The invention provides methods of using sequence based analysis and rational strategies to modify and improve the structural and biophysical properties of immunobinders, and in particular of single chain antibodies (scFvs), including such properties as stability, solubility, and/or antigen binding affinity. The invention provides methods of engineering immunobinders, and in particular scFvs, by performing one or more substitutions at amino acid positions identified by analysis of a database of selected, stable scFv sequences, wherein preferred amino acid residues for substitution have been identified. The invention also provides immunobinders prepared according to the engineering methods of the invention. The invention also provides preferred scFv framework scaffolds, into which CDR sequences can be inserted, as well as scFv antibodies made using these preferred framework scaffolds.
