Chromium Release Research Articles

Mineralogical determination and geo-chemical modeling of chromium release from AOD slag: Distribution and leachability aspects

AOD (argon oxygen decarburization) slag, which is the by-product of the stainless steel refining process, is a recyclable slag because of its high content of calcium and silicon. The leaching toxicity cannot be ignored in the recycling process because the slag contains a certain amount of Cr. In this study, the mineral analysis, batch leaching tests and thermodynamic and kinetic modeling by PHREEQC combined with FactSage software were performed to explore the influence of the dissolution of primary minerals and the precipitation of secondary minerals on the elution of Cr from AOD slag. The results indicated that the main minerals in the original AOD slag are larnite, merwinite, pyroxene and periclase. Cr was dispersed in the mineral phases mentioned above. The simulation of Cr leaching controlled by Cr(III)-hydroxide corresponded better to the batch leaching tests, while the Cr leaching controlled by chromite or double control was underestimated. Increasing the L/S ratio enhances the pH of the leachate and restrains the elution of Cr from the AOD slag.

Acid-Free Electrochemical Chromium Etch and Release of Nanoscale Gold Films

We describe a method for releasing coated and uncoated 100-nm gold films from a 5-nm chromium-coated glass substrate. The method is rapid at room temperature within a non-toxic solution of sodium phosphate or sodium hydroxide. Etching processes using hydroxide successfully etch the chromium layer without affecting a ZnO coating layer deposited on the gold film. Etching using sodium phosphate at neutral pH causes some surface damage to the ZnO surface while leaving the bulk of the film intact. Other anions tested include nitrates and chlorides, both demonstrating poor etching selectivity, damaging the gold as well as the chromium layers. A copper counter electrode is used to collect the chromium from the solution, minimizing chromium persistence in the etching solution and any contamination of the ZnO surface. This single step acid-free process can be applied to a variety of materials and structures formed on gold, where insulating materials and many oxides do not require any special protection from the etching solution. An uncoated or polyimide coated $2~\text {cm}\times 1.5$ cm film can be released in under 1 h, while ZnO-coated films require up to 7 h to to release films 1 cm $\times $ 1 cm in size. [2015-0331]

Abstract 2294: Reversible regulation of chimeric antigen receptor surface expression

Abstract The purpose of this study is to develop and characterize a chimeric antigen receptor (CAR) T cell platform that can be rapidly and reversibly down-regulated. The rationale for this approach is to mitigate the risk of tissue toxicity as CAR T cell therapies are expanded from hematologic malignancies to solid tumors. Whereas off-tumor/on-target activity can be tolerable in the case of the B cell hypoplasia that occurs with CART therapy for ALL, targeting of other normal tissues predicted to occur in the solid tumor setting likely will be more clinically challenging. Currently implemented design approaches to arresting CAR-mediated toxicity have involved suicide domains. While effective in triggering CAR T cell apoptosis, these approaches are not reversible, and additional therapy would require repeated infusions, which may not be feasible under certain circumstances. A system of temporary down-regulation could overcome this obstacle and provide a useful alternative to current strategies. To this end, we have fused a mouse anti-human GD2 neuroblastoma antigen-specific CAR to a LID (ligand-induced degradation) domain. The LID domain, developed at Stanford University, targets the polypeptide for degradation upon the addition of rapamycin or a similar ligand, “shield.” In the study presented here, bulk human T cells were transduced with lentivirus encoding either the control anti-GD2 CAR or the anti-GD2-CAR-LID fusion construct. Transduced T cells, after a standard procedure of stimulation and expansion, were then incubated in either standard media alone or media with shield ligand. CAR expression was measured at a range of shield ligand concentrations and at various time points up to 24 hours by flow cytometry using goat-anti mouse antibody. We observed a shield dose-response reduction in CAR expression and found that this shield-induced reduction in expression started at 8 hours and approached ∼ 20% of baseline by 24 hours. As early as 24 hours following the removal of the shield-containing media and washing, CAR surface expression had returned to ∼65% of baseline. Next, we assessed in vitro T cell effector functions including proliferation as measured by flow cytometric cell counting, IFN-gamma release as measured by ELISA, and cytotoxicity as measured by chromium release. We observed that in the presence of shield ligand, T cell effector functions were reduced to near background levels. The shield compound appeared to have no effect on the control anti-GD2 CAR that did not contain the LID domain. Our conclusion from these in vitro experiments is that the CAR-LID construct has the potential to allow for temporary and tunable down-regulation of CAR T cell activity in a clinical setting and warrants further characterization. In vivo experiments designed to test the CAR-LID T cells in a mouse model of xenografted disseminated neuroblastoma are ongoing, and we plan to expand this approach to other tumor systems. Citation Format: Sarah A. Richman, Liang-Chuan Wang, Edmund K. Moon, Steven M. Albelda, Michael C. Milone. Reversible regulation of chimeric antigen receptor surface expression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2294.

