(427d) A New Platform Technology for Engineering Intracellular Antibodies Based on the Bacterial Tat Pathway

Advanced techniques in antibody engineering now allow for the manipulation of genes encoding antibodies such that the antigen-binding domain can be expressed intracellularly. These intracellular antibodies are termed ‘intrabodies'. Single-chain 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 of 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 strategy useful for the discovery and engineering of scFv that function inside cells, i.e. fold within the reducing environment and still retain antigen affinity. This proposed selection is founded upon the recently discovered bacterial twin-arginine translocation (Tat) pathway, a remarkable secretion system that has the ability to (1) transport folded proteins across biological membranes, (2) proofread a protein substrate prior to export and (3) process heterodimeric protein complexes. 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. As proof-of-principle, we have isolated intrabodies that bind with high affinity to the Alzheimer's amyloid beta-peptide (Abeta42) and represent putative protein therapeutics for treating Alzheimer's disease.