Antibody variable domains represent potential structural models for the rational design of therapeutic molecules that bind cellular proteins with high affinity and specificity. The Activating Transcription Factor 1 (ATF1)/Cyclic AMP Response Element Binding Protein (CREB) family of transcription factors are particularly relevant targets due to their strong association with melanoma and clear cell sarcoma. Biochemical and structural investigations were performed to optimize a single-chain antibody fragment (scFv), scFv41.4, that disrupts the binding of ATF1/CREB to cyclic-AMP response elements (CRE) in vitro and inhibits transcriptional activation in cells. Molecular modeling and ligand docking simulations suggested that scFv41.4 could function as a disulfide-deficient single domain scFv. Functional studies verified that deletion of the light chain did not result in reduced inhibitory activity. The isolated heavy chain was predicted to assume a relaxed structural conformation that maintained a functional antigen binding pocket. The minimal structural elements necessary for intracellular function were further analyzed by selective deletion of CDR1 and CDR2. VH-CDR1 and VH-CDR3 were shown to play a key role in antigen binding activity, but VH-CDR2 was dispensable. Thus, scFv41.4 represents a unique molecule with potential for use in the design of peptidomimetic derivatives having therapeutic application to human cancer.
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