Selection of recombinant anti-SH3 domain antibodies by high-throughput phage display

Abstract

Antibodies are indispensable tools in biochemical research and play an expanding role as therapeutics. While hybridoma technology is the dominant method for antibody production, phage display is an emerging technology. Here, we developed and employed a high-throughput pipeline that enables selection of antibodies against hundreds of antigens in parallel. Binding selections using a phage-displayed synthetic antigen-binding fragment (Fab) library against 110 human SH3 domains yielded hundreds of Fabs targeting 58 antigens. Affinity assays demonstrated that representative Fabs bind tightly and specifically to their targets. Furthermore, we developed an efficient affinity maturation strategy adaptable to high-throughput, which increased affinity dramatically but did not compromise specificity. Finally, we tested Fabs in common cell biology applications and confirmed recognition of the full-length antigen in immunoprecipitation, immunoblotting and immunofluorescence assays. In summary, we have established a rapid and robust high-throughput methodology that can be applied to generate highly functional and renewable antibodies targeting protein domains on a proteome-wide scale.

Keywords: SH3 domain; antibodies; high throughput method; phage display.

Figure 1

CDR sequences and binding kinetics of Fabs. Positions randomized within each of the CDRS are shown at the top of each column numbered according to IMGT standards. Each Fab is identified with a unique identifier (Fab ID #) used throughout the manuscript. For each SH3 domain antigen, abbreviated protein codes corresponding to the Uniprot recommended gene codes are provided to unambiguously identify the parent protein, and its position relative to the total number of SH3 domains in the parent protein is denoted numerically (e.g., ITSN2-2/5 represents the second of five SH3 domains in the protein ITSN2). Binding parameters (k_on, k_off, K_d) were determined from kinetic analysis of Fabs binding to immobilized antigens measured by surface plasmon resonance. Sequences are shown for Fabs raised against 12 antigens discussed in detail in the text and other Fab sequences are shown in Supporting Information Figure S2.

Figure 2

Evaluation of Fab specificities. The binding specificities of Fab-phage clones were evaluated by phage ELISAs. Signals were measured for Fab-phage binding to cognate SH3 domains (white bars) or homologous SH3 domains (black bars). Asterisks denote Fab-phage exhibiting high affinities by competitive phage ELISAs (IC₅₀ < 50 nM).

Figure 3

Evaluation of anti-RASA1 fabs in IP and western blot applications. (A) Fabs F003.1 and F003.2 (but not a negative control Fab) immunoprecipitated full-length RASA1 protein from lysates of both untransfected HEK293T cell lysates and from lysates of HEK293T cells transfected with a vector designed to over-express RASA1. Immunoprecipitated RASA1 (MW ∼116 kDa) was visualized using a commercial anti-RASA1 antibody (B4F8). (B) Fabs F003.1 and F003.2, and the commercial anti-RASA1 antibody B4F8, recognize a common 116 kDa species corresponding to the molecular weight of full-length RASA1 in western blot experiments with SDS-PAGE of cell lysates from untransfected HEK293T cells or HEK293T cells transfected with a vector designed to over-express RASA1.

Figure 4

Evaluation of anti-RASA1 Fab F003.2 in immunofluorescence applications. HEK-293T cells transfected with a vector for expression of a GFP-tagged full length RASA1 protein were stained with Fab F003.2 and an anti-FLAG Alexa Fluor 647 secondary (recognizing the FLAG tag on the C-terminus of the Fab light chain). Fluorochrome emission from Fab F003.2 staining of cells transfected with the RASA1 expression construct is shown in “A” (red), emission from the GFP tag shown in “B” (green) and an overlay of the two images shown in “C.” The resultant yellow staining pattern observed upon merger of the two images serves as evidence of co-localization between the GFP fusion tag on RASA1 and Fab F003.2 bound to RASA1, confirming target specificity. HEK293T cells transfected with a vector for expression of a negative control GFP-tagged protein (PTK7) were stained as above. In contrast to the staining observed in cells expressing RASA1, no red fluorochrome emission was observed in cells incubated with Fab F003.2 (panel D), despite that GFP emission confirms robust expression of the control PTK7 fusion protein as observed in “E.” Further, upon merger of the two images, only green pseudo-coloured GFP emission is observed in “F,” further attesting to the specificity of the anti-RASA1 Fab F003.2 for it's target only. For contrast, cells were counter-stained with DAPI and shown in the overlays (C and F).