Design and construction of a naive mouse antibody phage display library
Design and construction of a naive mouse antibody phage display library
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DOI：
10.1016/j.jim.2010.01.003
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发表时间：
2010-02-28
影响因子：
2.2
通讯作者：
Neri, D.
中科院分区：
文献类型：
--
作者：
Sommavilla, R.;Lovato, V.;Neri, D.
抗体噬菌体技术极大地促进了针对几乎任何靶抗原的高质量单克隆抗体的分离。人抗体的大型组合噬菌体展示文库通常用于鉴定用于临床应用的抗体候选物。然而，啮齿类动物的临床前研究将受益于高质量的单罐小鼠抗体库的可用性，目前还没有。在这篇文章中，我们报告了三个小鼠抗体噬菌体展示文库的构建，所有文库均包含超过10亿个抗体克隆，并且所有文库均基于类似的文库设计，其特征在于给定抗体支架的CDR 3环中的残基的组合诱变。虽然发现所有三个文库都在细菌上清液中表达抗体，但其中只有一个文库（称为“PHPtop”）显示出可靠地产生针对迄今为止在选择实验中使用的所有蛋白质抗原（包括三种肿瘤相关抗原）的优质抗体。PHYRTOP文库的模块化结构促进了基于VH结构域的CDR 1和CDR 2环的组合诱变的简单亲和力成熟程序，这导致了对纤连蛋白的EDB结构域（血管生成的标志物）特异性的高亲和力抗体（“H7”：Kd = 6 nM）的分离。因此，单罐抗体库PHYSTOPTOP可以代表结合特异性的有用来源，促进在免疫活性同基因小鼠病理模型中的临床前研究。(C)2010 Elsevier B.V.保留所有权利。
Antibody phage technology has greatly facilitated the isolation of good-quality monoclonal antibodies to virtually any target antigen. Large combinatorial phage display libraries of human antibodies are routinely being used for the identification of antibody candidates for clinical applications. However, preclinical studies in rodents would benefit from the availability of good-quality single-pot mouse antibody libraries, which at present are not available. In this article, we report on the construction of three mouse antibody phage display libraries, all containing over I billion antibody clones and all based on a similar library design, which featured the combinatorial mutagenesis of residues in the CDR3 loops of a given antibody scaffold. While all three libraries were found to express antibodies in bacterial supernatants, only one of them (termed "PHILOtop") was shown to reliably yield good-quality antibodies towards all protein antigens used so far in selection experiments, including three tumor-associated antigens. The modular structure of the PHILOtop library facilitates a simple affinity-maturation procedure based on the combinatorial mutagenesis of CDR1 and CDR2 loops of the VH domain, which has led to the isolation of a high-affinity antibody ("H7": Kd = 6 nM) specific to the EDB domain of fibronectin, a marker of angiogenesis. The single-pot antibody library PHILOtop may thus represent a useful source of binding specificities, facilitating preclinical studies in immunocompetent syngeneic mouse models of pathology. (C) 2010 Elsevier B.V. All rights reserved.