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

目的：研究重组免疫毒素dsFv抗叶酸受体β (FRβ)对类风湿关节炎滑膜组织炎症和降解过程中起作用的细胞活化和增殖的影响。20051)重组型抗人FRβ免疫毒素的制备：在大肠杆菌中制备了Ig VH-PE38融合蛋白和Ig VL蛋白，并在其框架区通过工程半胱氨酸残基与二硫键连接。2)体外实验：免疫毒素显著诱导fr β转染的巨噬细胞和贴壁RA滑膜单核细胞凋亡。3)体内实验：免疫毒素可降低SCID小鼠移植RA滑膜组织中CD68^+巨噬细胞的数量，激活成纤维细胞样细胞（VCAM-1^+、血管生成素-1^+）、炎症因子（TNF-α、IL-6）和血管生成（CD34^+细胞）。FRβ在恒河猴体内的组织定位：恒河猴肝脏和皮肤中FRβ表达较弱。2)大鼠抗小鼠FRβ单克隆抗体的制备：将小鼠FRβ cDNA转染大鼠乳腺，注射至WKY大鼠足部。获得9个单克隆抗体。单克隆抗体与小鼠FRα无交叉反应。3) FRβ在小鼠的组织定位：FRβ在巯基乙酸诱导的腹膜巨噬细胞（++++）、肝脏（+ ~ ++）、肺（+ ~ ++）和结肠（+）中表达，提示炎症刺激可能上调；小鼠和恒河猴的FRβ^+细胞。结论：免疫毒素是治疗类风湿关节炎的有效手段。前景：目前，研究人员正在生产抗小鼠FRβ免疫毒素，并将研究其对小鼠关节炎模型（cia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).