Site-specific chemical modification with polyethylene glycol of recombinant immunotoxin anti-Tac(Fv)-PE38 (LMB-2) improves antitumor activity and reduces animal toxicity and immunogenicity
Site-specific chemical modification with polyethylene glycol of recombinant immunotoxin anti-Tac(Fv)-PE38 (LMB-2) improves antitumor activity and reduces animal toxicity and immunogenicity
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DOI：
10.1073/pnas.140210597
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发表时间：
2000-07-18
影响因子：
11.1
通讯作者：
Pastan, I
中科院分区：
文献类型：
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作者：
Tsutsumi, Y;Onda, M;Pastan, I
用聚乙二醇（PEG化）对蛋白质进行化学修饰可以增加血浆半衰期、稳定性和治疗效力。为了制备具有改善的治疗特性的聚乙二醇化重组免疫毒素，我们制备了抗Tac（Fv）-PE 38（LMB-2）的突变体，其是由抗人Tac单克隆抗体的针对IL-2受体cu亚基的单链Fv片段与假单胞菌外毒素的38-kDa片段融合组成的重组免疫毒素。对于LMB-2的位点特异性PEG化，将一个半胱氨酸残基引入Fv和毒素之间的肽连接子（ASGCGPE）中。然后将保留完全细胞毒性活性的该突变体LMB-2（cys 1-LMB-2）与5或20 kDa的聚乙二醇-马来酰亚胺位点特异性缀合。当与未修饰的LMB-2相比时，两种PEG化免疫毒素在体外显示出相似的细胞毒性活性，但在小鼠血清中在37 ℃下具有上级稳定性，小鼠血浆半衰期增加5至8倍，抗肿瘤活性增加3至4倍。这伴随着动物毒性和免疫原性的大幅降低。重组免疫毒素的位点特异性PEG化可以增加其在人体中的治疗效力。
Chemical modification of proteins with polyethylene glycol (PEGy-lation) can increase plasma half-lives, stability, and therapeutic potency. To make a PEGylated recombinant immunotoxin with improved therapeutic properties, we prepared a mutant of anti-Tac(Fv)-PE38 (LMB-2), a recombinant immunotoxin composed of a single-chain Fv fragment of the anti-human Tac monoclonal antibody to the IL-2 receptor cu subunit fused to a 38-kDa fragment of Pseudomonas exotoxin. For site-specific PEGylation of LMB-2, one cysteine residue was introduced into the peptide connector (ASGCGPE) between the Fv and the toxin. This mutant LMB-2 (cys1-LMB-2), which retained full cytotoxic activity, was then site-specifically conjugated with 5 or 20 kDa of polyethylene glycol-maleimide. When compared with unmodified LMB-2, both PEGylated immunotoxins showed similar cytotoxic activities in vitro but superior stability at 37 degrees C in mouse serum, a 5- to 8-fold increase in plasma half-lives in mice, and a 3- to 4-fold increase in antitumor activity. This was accompanied by a substantial decrease in animal toxicity and immunogenicity. Site-specific PEGylation of recombinant immunotoxins may increase their therapeutic potency in humans.