Spitzer Dirk, Unsinger Jacqueline, Bessler Monica, Atkinson John P
Division of Rheumatology, Washington University School of Medicine, 660 S. Euclid Avenue, Campus Box 8045, St. Louis, MO 63110, USA.
Mol Immunol. 2004 Feb;40(13):911-9. doi: 10.1016/j.molimm.2003.10.017.
Antibodies or antibody fragments represent a powerful class of targeting moieties to specifically attach proteins to the surface of a given cell or tissue. Since the presence of constant antibody domains in these targeted fusion proteins may have certain disadvantages, we report here the reduction of the targeting epitope to the variable regions of an Ab by the generation of a single chain antibody fragment (scFv). As an effector molecule, we attached the human complement regulatory protein (CRP) decay-accelerating factor (DAF) at its amino-terminus with a scFv specific for TER-119, a red blood cell (RBC) restricted surface antigen of the mouse. This heterologous system enabled us to study (a) the applicability of a scFv as a targeting domain, (b) the functionality of the effector molecule with respect to regulation of the complement cascade in vitro, and (c) the in vivo biodistribution characteristics of a scFv-DAF fusion protein attached to a clinically relevant target cell type. RBCs from C57BL/6 mice loaded in vitro or in vivo with this fusion protein were significantly protected against lysis by human complement. After intravenous injection, a homogeneous population of in vivo tagged RBCs was maintained throughout a 6-day follow-up. This result and in vitro mixing experiments indicated that there was an equilibration of the fusion protein between tagged and non-tagged RBCs. Thus, scFv-mediated targeting of proteins to a selected cell or tissue surface has promise as a means to supplement absent or defective plasma membrane constituents. This approach should therefore be applicable for diseases caused by a membrane protein deficiency such as paroxysmal nocturnal hemoglobinuria (PNH).
抗体或抗体片段是一类强大的靶向部分，可将蛋白质特异性附着于特定细胞或组织的表面。由于这些靶向融合蛋白中恒定抗体结构域的存在可能有某些缺点，我们在此报告通过产生单链抗体片段（scFv）将靶向表位减少到抗体的可变区。作为效应分子，我们在人补体调节蛋白（CRP）衰变加速因子（DAF）的氨基末端连接了一种对TER-119特异的scFv，TER-119是小鼠红细胞（RBC）限制性表面抗原。这个异源系统使我们能够研究：（a）scFv作为靶向结构域的适用性；（b）效应分子在体外调节补体级联反应方面的功能；（c）附着于临床相关靶细胞类型的scFv-DAF融合蛋白的体内生物分布特征。体外或体内加载这种融合蛋白的C57BL/6小鼠的红细胞受到人补体显著保护而不被裂解。静脉注射后，在为期6天的随访中体内标记的红细胞群体保持均匀。这一结果和体外混合实验表明，标记和未标记的红细胞之间存在融合蛋白的平衡。因此，scFv介导的蛋白质靶向选定细胞或组织表面有望作为补充缺失或有缺陷的质膜成分的一种手段。因此，这种方法应该适用于由膜蛋白缺乏引起的疾病，如阵发性夜间血红蛋白尿（PNH）。
