The development of non-animal based methods of antibody production has become increasingly favored in recent years. In addition, the development of methods of producing human-derived or humanized versions of murine- derived antibodies has paralleled the attempts to develop the non-animal systems. Concurrently, other groups of investigators studying primarily signal transduction pathways and/or transcriptional regulation have developed several methods for assessing the protein/protein interactions inherent in these systems. In this application, we propose to combine the technological advances and existing needs of both of the aforementioned groups to produce a genetic selection system in yeast that is based on a transcriptional readout assay of reporter gene function with the goal of isolating single-chain fragments of immunoglobulin variable domains (sFv's) with high affinity for desired antigens. The system is based on the two-hybrid system described originally by Fields and Song (Nature 340:245, 1989). A library of human-derived sFv's will be cloned into vectors that will encode fusion proteins linking these sFv's with constitutive transactivation domains and nuclear localization signals. The goal of these initial studies will be to isolate a fusion molecule from this library that will target a transcriptional activation domain to a specific DNA-bound factor.
The technology will be patented and could be licensed to companies for non-animal means of producing antibodies. Similarly, the assay could be used to isolate molecules to be used in therapeutic applications of gene therapy. Specific targeting molecules will also have commercial potential as research reagents.
