Isolation of TGF-β-neutralizing single-domain antibodies of predetermined epitope specificity using next-generation DNA sequencing

The epitope specificity of therapeutic antibodies is often critical to their efficacy and mode of action. Here, we report the isolation of single-domain antibodies (sdAbs) against a pre-specified epitope of TGF-beta 3: namely, the site of interaction between the cytokine and its cell-surface type II receptor. By panning a phage-displayed immune llama VhH library against TGF-beta 3 using competitive elution with soluble dimeric type II receptor ectodomain in tandem with next-generation DNA sequencing, we identified several sdAbs that competed with the receptor for TGF-beta 3 binding and neutralized TGF-beta 3 in in vitro cellular assays. In contrast, all other sdAbs identified using conventional panning approaches (i.e., without regard to epitope specificity) did not target the site of receptor:cytokine interaction. We expect this strategy to be generally applicable for identifying epitope-specific sdAbs when binding reagents directed against the epitope of interest are available. The sdAbs identified here are of potential interest as cancer immunotherapeutics.