Generating conformation-specific synthetic antibodies to trap proteins in selected functional states

Abstract

A set of phage display sorting strategies and validation methodologies are presented that are capable of producing high performance synthetic antibodies (sABs) with customized properties. Exquisite control of antigen and conditions during the phage display selection process can yield sABs that: (1) recognize conformational states, (2) target specific regions of the surface of a protein, (3) induce conformational changes, and (4) capture and stabilize multi-protein complexes. These unique capabilities open myriad opportunities to study complex macromolecular processes inaccessible to traditional affinity reagent technology. We present detailed protocols for de novo isolation of binders, as well as examples of downstream biophysical characterization. The methods described are generalizable and can be adapted to other in vitro direct evolution approaches based on yeast or mRNA display.

Fig. 1. sAB phage display

A) sAB binders undergo standard phage affinity selection and amplification in host E. coli cells. B) sAB - optimized Fab fragment of anti-Her2 mAB scaffold where CDR loops L3, H1, H2, H3 are randomized, and the entire antibody fragment is encoded on the phagemid. The sAB is fused to the p3 coat protein, allowing for display on the surface of the phage.

Fig. 2. sAB binding characteristics

Phage display selections can be tuned to generate sABs with multiple activities A), B), C) based on either targeting a particular surface epitope D), conformational state E) or stabilizing a transient complex F).

Fig. 3. sAB selection strategies

A) Subtractive selection - competitor (red diamond) in step 1 (negative selection) is added to the beads, library is pre-cleared and added in step 2 (positive selection) to the new beads containing target of interest (blue circle), B) competition selection - competitor is added in excess in solution along with captured biotinylated target.

Fig. 4. Process work flow chart

sAB screening starts with selection design (Section 2) which is followed by phage display selection (Section 3). Information acquired during selections can be used to further enhance experiment design and introduce necessary modifications (for troubleshooting see Section 4). Primary and secondary screening proceeds immediately after (Sections 3.2 and 3.3).

Fig. 5. Examples of primary and secondary validation

A) Pull-down assay; SDS-PAGE gel: T -total biotinylated protein before loading on the beads, S - supernatant (fraction of the protein not captured by streptavidin), BW - bead wash, E - DTT elution, EW - wash after elution, B - beads, M - protein marker. Almost 90 % of the protein is captured and eluted in pull-down. B) phage selection enrichment check; bars marked with “+” represent count of the phages eluted in selection experiment"" phages obtained from negative control where protein of interest was not added. C) Single point competition ELISA - scatter plot showing ELISA signals of target binding (x-axis) and competition with soluble protein (y-axis) - clones located in top left corner of the plot (high ELISA 450 nm signal and low competition ratio; positive clone threshold - dashed line in gray) are picked for secondary screening (marked in blue). D) SPR - affinity measurement; plot represents SPR trace of sAB injection (3 analyte concentrations) over 4 channels on a chip - binding is detected only when target protein (Abh3) is crosslinked to its ligand (DNA) - immobilized on channel 2 (blue lines), sABs do not bind to the reference channel or ligand alone and only residual binding (small signal change) is observed for protein without the ligand (red lines). E) DSF - thermal shift assay: red line - target, blue line - target-sAB complex melt curves, increased stability of target protein is observed in the presence of the excess of sAB indicated by thermal shift (black arrow, 15 °C shift). F) size exclusion chromatography - complex formation is observed by large volumetric shift (black arrow) of target-sAB (blue line) complex on elution profile.