Generation of a monoclonal antibody agonist to toll-like receptor 4.

Toll-Like Receptor 4 (TLR4) has recently been identified as the lipopolysaccharide (LPS) receptor. Upon stimulation of TLR4 with LPS, a Th1 type cytokine response is observed. This immune modulation is thought to have a potential benefit in anti-tumor therapy. We have generated a TLR4 antibody agonist (5D24.D4) that mimics the action of the natural ligand. 5D24.D4 and LPS stimulation of TLR4 leads to a similar response, including IL-8 secretion, activation of NF-kB-mediated transcription, and the induced expression of an overlapping set of target genes. 5D24.D4 may be beneficial as an adjuvant anti-tumor therapy

Synthetic lethal targeting of MYC by activation of the DR5 death receptor pathway.

The genetic concept of synthetic lethality provides a framework for identifying genotype-selective anticancer agents. In this approach, changes in cellular physiology that arise as a consequence of oncogene activation or tumor suppressor gene loss, rather than oncoproteins themselves, are targeted to achieve tumor selectivity. Here we show that agonists of the TRAIL death receptor DR5 potently induce apoptosis in human cells overexpressing the MYC oncogene, both in vitro and as tumor xenografts in vivo. MYC sensitizes cells to DR5 in a p53-independent manner by upregulating DR5 cell surface levels and stimulating autocatalytic processing of procaspase-8. These results identify a novel mechanism by which MYC sensitizes cells to apoptosis and validate DR5 agonists as potential MYC-selective cancer therapeutics

Differential regulation of the TRAIL death receptors DR4 and DR5 by the signal recognition particle.

TRAIL (TNF-related apoptosis-inducing ligand) death receptors DR4 and DR5 facilitate the selective elimination of malignant cells through the induction of apoptosis. From previous studies the regulation of the DR4 and DR5 cell-death pathways appeared similar; nevertheless in this study we screened a library of small interfering RNA (siRNA) for genes, which when silenced, differentially affect DR4- vs. DR5-mediated apoptosis. These experiments revealed that expression of the signal recognition particle (SRP) complex is essential for apoptosis mediated by DR4, but not DR5. Selective diminution of SRP subunits by RNA interference resulted in a dramatic decrease in cell surface DR4 receptors that correlated with inhibition of DR4-dependent cell death. Conversely, SRP silencing had little influence on cell surface DR5 levels or DR5-mediated apoptosis. Although loss of SRP function in bacteria, yeast and protozoan parasites causes lethality or severe growth defects, we observed no overt phenotypes in the human cancer cells studied--even in stable cell lines with diminished expression of SRP components. The lack of severe phenotype after SRP depletion allowed us to delineate, for the first time, a mechanism for the differential regulation of the TRAIL death receptors DR4 and DR5--implicating the SRP complex as an essential component of the DR4 cell-death pathway

Differential Regulation of the TRAIL Death Receptors DR4 and DR5 by the Signal Recognition Particle

TRAIL (TNF-related apoptosis-inducing ligand) death receptors DR4 and DR5 facilitate the selective elimination of malignant cells through the induction of apoptosis. From previous studies the regulation of the DR4 and DR5 cell-death pathways appeared similar; nevertheless in this study we screened a library of small interfering RNA (siRNA) for genes, which when silenced, differentially affect DR4- vs. DR5-mediated apoptosis. These experiments revealed that expression of the signal recognition particle (SRP) complex is essential for apoptosis mediated by DR4, but not DR5. Selective diminution of SRP subunits by RNA interference resulted in a dramatic decrease in cell surface DR4 receptors that correlated with inhibition of DR4-dependent cell death. Conversely, SRP silencing had little influence on cell surface DR5 levels or DR5-mediated apoptosis. Although loss of SRP function in bacteria, yeast and protozoan parasites causes lethality or severe growth defects, we observed no overt phenotypes in the human cancer cells studied—even in stable cell lines with diminished expression of SRP components. The lack of severe phenotype after SRP depletion allowed us to delineate, for the first time, a mechanism for the differential regulation of the TRAIL death receptors DR4 and DR5—implicating the SRP complex as an essential component of the DR4 cell-death pathway

IL-2 Immunotherapy Reveals Potential for Innate Beta Cell Regeneration in the Non-Obese Diabetic Mouse Model of Autoimmune Diabetes

<div><p>Type-1 diabetes (T1D) is an autoimmune disease targeting insulin-producing beta cells, resulting in dependence on exogenous insulin. To date, significant efforts have been invested to develop immune-modulatory therapies for T1D treatment. Previously, IL-2 immunotherapy was demonstrated to prevent and reverse T1D at onset in the non-obese diabetic (NOD) mouse model, revealing potential as a therapy in early disease stage in humans. In the NOD model, IL-2 deficiency contributes to a loss of regulatory T cell function. This deficiency can be augmented with IL-2 or antibody bound to IL-2 (Ab/IL-2) therapy, resulting in regulatory T cell expansion and potentiation. However, an understanding of the mechanism by which reconstituted regulatory T cell function allows for reversal of diabetes after onset is not clearly understood. Here, we describe that Ab/IL-2 immunotherapy treatment, given at the time of diabetes onset in NOD mice, not only correlated with reversal of diabetes and expansion of Treg cells, but also demonstrated the ability to significantly increase beta cell proliferation. Proliferation appeared specific to Ab/IL-2 immunotherapy, as anti-CD3 therapy did not have a similar effect. Furthermore, to assess the effect of Ab/IL-2 immunotherapy well after the development of diabetes, we tested the effect of delaying treatment for 4 weeks after diabetes onset, when beta cells were virtually absent. At this late stage after diabetes onset, Ab/IL-2 treatment was not sufficient to reverse hyperglycemia. However, it did promote survival in the absence of exogenous insulin. Proliferation of beta cells could not account for this improvement as few beta cells remained. Rather, abnormal insulin and glucagon dual-expressing cells were the only insulin-expressing cells observed in islets from mice with established disease. Thus, these data suggest that in diabetic NOD mice, beta cells have an innate capacity for regeneration both early and late in disease, which is revealed through IL-2 immunotherapy. </p> </div

