Advanced techniques in antibody engineering now allow for the manipulation of genes encoding antibodies such that the antigen binding process can proceed inside living cells. Such intracellular antibodies (intrabodies) are capable of interfering with a wide range of molecular targets in various intracellular compartments with high specificity and affinity. Single-chain variable region antibody fragments (scFv) are a suitable format for intrabodies as they are small molecules with high-affinity ligand-binding capability and minimal assembly requirements. Unfortunately, the reducing environment in the cytoplasm inhibits formation of the intrachain disulfide bonds of the variable domains of the heavy and light chains and, as a result, blocks their proper folding. Consequently, the scFv is rendered nonfunctional and exhibits poor expression levels, low solubility and/or a short half-life.

To address this shortcoming, we have developed a genetic selection technology useful for the discovery and engineering of scFv that function inside cells, i.e. fold under the reducing environment and still retain antigen affinity. Our selection strategy is founded upon the recently discovered bacterial twin-arginine translocation (Tat) pathway that has the ability to transport folded protein complexes across biological membranes and proofread these complexes prior to export. By exploiting these unique features, our selection allows for single-step isolation of scFv that are compatible with folding in the reducing cytoplasm and that bind with high-affinity to virtually any peptide or protein target antigen. Preliminary experiments have demonstrated that the efficiency of transport through the Tat system is proportional to binding affinity between the scFv and its antigen. As a result, selection conditions have been established that allow for rapid isolation of scFv-antigen pairs that bind with exquisitely tailored affinities. As proof-of-principle, we will discuss our efforts to isolate intrabodies that bind with high affinity to the cytosolic Rex-1 transcription factor that plays a key role in regulating stem cell differentiation into different lineages.

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