Phage-displayed antibody libraries of synthetic heavy chain complementarity determining regions
Phage-displayed antibody libraries of synthetic heavy chain complementarity determining regions
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DOI：
10.1016/j.jmb.2004.02.050
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发表时间：
2004-04-23
影响因子：
5.6
通讯作者：
Fuh, G
中科院分区：
文献类型：
--
作者：
Sidhu, SS;Li, B;Fuh, G
使用基于结构的方法来设计合成重链互补决定区（CDR）的文库。CDR文库展示为具有单个重链可变结构域支架和固定的轻链可变结构域的单价或二价单链可变片段（scFv）。使用亲本抗体的结构作为指导，我们将文库多样性限制在显著暴露于溶剂的CDR位置。我们用定制的简并密码子引入了多样性，理想情况下仅编码天然抗体CDR中常见的氨基酸。有了这些设计原则，我们推断，我们将产生不同的溶剂暴露的表面库显示在稳定的支架与最小的结构扰动。针对一组蛋白质对文库进行分选，并产生针对所有六种测试抗原的多个独特结合克隆。二价文库产生许多独特的序列，而单价文库产生较少的独特克隆。将选择的scFv转化为Fab形式，并且纯化的Fab蛋白保留对抗原的高亲和力。结果支持这样的观点，即单独的合成重链多样性可能足以从噬菌体展示文库产生高亲和力抗体;因此，可能完全免除轻链，就像天然骆驼免疫球蛋白的情况一样。(C)2004爱思唯尔有限公司保留所有权利。
A structure-based approach was used to design libraries of synthetic heavy chain complementarity determining regions (CDRs). The CDR libraries were displayed as either monovalent or bivalent single-chain variable fragments (scFvs) with a single heavy chain variable domain scaffold and a fixed light chain variable domain. Using the structure of a parent antibody as a guide, we restricted library diversity to CDR positions with significant exposure to solvent. We introduced diversity with tailored degenerate codons, that ideally only encoded for amino acids commonly observed in natural antibody CDRs. With these design principles, we reasoned that we would produce libraries of diverse solvent-exposed surfaces displayed on stable scaffolds with minimal structural perturbations. The libraries were sorted against a panel of proteins and yielded multiple unique binding clones against all six antigens tested. The bivalent library yielded numerous unique sequences, while the monovalent library yielded fewer unique clones. Selected scFvs were converted to the Fab format, and the purified Fab proteins retained high affinity for antigen. The results support the view that synthetic heavy chain diversity alone may be sufficient for the generation of high-affinity antibodies from phage-displayed libraries; thus, it may be possible to dispense with the light chain altogether, as is the case in natural camelid immunoglobulins. (C) 2004 Elsevier Ltd. All rights reserved.