The objective of the proposed research is to engineer the structure of anti-fluorescein (anti-FL) single chain antigen binding (SCA TM) proteins for improved stability and modification of binding affinity and specificity. In Phase I, Genex developed an SCA protein against the hapten fluorescein because the binding of this antigen can be easily assayed quantitatively. SCA proteins are composed of the VL and VH domains of an immunoglobulin fused into a single polypeptide chain via a designed peptide linker. These SCA proteins retain the binding specificity and affinity of antibodies but in a smaller, simpler form and therefore have several advantages for in vivo and in vitro health care applications. In Phase II, the antigen binding site of the anti- FL SCA protein will be engineered to alter binding affinity and specificity, and the framework and linker peptide will be engineered to improve stability and add specific functionalization sites. This protein engineering research will involve (a) x-ray crystallographic structure determinations; (b) protein design; (c) construction and expression of modified SCA genes; and (d) purification and analysis of the modified proteins. The techniques learned in engineering the anti-FL SCA proteins should be applicable for designing and producing other SCA affinity products for use in various health-care applications, such as anti-tumor imaging and therapy.