Chilukuri Nageswararao, Sun Wei, Naik Ramachandra S, Parikh Kalpana, Tang Lin, Doctor Bhupendra P, Saxena Ashima
Division of Biochemistry, Walter Reed Army Institute of Research, Silver Spring, MD 20910-7500, USA.
Chem Biol Interact. 2008 Sep 25;175(1-3):255-60. doi: 10.1016/j.cbi.2008.05.020. Epub 2008 May 21.
The therapeutic value of human serum butyrylcholinesterase (Hu BChE) as a bioscavenger of chemical warfare agents is due to its high reactivity with organophosphorus compounds and prolonged circulatory stability. Native Hu BChE is mostly tetrameric in form while the enzyme produced using molecular cloning technology is a mixture of tetramers, dimers, and monomers. Previous studies revealed that monomers and dimers of recombinant human (rHu) BChE cleared rapidly from the circulation of mice compared to tetrameric rHu BChE and native Hu BChE, which have mean residence times (MRTs) of 18h and 45h, respectively. It was also shown that polyethylene glycol-20K (PEG) modification of tetrameric rHu BChE prolonged its circulatory stability and bioavailability in vivo. The goal of this study was to determine if modification with PEG could prolong the circulatory stability and eliminate the immunogenicity of monomeric rHu BChE. Monomeric rHu BChE was expressed in human 293A cells using a cDNA lacking the 45 amino acid tetramerization domain from the carboxyl terminus and the adenovirus expression system. The catalytic and inhibitory properties of purified monomeric rHu BChE were similar to those for native Hu BChE and were not affected by PEG modification. As expected, monomeric rHu BChE rapidly cleared from the circulation of mice (MRT=3.2+/-0.3h) while monomeric PEG-rHu BChE demonstrated significant improvement in its bioavailability and circulatory stability in blood (MRT=31.4+/-5.4h). However, a second injection of monomeric PEG-rHu BChE, 28 days after the first, displayed a much shorter MRT=11.6+/-0.4h, and circulating anti-monomeric PEG-rHu BChE antibodies were detected in the blood of mice. These results suggest that PEG modification increased the circulatory stability of monomeric rHu BChE but failed to reduce or eliminate its immunogenicity.
人血清丁酰胆碱酯酶（Hu BChE）作为化学战剂的生物清除剂，其治疗价值在于它与有机磷化合物具有高反应性以及循环稳定性延长。天然Hu BChE大多呈四聚体形式，而使用分子克隆技术生产的酶是四聚体、二聚体和单体的混合物。先前的研究表明，与四聚体rHu BChE和天然Hu BChE相比，重组人（rHu）BChE的单体和二聚体从小鼠循环中清除得很快，四聚体rHu BChE和天然Hu BChE的平均驻留时间（MRT）分别为18小时和45小时。还表明，对四聚体rHu BChE进行聚乙二醇-20K（PEG）修饰可延长其循环稳定性和体内生物利用度。本研究的目的是确定PEG修饰是否可以延长单体rHu BChE的循环稳定性并消除其免疫原性。使用来自羧基末端缺少45个氨基酸四聚化结构域的cDNA和腺病毒表达系统，在人293A细胞中表达单体rHu BChE。纯化的单体rHu BChE的催化和抑制特性与天然Hu BChE相似，并且不受PEG修饰的影响。正如预期的那样，单体rHu BChE从小鼠循环中迅速清除（MRT = 3.2±0.3小时），而单体PEG-rHu BChE在血液中的生物利用度和循环稳定性有显著改善（MRT = 31.4±5.4小时）。然而，在第一次注射28天后第二次注射单体PEG-rHu BChE时，其MRT短得多，为11.6±0.4小时，并且在小鼠血液中检测到循环抗单体PEG-rHu BChE抗体。这些结果表明，PEG修饰增加了单体rHu BChE的循环稳定性，但未能降低或消除其免疫原性。
