Isolation of Antigen-Specific Intracellular Antibody Fragments as Single Chain Fv for Use in Mammalian Cells

The control of gene function by the modulation of mRNA translation, stability, or protein activity has important implications for the treatment of disease, as well as in research programs designed to study the biological role of proteins. For instance, preventing protein function in specific cell types in development can produce a phenotypic knock-out due to the effective protein loss. Antibodies are ideally suited for this purpose, as they have evolved to bind macromolecules with high affinity and can neutralize their function as a result. Ablation of protein function in vivo with antibodies has been achieved by microinjecting whole antigen-specific immunoglobulin molecules into the cell cytoplasm (1 ). However, complete antibody comprises four chains (two heavy and two light chains) held by interchain disulfide bonds. This is not suitable for expression from DNA vectors, as the individual chains would not assemble in the cytoplasm. Therefore, antibody fragments have been employed in the single chain Fv (scFv) format (2 ), which are single polypeptide chains comprising a heavy chain variable region (VH) and a light chain variable region (VL) held by a short linker sequence. ScFv folds into an antibody combining site (which binds antigen) but no effector function is present. Many uses of scFv expressed inside cells (herein called intracellular antibody or ICAbs) have been described in cell systems (3 ,4 ) and in whole organisms. For example, in vivo expression of a scFv directed against the coat protein of artichoke mottle crinkle virus has conferred resistance to viral infection in plant cells (5 ).