Effect of domain order on the activity of bacterially produced bispecific single-chain Fv antibodies
Effect of domain order on the activity of bacterially produced bispecific single-chain Fv antibodies
复制标题
DOI：
10.1016/s0022-2836(03)00526-6
复制
发表时间：
2003-06-27
影响因子：
5.6
通讯作者：
Little, M
中科院分区：
文献类型：
--
作者：
Kipriyanov, SM;Moldenhauer, G;Little, M
双特异性单链 Fv 抗体包含两个不同特异性的四个共价连接的免疫球蛋白可变区（VH 和 VL）结构域。根据 VH 和 VL 结构域的顺序以及分隔它们的肽的长度，单链分子要么从具有相同特异性的相邻结构域形成两个单链 Fv (scFv) 模块，即所谓的 scFv-scFv 串联 [(scFv)2]，要么头尾相连形成双抗体样结构，即所谓的双特异性单链双抗体 (scBsDb)。我们生成了许多四结构域构建体，这些结构由对人类 CD19 或 CD3 具有特异性的相同 VH 和 VL 结构域组成，但排列顺序不同。当在细菌中表达时，所有（scFv）变体似乎仅具有一半功能，与 CD19 结合并且没有表现出 CD3 结合活性。只有双抗体样 scBsDb 才能结合这两种抗原。 scBsDb 与结构相似的非共价二聚体（双抗体）的比较证明了 scBsDb 分子中间的接头具有稳定作用。我们证明了生理条件下 CD19 X CD3 双抗体的失活机制是通过较弱（抗 CD3）VH/VL 界面的解离引发的，随后进行结构域交换并形成非活性同二聚体。一种同型二聚体的不稳定性使得双抗体解离/重结合过程不可逆，从而逐渐减少活性分子的比例。指出了影响功能性双特异性单链抗体形成的结构参数，并提出了制备相对稳定的双特异性分子的方法。 (C) 2003 Elsevier Science Ltd. 保留所有权利。
Bispecific single-chain Fv antibodies comprise four covalently linked immunoglobulin variable (VH and VL) domains of two different specificities. Depending on the order of the VH and VL domains and on the length of peptides separating them, the single-chain molecule either forms two single-chain Fv (scFv) modules from the adjacent domains of the same specificity, a so-called scFv-scFv tandem [(scFv)2], or folds head-to-tail with the formation of a diabody-like structure, a so-called bispecific single-chain diabody (scBsDb). We generated a number of four-domain constructs composed of the same VH and VL domains specific either for human CD19 or CD3, but arranged in different orders. When expressed in bacteria, all (scFv), variants appeared to be only half-functional, binding to CD19 and demonstrating no CD3-binding activity. Only the diabody-like scBsDb could bind both antigens. Comparison of the scBsDb with a structurally similar non-covalent dimer (diabody) demonstrated a stabilizing effect of the linker in the middle of the scBsDb molecule. We demonstrated that the mechanism of inactivation of CD19 X CD3 diabody under physiological conditions is initiated by a dissociation of the weaker (anti-CD3) VH/VL interface followed by domain swapping with the formation of non-active homodimers. The instability of one homodimer makes the process of diabody dissociation/reassociation irreversible, thus gradually decreasing the fraction of active molecules. The structural parameters influencing the formation of functional bispecific single-chain antibodies are indicated and ways of making relatively stable bispecific molecules are proposed. (C) 2003 Elsevier Science Ltd. All rights reserved.