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.
Hu
R
R
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
Bekisz
J
J
Hayes
M
M
Audet
S
S
Beeler
J
J
Petricoin
E
E
Zoon
K
K
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