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首页> 外文期刊>Biomolecular engineering >A human biotin acceptor domain allows site-specific conjugation of an enzyme to an antibody-avidin fusion protein for targeted drug delivery
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A human biotin acceptor domain allows site-specific conjugation of an enzyme to an antibody-avidin fusion protein for targeted drug delivery

机译：人类生物素受体域可将酶与抗体-亲和素融合蛋白进行位点特异性缀合，以靶向药物递送

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We have previously constructed an antibody-avidin (Av) fusion protein, anti-transferrin receptor (TfR) IgG3-Av, which can deliver biotinylated molecules to cells expressing the TfR. We now describe the use of the fusion protein for antibody-directed enzyme prodrug therapy (ADEPT). The 67 amino acid carboxyl-terminal domain (P67) of human propionyl-CoA carboxylase a subunit can be metabolically biotinylated at a fixed lysine residue. We genetically fused P67 to the carboxyl terminus of the yeast enzyme FCU1, a derivative of cytosine deaminase that can convert the non-toxic prodrug 5-fluorocytosine to the cytotoxic agent 5-fluorouracil. When produced in Escherichia coli cells overexpressing a biotin protein ligase, the FCU1-P67 fusion protein was efficiently mono-biotinylated. In the presence of 5-fluorocytosine, the biotinylated fusion protein conjugated to anti-rat TfR IgG3-Av efficiently killed rat Y3-Ag1.2.3 myeloma cells in vitro, while the same protein conjugated to an irrelevant (anti-dansyl) antibody fused to Av showed no cytotoxic effect. Efficient tumor cell killing was also observed when E. coli purine nucleoside phosphorylase was similarly targeted to the tumor cells in the presence of the prodrug 2-fluoro-2'-deoxyadenosine. These results suggest that when combined with P67-based biotinylation, anti-TfR IgG3-Av could serve as a universal delivery vector for targeted chemotherapy of cancer. (c) 2004 Elsevier B.V. All rights reserved.
机译：我们先前已经构建了抗体-亲和素（Av）融合蛋白，抗运铁蛋白受体（TfR）IgG3-Av，它可以将生物素化的分子传递到表达TfR的细胞中。现在我们描述融合蛋白在抗体指导的酶前药治疗（ADEPT）中的用途。人丙酰辅酶A羧化酶α亚基的67个氨基酸的羧基末端结构域（P67）可以在固定的赖氨酸残基上被代谢生物素化。我们将P67遗传融合到酵母酶FCU1的羧基末端，该酶是胞嘧啶脱氨酶的衍生物，可以将无毒的前药5-氟胞嘧啶转化为细胞毒剂5-氟尿嘧啶。当在过表达生物素蛋白连接酶的大肠杆菌细胞中产生时，FCU1-P67融合蛋白被有效地单生物素化。在存在5-氟胞嘧啶的情况下，偶联至抗大鼠TfR IgG3-Av的生物素化融合蛋白可在体外有效杀死大鼠Y3-Ag1.2.3骨髓瘤细胞，而偶联至无关的（抗丹磺酰）抗体的相同蛋白则融合至Av无细胞毒性作用。当在前药2-氟-2'-脱氧腺苷的存在下类似地将大肠杆菌嘌呤核苷磷酸化酶靶向肿瘤细胞时，也观察到有效的肿瘤细胞杀伤。这些结果表明，当与基于P67的生物素化结合使用时，抗TfR IgG3-Av可以作为癌症靶向化疗的通用载体。 （c）2004 Elsevier B.V.保留所有权利。

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