A RECOMBINANT IMMUNOTOXIN CONTAINING A DISULFIDE-STABILIZED FV FRAGMENT
A RECOMBINANT IMMUNOTOXIN CONTAINING A DISULFIDE-STABILIZED FV FRAGMENT
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DOI：
10.1073/pnas.90.16.7538
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发表时间：
1993-08-15
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：
BRINKMANN, U;REITER, Y;PASTAN, I
B3（dsFv）-PE 38 KDEL是一种重组免疫毒素，由单克隆抗体B3的Fv区与截短形式的假单胞菌外毒素（PE 38 KDEL）连接组成，其中不稳定的Fv异源二聚体（由重链和轻链可变区组成）通过二硫键保持在一起并稳定[称为二硫键稳定的Fv（dsFv）]。通过突变和能量最小化McPC 603的氨基酸序列和结构，计算机模拟了B3（Fv）的结构，使我们能够鉴定保守框架区中“假设”可用于二硫键稳定而不改变结构或影响抗原结合的位置。通过构建在大肠杆菌中产生的二硫键连接的双链dsFv免疫毒素，对该预测进行了实验评估。该免疫毒素的活性和特异性与其单链Fv（scFv）对应物没有区别，表明与B3（scFv）一样，B3（dsFv）中保留了结合区的结构。因为我们在大多数Fv分子中保守的位置中的两个框架残基之间引入了稳定的二硫键，所以重链和轻链可变区之间的这种连接方法应该普遍适用于使用其他抗体构建免疫毒素和dsFv分子。此外，B3（dsFv）在37 ℃下在人血浆中比B3（scFv）稳定得多的发现表明dsFv可能比scFv更通用于治疗应用。
B3(dsFv)-PE38KDEL is a recombinant immunotoxin composed of the Fv region of monoclonal antibody B3 connected to a truncated form of Pseudomonas exotoxin (PE38KDEL), in which the unstable Fv heterodimer (composed of heavy- and light-chain variable regions) is held together and stabilized by a disulfide bond [termed disulfide-stabilized Fv (dsFv)]. A computer modeled structure of the B3(Fv), made by mutating and energy minimizing the amino acid sequence and structure of McPC603, enabled us to identify positions in conserved framework regions that ''hypothetically'' could be used for disulfide stabilization without changing the structure or affecting antigen binding. This prediction was evaluated experimentally by constructing a disulfide-linked two-chain dsFv-immunotoxin that was produced in Escherichia coli. The activity and specificity of this immunotoxin was indistinguishable from its single-chain Fv (scFv) counterpart, indicating that, as in B3(scFv), the structure of the binding region is retained in B3(dsFv). Because we introduced the stabilizing disulfide bond in between two framework residues in a position that is conserved in most Fv molecules, this method of linkage between the heavy- and light-chain variable regions should be generally applicable to construct immunotoxins and dsFv molecules using other antibodies. Furthermore, the finding that B3(dsFv) was much more stable at 37-degrees-C in human plasma than B3(scFv) indicates that dsFvs are possibly more versatile for therapeutic application than scFvs.