Amino Acid Substitutions in Loop BC and Helix C Affect Antigenic Properties of Helix D in Hybrid IFN- alpha 21a/ alpha 2c Molecules

We compared the antigenic properties of human interferon- alpha 2c (IFN- alpha 2c), IFN- alpha 21a, hybrids IFN- alpha 21a/ alpha 2c, and their mutants, using a panel of 27 anti-IFN- alpha 1, anti-IFN- alpha 2, and anti-IFN- alpha 8/1/8 monoclonal antibodies (mAb). After immunoanalysis by ELISA, we found parental IFN- alpha 2c and IFN- alpha 21a to be antigenically distinct. Lack of reactivity of anti-IFN- alpha 1 mAb with IFN- alpha 21a indicated an antigenic distinction between subtypes alpha 1 and alpha 21a. The antigenic properties of hybrid IFNs consisting of the N-terminal portion (1-75) of IFN- alpha 21a and the C-terminal portion (76-166) of IFN- alpha 2c were analyzed with mAb recognizing defined regions of IFN- alpha 2c, IFN- alpha 1, and IFN- alpha 8/1/8. We found that extending the sequence of IFN- alpha 21a up to position 95 in hybrid molecule decreased the immunoreactivity of mAb specific for the antigenic structure formed by residues similar to 112-132 similar to (helix D) of IFN- alpha 2c. Inserting the sequence 76-81 (loop BC) of IFN- alpha 2c into the sequence of 1-95 of IFN- alpha 21a restored the reactivity of anti-IFN- alpha 2c mAb. Some amino acid substitutions at positions 86 and 90 (helix C) of hybrid IFN- alpha 21a/ alpha 2c also affected the immunoreactivity of C-terminal-specific mAb, which recognize helix D, but did not influence the structure of C-terminus of IFN (aa 151-165). Changes in the structure of constructs affected not only their antiproliferative activity but also their antiviral activity on human cells.