C-Terminal Cysteine PEGylation of Adalimumab Fab with an Engineered Interchain SS Bond
C-Terminal Cysteine PEGylation of Adalimumab Fab with an Engineered Interchain SS Bond
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DOI：
10.1248/bpb.b19-00612
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发表时间：
2020-03-01
影响因子：
2
通讯作者：
Ohkuri, Takatoshi
中科院分区：
文献类型：
--
作者：
Nakamura, Hitomi;Anraku, Makoto;Ohkuri, Takatoshi
与聚乙二醇（PEG）偶联以增加Fab的血清半衰期，用于临床应用。然而，目前重组Fab的设计只允许在Fab的重链和轻链c端的链间SS键（二硫键）上进行聚乙二醇化，这是由于链间SS键的部分减少而导致热稳定性下降。阿达木单抗Fab突变体具有新的链间SS键（CH,: C177-CL: C160）和c端一个半胱氨酸（mutSS Fab(SH)），用于维持Fab的热稳定性和位点特异性PEGylation。在毕赤酵母中表达MutSS Fab(SH)，纯化后的MutSS Fab（SH）与靶向游离半胱氨酸的20 kda PEG偶联。基于酶联免疫吸附试验（ELISA）， PEGylation不影响mutSS Fab的结合能力。为了证实PEGylation对Fab的药代动力学行为的影响，我们通过尾静脉注射给鼠PEGylation的mutSS Fab。通过ELISA分析聚乙二醇化的mutSS Fab（SH）的平均血清浓度与时间的关系表明，与未聚乙二醇化的野生型Fab相比，其半衰期增加。因此，我们已经成功地证明了具有新的链间SS键和c端一个游离半胱氨酸的Fab突变体可以被聚乙二醇化而不改变功能。该设计可作为其他重组晶圆厂改造的潜在平台。
Conjugation with polyethylene glycol (PEG) is performed to increase serum half-life of the Fab for clinical applications. However, current designs for recombinant Fab only allow PEGylation at the interchain SS bond (disulfide bond) at the C-terminal end of the heavy chain and light chain of the Fab, which the decrease of thermostability occurred by partial reduction of the interchain SS bond. An adalimumab Fab mutant with a novel interchain SS bond (CH, : C177-CL : C160) and one cysteine at the C-terminal end (mutSS Fab(SH)) was designed to maintain Fab thermostability and for site-specific PEGylation. MutSS Fab(SH) was expressed in Pichia pastoris and purified mutSS Fab(SH) was conjugated with 20-kDa PEG targeted at the free cysteine. Based on enzyme-linked immunosorbent assay (ELISA), PEGylation did not affect the binding capacity of the mutSS Fab. To confirm the influence of PEGylation on the pharmacokinetic behavior of the Fab, PEGylated mutSS Fab was administered to rats via tail vein injection. Analysis of the mean serum concentration of the PEGylated mutSS Fab(SH) versus time through ELISA indicated an increase in half-life compared to that of non-PEGylated wild-type Fab. Consequently, we have successfully demonstrated that a Fab mutant with a novel interchain SS bond and one free cysteine at the C-terminal end can be PEGylated without changes in functionality. This design can potentially be used as a platform for modification of other recombinant Fabs.