Technical Highlight - June 2014
Short description: Protocols for phage display enable the generation of highly customizable synthetic antibodies against specific molecular complexes or conformational states.
Phage display has emerged as a powerful alternative to hybridoma-based methods for generating antibodies with specified properties. Since antibody fragments are displayed on the phage surface for selection, there is a direct link between phenotype and genotype; the latter can be obtained by sequencing phage DNA. In silico libraries contain substantial variation, allowing for the selection of highly customizable antibodies.
Kossiakoff and colleagues have presented integrated protocols for library design and phage selection methods to generate synthetic antibodies (sABs). sABs have several advantages over monoclonal antibodies (mABs), including the ability to precisely specify changes in the complementarity determining regions (CDRs), which are critical for antigenic binding activity. Additionally, the coding DNA is easily recovered for subsequent heterologous expression, and issues such as clonal drift and animal experimentation are avoided. While sABs can be used for the same applications as mABs, the phage display method can more readily target protein–protein or protein–nucleic acid complexes and specific conformational states. A particularly interesting example is the use of sABs as chaperones for crystallization and molecular replacement in challenging protein and RNA systems.
The authors utilized synthetic fragment antigen-binding (Fab) libraries in which amino acid diversity is introduced into the CDRs to yield a naïve library containing ∼1010 unique clones. Next, they provided detailed protocols for sAB selection, library sorting and screening. Competition selection strategies require fewer steps and generate more clones, and are therefore more suitable for systems with defined conformational states or high-affinity interactions. Primary screening of clones can be accomplished by DNA sequencing, while secondary screening requires expression, purification and biophysical characterization of the sABs.
Depending on the set thresholds, up to 50% of isolated clones may have the required properties. The authors highlighted potential pitfalls and solutions for isolating antibodies against complexes, low-affinity interactions and proteins with antigenic hot-spots that generate sABs against a single epitope. Despite the challenges associated with these and other systems, the pipeline can be automated and run in parallel, allowing investigators to screen more clones in the search for effective antibodies.
M. Paduch et al. Generating conformation-specific synthetic antibodies to trap proteins in selected states.
Methods. 60, 3-14 (2013). doi:10.1016/j.ymeth.2012.12.010