Miclea Razvan D, Purohit Vivek S, Balu-Iyer Sathy V
Department of Molecular & Cellular Biophysics and Biochemistry, Roswell Park Cancer Institute, Buffalo, NY, USA.
AAPS J. 2007 Jun 29;9(2):E251-9. doi: 10.1208/aapsj0902028.
Factor VIII (FVIII) is an important cofactor in the blood coagulation cascade. A deficiency or dysfunction of FVIII causes hemophilia A, a life-threatening bleeding disorder. FVIII circulates in plasma as a heterodimer comprising 6 domains (heavy chain, A1-A2-B and light chain, A3-C1-C2). Replacement therapy using FVIII is the leading therapy in the management of hemophilia A. However, approximately 15% to 30% of patients develop inhibitory antibodies that neutralize the activity of the protein. Neutralizing antibodies to epitopes in the lipid binding region of FVIII are commonly identified in patients' plasma. In this report, we investigated the effect of O-phospho-L-serine (OPLS), which binds to the lipid binding region, on the immunogenicity of B domain deleted recombinant factor VIII (BDDrFVIII). Sandwich enzyme-linked immunosorbent assay (ELISA) studies showed that OPLS specifically bind to the lipid binding region, localized in the C2 domain of the coagulation factor. Size exclusion chromatography and fluorescence anisotropy studies showed that OPLS interfered with the aggregation of BDDrFVIII. Immunogenicity of free- vs BDDrFVIII-OPLS complex was evaluated in a murine model of hemophilia A. Animals administered subcutaneous (sc) injections of BDDrFVIII-OPLS had lower neutralizing titers compared with animals treated with BDDrFVIII alone. Based on these studies, we hypothesize that specific molecular interactions between OPLS and BDDrFVIII may improve the stability and reduce the immunogenicity of BDDrFVIII formulations.
凝血因子VIII（FVIII）是血液凝固级联反应中的一种重要辅助因子。FVIII缺乏或功能异常会导致甲型血友病，这是一种危及生命的出血性疾病。FVIII以异源二聚体形式在血浆中循环，该异源二聚体由6个结构域组成（重链，A1 - A2 - B和轻链，A3 - C1 - C2）。使用FVIII的替代疗法是治疗甲型血友病的主要疗法。然而，约15%至30%的患者会产生中和该蛋白活性的抑制性抗体。在患者血浆中通常可检测到针对FVIII脂质结合区域表位的中和抗体。在本报告中，我们研究了与脂质结合区域结合的O - 磷酸 - L - 丝氨酸（OPLS）对B结构域缺失重组凝血因子VIII（BDDrFVIII）免疫原性的影响。夹心酶联免疫吸附测定（ELISA）研究表明，OPLS特异性结合于位于凝血因子C2结构域的脂质结合区域。尺寸排阻色谱和荧光各向异性研究表明，OPLS干扰了BDDrFVIII的聚集。在甲型血友病小鼠模型中评估了游离BDDrFVIII与BDDrFVIII - OPLS复合物的免疫原性。与单独接受BDDrFVIII治疗的动物相比，皮下注射BDDrFVIII - OPLS的动物中和滴度更低。基于这些研究，我们推测OPLS与BDDrFVIII之间的特定分子相互作用可能会提高BDDrFVIII制剂的稳定性并降低其免疫原性。