Ab/IL-2 immunotherapy enhances a rare dual-expressing insulin+/glucagon+ cell population in diabetic NOD mice.

<p>Established or recently diabetic NOD mice were treated with Ab/IL-2 or isotype control Ab for 3 weeks, as described. Pancreata were harvested and processed for immunofluorescence staining for insulin (red), glucagon (green), and DAPI (blue). Insulin+/glucagon+ cell numbers appeared significantly increased by Ab/IL-2 treatment in established diabetic (<b>A</b>-<b>G</b>) and recent onset diabetic (<b>H</b>-<b>N</b>) NOD pancreata. Notably, in established diabetic NOD mice, insulin+/glucagon+ cells were the only detectable source of insulin. (<b>G</b>, <b>N</b>) Percentage of insulin+, glucagon+, and dual-expressing insulin+/glucagon+ (yellow) cells were quantitated by counting all islet cells from each of two representative, non-consecutive sections per pancreas per group. <b>G</b>. In established diabetic mice, a total of n=2898 Ab/IL-2 islet cells and n=1498 control islet cells (n=4-8 mice per group) were counted; (<b>N</b>) in recent onset diabetic mice, n=2264 Ab/IL-2 islet cells and n=961 control islet cells (n=3-4 mice per group) were counted. Due to the rarity of islets themselves and insulin+/glucagon+ cells within the islet, cells from all pancreata within each group were pooled in order to determine the overall percentage. Thus, statistical comparison between individual animals could not be performed. </p

Ab/IL-2 immunotherapy enhances survival and preserves beta cells in diabetic NOD mice.

<p>NOD mice were diagnosed with recent-onset diabetes after two consecutive days with measurements of blood glucose between 200-400mg/dl. Diabetic mice were treated daily with intra-peritoneal injections of either control (PBS or isotype Ab) or Ab/IL-2 for 21 days (grey shaded area). Blood glucose (<b>A</b>), and survival (<b>B</b>) were monitored. (n=12 for isotype control; n=13 for Ab/IL-2). <b>C</b>-<b>H</b>. Pancreata from mice of Ab/IL-2 and control groups were processed for immunofluorescence staining for insulin (red), glucagon (green), and DAPI (blue) at the indicated number of days post-onset. <b>I</b>. Islets from Ab/IL2 treated mice retained a higher absolute number of insulin+ cells/islet compared with controls. Insulin+ beta cells in recently diabetic NOD pancreata were counted from n=3-4 mice per treatment group, from each of two representative non-consecutive sections per pancreas, with a minimum of 30 islets and 900 beta cells/group analyzed. Mean insulin+ beta cell number per islet ± standard deviation between individual mice is presented. (**P<0.009, ***P<0.0001) .</p

Insulin+/glucagon+ cells do not reflect mature endocrine cells.

<p>Recent onset diabetic NOD pancreata treated with Ab/IL-2 were processed and stained for insulin (red), glucagon (green), DAPI (blue) and mature endocrine markers, Brn4, Pdx1, or Nkx6.1 (white). (<b>A</b>) Staining for Brn4, a transcription factor expressed in mature alpha cells, showed expression in glucagon+ (arrowhead) and insulin+/glucagon+ (arrow) cells. (<b>B</b>) Staining for Pdx1, a beta cell specific transcription factor, showed weak expression in insulin+/glucagon+ (arrow) cells, as well some hormone negative cells (arrowhead). (<b>C</b>) Staining for Nkx6.1 showed only weak or absent nuclear staining (arrows) in insulin+/glucagon+ cells, as well as abnormal cytoplasmic staining (arrowhead). (<b>D</b>) Graph represents quantitation of the percent of insulin+/glucagon+ cells that also express nuclear Brn4, Pdx1 or Nkx6.1 (n=97+ insulin+/glucagon+ cells analyzed per transcription factor, from n≥4 individual samples).</p

Ab/IL-2 immunotherapy augments regeneration through beta cell proliferation.

<p><b>A</b>-<b>D</b>. Recently diabetic NOD mice were treated with Ab/IL-2 or control isotype Ab for one to two weeks, and pancreata were processed for immunofluorescence staining for insulin (green), Ki67 (red), and DAPI (blue). <b>E</b>. Mean percent of proliferating Ki67+/insulin+ beta cells of total insulin+ beta cells was calculated from mouse pancreata samples collected during the first week of treatment by counting beta cells (blinded to treatment group) from n=4-8 mice per group, from each of two representative non-consecutive sections per pancreas, with a minimum of 1800 insulin+ cells per group counted <u>+</u> standard error of the mean. (*P<0.013, *** P<0.0008).</p