Newton D L, Xue Y, Olson K A, Fett J W, Rybak S M
BCDP, SAIC Frederick, Maryland 21702, USA.
Biochemistry. 1996 Jan 16;35(2):545-53. doi: 10.1021/bi951650w.
The gene for human angiogenin (Ang), a member of the ribonuclease superfamily, was fused to a gene encoding a single-chain antibody (sFv) against the human transferrin receptor. Three Ang single-chain immunofusion proteins (AngsFvs) were constructed with variations in the type of linker connecting the VL and VH chain [EGKSSGSGSESKEF, L1 or (GGGGS)3, L2] as well as with or without a spacer (FB) connecting the Ang and sFv (AngFBsFvL1 or L2; AngsFv(L2)]. Although the nature of the linker did not affect the enzymatic activity of the FB-containing fusion proteins, the fusion protein containing the L2 linker was 2.3-fold more effective than the L1 linker in competing with the labeled monoclonal IgG1 antibody for binding to the transferrin receptor. The fusion protein containing the L2 linker without the FB spacer exhibited a 13-fold decrease in binding to the transferrin receptor as well as a decrease in its capacity to degrade tRNA and to inhibit translation in the rabbit reticulocyte lysate compared to its counterpart containing the FB spacer. Binding of placental ribonuclease inhibitor (PRI) to Ang also was affected by the nature of the linker and by the presence or absence of a spacer. PRI bound to Ang and AngFBsFv(L2) and inhibited their ribonuclease activity. A 3-fold greater concentration of PRI, however, did not affect the activity of AngFBsFv(L1) or AngsFv(L2), suggesting that the conformation of these fusion proteins was altered. Binding of monoclonal and polyclonal anti-Ang antibodies to AngsFvs was also used to investigate conformational alterations of the fusion proteins. AngFBsFv(L2) was the least altered while AngFBsFv(L1) exhibited the greatest change in structure. Yet maximal concentrations of all AngsFvs elicited angiogenesis in the chick chorioallantoic membrane assay, demonstrating that Ang in all three fusion proteins remained functionally active. Consistent with all the activities, the fusion protein containing the FB spacer and L2 linker was the most cytotoxic to three different human tumor cell lines. The fusion protein lacking the FB spacer exhibited the least cytotoxicity. These data demonstrate that the linker connecting the VH-VL chains can affect the binding and cellular cytotoxicity of Ang immunofusions and that placement of a spacer between the antibody binding domains and Ang is necessary for optimal activity. Thus, a new class of targeted therapeutic agents containing Ang as the toxic moiety can be designed that potentially will be less immunogenic and less toxic than immunotoxins available currently.
人血管生成素（Ang）基因属于核糖核酸酶超家族成员，与编码抗人转铁蛋白受体的单链抗体（sFv）的基因融合。构建了三种Ang单链免疫融合蛋白（AngsFvs），连接VL和VH链的接头类型有所不同[EGKSSGSGSESKEF，L1或(GGGGS)3，L2]，并且在Ang和sFv之间有或没有间隔序列（FB）（AngFBsFvL1或L2；AngsFv(L2)）。尽管接头的性质不影响含FB融合蛋白的酶活性，但含L2接头的融合蛋白在与标记的单克隆IgG1抗体竞争结合转铁蛋白受体方面比含L1接头的融合蛋白有效2.3倍。与含FB间隔序列的对应物相比，不含FB间隔序列的含L2接头的融合蛋白与转铁蛋白受体的结合减少了13倍，其降解tRNA和抑制兔网织红细胞裂解物中翻译的能力也降低。胎盘核糖核酸酶抑制剂（PRI）与Ang的结合也受接头性质以及间隔序列有无的影响。PRI与Ang和AngFBsFv(L2)结合并抑制它们的核糖核酸酶活性。然而，3倍更高浓度的PRI并不影响AngFBsFv(L1)或AngsFv(L2)的活性，这表明这些融合蛋白的构象发生了改变。单克隆和多克隆抗Ang抗体与AngsFvs的结合也用于研究融合蛋白的构象改变。AngFBsFv(L2)的构象改变最小，而AngFBsFv(L1)的结构变化最大。然而，所有AngsFvs的最大浓度在鸡绒毛尿囊膜试验中都能诱导血管生成，这表明所有三种融合蛋白中的Ang仍然具有功能活性。与所有活性一致，含FB间隔序列和L2接头的融合蛋白对三种不同的人肿瘤细胞系细胞毒性最大。缺乏FB间隔序列的融合蛋白细胞毒性最小。这些数据表明，连接VH-VL链的接头可影响Ang免疫融合物的结合和细胞毒性，并且在抗体结合域和Ang之间放置间隔序列对于最佳活性是必要的。因此，可以设计出一类新的以Ang作为毒性部分的靶向治疗剂，其潜在的免疫原性和毒性可能比目前可用的免疫毒素更低。
