Fusion of a recombinant antibody fragment with a homo-amino-acid polymer: effects on biophysical properties and prolonged plasma half-life
Fusion of a recombinant antibody fragment with a homo-amino-acid polymer: effects on biophysical properties and prolonged plasma half-life
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DOI：
10.1093/protein/gzm020
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发表时间：
2007-06-01
影响因子：
2.4
通讯作者：
Skerra, Arne
中科院分区：
文献类型：
--
作者：
Schlapschy, Martin;Theobald, Ina;Skerra, Arne
小重组蛋白与聚乙二醇（PEG）的化学缀合是将其通常较短的循环时间延长至治疗有用范围的既定策略。我们已经研究了与富含甘氨酸的同源氨基酸聚合物（HAP）的基因融合的生产，作为连接具有大流体动力学体积的溶剂化随机链的替代方式。抗HER 2 Fab片段4D 5用作模型系统，并与重复序列（Gly（4）Ser）（n）的100或200个残基聚合物融合至其轻链。这两种融合蛋白在大肠杆菌的周质中成功地产生，并在两步亲和层析后通过融合至重链的His（6）标签和融合至延伸的轻链的Strep标签II获得均质制剂。两种修饰的Fab片段均显示出与常规重组Fab片段不可区分的针对HER 2抗原的结合活性。当与未融合的Fab片段相比时，显著增加的流体动力学体积，约为100 μ g/ml。在凝胶过滤期间，对于200个残基的HAP融合蛋白，观察到120%，并且在较小程度上，在100个残基的HAP的情况下。差异CD测量揭示了100和200残基HAP融合部分的特征性无规卷曲光谱。最后，在放射性碘标记重组Fab片段后，在小鼠中进行药代动力学实验。尽管100个残基的HAP融合体显示出与未融合的Fab片段非常相似的行为，具有约100个残基的终末血浆半衰期。2 h后，200个残基的HAP化Fab片段引起ca. 6小时虽然这种适度的效果到目前为止可能对于专门的医学应用（例如体内成像）是最有益的，但是优化的HAP序列的基因工程应该产生类似于PEG化的药代动力学性质，而不需要体外修饰步骤。
Chemical conjugation of small recombinant proteins with polyethylene glycol (PEG) is an established strategy to extend their typically short circulation times to a therapeutically useful range. We have investigated the production of a genetic fusion with a glycine-rich homo-amino-acid polymer (HAP) as an alternative way to attach a solvated random chain with large hydrodynamic volume. The anti-HER2 Fab fragment 4D5 was used as a model system and fused with either 100 or 200 residue polymers of the repetitive sequence (Gly(4)Ser)(n) to its light chain. Both fusion proteins were successfully produced in the periplasm of Escherichia coli and obtained as homogeneous preparations after two-step affinity chromatography via the His(6) tag fused to the heavy chain and the Strep-tag II fused to the extended light chain. Both modified Fab fragments showed binding activity towards the HER2 antigen indistinguishable from the conventional recombinant Fab fragment. When compared with the unfused Fab fragment, a significantly increased hydrodynamic volume, by ca. 120%, was observed during gel filtration for the 200 residue HAP fusion protein and, to a lesser extent, in the case of the 100 residue HAP. Difference CD measurements revealed a characteristic random coil spectrum for the 100 and 200 residue HAP fusion moieties. Finally, pharmacokinetic experiments were carried out in mice after radioiodination of the recombinant Fab fragments. Although the 100 residue HAP fusion showed a behavior very similar to the unfused Fab fragment, with a terminal plasma half-life of ca. 2 h, the 200 residue HAPylated Fab fragment gave rise to a significantly prolonged half-life of ca. 6 h. While this moderate effect may so far be most beneficial for specialized medical applications, such as in vivo imaging, the genetic engineering of optimized HAP sequences should yield pharmacokinetic properties similar to PEGylation, yet without necessitating in vitro modification steps.