title: Amino acid substitutions in loop BC and helix C affect antigenic properties of helix D in hybrid IFN-alpha21a/alpha2c molecules.
abstract: We compared the antigenic properties of human interferon-alpha2c (IFN-alpha2c), IFN-alpha21a, hybrids IFN-alpha21a/alpha2c, and their mutants, using a panel of 27 anti-IFN-alpha1, anti-IFN-alpha2, and anti-IFN-alpha8/1/8 monoclonal antibodies (mAb). After immunoanalysis by ELISA, we found parental IFN-alpha2c and IFN-alpha21a to be antigenically distinct. Lack of reactivity of anti-IFN-alpha1 mAb with IFN-alpha21a indicated an antigenic distinction between subtypes alpha1 and alpha21a. The antigenic properties of hybrid IFNs consisting of the N-terminal portion (1-75) of IFN-alpha21a and the C-terminal portion (76-166) of IFN-alpha2c were analyzed with mAb recognizing defined regions of IFN-alpha2c, IFN-alpha1, and IFN-alpha8/1/8. We found that extending the sequence of IFN-alpha21a up to position 95 in hybrid molecule decreased the immunoreactivity of mAb specific for the antigenic structure formed by residues --112-132-- (helix D) of IFN-alpha2c. Inserting the sequence 76-81 (loop BC) of IFN-alpha2c into the sequence of 1-95 of IFN-alpha21a restored the reactivity of anti-IFN-alpha2c mAb. Some amino acid substitutions at positions 86 and 90 (helix C) of hybrid IFN-alpha21a/alpha2c 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.
authors.affiliation: Division of Therapeutic Proteins, Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, MD 20892, USA. schmeisserh@cber.fda.gov
authors.forename: Hana
authors.initials: H
authors.lastname: Schmeisser
authors.forename: Renqiu
authors.initials: R
authors.lastname: Hu
authors.forename: Peter
authors.initials: P
authors.lastname: Kontsek
authors.forename: Joseph
authors.initials: J
authors.lastname: Bekisz
authors.forename: Kathryn
authors.initials: K
authors.lastname: Zoon
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000911:Antibodies, Monoclonal / Q000276:immunology; D000941:Antigens / Q000276:immunology; D000998:Antiviral Agents / Q000737:chemistry* / Q000276:immunology* / Q000494:pharmacology; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D006131:Growth Inhibitors / Q000737:chemistry* / Q000276:immunology* / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D007370:Interferon Type I / Q000737:chemistry / Q000235:genetics* / Q000276:immunology* / Q000494:pharmacology; D016898:Interferon-alpha / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000494:pharmacology; D011994:Recombinant Proteins; D016415:Sequence Alignment
chemical_list: D000911:Antibodies, Monoclonal; D000941:Antigens; D000998:Antiviral Agents; D006131:Growth Inhibitors; D007370:Interferon Type I; D016898:Interferon-alpha; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C554129:interferon alfa-2c