A high-throughput platform for population reformatting and mammalian expression of phage display libraries to enable functional screening as full-length IgG

Abstract

Phage display antibody libraries are a rich resource for discovery of potential therapeutic antibodies. Single-chain variable fragment (scFv) libraries are the most common format due to the efficient display of scFv by phage particles and the ease by which soluble scFv antibodies can be expressed for high-throughput screening. Typically, a cascade of screening and triaging activities are performed, beginning with the assessment of large numbers of E. coli-expressed scFv, and progressing through additional assays with individual reformatting of the most promising scFv to full-length IgG. However, use of high-throughput screening of scFv for the discovery of full-length IgG is not ideal because of the differences between these molecules. Furthermore, the reformatting step represents a bottle neck in the process because each antibody has to be handled individually to preserve the unique VH and VL pairing. These problems could be resolved if populations of scFv could be reformatted to full-length IgG before screening without disrupting the variable region pairing. Here, we describe a novel strategy that allows the reformatting of diverse populations of scFv from phage selections to full-length IgG in a batch format. The reformatting process maintains the diversity and variable region pairing with high fidelity, and the resulted IgG pool enables high-throughput expression of IgG in mammalian cells and cell-based functional screening. The improved process led to the discovery of potent candidates that are comparable or better than those obtained by traditional methods. This strategy should also be readily applicable to Fab-based phage libraries. Our approach, Screening in Product Format (SiPF), represents a substantial improvement in the field of antibody discovery using phage display.

Intein mediated high throughput screening for bispecific antibodies

“…Therefore, the number of combinations or bsAbs finally generated is usually only a small fraction of the selected repertoire. [20] Moreover, subsequent optimization of each binding moiety, such as affinity maturation, might need to be performed after combining hit candidates and re-engineering into a bispecific format. [21] Finally, most bsAb engineering approaches include comparisons of several formats to further optimize the intended biological mode of action, which also enlarge the screening space or efforts.…”

Periplasmic Expression of SpyTagged Antibody Fragments Enables Rapid Modular Antibody Assembly

“…Being able to effortlessly change the valency of uncharacterized antibodies after expression offers a new degree of freedom for the screening of selection outcomes, allowing for bivalent cross-reactivity profiling while still maintaining monovalency for affinity determination. Adding an FcCatcher to the lysates will similarly allow high-throughput functional screening in assays that require the Fc moiety such as antibodydependent cellular cytotoxicity (ADCC) assays 44 . Furthermore, the addition of an antibody-SpyCatcher fusion protein during the selection step could be used to select for bispecific antibodies, similarly to a recently developed intein-based approach 45 .…”

Screening Strategies for the Discovery of Ion Channel Monoclonal Antibodies

“…This provides greater flexibility in testing multiple replicates of the same sample at multiple concentrations within the same assay or in additional assays without the need to regenerate new batches of antibody. Alternatively, hundreds of antibodies can be expressed simultaneously from HEK or CHO cells in 96 well format with the conditioned medium being tested directly in a secondary assay, provided the assay signal is not adversely affected by the cell medium and the antibody concentration is adequate to enable detection of function (Xiao et al., ). If the conditioned medium reduces the assay signal, antibodies can be purified from the medium to mg/ml concentrations using automated liquid handlers and 96 well protein A tips.…”