Therapeutic Antibody‐Based Drugs in the Treatment of Human Inflammatory Disorders

Inflammation causes debilitating human conditions and older treatments rely on global immunosuppression that non‐specifically alleviates symptoms. Currently, several monoclonal antibodies (mAbs) are available that specifically block pro‐inflammatory cytokines. These include mAbs specific to tumour necrosis factor (TNF), interleukin (IL)‐1β, IL‐6, IL‐17 and IL‐12/IL‐23. The chapter summarises the key elements in human inflammatory disease conditions, including various forms of arthritis, psoriasis, Crohn\u27s disease and ulcerative colitis, plus pyrin‐associated inflammatory syndromes and periodic fevers, to explain the benefit of cytokine neutralisation through mAb‐type reagents. The chapter reviews the efficacy and safety of the current repertoire of anti‐cytokine/cytokine receptor mAbs. It also discusses the known side effects and adverse events that are sometimes associated with systemic blockade of cytokines in vivo, and concludes that the accumulating knowledge of treatment failures can reveal unappreciated aspects of cytokine biology and even new treatment opportunities. The chapter includes mention of the rapidly expanding cohort of biosimilar mAbs and the mAbs to IL‐4, IL‐5 and IL‐13 that are now emerging, in addition to the need for treatments for disorders that remain refractory to the current repertoire of anti‐cytokine mAbs and conventional treatments. Thus, here we summarise the current status of anti‐cytokine mAbs for human inflammatory diseases

Intercellular Vesicular Transfer by Exosomes, Microparticles and Oncosomes - Implications for Cancer Biology and Treatments

Intercellular communication is a normal feature of most physiological interactions between cells in healthy organisms. While cells communicate directly through intimate physiology contact, other mechanisms of communication exist, such as through the influence of soluble mediators such as growth factors, cytokines and chemokines. There is, however, yet another mechanism of intercellular communication that permits the exchange of information between cells through extracellular vesicles (EVs). EVs are microscopic (50 nm−10 μM) phospholipid bilayer enclosed entities produced by virtually all eukaryotic cells. EVs are abundant in the intracellular space and are present at a cells' normal microenvironment. Irrespective of the EV “donor” cell type, or the mechanism of EV biogenesis and production, or the size and EV composition, cancer cells have the potential to utilize EVs in a manner that enhances their survival. For example, cancer cell EV overproduction confers benefits to tumor growth, and tumor metastasis, compared with neighboring healthy cells. Herein, we summarize the current status of knowledge on different populations of EVs. We review the situations that regulate EV release, and the factors that instruct differential packaging or sorting of EV content. We then highlight the functions of cancer-cell derived EVs as they impact on cancer outcomes, promoting tumor progression, metastases, and the mechanisms by which they facilitate the creation of a pre-metastatic niche. The review finishes by focusing on the beneficial (and challenging) features of tumor-derived EVs that can be adapted and utilized for cancer treatments, including those already being investigated in human clinical trials

Monocyte-mediated Tumoricidal Activity via the Tumor Necrosis Factor–related Cytokine, TRAIL

TRAIL (tumor necrosis factor [TNF]-related apoptosis-inducing ligand) is a molecule that displays potent antitumor activity against selected targets. The results presented here demonstrate that human monocytes rapidly express TRAIL, but not Fas ligand or TNF, after activation with interferon (IFN)-γ or -α and acquire the ability to kill tumor cells. Monocyte-mediated tumor cell apoptosis was TRAIL specific, as it could be inhibited with soluble TRAIL receptor. Moreover, IFN stimulation caused a concomitant loss of TRAIL receptor 2 expression, which coincides with monocyte acquisition of resistance to TRAIL-mediated apoptosis. These results define a novel mechanism of monocyte-induced cell cytotoxicity that requires TRAIL, and suggest that TRAIL is a key effector molecule in antitumor activity in vivo

Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand (Trail) Contributes to Interferon γ–Dependent Natural Killer Cell Protection from Tumor Metastasis

Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) is expressed by in vitro activated natural killer (NK) cells, but the relevance of this observation to the biological function of NK cells has been unclear. Herein, we have demonstrated the in vivo induction of mouse TRAIL expression on various tissue NK cells and correlated NK cell activation with TRAIL-mediated antimetastatic function in vivo. Expression of TRAIL was only constitutive on a subset of liver NK cells, and innate NK cell control of Renca carcinoma hepatic metastases in the liver was partially TRAIL dependent. Administration of therapeutic doses of interleukin (IL)-12, a powerful inducer of interferon (IFN)-γ production by NK cells and NKT cells, upregulated TRAIL expression on liver, spleen, and lung NK cells, and IL-12 suppressed metastases in both liver and lung in a TRAIL-dependent fashion. By contrast, α-galactosylceramide (α-GalCer), a powerful inducer of NKT cell IFN-γ and IL-4 secretion, suppressed both liver and lung metastases but only stimulated NK cell TRAIL-mediated function in the liver. TRAIL expression was not detected on NK cells from IFN-γ–deficient mice and TRAIL-mediated antimetastatic effects of IL-12 and α-GalCer were strictly IFN-γ dependent. These results indicated that TRAIL induction on NK cells plays a critical role in IFN-γ–mediated antimetastatic effects of IL-12 and α-GalCer

p14ARF post-transcriptional regulation of nuclear cyclin D1 in MCF-7 breast cancer cells: discrimination between a good and bad prognosis?

