Pivotal Involvement of Fcγ Receptor IIA in the Neutralization of Lipopolysaccharide Signaling via a Potent Novel Anti-TLR4 Monoclonal Antibody 15C1

NovImmune SA, Genève, Geneva, Switzerland
Journal of Biological Chemistry (Impact Factor: 4.57). 12/2007; 282(48):34817-27. DOI: 10.1074/jbc.M706440200
Source: PubMed


The mammalian Toll-like receptor (TLR) family has evolved to sense pathogens in the environment and protect the host against
infection. TLR4 recognizes lipopolysaccharide (LPS) from Gram-negative bacteria and induces a signaling cascade that, when
exaggerated, has been associated with severe sepsis. We have generated a TLR4-specific monoclonal antibody, 15C1, which neutralizes
LPS-induced TLR4 activation in a dose-dependent manner. 15C1 potently blocks the effects of LPS on a panel of primary cells
and cell lines in vitro. The binding of 15C1 was mapped to an epitope in the second portion of the extracellular region of TLR4, which has been shown
previously to be functionally important in the recognition of LPS. Furthermore, we demonstrate a novel mechanism of inhibition,
as the effects of 15C1 are partially Fc-dependent, involving the regulatory Fcγ receptor IIA (CD32A). In addition to introducing
15C1 as a potent clinical candidate for use in the treatment of LPS-mediated indications, our work demonstrates a newly discovered
pathway whose manipulation is pivotal in achieving optimal neutralizing benefit.

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    • "Leukocytes were resuspended in PBS containing 3% heat inactivated human plasma (stock from a single donor). To measure receptor surface expression, leukocytes were incubated with the following murine antibodies: anti-huCD14 28C5 MAb (a gift from P.S. Tobias, The Scripps Research Institute, La Jolla, USA), anti-huTLR2 T2.5 MAb (eBioscience, San Diego, CA), anti-huTLR4 15C1 MAb, anti-huMD-2 18H10 MAb (gifts from G. Elson, NovImmune SA, Geneva, Switzerland) and a secondary APC-labeled anti-mouse antibody (Molecular Probes, Leiden, The Netherlands) as described [6], [50], [51], [52]. Polymorphonuclear neutrophil (PMN), monocyte, and lymphocyte populations were gated according to their forward and side-scatter characteristics, their CD16 and HLA-DR expression, and analyzed by flow cytometry. "
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    • "bLF was checked for purity [52], iron saturation and endotoxin content as previously described [52]. Monoclonal Abs (mAbs) against TLR4 [53] (5 µg/ml, clone 15C1) and TLR2 [54] (5 µg/ml, clone T 2.5) were kindly provided by Greg Elson. Monoclonal Abs against CD14 (5 µg/ml, clone 134620) and IgG1k isotype control Abs (5 µg/ml, clone 11711) were purchased from R&D. MD-DC phenotype was characterised by using the following mAbs: FITC-CD1a and PE-CD1a (clone HI149), FITC-CD14 and PE-CD14 (clone MΦP9), FITC-CD40 (clone 5C3), FITC-CD80 (clone L307.4), "
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