Rat IgG1 kappa Isotype Control [Alexa Fluor® 647]

Expression of fibroblast growth factor (FGF)-inducible 14 (Fn14), a member of the tumor necrosis factor receptor superfamily, is typically low in healthy adult organisms, but strong Fn14 expression is induced in tissue injury and tissue remodeling. High Fn14 expression is also observed in solid tumors, which is why this receptor is under consideration as a therapeutic target in oncology. Here, we describe various novel mouse-human cross-reactive llama-derived recombinant Fn14-specific antibodies (586, 1801, 4G5) harboring the human IgG1 Fc domain. In contrast to recombinant variants of the established Fn14-specific antibodies PDL192 and P4A8, all three llama-derived antibodies efficiently bound to the W42A and R56P mutants of human Fn14. 18D1 and 4G5, but not 586, efficiently blocked TNF-like weak inducer of apoptosis (TWEAK) binding at low concentrations (0.2-2 mu g/ml). Oligomerization and Fey receptor (Fc gamma R) binding converted all antibodies into strong Fn14 agonists. Variants of 18D1 with enhanced and reduced antibody-dependent cell-mediated cytotoxicity (ADCC) activity were further analyzed in vivo with respect to their effect on metastasis. In a xenogeneic model using human colon carcinoma cancer cells, both antibody variants were effective in reducing metastasis to the liver. In contrast, only the 1801 variant with enhanced ADCC activity, but not its ADCC-defective counterpart, suppressed lung metastasis in the RENCA model. In sum, this suggests that Fn14 targeting might primarily act by triggering of antibody effector functions, but also by blockade of TWEAK-Fn14 interaction in some cases.

It has been recently shown that Toll-like receptor4 mediated nuclear factor kappa B (TLR4-NF-kappa B) signaling plays a critical role in the pathogenesis of rheumatoid arthritis mediated by pro-inflammatory cytokines in arthritic synovium. Here we evaluate the therapeutic potential of glycol-split non-anticoagulant heparin/D-erythro-sphingosine nanoparticles (NAHNPs), which have shown strong inhibitory effect against TLR4 induced inflammation, in an experimental arthritis model. NAHNP significantly inhibited the production of pro-inflammatory cytokines such as TNF-alpha, IL-6 and IL-1 beta in lipopolysaccharide (LPS)-induced primary mouse macrophages and DC2.4 dendritic cell line. The nanoparticles were administered to type II collagen-induced arthritis (CIA) mice by intraarticular injections once per day starting from onset of the disease symptoms. Treatment with NAHNP had a potent suppressive effect in CIA mice, observed with a decrease in arthritis score and footpad swelling. The animals treated with NAHNP significantly reduced levels of IgG1 and IgG2a antibodies against bovine type II collagen. Levels of proinflammatory cytokines -e.g., TNF-alpha, IL-6 and IL-1 beta in knee joints and sera were significantly inhibited compared to control mice. Moreover, nuclear localization of RelA in knee joints was significantly inhibited in NAHNP treatment, indicating down-regulation of the NF-kappa B signaling pathway. In addition, histological examination revealed significant suppression of inflammatory cell infiltration, joint destruction and synovial proliferation in synovium compared with control mice. These results suggest that selective inhibition of TLR4-NF-kappa B signaling with lipid modified heparin derivatives composited to nanostructures provides an effective therapeutic approach to inhibit chronic inflammation in an animal model of rheumatoid arthritis. (C) 2014 Elsevier B.V. All rights reserved.