Protein Engineering Unit, Life and Science Research Department, Anti-Doping Lab-Qatar (ADLQ), Doha, Qatar; Drug Design Group, Department of Pharmacy, University of Groningen, Groningen, the Netherlands.
Protein Engineering Unit, Life and Science Research Department, Anti-Doping Lab-Qatar (ADLQ), Doha, Qatar; University of Groningen University, Medical Center Groningen (UMCG), Groningen, the Netherlands.
Eur J Pharm Sci. 2019 Jan 15;127:79-91. doi: 10.1016/j.ejps.2018.10.014. Epub 2018 Oct 19.
Recombinant glucarpidase (formerly: Carboxypeptidase G2, CPG2) is used in Antibody Directed Enzyme Prodrug Therapy (ADEPT) for the treatment of cancer. In common with many protein therapeutics, glucarpidase has a relatively short half-life in serum and, due to the need for the repeated cycles of the ADEPT, its bioavailability may be further diminished by neutralizing antibodies produced by patients. PEGylation and fusion with human serum albumin (HSA) are two approaches that are commonly employed to increase the residency time of protein therapeutics in blood, and also to increase the half-lives of the proteins in vivo. To address this stability and the immunogenicity problems, 'biobetter' glucarpidase variants, mono-PEGylated glucarpidase, and HSA fused glucarpidase by genetic fusion with albumin, were produced. Biochemical and bioactivity analyses, including anti-proliferation, bioassays, circular dichroism, and in vitro stability using human blood serum and immunoassays, demonstrated that the functional activities of the designed glucarpidase conjugates were maintained. The immunotoxicity studies indicated that the PEGylated glucarpidase did not significantly induce T-cell proliferation, suggesting that glucarpidase epitopes were masked by the PEG moiety. However, free glucarpidase and HSA-glucarpidase significantly increased T-cell proliferation compared with the negative control. In the latter case, this might be due to the type of expression system used or due to trace impurities associated with the highly purified (99.99%) recombinant HSA-glucarpidase. Both PEGylated glucarpidase and HAS-glucarpidase exhibit more stability in human serum and were more resistant to key human proteases relative to native glucarpidase. To our knowledge, this study is the first to report stable and less immunogenic glucarpidase variants produced by PEGylation and fusion with HSA. The results suggest that they may have better efficacy in drug detoxification and ADEPT, thereby improving this cancer treatment strategy.
重组葡糖醛酸酶（以前称为羧肽酶 G2，CPG2）用于抗体导向酶药物前体疗法（ADEPT）治疗癌症。与许多蛋白治疗药物一样，葡糖醛酸酶在血清中的半衰期相对较短，并且由于需要重复 ADEPT 循环，其生物利用度可能会因患者产生的中和抗体而进一步降低。聚乙二醇化和与人血清白蛋白（HSA）融合是两种常用的方法，可增加蛋白治疗药物在血液中的停留时间，并增加蛋白质在体内的半衰期。为了解决稳定性和免疫原性问题，生产了“生物改良”葡糖醛酸酶变体、单聚乙二醇化葡糖醛酸酶和通过与白蛋白融合的 HSA 融合葡糖醛酸酶。生化和生物活性分析，包括抗增殖、生物测定、圆二色性和使用人血清的体外稳定性以及免疫测定，表明设计的葡糖醛酸酶缀合物的功能活性得以维持。免疫毒性研究表明，聚乙二醇化葡糖醛酸酶不会显著诱导 T 细胞增殖，这表明葡糖醛酸酶表位被聚乙二醇部分掩盖。然而，游离葡糖醛酸酶和 HSA-葡糖醛酸酶与阴性对照相比显著增加了 T 细胞增殖。在后一种情况下，这可能是由于使用的表达系统类型或与高度纯化的（99.99%）重组 HSA-葡糖醛酸酶相关的痕量杂质所致。与天然葡糖醛酸酶相比，聚乙二醇化葡糖醛酸酶和 HAS-葡糖醛酸酶在人血清中更稳定，对关键的人类蛋白酶更具抗性。据我们所知，这项研究首次报道了通过聚乙二醇化和与人血清白蛋白融合产生的稳定且免疫原性较低的葡糖醛酸酶变体。结果表明，它们在药物解毒和 ADEPT 中可能具有更好的疗效，从而改善了这种癌症治疗策略。
