Method for decreasing immunogenicity

A method for reducing the immunogenicity of a single chain Fv (scFv), the method comprising:

• (a) providing the amino acid sequence of a first scFv that comprises a heavy chain variable region that comprises Leu at position 12, Val at position 103, and Leu at position 144 (AHo numbering), wherein the scFv reacts with a scFv test serum that contains pre-existing antibodies to the scFv but does not contain antibodies to the IgG form of the scFv;
• (b) substituting a Ser or Thr for one or more of the Leu at position 12, the Val at position 103, or the Leu at position 144 to produce one or more substituted scFv;
• (c) comparing the ability of a substituted scFv produced in (b) to compete with the first scFv for binding to anti-scFv antibodies in the scFv test serum;
• and
• (d) selecting a substituted scFv that has a reduced ability to compete with the first scFv for binding to the anti-scFv antibodies in the test serum, thereby providing a scFv with reduced immunogenicity.