis (-83%, p<0.001, and -61%, p<0.001 vs BLM for 3 weeks, respectively). Both murine and humanized AX-202 were well tolerated in all study groups in both models.Conclusion:We demonstrate that AX-202 confers potent antifibrotic effects in complementary models of SSc. These results confirm and expand previous data showing that inhibition of S100A4 by AX-202 is a promising potential therapeutic candidate for disease modification in SSc or other fibrotic conditions.References:[1]Tomcik M et al. S100A4 amplifies TGF-beta-induced fibroblast activation in systemic sclerosis. Ann Rheum Dis. 2015;74(9):1748-55.[2]Cerezo LA et al. The metastasis-associated protein S100A4 promotes the inflammatory response of mononuclear cells via the TLR4 signalling pathway in rheumatoid arthritis. Rheumatology (Oxford). 2014;53(8):1520-6.[3]Fei F, et al. Role of metastasis-induced protein S100A4 in human non-tumor pathophysiologies. Cell Biosci. 2017;7:64.Acknowledgements:The study was supported by Arxx Therapeutics and MHCR 023728.Disclosure of Interests:Michal Tomčík: None declared, Thuong Trinh-Minh: None declared, Cuong Tran Manh: None declared, Hana Štorkánová: None declared, Lenka Štorkánová: None declared, Ladislav Šenolt: None declared, Jörg Klingelhöfer Employee of: Arxx Therapeutics, Rizwan I Hussain Employee of: Arxx Therapeutics, Jonas Hallén Employee of: Arxx Therapeutics, Jörg H.W. Distler Shareholder of: the stock owner of 4D Science, Consultant of: Actelion, Active Biotech, Anamar, ARXX, Bayer Pharma, Boehringer Ingelheim, Celgene, Galapagos, GSK, Inventiva, JB Therapeutics, Medac, Pfizer, RuiYi and UCB, Grant/research support from: Anamar, Active Biotech, Array Biopharma, ARXX, aTyr, BMS, Bayer Pharma, Boehringer Ingelheim, Celgene, Galapagos, GSK, Inventiva, Novartis, Sanofi-Aventis, RedX, UCB

Objectives Our previous studies have demonstrated that the Damage Associated Molecular Pattern (DAMP) protein, S100A4, is overexpressed in the involved skin and peripheral blood of patients with systemic sclerosis (SSc). It is associated with skin and lung involvement and disease activity. Contrary, lack of S100A4 prevented the development of experimental dermal fibrosis. Herein we aimed to evaluate the effect of murine anti-S100A4 monoclonal antibody (mAb, 6B12) in the treatment of preestablished experimental dermal fibrosis. Methods The effects of 6B12 were assessed at therapeutic dosages in a modified bleomycin-induced dermal fibrosis mouse model by evaluating fibrotic (dermal thickness, proliferation of myofibroblasts, hydroxyproline content, phosphorylated (p)Smad3-positive cell count) and inflammatory (leukocytes infiltrating the lesional skin, systemic levels of selected cytokines and chemokines) outcomes, and transcriptional profiling (RNASeq). Results Treatment with 7.5 mg/kg 6B12 attenuated and might even reduce pre-existing dermal fibrosis induced by bleomycin as evidenced by reduction in dermal thickness, myofibroblast count, and collagen content. These antifibrotic effects were mediated by the downregulation of transforming growth factor-β/Smad signaling and partially by reducing the number of leukocytes infiltrating the lesional skin and decrease in the systemic levels of interleukin-1α, eotaxin, CCL2, and CCL5. Moreover, transcriptional profiling demonstrated that 7.5 mg/kg 6B12 also modulated several profibrotic and proinflammatory processes relevant to the pathogenesis of SSc. Conclusion Targeting S100A4 by the 6B12 mAb demonstrated potent antifibrotic and anti-inflammatory effects on bleomycin-induced dermal fibrosis and provided further evidence for the vital role of S100A4 in the pathophysiology of SSc.

