Abrogation of viral interleukin-6 (vIL-6)-induced signaling by intracellular retention and neutralization of vIL-6 with an anti-vIL-6 single-chain antibody selected by phage display.
Human herpesvirus 8 (HHV-8) encodes several putative oncogenes, which are homologues to cellular host genes known to function in cell cycle regulation, control of apoptosis, and cytokine signaling. Viral interleukin (vIL-6) is believed to play an important role in the pathogenesis of Kaposi's sarcoma as well as primary effusion lymphoma and multicentric Castleman's disease. Therefore, vIL-6 is a promising target for novel therapies directed against HHV-8-associated diseases. By phage display screening of human synthetic antibody libraries, we have selected a specific recombinant antibody, called monoclonal anti-vIL-6 (MAV), binding to vIL-6. The epitope recognized by MAV was localized on the top of the D helix of the vIL-6 protein, which is a part of receptor binding site III. Consequently, MAV specifically inhibits vIL-6-mediated growth of the primary effusion lymphoma-derived cell line BCBL-1 and blocks STAT3 phosphorylation in the human hepatoma cell line HepG2. Since it was previously found that vIL-6 can also induce signals from within the cell, presumably within the endoplasmic reticulum, we fused the recombinant antibody MAV with the endoplasmic retention sequence KDEL (MAV-KDEL). As a result, COS-7 cells expressing MAV-KDEL and synthesizing vIL-6 ceased to secrete the cytokine. Moreover, we observed that vIL-6 that was bound to MAV-KDEL and retained in the endoplasmic reticulum did not induce STAT3 phosphorylation in HepG2 cells. We conclude that the activity of the intracellularly retained vIL-6 protein is neutralized by MAV-KDEL. Our results might represent a novel therapeutic strategy to neutralize virally encoded growth factors or oncogenes.
Kovaleva
Marina
M
Department of Biochemistry, Christian Albrechts Universität, Kiel, Germany.
Bussmeyer
Ingo
I
Rabe
Björn
B
Grötzinger
Joachim
J
Sudarman
Enge
E
Eichler
Jutta
J
Conrad
Udo
U
Rose-John
Stefan
S
Scheller
Jürgen
J
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D000914:Antibodies, Viral / Q000276:immunology / Q000378:metabolism*; D000918:Antibody Specificity; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000276:immunology; D015850:Interleukin-6 / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D009500:Neutralization Tests; D019151:Peptide Library; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D015398:Signal Transduction / Q000276:immunology; D014764:Viral Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D007135:Immunoglobulin Variable Region; D015850:Interleukin-6; D019151:Peptide Library; D011994:Recombinant Proteins; D014764:Viral Proteins