Safety, tolerability, pharmacokinetics, and pharmacodynamics of the afucosylated, humanized anti-EPHA2 antibody DS-8895a: a first-in-human phase I dose escalation and dose expansion study in patients with advanced solid tumors

BACKGROUND:Erythropoietin-producing hepatocellular receptor A2 (EPHA2) is overexpressed on the cell surface in many cancers and predicts poor prognosis. DS-8895a is a humanized anti-EPHA2 IgG1 monoclonal antibody afucosylated to enhance antibody-dependent cellular cytotoxicity activity. We conducted a two-step, phase I, multicenter, open-label study to determine the safety, tolerability, and pharmacokinetics of DS-8895a in patients with advanced solid tumors.METHODS:Step 1 was a dose escalation cohort in advanced solid tumor patients (six dose levels, 0.1-20 mg/kg) to determine Step 2 dosing. Step 2 was a dose expansion cohort in EPHA2-positive esophageal and gastric cancer patients. DS-8895a was intravenously administered every 2 weeks for the duration of the study, with a 28-day period to assess dose-limiting toxicity (DLT). Safety, pharmacokinetics, tumor response, and potential biomarkers were evaluated.RESULTS:Thirty-seven patients (Step 1: 22, Step 2: 15 [9: gastric cancer, 6: esophageal cancer]) were enrolled. Although one DLT (Grade 4 platelet count decreased) was observed in Step 1 (dose level 6, 20 mg/kg), the maximum tolerated dose was not reached; the highest dose (20 mg/kg) was used in Step 2. Of the 37 patients, 24 (64.9%) experienced drug-related adverse events (AEs) including three (8.1%) with Grade ≥ 3 AEs. Infusion-related reactions occurred in 19 patients (51.4%) but were manageable. All patients discontinued the study (evident disease progression, 33; AEs, 4). Maximum and trough serum DS-8895a concentrations increased dose-dependently. One gastric cancer patient achieved partial response and 13 patients achieved stable disease. Serum inflammatory cytokines transiently increased at completion of and 4 h after the start of DS-8895a administration. The proportion of CD16-positive natural killer (NK) cells (CD3-CD56+CD16+) decreased 4 h after the start of DS-8895a administration, and the ratio of CD3-CD56+CD137+ to CD3-CD56+CD16+ cells increased on day 3.CONCLUSIONS:Twenty mg/kg DS-8895a infused intravenously every 2 weeks was generally safe and well tolerated in patients (n = 21) with advanced solid tumors. The exposure of DS-8895a seemed to increase dose-dependently and induce activated NK cells

New Application of High Niobium Cast Iron as a Grain Refiner for Stainless Steels

In order to develop a new application of cast iron, high niobium cast iron has been developed as a grain refiner for stainless steel. High niobium cast iron was prepared by adding pure niobium to a commercial cast iron. Coarse primary niobium carbide crystals were observed in the microstructure of the cast iron. The effect of the high niobium cast iron as an inoculant on the grain size of austenitic and ferritic stainless steels was examined in various experimental conditions. When the amount of the cast iron inoculant more than 3 mass% was added into the steel melt, fine equiaxed grains were observed and grain size was significantly reduced to 210 μm. The results indicate that the high niobium cast iron is effective as a grain refiner for the austenitic and ferritic stainless steels. From the dissolution rate measurement, the grain refining mechanism was proposed.9th International Symposium on Science and Processing of Cast Iron, SPCI-9; Luxor; 10 November 2010 through 13 November 201

Autocrine Regulation of Macrophage Activation via Exocytosis of ATP and Activation of P2Y11 Receptor

<div><p>It is important to understand the mechanisms that regulate macrophage activation to establish novel therapies for inflammatory diseases, such as sepsis; a systemic inflammatory response syndrome generally caused by bacterial lipopolysaccharide (LPS). In this study, we investigated the involvement of extracellular ATP-mediated autocrine signaling in LPS-induced macrophage activation. We show here that ATP release via exocytosis, followed by activation of P2Y11 receptor, is a major pathway of the macrophage activation, leading to release of cytokines. Treatment of human monocyte THP-1 cells with LPS induced rapid ATP release from cells, and this release was blocked by knockdown of SLC17A9 (vesicular nucleotide transporter, VNUT), which is responsible for exocytosis of ATP. ATP-enriched vesicles were found in cytosol of THP-1 cells. These data suggest the involvement of vesicular exocytosis in the release of ATP. Knockdown of SLC17A9, the P2Y11 antagonist NF157 or knockdown of P2Y11 receptor significantly suppressed both M1-type polarization and IL-6 production in THP-1 cells, indicating an important role of activation of P2Y11 receptor by released ATP in macrophage activation. Next, the effect of NF157 on LPS-induced immune activation was examined <i>in vivo</i>. Administration of LPS to mice caused increase of serum IL-1ß, IL-6, IL-12 and TNF-alpha levels at 3–24 h after the administration. Pre-treatment of LPS-treated mice with NF157 suppressed both elevation of proinflammatory cytokines in serum and M1 polarization of peritoneal/spleen macrophages. Moreover, post-treatment with NF157 at 30 min after administration of LPS also suppressed the elevation of serum cytokines levels. We conclude that vesicular exocytosis of ATP and autocrine, positive feedback through P2Y11 receptors is required for the effective activation of macrophages. Consequently, P2Y11 receptor antagonists may be drug candidates for treatment of inflammatory diseases such as sepsis.</p> </div

LPS treatment induced increased serum levels of cytokines.

