Editing Phage Display from Synthetic Library (section)

=== Introduction ===

With the onset of the post-genomic age, the availability of high quality binding reagents to characterize and modulate proteins is essential to open new research and therapeutic avenues. Antibodies continue to be critical to both academic and industrial researchers as basic research and diagnostic tools and potential therapeutics. Not surprisingly, there has been an impressive growth of contract antibody development firms, most of which rely upon conventional hybridoma technologies to generate custom antibodies. Nevertheless,''in vitro'' selection using phage-displayed antibody libraries is becoming a powerful alternative technology that can offer unique advantages and success where conventional technologies can face limitations<sup>1, 2</sup>.

In light of the considerable demand for high quality antibodies as research tools, two primary challenges for generating renewable antibodies are 1) selection throughput and 2) antigen availability. A number of groups have now described in vitro selection pipelines aimed at increasing throughput and the rate of antibody identification. These descriptions detail a variety of viable approaches that include selecting upon either full-length targets<sup>3,4</sup>, or structurally related domains<sup>5,6,7</sup>, using either bead-based<sup>6,8</sup> or plate-based<sup>3,4</sup> antigen immobilization schemes. In addition, the growing adoption of gene synthesis technologies<sup>9</sup> has made systematic antigen generation, particularly of isolated domains, reasonably cost effective and can potentially alleviate the difficulty in obtaining sufficient quantities of purified, full-length antigen. By using the two technologies in tandem, a self-contained and scalable antigen generation and antibody selection pipeline was devised that would enable the parallel isolation of antibodies for large sets of expressed antigen domains and facilitate the development of reagents for characterizing entire classes of structurally or functionally related proteins.

Toward this aim, an integrated pipeline that couples in silico identification of expressible antigen domains, gene synthesis, high-throughput bacterial expression of antigens and scalable phage-displayed antibody selections has been developed. This pipeline requires only basic infrastructure available to most life science laboratories (including antibody libraries which are increasingly available through license or material transfer agreement), but is also amenable to automation for use on an industrial scale. Using this protocol, it is possible to generate hundreds of affinity-tagged antigen domains, and routinely isolate highly specific antibody fragments to many of these antigens.

Phage display technology has shown demonstrated compatibility with a wide variety of recombinant affinity reagent formats including Fab, scFv, and autonomous Fv domains and a growing array of small ‘alternative frameworks’ (designed ankyrin repeat proteins (DARPINS), fibronectin (Fn), lipocalin domains and more<sup>10</sup>). Discussion is restricted in this example to the isolation of Fab antibody fragments, although it is presumed these methods can be adapted to other types of libraries. Using this technology, Fabs with low nanomolar affinity to small, tagged protein domains including transcription factor domains, SH2 domains, RNA-binding proteins and others have been successfully selected, many of which bind full-length protein and are functional in immunoassays such as immunofluorescence, immunoprecipitation and immunohistochemistry. Importantly, recombinant binding clones are fully renewable and can be re-generated from expression constructs via bacterial production offering increased consistency, reproducibility and cost-effectiveness, thus justifying the expense of rigorous clone validation.

In this protocol and accompanying video, basic methods for antibody selection from phage-displayed libraries using immobilized antigen domains are demonstrated. This particular method employs GST-tagged protein domains immobilized by passive adsorption in microwell plates, although other tags<sup>11,12,13</sup> and selection formats<sup>13,14,2</sup> have also been used successfully. Critical considerations for the setup and conduct of selections with parallel monitoring of selection parameters aimed at identifying and isolating specifically enriched clonal antibodies for validation are detailed.

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