Divergence of binding, signaling, and biological responses to recombinant human hybrid IFN.
Three human IFN-alpha hybrids, HY-1 [IFN-alpha21a(1-75)/alpha2c(76-165)], HY-2 [IFN-alpha21a(1-95)/alpha2c(96-165)], and HY-3 [IFN-alpha2c(1-95)/alpha21a(96-166)], were constructed, cloned, and expressed. The hybrids had comparable specific antiviral activities on Madin-Darby bovine kidney (MDBK) cells but exhibited very different antiproliferative and binding properties on human Daudi and WISH cells and primary human lymphocytes. Our data suggest that a portion of the N-terminal region of the molecule is important for interaction with components involved in binding of IFN-alpha2b while the C-terminal portion of IFN is critical for antiproliferative activity. A domain affecting the antiproliferative activity was found within the C-terminal region from amino acid residues 75-166. The signal transduction properties of HY-2 and HY-3 were evaluated by EMSA and RNase protection assays. Both HY-2 and HY-3 induced activation of STAT1 and 2. However, HY-2 exhibited essentially no antiproliferative effects at concentrations that activated STAT1 and 2. Additionally, at concentrations where no antiproliferative activity was seen, HY-2 induced a variety of IFN-responsive genes to the same degree as HY-3. RNase protection assays also indicate that, at concentrations where no antiproliferative activity was seen for HY-2, this construct retained the ability to induce a variety of IFN-inducible genes. These data suggest that the antiproliferative response may not be solely directed by the activation of the STAT1 and STAT2 pathway in the cells tested.
Division of Cytokine Biology, Office of Therapeutics Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, MD 20892, USA. hu@cber.fda.gov
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Hu
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Bekisz
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Hayes
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Audet
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Beeler
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Petricoin
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Zoon
D000595:Amino Acid Sequence; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D006131:Growth Inhibitors / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects; D006801:Humans; D007370:Interferon Type I / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D016898:Interferon-alpha; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000187:drug effects / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology; D011994:Recombinant Proteins; D050794:STAT1 Transcription Factor; D050795:STAT2 Transcription Factor; D015398:Signal Transduction / Q000187:drug effects / Q000276:immunology*; D015534:Trans-Activators / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects / Q000276:immunology; D014407:Tumor Cells, Cultured
D000998:Antiviral Agents; D004268:DNA-Binding Proteins; D006131:Growth Inhibitors; C120238:IFN-alpha21a(1-75), IFN-alpha2c(76-165) fusion protein; C120239:IFN-alpha21a(1-95), IFN-alpha2c(96-165) fusion protein; D007370:Interferon Type I; D016898:Interferon-alpha; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D050795:STAT2 Transcription Factor; C494084:STAT2 protein, human; D015534:Trans-Activators; C120240:interferon alpha, HY-3