SBIR-STTR Award

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 will 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.National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)

This Phase II SBIR application discusses the rationale and approach for further development of the SMART reagent system. Phase I developed a modified two-hybrid system in yeast for isolating single-chain antibodies (sFv's) with high affinity for desired antigens in vivo. A library of human-derived sFv's was cloned into a yeast expression vector that encodes fusion proteins linking sFv's with a constitutive transactivation domain (VP16). The goal of the initial studies was to develop this technology and isolate sFv's from this library that exhibit the appropriate antigen binding specificity and are capable of targeting antigens in vivo. To expand the utility of this novel system, the aims for phase II are: 1) To construct an optimal library vector with zeocin selection to facilitate the isolation of the sFv/VP16 plasmids and to subclone a human sFv library into the new library vector. 2) To screen the new sFv/VP16zeo expression library with a variety of new baits (including transcription factors, intracellular signaling molecules and nuclear hormone receptors). 3) To use the family of human open reading frame HORF kinases that have been cloned and expressed as baits to isolate neutralizing antibodies. PROPOSED COMMERCIAL APPLICATION: Not available.

Thesaurus Terms:
antibody specificity, biological signal transduction, technology /technique development, transcription factor, transfection vector, yeast two hybrid system DNA binding protein, antigen antibody reaction, genetic marker, neutralizing antibody, open reading frame, plasmid, protein kinase, reagent /indicator genetic library, molecular cloning