Development of the dual-vector system-III (DVS-III), which facilitates affinity maturation of a Fab antibody via light chain shuffling
Development of the dual-vector system-III (DVS-III), which facilitates affinity maturation of a Fab antibody via light chain shuffling
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DOI：
10.1016/j.imlet.2010.05.002
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发表时间：
2010-08-16
影响因子：
4.4
通讯作者：
Cha, Sang-hoon
中科院分区：
文献类型：
--
作者：
Hur, Byung-ung;Choi, Hyo-jung;Cha, Sang-hoon
轻(L)链洗牌通常用于分析与特定重（Fd或H）链配对的最佳L链，最终导致特定抗体的体外亲和成熟。此过程的主要缺点之一是必须为每个不同的H链创建L链库，这涉及到复杂的克隆过程。为此，我们设计了一套由pLf1T-3噬菌体和pHg3A-3质粒组成的双载体系统- ii (DVS-III)，用于通过噬菌体展示技术对任意给定的人Fab抗体进行L链重组。为了证明我们系统的可行性，给出了一个人类朴素L链子库。在pLf1T-3噬菌体中构建的HuNL-D3与人抗il -15 Fab， 4H10的Fd结合，亚克隆于pHg3A-3质粒中作为模型系统。经过溶液分选和生物筛选，我们获得了8个Fab变体（4H10-LP1-7和4H10-LS）。其中，4H10- lp4的亲和力最高，约为亲本分子4H10的36倍（K-D = 6 nM比200 nM）。我们的研究结果表明，通过L链优化，DVS-III和HuNL-D3 L链亚库可以作为一种方便的方法，对任何给定的人Fab抗体进行亲和成熟。(C) 2010 Elsevier B.V.版权所有
Light (L) chain shuffling is routinely used to analyze optimal L chains that pair with a specific heavy (Fd or H) chain, which ultimately leads to in vitro affinity maturation of a particular antibody. One of the major drawbacks to this procedure is that L chain libraries have to be created for each distinct H chain, which involves complicated cloning procedures. Herein, we designed of the dual-vector system-Ill (DVS-III), which is composed of a set of pLf1T-3 phagemid and pHg3A-3 plasmid, for L chain shuffling of any given human Fab antibody via phage display technology. To demonstrate the feasibility of our system, a human naive L chain sublibrary. HuNL-D3, constructed in pLf1T-3 phagemid, was combined with the Fd of a human anti-IL-15 Fab, 4H10, subcloned in pHg3A-3 plasmid as a model system. After solution-phase sorting and biopanning the library we obtained eight Fab variants (4H10-LP1-7 and 4H10-LS). Among them, 4H10-LP4 exhibited the highest affinity which is about 36-fold higher than that of the parent molecule 4H10 (K-D = 6 nM versus 200 nM). Our results demonstrate that the DVS-III, along with the HuNL-D3 L chain sublibrary, can be served as a convenient approach for affinity maturation of any given human Fab antibody through L chain optimization. (C) 2010 Elsevier B.V. All rights reserved.