Department of Genetics and Advanced Medical Technology, Medical Biotechnology Research Center, Faculty of Medicine, AJA University of Medical Sciences, Tehran, Iran (F.K.); Department of Medical Laboratory, Kashan University of Medical Sciences, Kashan, Iran (M.S.); Department of Civil Engineering, Sharif University of Technology, Tehran, Iran (A.M.); Department of Rabies, Virology Research Group (S.K.) and Department of Nanobiotechnology, New Technologies Research Group (R.A.C.), Pasteur Institute of Iran, Tehran, Iran; and Department of Biology, School of Basic Science, Science and Research Branch, Islamic Azad University, Tehran, Iran (A.V., E.K.).
Department of Genetics and Advanced Medical Technology, Medical Biotechnology Research Center, Faculty of Medicine, AJA University of Medical Sciences, Tehran, Iran (F.K.); Department of Medical Laboratory, Kashan University of Medical Sciences, Kashan, Iran (M.S.); Department of Civil Engineering, Sharif University of Technology, Tehran, Iran (A.M.); Department of Rabies, Virology Research Group (S.K.) and Department of Nanobiotechnology, New Technologies Research Group (R.A.C.), Pasteur Institute of Iran, Tehran, Iran; and Department of Biology, School of Basic Science, Science and Research Branch, Islamic Azad University, Tehran, Iran (A.V., E.K.)
J Pharmacol Exp Ther. 2020 Oct;375(1):69-75. doi: 10.1124/jpet.120.000012. Epub 2020 Jul 15.
Therapeutic fragmented antibodies show a poor pharmacokinetic profile that leads to frequent high-dose administration. In the current study, for the first time, a novel proline, alanine, serine (PAS) repeat sequence called PAS#208 was designed to extend the plasma half-life of a nanosized anti-vascular endothelial growth factor-A single-domain antibody. Polyacrylamide gel electrophoresis, circular dichroism, dynamic light scattering, and electrophoretic light scattering were used to assess the physicochemical properties of the newly designed PAS sequence. The effect of PAS#208 on the biologic activity of a single-domain antibody was studied using an in vitro proliferation assay. The pharmacokinetic parameters, including terminal half-life, the volume of distribution, elimination rate constant, and clearance, were determined in mice model and compared with the native protein and PAS#1(200) sequence. The novel PAS repeat sequence showed comparable physicochemical, biologic, and pharmacokinetic features to the previously reported PAS#1(200) sequence. The PAS#208 increased the hydrodynamic radius and decreased significantly the electrophoretic mobility of the native protein without any change in zeta potential. Surprisingly, the fusion of PAS#208 to the single-domain antibody increased the binding potency. In addition, it did not alter the biologic activity and did not show any cytotoxicity on the normal cells. The PAS#208 sequence improved the terminal half-life (14-fold) as well as other pharmacokinetic parameters significantly. The simplicity as well as superior effects on half-life extension make PAS#208 sequence a novel sequence for in vivo pharmacokinetic enhancement of therapeutic fragmented antibodies. SIGNIFICANCE STATEMENT: In the current study, a new proline, alanine, serine (PAS) sequence was developed that showed comparable physicochemical, biological, and pharmacokinetic features to the previously reported PAS#1(200) sequence. The simplicity as well as superior effects on half-life extension make PAS#208 sequence a novel sequence for in vivo pharmacokinetic enhancement of recombinant small proteins.
治疗性片段抗体表现出较差的药代动力学特征，导致频繁的高剂量给药。在本研究中，首次设计了一种新的脯氨酸-丙氨酸-丝氨酸（PAS）重复序列，称为 PAS#208，用于延长纳米级抗血管内皮生长因子-A 单域抗体的血浆半衰期。聚丙烯酰胺凝胶电泳、圆二色性、动态光散射和电泳光散射用于评估新设计的 PAS 序列的物理化学性质。通过体外增殖试验研究 PAS#208 对单域抗体的生物学活性的影响。采用小鼠模型测定药代动力学参数，包括末端半衰期、分布容积、消除率常数和清除率，并与天然蛋白和 PAS#1（200）序列进行比较。新型 PAS 重复序列显示出与先前报道的 PAS#1（200）序列相当的物理化学、生物学和药代动力学特征。PAS#208 增加了天然蛋白的水动力半径，显著降低了电泳迁移率，而 Zeta 电位没有变化。令人惊讶的是，PAS#208 与单域抗体融合增加了结合效力。此外，它没有改变生物活性，对正常细胞也没有显示出任何细胞毒性。PAS#208 序列显著延长了末端半衰期（14 倍）和其他药代动力学参数。其简单性以及对半衰期延长的卓越效果使 PAS#208 序列成为治疗性片段抗体体内药代动力学增强的新型序列。
在本研究中，开发了一种新的脯氨酸-丙氨酸-丝氨酸（PAS）序列，与先前报道的 PAS#1（200）序列具有相当的物理化学、生物学和药代动力学特征。其简单性以及对半衰期延长的卓越效果使 PAS#208 序列成为治疗性片段抗体体内药代动力学增强的新型序列。
