The stable cell line was obtained by glutamine deficiency cultivation and by adding 25 M MSX

The stable cell line was obtained by glutamine deficiency cultivation and by adding 25 M MSX. 4.1.2. HM-3-Fc inhibited the proliferation of splenic lymphocytes and reduced the release of TNF- from macrophages. The pharmacodynamics studies on mice paw in Collagen-Induced Arthritis (CIA) model demonstrated that HM-3-Fc administered once in 5 days in the 50 and 25 mg/kg groups, or once in 7 days in the 25 mg/kg group showed a better protective effect within two weeks than the positive control adalimumab and HM-3 group. Preliminary pharmacokinetic studies in cynomolgus confirmed that the in vivo half-life of HM-3-Fc was 15.24 h in comparison with 1.32 min that of HM-3, which demonstrated that an Fc fusion can effectively increase the half-life of HM-3 and make it possible for further reduction of subcutaneous injection frequency. Fc-HM-3 is a long-acting active molecule for RA treatment. Keywords: rheumatoid arthritis, HM-3, Fc-domain of immunoglobulin G4, synovial angiogenesis, inflammatory response, TNF-, half-life, pharmacodynamics 1. Introduction Rheumatoid arthritis (RA), a multi-systemic autoimmune disease, is characterized by joint CDK4/6-IN-2 synovitis, pannus formation and symmetrical destructive joint disease [1]. The microenvironment of RA involves hypoxia within the joint cavity with a large number of inflammatory factors and angiogenic active molecules [2]. These factors together contribute to the formation of characteristic angiogenesis [3,4]. It provides nutrients for proliferating synovial cells and it infiltrates the lubricating membrane with more inflammatory cells and mediators as well, maintaining and promoting the abnormal angiogenesis. Therefore, RA is considered as a vicious cycle with inflammation-angiogenesis process and it is called an angiogenesis disease [5,6]. HM-3 is an anti-angiogenic polypeptide with 18 amino acid residues, which is generated by the connection of an integrin-targeting RGD (Arg-Gly-Asp) sequence to the C-terminus of an endostatin fragment (IVRRADRAAVP). It targets integrin v3 and 51 [7]. Several studies have shown that as an integrin inhibitor, HM-3 directly reduced the expression of vascular endothelial growth factor (VEGF) and platelet derived growth factor A (PDGF-A) in endothelial cells and down-regulated the corresponding signal transduction pathways [8]. HM-3 achieved its anti-RA activity via the anti-inflammatory and anti-angiogenic effects. And it exhibited anti-RA effects in both adjuvant-induced and collagen-induced arthritis models [9]. PEGylated HM-3 (PEG-HM-3) also possessed anti-angiogenesis and anti-rheumatic activity [10]. In vitro, it decreased splenocyte viability and the levels of tumor necrosis factor- (TNF-) in macrophage supernatant. It also decreased the expression of toll-like receptor (TLR-4) protein in LPS-induced synoviocytes. In the adjuvant-induced arthritis model, mPEG-SC20K-HM-3 (PEG-HM-3) treatment decreased the levels of IL-6 in spleens, TNF-, cluster of differentiation 31 CDK4/6-IN-2 (CD31) and CD105 in the joint cavity by immunohistochemistry analysis [10]. Therefore, HM-3 and PEG-HM-3 are novel and promising multi-target anti-RA molecules. However, as a polypeptide, HM-3 is naturally prone to enzymatic hydrolysis by proteolytic enzymes in CD40 vivo and it has a short half-life [11]. In vivo pharmacokinetic studies showed that the half-life of HM-3 is only 27 min in male Sprague-Dawley (SD) rats [12]. Hence, it needs frequent dosing to maintain a sufficient drug concentration in clinical trials, which affects the living quality of clinical patients. In order to extend the in vivo half-life of peptides, various pharmaceutical technologies have been developed, such as fusion protein formation, chemical modification and glycosylation modification, among which PEG modification and fusion protein technology represented by Fc-fusion are the most prominent [13,14]. Both methods are applied in many successfully listed drugs. Compared with chemical modification, fusion protein technology greatly prolongs the half-life of drugs while obtaining more uniform product, higher yield and easier purification [15]. Moreover, the fusion protein is a bi-functional molecule that can lead to new biological functions to effector molecules. For instance, the FcRn receptor-mediated recycling mechanism can further extend the in vivo half-life of the fusion proteins, which has a wider application prospect than chemical modification methods. Currently, Fc fusion is the most popular and fastest-growing protein fusion technology [16]. It uses the crystalline fragment (Fc) segment of immunoglobulin (IgG) as a molecular chaperone to fuse functional proteins by means of molecular biology, which maintains the activity of functional proteins and keeps the long half-life of immunoglobulins as well [17]. Its fusion targets involve receptor domains, ligands, antibody fragments and peptides and it has evolved as a reliable drug development instrument [18]. Fc-fusion proteins greatly increase the molecular weight of proteins and peptides and CDK4/6-IN-2 reduce the glomerular filtration rate [19]. FcRn-mediated recycling mechanisms successfully avoid protein degradation and effectively increase half-life [20,21]. In addition, human Fc fragments reduce the immunogenicity of the fusion protein, thereby elimination of drugs by the immune system is prevented. The half-life of the Fc fusion protein has been greatly improved in drugs listed in the market [22,23]. As it has been reported, the half-life of ahatacept is up to CDK4/6-IN-2 13.1 days and that of aldfacept.