<p>(<b>A</b>–<b>D</b>) Male C57BL/6 mice at 6 weeks of age were given LPS (400 µg/head i.p.), and blood samples were collected at 3, 6, 12, and 24 h after LPS injection. Serum levels of IL-1ß, IL-6, IL-12 and TNF-alpha were determined as described in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059778#s2" target="_blank">Materials and Methods</a></i>.</p

Suppressive effect of post-treatment with NF157 on serum levels of cytokines in LPS-treated mice.

<p>(<b>A</b>–<b>D</b>) Male C57BL/6 mice at 6 weeks of age were given LPS (400 µg/head i.p.). Treatment with NF157 was initiated 15 min before to 180 min after the onset of LPS injection. Blood samples were collected at 6 h after LPS injection. Serum levels of IL-1ß, IL-6, IL-12 and TNF-alpha were determined as described in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059778#s2" target="_blank">Materials and Methods</a></i>. Each value represents the mean ± SE (n = 4). Significant differences between a test group and control group (LPS alone) are indicated by *(p<0.05) and **(p<0.01), respectively.</p

Effect of P2Y11 receptor antagonist on LPS-induced IL-6 production in THP-1 cells.

<p>(<b>A</b>) THP-1 cells were pretreated with NF449 (10 µM), NF157 (50 µM), or KH7 (100 µM) for 30 min. (<b>B</b>) Treatment with NF157 was initiated 30 min before to 180 min after the treatment with LPS (10 µg/mL). At 24 h after LPS stimulation, supernatants were collected and the concentration of IL-6 was measured by ELISA. (<b>C</b>) Cells were pre-treated with NF157 for 30 min, and then incubated with 100 µM ATP for 24 h. Supernatants were collected and the concentration of IL-6 was measured by ELISA. (<b>D–E</b>) Cells were incubated with LPS (10 µg/mL) for 1–6 h (<b>D</b>). Cells were pre-incubated with NF157, and incubated with LPS for 6 h (<b>E</b>). At the end of incubation, mRNA level of IL-6 was determined by RT²-PCR as described under <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059778#s2" target="_blank">Materials and Methods</a></i>. Each value represents the mean ± SE (n = 4–11). Significant differences between a test group and control group (LPS or ATP alone) are indicated by **(p<0.01) and ***(p<0.001), respectively.</p

Extracellular ATP contributes to M1 macrophage polarization via activation of P2 receptors.

<p>(<b>A–C</b>) THP-1 cells were cultured for 24 h in medium or in medium supplemented with LPS and IFN-gamma in the absence or presence of ATP (<b>A</b>), or bafilomycin A (50 nM), BFA (10 µM) (<b>B</b>), apyrase (25 U/mL), PPADS (100 µM), A438079 (100 µM), suramin (100 µM), NF449 (10 µM), MRS2179 (100 µM), MRS2578 (10 µM), NF157 (50 µM), clopidogrel (30 µM), or MRS2211 (100 µM) (<b>C</b>). Expression of M1 macrophage marker CCR7 was measured by flow cytometry. The supernatants were assayed for IL-6 (n = 8–10). Each value represents the mean ± SE. A significant difference between the control group (non-treated or LPS alone) and a test group is indicated by ***(p<0.001).</p

P2Y11 antagonist NF157 blocked ATP or LPS-induced increase of cAMP levels in THP-1 cells.

<p>(A) THP-1 cells were incubated with 10–100 µM of ATP for 10 min. (B) Cells were pre-incubated with NF157 (50 µM) for 30 min, and then incubated with 100 µM ATP for 10 min. (C) Cells were incubated with LPS (10 µg/mL) for indicated times. (D) Cells were pr)e-incubated with NF157 for 30 min, and then incubated with LPS. Intracellular cAMP level was measured as described in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059778#s2" target="_blank">Materials and Methods</a></i>. Data is expressed as fold increase in cAMP compared with control. Each value represents the mean ± SE (n = 4). Significant differences between the positive control group (ATP or LPS alone) and the indicated group are indicated by ***(p<0.001).</p

P2Y11 antagonist NF157 suppressed the increase in serum levels of cytokines in LPS-treated mice.

<p>(<b>A</b>–<b>D</b>) Male C57BL/6 mice at 6 weeks of age were given LPS (400 µg/head i.p.). NF157 (100 µL) doses of 5, 50 and 500 µM were administered intraperitoneally at 2 h before LPS was injected. Blood samples were collected at 6 h after LPS injection. Serum levels of IL-1ß, IL-6, IL-12 and TNF-alpha were determined as described in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059778#s2" target="_blank">Materials and Methods</a></i>. Each value represents the mean ± SE (n = 4). Significant differences between a test group and control group (LPS alone) are indicated by *(p<0.05) and **(p<0.01), respectively.</p

NF157 blocked M1 polarization of macrophages in LPS-treated mice.

<p>(<b>A</b>, <b>B</b>) Peritoneal and splenic macrophages were isolated from mice 24 h after LPS injection, and analyzed by flow cytometry for the expression of CCR7. Each value represents the mean ± SE. A significant difference between the positive control group (LPS alone) and the indicated group is indicated by **(p<0.01).</p