Abstract 2339: RNAi discovery platform to identify novel genes that prevent immune surveillance in pancreatic ductal adenocarcinoma (PDAC)

Abstract BACKGROUND: Being one of the most treatment-resistant cancer types, pancreatic ductal adenocarcinoma (PDAC) is characterized by its ability to escape immune surveillance by developing many immunological obstacles. These include a plethora of mechanisms that either dampen immune cell functionality, or foster tumor cell resistance towards immune attack. Immunotherapeutic strategies, such as immune checkpoint blockade, have proven clinical success in many cancer entities, but showed little clinical benefit in PDAC patients, emphasizing the need to identify more key players that could radically improve immunotherapy. AIM: We aim to systematically identify the whole arsenal of tumor-associated immune modulators by performing a high-throughput RNAi screen and subsequently validate novel therapeutic targets, whose blockade could potentially enhance anti-tumor immune response in PDAC patients. METHODS: We generated a luciferase-expressing PANC-1 cell line and knocked down 2514 genes using a siRNA library. Our library includes G-protein coupled receptors, protein kinases and 1117 surface proteins. We co-cultured HLA-A201+ matched tumor infiltrating lymphocytes (TILs) derived from a PDAC patient with the transfected tumor cells. We then measured the remaining luciferase intensity of the tumor cells as an estimation of TIL-mediated cytotoxicity. In order to exclude genes whose knock-down affected cell viability per se, we cultivated tumor cells with the siRNA library in the absence of TILs. RESULTS: We identified 155 candidate genes whose knock-down enhances TIL-mediated killing more efficiently than PD-L1 down-regulation. 35% of these genes are surface molecules and are most likely to directly mediate tumor immune evasion. Beside novel undescribed genes, our list contains well characterized immune modulators, supporting the reliability of our approach. Of note 13 of our hits were also found in a related melanoma screen and might play a role in the regulation of immune surveillance of different tumor entities. Among our candidates, 4 hits were chosen for further validation. We confirmed the expression of our selected candidates in several tumor cell lines and assessed the siRNA on-target effect using several non-overlapping siRNA sequences targeting the same hit. Transfection of PANC-1 with different siRNA sequences showed knock-down of the target gene as assessed via qPCR. Additionally we observed increased T-cell mediated killing as measured via luciferase-based killing assay and Chromium release assay. CONCLUSION: We set up a robust and systematic method to unravel novel key players of pancreatic cancer immune surveillance. Further functional validation of our candidate genes will prove their potential to be used as relevant therapeutic targets in the clinic. Citation Format: Antonio Sorrentino, Ayse Nur Menevse, Tillmann Michels, Nisit Khandelwal, Marco Breinig, Isabel Poschke, Valentina Volpin, Sabrina Wagner, Rienk Offringa, Michael Boutros, Philipp Beckhove. RNAi discovery platform to identify novel genes that prevent immune surveillance in pancreatic ductal adenocarcinoma (PDAC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2339.

