Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, 24 Rueven St., P.O. Box 19, Ness-Ziona 74100, Israel.
Chem Biol Interact. 2010 Sep 6;187(1-3):253-8. doi: 10.1016/j.cbi.2009.12.004. Epub 2009 Dec 11.
We have shown previously that conjugation of polyethylene glycol (PEG) chains to recombinant human acetylcholinesterase (rHuAChE) results in the extension of its residence time in the circulation of mice and monkeys [1,2]. By profiling the pharmacokinetic behavior of an array of well-defined hypolysine human mutant AChE molecules following PEGylation, we now determine that the duration of these enzyme forms in the circulation of rhesus macaques correlates with their number of appended PEG moieties, and is influenced by the actual location of the PEG chains at the molecule surface, as well. These findings, which concur with those we have previously established in mice, indicate that a common set of rules dictates the circulatory fate of PEGylated HuAChEs in rodents and non-human primates. In addition to its effect on circulatory residence, PEGylation reduces the ability of the rHuAChE bioscavenger to elicit an immune response in the heterologous mouse animal system. Thus, an inverse relationship between anti-AChE antibody production and PEG loading was observed following repeated administration of the different PEGylated hypolysine human AChEs to mice. We note however, that in rhesus macaques, the essentially homologous (human) AChE does not induce specific anti-AChE antibodies after repeated administration of high doses of the enzyme in its PEGylated form, and even in its non-PEGylated form. Taken together, these findings indicate that PEG acts by veiling enzyme-related epitopes, which would otherwise interact with host circulatory elimination pathways and immune system. The barring of such interactions by obstructive PEGs, confers the enzyme molecule with both extended circulatory residence and mitigated immunogenic properties. Further modulation by incorporation of the F338A mutation into the PEGylated hypolysine rHuAChE enzyme mold, resulted in the generation of an OP-bioscavenger that displayed reduced aging rates and could effectively protect mice against repeated exposure to CW agents. This selected 4-PEG F338A-AChE can serve as a paradigm for new generation OP-bioscavengers, specifically tailored for prophylactic treatment against toxic OP-agents.
我们之前已经证明，将聚乙二醇（PEG）链与重组人乙酰胆碱酯酶（rHuAChE）缀合，可延长其在小鼠和猴子体内的循环时间[1,2]。通过对一系列经过 PEG 化的低赖氨酸人突变 AChE 分子的药代动力学行为进行分析，我们现在确定，这些酶形式在恒河猴体内的循环时间与附着的 PEG 数量相关，并且受到分子表面 PEG 链的实际位置的影响。这些发现与我们之前在小鼠中建立的发现一致，表明一套共同的规则决定了 PEG 化 HuAChE 在啮齿动物和非人类灵长类动物中的循环命运。除了对循环停留时间的影响外，PEG 化还降低了 rHuAChE 生物清除剂在异种小鼠动物系统中引发免疫反应的能力。因此，在向小鼠重复给予不同 PEG 化低赖氨酸人 AChE 后，观察到抗 AChE 抗体产生与 PEG 加载呈反比关系。然而，我们注意到，在恒河猴中，即使在其 PEG 化形式或非 PEG 化形式下，重复给予高剂量酶后，基本上同源的（人）AChE 也不会诱导特异性抗 AChE 抗体。综上所述，这些发现表明 PEG 通过掩盖酶相关表位来发挥作用，否则这些表位会与宿主循环消除途径和免疫系统相互作用。PEG 的这种阻塞作用使酶分子具有延长的循环停留时间和减轻的免疫原性。通过将 F338A 突变纳入 PEG 化低赖氨酸 rHuAChE 酶模型进一步调节，产生了一种 OP 生物清除剂，其显示出降低的老化率，并能有效保护小鼠免受重复接触 CW 剂的影响。这种经过选择的 4-PEG F338A-AChE 可作为新一代 OP 生物清除剂的范例，专门针对有毒 OP 剂的预防性治疗进行了定制。
