King David J, Bowers Peter M, Kehry Marilyn R, Horlick Robert A
AnaptysBio Inc., 10421 Pacific Center Court, San Diego, CA 92121, USA.
Curr Drug Discov Technol. 2014 Mar;11(1):56-64. doi: 10.2174/15701638113109990037.
Human therapeutic antibody discovery has utilized a variety of systems, from in vivo immunization of human immunoglobulin-expressing mice, to in vitro display of antibody libraries. Of the in vitro antibody display technologies, mammalian cell display provides a number of advantages with the ability to co-select immunoglobulin molecules for high expression level in mammalian cells, native folding, and biophysical properties appropriate for drug development. Mammalian cell display has been achieved using either transient or stable expression systems, using a number of alternate transmembrane domains to present antibody on the cell surface. The unique capability of mammalian cells to present IgG in its fully post-translationally modified format also allows selection of antibodies for functional properties. One limitation of mammalian cell based systems, however, has been the smaller library size that can be presented compared to phage display approaches. Until recently, this has necessitated the use of libraries biased toward a particular antigen, such as libraries derived from immunized donors, to achieve success. An alternative approach has now been developed which recapitulates key aspects of the in vivo immune system through reproducing somatic hypermutation (SHM) in vitro. Libraries representing a naïve human B lymphocyte antibody repertoire are created by PCR amplification of the rearranged (D)J segments of heavy and light chain variable regions from human donors and incorporating the resulting sequence diversity into panels of human germline VH and VL genes. The resulting antibodies are presented as full length IgG on the surface of HEK293 cells. After isolation of antibodies binding to individual target antigens, subsequent affinity maturation using in vitro SHM is induced by expression of activation-induced cytidine deaminase (AID). Selection of antibodies from naïve fully human libraries using mammalian cell display coupled with in vitro SHM is an efficient methodology for the generation of high affinity human antibodies with excellent properties for drug development.
人类治疗性抗体的发现利用了多种系统，从对表达人免疫球蛋白的小鼠进行体内免疫，到抗体文库的体外展示。在体外抗体展示技术中，哺乳动物细胞展示具有许多优势，它能够共同选择在哺乳动物细胞中高表达、天然折叠且具有适合药物开发的生物物理特性的免疫球蛋白分子。哺乳动物细胞展示可通过瞬时或稳定表达系统来实现，使用多种替代跨膜结构域将抗体呈现在细胞表面。哺乳动物细胞以完全翻译后修饰形式呈现IgG的独特能力，也使得能够选择具有功能特性的抗体。然而，基于哺乳动物细胞的系统的一个局限性是，与噬菌体展示方法相比，其可展示的文库规模较小。直到最近，这都需要使用偏向特定抗原的文库，例如来自免疫供体的文库，才能取得成功。现在已经开发出一种替代方法，通过在体外重现体细胞超突变（SHM）来概括体内免疫系统的关键方面。通过从人类供体的重链和轻链可变区的重排（D）J片段进行PCR扩增，并将产生的序列多样性整合到人类种系VH和VL基因库中，创建代表天然人类B淋巴细胞抗体库的文库。产生的抗体以全长IgG的形式呈现在HEK293细胞表面。在分离出与各个靶抗原结合的抗体后，通过激活诱导的胞苷脱氨酶（AID）的表达诱导使用体外SHM进行后续的亲和力成熟。使用哺乳动物细胞展示结合体外SHM从天然全人类文库中选择抗体，是一种高效的方法，可用于生成具有优异药物开发特性的高亲和力人类抗体。
