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Abstract
We describe a novel cloning method, referred to as insert-tagged (InTag) positive selection, for the rapid one-step reformatting of phage-displayed antibody fragments to full-length immunoglobulin Gs (IgGs). InTag positive selection enables recombinant clones of interest to be directly selected without cloning background, bypassing the laborious process of plating out cultures and colony screening and enabling the cloning procedure to be automated and performed in a high-throughput format. This removes a significant bottleneck in the functional screening of phage-derived antibody candidates and enables a large number of clones to be directly reformatted into IgG without the intermediate step of Escherichia coli expression and testing of soluble antibody fragments. The use of InTag positive selection with the Dyax Fab-on-phage antibody library is demonstrated, and optimized methods for the small-scale transient expression of IgGs at high levels are described. InTag positive selection cloning has the potential for wide application in high-throughput DNA cloning involving multiple inserts, markedly improving the speed and quality of selections from protein libraries.
Highlights
Since the initial finding that filamentous phages were capable of expressing heterologous peptides on their surface [1] and that functional antibody fragments could be assembled in Escherichia coli [2,3] and expressed on the surface of fd bacteriophage [4], phage display of antibody fragments has evolved as an important tool in the discovery of human therapeutic antibodies
Owing to the time-consuming and laborious nature of current immunoglobulin Gs (IgGs) reformatting methods, most of the initial screening of phage-derived candidates is performed with phage clones or antibody fragments expressed in E. coli before selection of a small number of leads for IgG reformatting and functional characterization
This is a major bottleneck for a process that has been highly automated for HTP screening before the important step of testing as IgGs, often the final clinical format
Summary
Since the initial finding that filamentous phages were capable of expressing heterologous peptides on their surface [1] and that functional antibody fragments could be assembled in Escherichia coli [2,3] and expressed on the surface of fd bacteriophage [4], phage display of antibody fragments has evolved as an important tool in the discovery of human therapeutic antibodies. Over the past three decades, a number of methods have been used to generate large Fab or scFv-based phage display libraries of human antibodies, which attempt to mimic the sequence and structural diversity of the human immunological repertoire [5]. The functional evaluation of antibodies while still fused to the bacteriophage is limited and generally requires the re-engineering of phage clones to enable expression and purification of soluble recombinant antibody fragments for analysis, typically in E. coli. This process is time-consuming and can be problematic in regard to yield, for mammalian-derived antibody libraries where codon usage favours mammalian expression. Owing to the lack of rapid and HTP IgG reformatting methods, the E. coli expression step is currently required to narrow the number of lead candidates before IgG reformatting and mammalian expression
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