It is very popular to fuse a protein drug or drug candidate to the Fc domain of immunoglobulin G (IgG) in order to prolong the in vivo half-life. In this study, we have designed, prepared, and tested an Fc-fused thermostable cocaine esterase (CocE) mutant (known as E196-301, with the T172R/G173Q/L196C/I301C substitutions on CocE) expressed in E. coli. As expected, Fc-fusion does not affect the in vitro enzyme activity and thermal stability of the enzyme and that Fc-E196-301 can favorably bind FcRn with Kd = 386 ± 35 nM. However, Fc-fusion does not prolong the in vivo half-life of E196-301 at all; Fc-E196-301 and E196-301 have essentially the same PK profile (t1/2 = 0.4 ± 0.1 h) in rats. This is the first time demonstrating that Fc-fusion does not prolong in vivo half-life of a protein. This finding is consistent with the mechanistic understanding that E196-301 and Fc-E196-301 are all degraded primarily through rapid proteolysis in the body. The Fc fusion cannot protect E196-301 from the proteolysis in the body. Nevertheless, it has been demonstrated that PEGylation can effectively protect E196-301, as the PEGylated E196-301, i.e., PEG-E196-301, has a significantly prolonged in vivo half-life. It has also been demonstrated that both E196-301 and PEG-E196-301 have dose-dependent in vivo half-lives (e.g., 19.9 ± 6.4 h for the elimination t1/2 of 30 mg/kg PEG-E196-301), as the endogenous proteolytic enzymes responsible for proteolysis of E196-301 (PEGylated or not) are nearly saturated by the high plasma concentration produced by a high dose of E196-301 or PEG-E196-301.

中文翻译：

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聚乙二醇化而非Fc融合改善了细菌可卡因酯酶的热稳定突变体的体内滞留时间。

为了延长体内半衰期，将蛋白药物或候选药物与免疫球蛋白G（IgG）的Fc结构域融合是非常普遍的。在这项研究中，我们设计，制备和测试了在大肠杆菌中表达的Fc融合热稳定可卡因酯酶（CocE）突变体（称为E196-301，在CocE上具有T172R / G173Q / L196C / I301C替代）。如预期的那样，Fc融合不会影响酶的体外酶活性和热稳定性，并且Fc-E196-301可以以Kd = 386±35 nM良好地结合FcRn。但是，Fc融合根本不会延长E196-301的体内半衰期；Fc-E196-301和E196-301在大鼠中具有相同的PK分布（t1 / 2 = 0.4±0.1 h）。这是首次证明Fc融合不会延长蛋白质的体内半衰期。该发现与以下机理理解一致：E196-301和Fc-E196-301均主要通过体内的快速蛋白水解而降解。Fc融合不能保护E196-301免受体内蛋白水解的影响。然而，已经证明聚乙二醇化可以有效地保护E196-301，因为聚乙二醇化的E196-301（即PEG-E196-301）具有显着延长的体内半衰期。还已经证明E196-301和PEG-E196-301均具有剂量依赖性的体内半衰期（例如，对于30 mg / kg PEG-E196-301的t1 / 2消除，为19.9±6.4小时），因为负责高剂量E196-301或PEG-E196-301产生的高血浆浓度使负责E196-301蛋白水解的内源蛋白水解酶（已聚乙二醇化或未聚乙二醇化）几乎饱和。