A preclinical study using recombinant anti-folate receptor-beta immunotoxin for the treatment of rheumatoid arthritis

目的：研究抗叶酸受体β（FRβ）的重组免疫毒素dsFv对类风湿关节炎滑膜组织中参与炎症和降解过程的细胞活化和增殖的影响。20051）重组型抗人FRβ免疫毒素的生产：在大肠杆菌中生产IG VH-PE 38融合蛋白和IG VL蛋白，然后通过在这些蛋白质的框架区中改造半胱氨酸残基以二硫键连接。2）体外研究：免疫毒素显著诱导FRβ转染的巨噬细胞和粘附的RA滑膜单核细胞凋亡。3）体内研究：免疫毒素的施用减少了CD 68 ^+巨噬细胞、活化的成纤维细胞样细胞（VCAM-1^+、血管生成素-1 ^+）、炎症细胞因子（TNF-α、IL-6）、血管生成20061）FRβ在恒河猴中的组织定位：2）大鼠抗小鼠FRβ单克隆抗体的制备：将小鼠FRβ cDNA转染大鼠肥大细胞，注射于WKY大鼠足垫。获得了9株单克隆抗体。3）FRβ在小鼠体内的组织定位：在巯基乙酸诱导的小鼠腹腔巨噬细胞（+）、肝（+）、肺（+）和结肠（+）中均有FRβ的表达，提示炎症刺激可上调小鼠和恒河猴的FRβ^+细胞。免疫毒素有望成为治疗类风湿关节炎的有效药物。前景：目前，研究人员正在研制抗小鼠FRβ免疫毒素，并将检测其对小鼠关节炎模型（CIIA诱导的关节炎和血清转移诱导的关节炎）的影响。

OBJECTIVE: To investigate the effects of the recombinant immunotoxin dsFv anti-folate receptor beta (FRβ) on the activation and proliferation of the cell that function in inflammatory and degradative processes in rheumatoid arthritis synovial tissue.20051) Productior of recombinant type anti-human FRβ immunotoxin: The Ig VH-PE38 fusion protein and the Ig VL protein were produced in Escherichia coli, and then joined with a disulfide bond by engineering cysteine residues in the framework regions of these proteins.2) In vitro study: The immunotoxin significantly induced the apoptosis of FRβ-transfected macrophages and adherent RA synovial mononuclear cells.3) In vivo study: Administration of immunotoxin reduced the numbers of CD68^+ macrophages, activated fibroblast-like cells (VCAM-1^+, Angiopoietin-1^+), inflammatory cytokines (TNF-α, IL-6), angiogenesis (CD34^+ cells) in RA synovial tissue engrafted into SCID mice.20061) Tissue localization of FRβ in rhesus monkey: The liver and skin weakly expressed FRβ in rhesus monkey.2) Productior of rat-anti mouse FRβ monoclonal antibody: Murine FRβ cDNA was transfected into rat mastcytorra, and injected into foot-pad of WKY rats. Nine monoclonal antibodies were obtained. Any monoclonal antibodies were not cross-reacted with mouse FRα.3) Tissue localization of FRβ in mouse: FRβ were expressed in thioglycolate-induced peritoneal macrophages (++++), liver (+ 〜 ++), lung (+ 〜 ++) and colon (+), suggesting that inflammatory stimulation may up-regulate; FRβ^+ cells in mouse and rhesus monkey.Conclusion: Immunotoxin would be a promising tool for the treatment of RA. Prospects: Now, investigators are producing anti-mouse FRβ immunotoxin and will examine the effects on murine arthritis model (CIIA-induced arthritis and serum transfer-induced arthritis).