Guttieri Mary C, Sinha Tanima, Bookwalter Carol, Liang Mifang, Schmaljohn Connie S
Virology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702-5011, USA.
Hybrid Hybridomics. 2003 Jun;22(3):135-45. doi: 10.1089/153685903322286548.
Phage display technology allows for the production and rapid selection of antigen-specific, Fab antibody fragments. For purposes of immune therapy, though, complete antibodies that retain the Fc domain are often required. In this regard, we designed cassette vectors for converting human Fab fragments selected from combinatorial phage display libraries into full-length IgG(1) monoclonal antibodies (MAbs). Two expression vectors, pIEI-Light and pIEI-Heavy, were engineered to contain respective light- and heavy-chain human signal sequences downstream of the baculovirus immediate early gene promoter, IEI. Vector pIEI-Heavy also contains the coding region for each of the human IgG(1) constant domains. To generate complete antibody genes, the cassette vectors possess convenient restriction enzyme sites for rapid in-frame cloning of coding regions for full-length light chains in pIEI-Light and for the heavy-chain variable domains in pIEI-Heavy of Fab fragments. Using these constructs and a method that allows for stable transformation of insect cells, complete light- and heavy-chain genes can be inserted into the insect cell genome and subsequently expressed under the control of the baculovirus IEI promoter. This cassette vector system was used to generate stably transformed insect cells that continuously secreted functional full-length, IgG(1) MAbs. The expressed antibodies exhibited light and heavy chains of the appropriate molecular sizes and retained the ability to bind antigen. We conclude that our cassette vectors could serve as valuable tools for generating human IgG(1) antibodies.
噬菌体展示技术能够生产并快速筛选出抗原特异性的Fab抗体片段。然而，对于免疫治疗而言，通常需要保留Fc结构域的完整抗体。在这方面，我们设计了盒式载体，用于将从组合噬菌体展示文库中筛选出的人Fab片段转化为全长IgG(1)单克隆抗体（MAb）。构建了两个表达载体pIEI-Light和pIEI-Heavy，使其在杆状病毒立即早期基因启动子IEI的下游分别包含人轻链和重链信号序列。载体pIEI-Heavy还包含每个人IgG(1)恒定结构域的编码区。为了生成完整的抗体基因，盒式载体具有方便的限制性酶切位点，用于将全长轻链编码区快速框内克隆到pIEI-Light中，以及将Fab片段的重链可变区编码区克隆到pIEI-Heavy中。利用这些构建体和一种允许昆虫细胞稳定转化的方法，完整的轻链和重链基因可以插入昆虫细胞基因组，并随后在杆状病毒IEI启动子的控制下表达。该盒式载体系统用于生成稳定转化的昆虫细胞，这些细胞持续分泌功能性全长IgG(1)单克隆抗体。所表达的抗体显示出适当分子大小的轻链和重链，并保留了结合抗原的能力。我们得出结论，我们的盒式载体可作为生成人IgG(1)抗体的有价值工具。
