581470 SEQUENCE BASED ENGINEERING AND OPTIMIZATION OF SINGLE CHAIN ANTIBODIES Provided is a method for identifying one or more amino acid residues for substitution in a particular position in an immunobinder for improving a functional property of an immunobinder, with the proviso that the functional property is not an improvement in antigen binding affinity, the method comprising: a) providing a first database of VH and/or VL amino acid sequences grouped according to subtype, wherein the database comprises or consists of germ line or mature amino acid sequences； b) providing a second database of VH and/or VL amino acid sequences grouped according to subtype and selected as having at least one desirable functional property； c) determining amino acid frequency for an amino acid residue at a framework position of the first database and at a corresponding framework position of the second database； d) determining the enrichment factor (RF2:RF1) of said amino acid residue by building the ratio between the relative frequency of said amino acid residue within the second database (RF2) and the relative frequency of said amino acid residue within the first database (RF1)； e) identifying the amino acid residue (i) as a preferred amino acid residue for substitution at the corresponding amino acid position of the immunobinder when the amino acid has an enrichment factor 20 of greater than 1 , or (ii) as an amino acid residue to be excluded at the corresponding amino acid position of the immunobinder when the amino acid residue has an enrichment factor of less than 1.