As part of a cell's inherent protection against carcinogenesis, p14ARF is upregulated in response to hyperproliferative signalling to induce cell cycle arrest. This property makes p14ARF a leading candidate for cancer therapy. This study explores the consequences of reactivating p14ARF in breast cancer and the potential of targeting p14ARF in breast cancer treatment. Our results show that activation of the p14ARF-p53-p21-Rb pathway in the estrogen sensitive MCF-7 breast cancer cells induces many hallmarks of senescence including a large flat cell morphology, multinucleation, senescence-associated-β-gal staining, and rapid G1 and G2/M phase cell cycle arrest. P14ARF also induces the expression of the proto-oncogene cyclin D1, which is most often associated with a transition from G1-S phase and is highly expressed in breast cancers with poor clinical prognosis. In this study, siRNA knockdown of cyclin D1, p21 and p53 show p21 plays a pivotal role in the maintenance of high cyclin D1 expression, cell cycle and growth arrest post-p14ARF induction. High p53 and p14ARF expression and low p21/cyclin D1 did not cause cell-cycle arrest. Knockdown of cyclin D1 stops proliferation but does not reverse senescence-associated cell growth. Furthermore, cyclin D1 accumulation in the nucleus post-p14ARF activation correlated with a rapid loss of nucleolar Ki-67 protein and inhibition of DNA synthesis. Latent effects of the p14ARF-induced cellular processes resulting from high nuclear cyclin D1 accumulation included a redistribution of Ki-67 into the nucleoli, aberrant nuclear growth (multinucleation), and cell proliferation. Lastly, downregulation of cyclin D1 through inhibition of ER abrogated latent recurrence. The mediation of these latent effects by continuous expression of p14ARF further suggests a novel mechanism whereby dysregulation of cyclin D1 could have a double-edged effect. Our results suggest that p14ARF induced-senescence is related to late-onset breast cancer in estrogen responsive breast cancers and/or the recurrence of more aggressive breast cancer post-therapy

Poxvirus Tumor Necrosis Factor Receptor (TNFR)-Like T2 Proteins Contain a Conserved Preligand Assembly Domain That Inhibits Cellular TNFR1-Induced Cell Death

The poxvirus tumor necrosis factor receptor (TNFR) homologue T2 has immunomodulatory properties; secreted myxoma virus T2 (M-T2) protein binds and inhibits rabbit TNF-α, while intracellular M-T2 blocks virus-induced lymphocyte apoptosis. Here, we define the antiapoptotic function as inhibition of TNFR-mediated death via a highly conserved viral preligand assembly domain (vPLAD). Jurkat cell lines constitutively expressing M-T2 were generated and shown to be resistant to UV irradiation-, etoposide-, and cycloheximide-induced death. These cells were also resistant to human TNF-α, but M-T2 expression did not alter surface expression levels of TNFRs. Previous studies indicated that T2's antiapoptotic function was conferred by the N-terminal region of the protein, and further examination of this region revealed a highly conserved N-terminal vPLAD, which is present in all poxvirus T2-like molecules. In cellular TNFRs and TNF-α-related apoptosis-inducing ligand (TRAIL) receptors (TRAILRs), PLAD controls receptor signaling competency prior to ligand binding. Here, we show that M-T2 potently inhibits TNFR1-induced death in a manner requiring the M-T2 vPLAD. Furthermore, we demonstrate that M-T2 physically associates with and colocalizes with human TNFRs but does not prevent human TNF-α binding to cellular receptors. Thus, M-T2 vPLAD is a species-nonspecific dominant-negative inhibitor of cellular TNFR1 function. Given that the PLAD is conserved in all known poxvirus T2-like molecules, we predict that it plays an important function in each of these proteins. Moreover, that the vPLAD confers an important antiapoptotic function confirms this domain as a potential target in the development of the next generation of TNF-α/TNFR therapeutics

Comparative analysis of p14ARF transcriptional and post-transcriptional regulation of cyclin D1, p53 and p21 by p14ARF.

<p>MCF-7p14ARF 4a cells were treated with IPTG (lanes, 2, 4, 5, 8) or control (lanes 1, 3, 5, 7). Cells were subsequently treated with CHX, Act D, MG132 or vehicle (control). <b>A.</b> Cyclin D1, p21 and p53 protein expression was analysed by Western blot, post-treatments as indicated. <i>Column graph:</i> Protein expression was analysed by densitometry and expressed as arbitrary units (AU). <b>B.</b> Time course of p53, cyclin D1 expression in IPTG induced MCF-7p14ARF cells with MG132, CHX and Act D. Representative Western blot from three experiments show relative protein expression of p14ARF, p53 and cyclin D1. C. Line graph of % change in cyclin D1 and p53 protein expression when compared to control post MG132 and CHX treatments.</p