Abstract 4995: anti-ROR1 x anti-CD3 ADAPTIR™ molecule, ES425, redirects T-cell cytotoxicity and inhibits tumor growth in preclinical models of triple-negative breast cancer

Abstract Background: Effective treatment of metastatic, triple-negative breast cancer (TNBC) remains a highly unmet medical need. We have developed ES425, a bispecific ADAPTIR™ (modular protein technology) molecule that redirects T-cell cytotoxicity to tumor cells expressing ROR1 (receptor tyrosine kinase-like orphan receptor 1), an oncofetal antigen expressed on TNBC and other malignancies. Results are presented for studies run to examine in vitro and in vivo activity of ES425 in preclinical models of TNBC. Materials and Methods: Target-dependent cytotoxic activity was examined in vitro by treating ROR1(+) cell lines and ROR1(−) cell lines with ES425 in the presence of purified human T cells or human peripheral blood mononuclear cells (PBMCs). Cytotoxic activity was determined using chromium release assays. T cells were assessed for activation and proliferation using multi-color flow cytometry. Pharmacokinetics of ES425 in NOD/SCID gamma (NSG) mice was determined using single intravenous dose of approximately 10 mg/kg. Serum concentrations at time points ranging from 15 minutes to 504 hours were used to calculate the terminal elimination half-life of ES425. To assess activity in vivo, NOD/SCID mice were implanted subcutaneously with the ROR1(+) TNBC tumor cell line MDA-MB-231 and purified human T cells and treated with ES425. This model was run twice with T cells from different human donors. Tumor growth was assessed by measuring tumor volume. Results: ES425 efficiently redirected T cell cytotoxicity against ROR1(+) cell lines at low picomolar concentrations in vitro. Cytotoxic activity was dependent on expression of ROR1 by the target cells. T cells were activated and proliferated in response to ES425 in the presence of ROR1(+) target cells; proliferation was not observed in response to ROR1(−) cells. In vivo, pharmacokinetic analysis showed a serum half-life of approximately 7 days in NSG mice, and ES425 inhibited growth of MDA-MB-231 tumors in mouse xenografts. Repeat experiments showed similar inhibition of tumor growth and an improvement in overall survival. Conclusions: These studies show that ES425 may be an efficient agent for redirecting T cell cytotoxicity in preclinical TNBC models and merits investigation as a potential therapeutic in TNBC and other malignancies. Citation Format: John W. Blankenship, Lynda Misher, Danielle Mitchell, Nicole Zhang, Philip Tan, Gabriela H. Hoyos, Padma Ravikumar, Robert Bader, Catherine J. McMahan, Robert E. Miller, Jeannette Bannink, Hang Fang, Lara Parr, Maria Dasovich, David Bienvenue, Megan Aguilar, Carina Xu, Mollie Daugherty, Brian Woodruff, Jane A. Gross. anti-ROR1 x anti-CD3 ADAPTIR™ molecule, ES425, redirects T-cell cytotoxicity and inhibits tumor growth in preclinical models of triple-negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4995.

Abstract 280: Synergistic in vitro activity of MOR209/ES414 in combination with enzalutamide

