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.
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