Inhibition of synovitis and joint destruction by a new single domain antibody specific for cyclophilin A in two different mouse models of rheumatoid arthritis

Li Wang 1 Junfeng Jia 0 Conghua Wang 0 Xiaokui Ma Chenggong Liao 1 Zhiguang Fu 1 Bin Wang 1 Xiangmin Yang 1 Ping Zhu 0 Yu Li 1 Zhinan Chen 1 0 Department of Clinical Immunology, First Affiliated Hospital, Fourth Military Medical University , No. 15 West Changle Road, Xi'an 710032, Shaanxi , PR China 1 Cell Engineering Research Centre, Cancer Biology of State Key Laboratory, Fourth Military Medical University , No.17 West Changle Road, Xi'an 710032, Shaanxi , PR China Introduction: Cyclophilin A (CypA) is implicated in rheumatoid arthritis (RA) pathogenesis. We studied whether a novel anti-CypA single domain antibody (sdAb) treatment would modulate the severity of the disease in two different animal models of RA. Methods: A novel sdAb, named sdAbA1, was screened from an immunized camel sdAb library and found to have a high binding affinity (KD = 6.9 10-9 M) for CypA. The SCID-HuRAg model and the collagen-induced arthritis (CIA) in mice were used to evaluate the effects of sdAbA1 treatment on inflammation and joint destruction. For in vitro analysis, monocytes/macrophages were purified from synovial fluid and peripheral blood of patients with RA and were tested for the effect of anti-CypA sdAb on metalloproteinase (MMP) production. Human monocyte cell line THP-1 cells were selected and western blot analyses were performed to examine the potential signaling pathways. Results: In the CIA model of RA, the sdAbA1 treatment resulted in a significant decrease in clinical symptoms as well as of joint damage (P <0.05). In the SCID-HuRAg model, treatment with anti-CypA antibody sdAbA1 significantly reduced cartilage erosion, inflammatory cell numbers and MMP-9 production in the implanted tissues (P <0.05). It also significantly reduced the levels of human inflammatory cytokines IL-6 and IL-8 in mouse serum (P <0.05). No toxic effects were observed in the two animal models. In vitro results showed that sdAbA1 could counteract CypA-dependent MMP-9 secretion and IL-8 production by interfering with the ERK-NF-B pathway. Conclusions: Blockade of CypA significantly inhibited synovitis and cartilage/bone erosion in the two tested animal models of RA. Our findings provide evidence that sdAbA1 may be a potential therapeutic agent for RA. - Introduction Rheumatoid arthritis (RA) is a chronic and debilitating disease of the joints characterized by synovial inflammation and progressive destruction of articular cartilage and bone [1]. The number of inflammatory cells and the level of inflammatory cytokines in the joints correlate with the extent of synovitis, and matrix metalloproteinases (MMPs) at the cartilagepannus junction of RA patients are the main proteases involved in the invasion and degradation of cartilage [2]. In RA, the number of monocytes/macrophages, which secrete multiple cytokines [3] and MMPs, is significantly increased in both the lining and sublining areas of the RA synovium, where they play a critical role in inflammation and joint destruction. Cyclophilins are a novel family of proteins exerting potent chemotactic capacity that have been well researched recently. Cyclophilins are widely expressed intracellular proteins, well known as receptors for the immunosuppressive drug cyclosporine A (CsA). Cyclophilin A (CypA) is the most abundant cyclophilin and can be actively released into extracellular tissue spaces in response to inflammatory stimuli [4]. Extracellular CypA is not only a strong in vitro chemoattractant for neutrophils, T cells and monocytes, but can also induce a rapid influx of leukocytes in vivo [5]. High levels of CypA have been detected in the serum and synovial fluid of RA patients and the amount of CypA was closely related to disease severity [6]. In RA synovium, CypA has been detected in most of macrophages in the lining layer and sublining area, and CypA staining overlaps with MMP-9 staining [7]. Our previous study claims that CypA upregulates MMP-9 expression and adhesion of monocytes/macrophages [8], and may aggravate cartilage erosion when injected in vivo [9]. Taken together, these results show that CypA plays an important role in the pathogenesis of RA and that reagents targeting CypA could be beneficial in the treatment of this disease. Monoclonal antibodies have played vital roles in antibody-based therapies against various diseases including RA [10]. Nevertheless, difficulties such as the costs of production and protein stability can be encountered. Recent major progress involved the use of single-domain antibodies (sdAbs) derived from camelids. In addition to conventional antibodies, camelids produce antibodies composed of heavy chains only, with a single variable domain (referred to as VHH) capable of recognizing specific antigens. These variable domains, named sdAbs, are the smallest naturally occurring, intact antigen-binding units and are highly valuable for their unique features. SdAbs are easily produced by bacterial fermentation. This approach implies an estimated 10-fold reduction in production costs as compared with conventional therapeutic antibodies, which are all produced in mammalian systems [11]. The long CDR3 in sdAbs recognizes structures such as pockets and clefts on the surface of antigens that are inaccessible for conventional antibodies. Other attractive features of sdAbs include high solubility, thermal stability, and low immunogenicity to humans [12]. A number of sdAbs have also been developed to treat a spectrum of human diseases, and some are currently in the late stages of clinical trials. For example, two antibodies, ozoralizumab (an anti-tumor necrosis factor (TNF)-alpha sdAb) and ALX-0061 (an interleukin (IL)-6R sdAb), are also in phase II trials [13]. The efficacy of CsA on animal models for RA is so far not satisfactory, even paradoxical [14]. A few other CypA inhibitors, such as the CsA derivative SDZ NIM811, capable of inhibiting neutrophil influx in vivo have been reported [5]. However, previous studies focused on the ability of CypA to regulate chemotaxis, and did not investigate other critical functions of CypA, such as the stimulation of MMP secretion that leads to cartilage destruction. Until now, there have been no reports of CypA-specific antibodies used for the treatment of RA. In this study, we characterized a new sdAb that was shown to inhibit important biological functions of CypA both in vitro and in vivo. Materials Animals and patient samples The DBA/1 mice and the NOD/SCID mice were purchased from Shanghai Laboratory Animal Co. Ltd (Shanghai, China). Peripheral blood and synovial fluid were obtained from patients with active RA and the synovium tissues were obtained from RA patients undergoing joint replacement surgery or synovectomy. All of the RA patients met the 1987 revised diagnostic criteria of the American College of Rheumatology [15]. The normal human cartilage specimens were obtained from nonarthritis patients with femoral head fractures. Ethics approval was granted from the E (...truncated)