Abstract Metastatic castration-resistant prostate cancer (mCRPC) presents a daunting therapeutic challenge for patients who eventually progress after androgen ablation therapy. Although newer AR antagonists show a survival benefit, these patients ultimately relapse. Thus, it is critical to identify new therapies that will work in combination with AR antagonists or in AR-antagonist resistant settings. MOR209/ES414 is a bispecific ADAPTIR™ (modular protein technology) molecule currently under investigation in a phase 1 clinical trial in mCRPC, including patients that have progressed on enzalutamide. In pre-clinical studies, MOR209/ES414 has previously been shown to redirect T cell cytotoxicity against cells expressing Prostate Specific Membrane Antigen (PSMA). Here, we aimed to determine whether enzalutamide affects PSMA expression on CRPC cells in vitro, and could alter the effectiveness of MOR209/ES414 at inducing T-cell mediated tumor cell death. To address these questions, we utilized a CRPC cell line, 22Rv1, which has low expression of PSMA and is resistant to enzalutamide. Treatment of 22Rv1 cells with 10 μM enzalutamide for 3 weeks resulted in increased PSMA cell surface expression as measured by flow cytometry. 22Rv1 cells exposed to enzalutamide were also more sensitive to MOR209/ES414-mediated T-cell lysis, with a 25% increase in cytotoxicity over a 4 hour chromium release assay compared to 22Rv1 cells that were not exposed to enzalutamide. A concomitant decrease in EC50 for MOR209/ES414-mediated lysis from 0.8 pM to 0.5 pM was also observed in 22Rv1 cells exposed to enzalutamide. MOR209/ES414 and enzalutamide were next tested in combination using a PSMA-positive, enzalutamide-sensitive cell line, LNCaP cultured with primary T cells over several days. Suboptimal doses of MOR209/ES414 and enzalutamide were added to the cultures, and surviving LnCaP cells were quantitated 4 days later. Enzalutamide did not interfere with the ability of MOR209/ES414 to target T cells to LNCaP cells, and Combination Index analysis showed the activity of MOR209/ES414 and enzalutamide to be synergistic in vitro. These studies provide a rationale for further examination of combining MOR209/ES414 with enzalutamide, even in enzalutamide-resistant settings. Citation Format: Toddy Sewell, Michelle Blake, Jane A. Gross, Jan Endell, Johannes Weirather, John W. Blankenship. Synergistic in vitro activity of MOR209/ES414 in combination with enzalutamide. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 280.

Assessment of chromium(VI) release from 848 jewellery items by use of a diphenylcarbazide spot test.

Keywords: allergic chromium dermatitis; chromium; chromium allergy; dermatitis; metals; potassium dichromate; screening; spot test

Chromium Release from a COPR-Contaminated Soil at Varying Water Content and Redox Conditions.

Many soils in the region of Kanpur, North India, are heavily affected by the leather industry and its upstream supplier sector, as indicated by elevated chromium (Cr) contents. Under reducing conditions-for instance, at water saturation after monsoon rain or flood irrigation-the dynamic and species distribution of Cr may be affected due to changes in redox potential (E). In this study, the influence of E on the speciation and release of Cr from a contaminated agricultural soil was investigated. A soil sample that was affected by hyperalkaline leachate from chromite ore processing residue, was taken and packed in soil columns, and subjected to a saturation-drainage-saturation cycle. After initial water saturation, the E dropped slowly to minimum values of around ‒100 mV (calculated to pH 7), while E was controlled by CrO/CrO(s), or CrO/(Fe,Cr)OOH redox couples. Soil drainage resulted in a quick return to oxidizing conditions; i.e., E > 300 mV. The Cr species distribution and release showed a clear trend with E. At the beginning of the experiment, under oxidizing and weakly reducing conditions (E range from >100 to 300 mV), Cr(VI) was released in particular. However, under moderately reducing conditions (E range from 100 to -100 mV), Cr was gradually immobilized and irreversible sequestered via reductive precipitation. The results presented in this study provide an improved understanding of the mobility of Cr(VI) in contaminated soils at varying water contents, which is essential for the evaluation of environmental risks in this region.

