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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Available from: Bruno Daubeuf, Oct 04, 2015
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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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    ABSTRACT: Bacterial sepsis is a major threat in neonates born prematurely, and is associated with elevated morbidity and mortality. Little is known on the innate immune response to bacteria among extremely premature infants. We compared innate immune functions to bacteria commonly causing sepsis in 21 infants of less than 28 wks of gestational age, 24 infants born between 28 and 32 wks of gestational age, 25 term newborns and 20 healthy adults. Levels of surface expression of innate immune receptors (CD14, TLR2, TLR4, and MD-2) for Gram-positive and Gram-negative bacteria were measured in cord blood leukocytes at the time of birth. The cytokine response to bacteria of those leukocytes as well as plasma-dependent opsonophagocytosis of bacteria by target leukocytes was also measured in the presence or absence of interferon-γ. Leukocytes from extremely premature infants expressed very low levels of receptors important for bacterial recognition. Leukocyte inflammatory responses to bacteria and opsonophagocytic activity of plasma from premature infants were also severely impaired compared to term newborns or adults. These innate immune defects could be corrected when blood from premature infants was incubated ex vivo 12 hrs with interferon-γ. Premature infants display markedly impaired innate immune functions, which likely account for their propensity to develop bacterial sepsis during the neonatal period. The fetal innate immune response progressively matures in the last three months in utero. Ex vivo treatment of leukocytes from premature neonates with interferon-γ reversed their innate immune responses deficiency to bacteria. These data represent a promising proof-of-concept to treat premature newborns at the time of delivery with pharmacological agents aimed at maturing innate immune responses in order to prevent neonatal sepsis.
    PLoS ONE 03/2012; 7(3):e32863. DOI:10.1371/journal.pone.0032863 · 3.23 Impact Factor
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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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    ABSTRACT: Lactoferrin (LF), a key element in mammalian immune system, plays pivotal roles in host defence against infection and excessive inflammation. Its protective effects range from direct antimicrobial activities against a large panel of microbes, including bacteria, viruses, fungi and parasites, to antinflammatory and anticancer activities. In this study, we show that monocyte-derived dendritic cells (MD-DCs) generated in the presence of bovine LF (bLF) fail to undergo activation by up-modulating CD83, co-stimulatory and major histocompatibility complex molecules, and cytokine/chemokine secretion. Moreover, these cells are weak activators of T cell proliferation and retain antigen uptake activity. Consistent with an impaired maturation, bLF-MD-DC primed T lymphocytes exhibit a functional unresponsiveness characterized by reduced expression of CD154 and impaired expression of IFN-γ and IL-2. The observed imunosuppressive effects correlate with an increased expression of molecules with negative regulatory functions (i.e. immunoglobulin-like transcript 3 and programmed death ligand 1), indoleamine 2,3-dioxygenase, and suppressor of cytokine signaling-3. Interestingly, bLF-MD-DCs produce IL-6 and exhibit constitutive signal transducer and activator of transcription 3 activation. Conversely, bLF exposure of already differentiated MD-DCs completely fails to induce IL-6, and partially inhibits Toll-like receptor (TLR) agonist-induced activation. Cell-specific differences in bLF internalization likely account for the distinct response elicited by bLF in monocytes versus immature DCs, providing a mechanistic base for its multiple effects. These results indicate that bLF exerts a potent anti-inflammatory activity by skewing monocyte differentiation into DCs with impaired capacity to undergo activation and to promote Th1 responses. Overall, these bLF-mediated effects may represent a strategy to block excessive DC activation upon TLR-induced inflammation, adding further evidence for a critical role of bLF in directing host immune function.
    PLoS ONE 07/2011; 6(7):e22504. DOI:10.1371/journal.pone.0022504 · 3.23 Impact Factor
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