Background:Our previous study demonstrated that Heat shock protein 90 (Hsp90) is overexpressed in the skin of patients with systemic sclerosis (SSc), in cultured SSc fibroblasts and preclinical models of SSc. HSP90 is a new regulator of canonical TGF-β signalling and its inhibition prevents the stimulatory effects of TGF-β on collagen synthesis and dermal fibrosis in three preclinical models of SSc.Objectives:Herein, we aimed to evaluate the efficacy of Hsp90 inhibitor (17-DMAG) in the treatment of established experimental dermal fibrosis induced by bleomycin.Methods:Design consisted of three control groups, I (NaCl-s.c./6 weeks), II (bleomycin-s.c./3w and NaCl-s.c./3w), III (bleomycin-s.c./6w), and 2 treatment groups (bleomycin-s.c./6w). During the last 3 weeks, one group was treated with 17-DMAG 0.5mg/kg-i.p. every third day, whereas one group (with nintedanib 50mg/kg-p.o. twice daily) served as a comparator with already published efficacy in this setting. Total of 40 BL6 mice were examined weekly for weight, activity and fur texture. The effects of 17-DMAG were determined by assessment of dermal thickness (HE-staining), collagen content (hydroxyproline assay), myofibroblast counts (α-SMA staining) and of 23 serum inflammatory cytokines/chemokines (Mouse-Cytokine-23-plex, Bio-Rad-Laboratories).Results:17-DMAG decreased dermal thickening by 53±3% (p<0.001) (nintedanib by 46±2%,p<0.001), collagen content by 48±5% (p=0.004) (nintedanib by 50±4%,p=0.003), myofibroblast counts by 42±9% (p<0.001) (nintedanib by 44±7%,p<0.001), and levels of IL-1α, IL-6, IL-12(p40), CXCL1, MCP-1, MIP-1α/β, RANTES (in all: p<0.05) compared to vehicle-treated mice injected with bleomycin for 6w. Moreover, 17-DMAG also induced regression of pre-established fibrosis to below the levels of vehicle-treated mice injected with bleomycin for 3w and NaCl for 3w (dermal thickness by 14±3%, collagen content by 20±5%, myofibroblast counts by 13±9%; whereas in nintedanib by 10±3%, 21±4%, 17±7%, respectively; in all: p<0.05), and levels of IL-12(p40), CXCL1, MCP-1, MIP-1β, RANTES (in all: p<0.05). No significant weight loss, decrease in activity or changes in fur texture were observed upon 17-DMAG treatment.Conclusion:This is the first study on effects of Hsp90 inhibitor 17-DMAG in the treatment of established dermal fibrosis. We demonstrate that 17-DMAG effectively prevents the progression and induces regression of established bleomycin-induced dermal fibrosis, in an extent that was comparable to nintedanib in this study (which was recently FDA approved for slowing the rate of decline in lung function in adults with SSc-ILD). 17-DMAG was well tolerated without obvious clinical signs of toxicity. These data suggest that Hsp90 could be a novel potential target in the treatment of SSc dermal fibrosis.Acknowledgments:Supported by AZV-16-33542A, MHCR023728, SVV260373, Boehringer Ingelheim.Disclosure of Interests:Hana Štorkánová: None declared, Lenka Štorkánová: None declared, Sabina Oreska: None declared, Maja Špiritović: None declared, Barbora Heřmánková: None declared, Radim Bečvář Consultant of: Actelion, Roche, Karel Pavelka Consultant of: Abbvie, MSD, BMS, Egis, Roche, UCB, Medac, Pfizer, Biogen, Speakers bureau: Abbvie, MSD, BMS, Egis, Roche, UCB, Medac, Pfizer, Biogen, Jiří Vencovský: None declared, Jörg Distler Grant/research support from: Boehringer Ingelheim, Consultant of: Boehringer Ingelheim, Paid instructor for: Boehringer Ingelheim, Speakers bureau: Boehringer Ingelheim, Ladislav Šenolt: None declared, Michal Tomcik: None declared