Removal of chromium by Staphylococcus saprophyticus subsp. bovis strain 1

The present study was taken up to carry out the removal of chromium by bacteria. Chromium is a heavy metal which is of concern as it causes chromium toxicity in both plants and animals. The tanneries are a major source of chromium release into the water bodies, and hence this study is aimed at identifying the potential chromium-tolerant bacterium and at studying the bacterium for its maximum tolerance and chromium removal capacity. A total of 20 samples each of soil and industrial effluents were collected from the Peenya industrial area, Bangalore, India. The bacterial isolates were screened for chromium resistance by supplementing the nutrient media with 300 µg/ml of chromium. The growth of the bacteria was measured in terms of O.D. at 670 nm. The isolates were further screened by increasing the concentration of chromium from 300 to 3000 µg/ml. The isolate S105c which showed better resistance to chromium compared to other isolates was identified as Staphylococcus saprophyticus subsp. bovis strain 1 based on 16S rRNA sequencing. Optimization of temperature, pH, carbon sources, and nitrogen sources was carried out for the removal of chromium by S. saprophyticus subsp. bovis strain 1. Atomic absorption spectrophotometric analysis was carried out for the removal of chromium by S. saprophyticus subsp. bovis strain 1 in the mineral salts medium supplemented with 300 µg/ml of chromium. Out of 40 soil and industrial effluent samples collected, a total of 51 isolates were obtained. Out of 51 isolates, 7 isolates gave the highest O.D. values in the presence of 300 µg/ml of chromium. The isolate S10-5c showed maximum tolerance up to 3000 µg/ml of chromium. S. saprophyticus subsp. bovis strain 1 was able to remove 2% of chromium in the medium at the end of 24 hours under optimized conditions. It can be concluded that the organism Staphylococcus saprophyticus subsp. bovis strain 1 can be used for the removal of chromium from industrial effluents.

Chromium(III) and chromium(VI) release from leather during 8 months of simulated use.

SummaryBackgroundChromium (Cr) release from Cr‐tanned leather articles is a major cause of Cr contact dermatitis. It has been suggested that Cr(VI) release from leather is not necessarily an intrinsic property of the leather, but is strongly dependent on environmental conditions.ObjectivesTo test this hypothesis for long‐term (8 months) simulated use.Materials and methodsThe release of total Cr and Cr(VI) from Cr‐tanned, unfinished leather was analysed in subsequent phosphate buffer (pH 8.0) immersions for a period of 7.5 months. The effect of combined ultraviolet treatment and alkaline solution (pH 12.1) was tested. Dry storage [20% relative humidity (RH)] was maintained between immersions. Atomic absorption spectroscopy, X‐ray fluorescence and diphenylcarbazide tests were used.Results Cr(VI) release was dependent on previous dry storage or alkaline treatment, but not on duration or number of previous immersions. Cr(III) release decreased with time. Fifty‐two percent of the total Cr released during the last immersion period was Cr(VI). Cr(VI) release exceeded 9 mg/kg in all immersion periods except in the first 10‐day immersion (2.6 mg/kg).Conclusions Cr(VI) release is primarily determined by environmental factors (RH prior to immersion, solution pH, and antioxidant content). The RH should be kept low prior to testing Cr(VI) release from leather.

A high-throughput image cytometry-based screening method for the detection of IL2-induced peripheral blood mononuclear cell-mediated cytotoxicity

Abstract Cell-mediated cytotoxicity assays have been an important functional test for investigating the cytotoxic effect of immune effector on target cancer cells. Cytotoxicity assays have traditionally been performed using the 51Chromium (51Cr) release assay, which involves labeling the tumor cells (target) with radioisotopes. The 51Chromium release assay is highly hazardous, time-consuming, and can only acquire end point readout. Furthermore, it can generate inconsistent results due to batch variation, ability of the target cell for 51Cr uptake, and low sensitivity. In this work, we demonstrate a novel high-throughput cytotoxicity screening assay using the Celigo imaging-cytometry method. First the live K562 target cells were stained with Calcein AM, and were co-cultured with PBMCs from two healthy donors in a flat- bottom 96-well plate in the presence or absence of IL-2. Direct cell counting of Calcein positive cells was implied to calculate the changes in number of live target cells in the same well from T = 0 to T = 4 hours. Thus, accurate cell-mediated killing was determined using the “self-referencing” calculation, which could be a more direct method for assessing cytotoxicity than 51Cr release. The donor PBMCs were added to each well with different Calcein-stained target cells at Effector-to-Target (E:T) ratios. The 96-well microplate was scanned and analyzed using the Celigo image cytometer at different time points to measure the % lysis of target cell. The proposed image cytometry method can scan and analyze in 7 min, which can be utilized to perform high-throughput screening of potential antibody- or chemical-based cancer drug, which could be a more efficient method for academic, industry, and clinical research.

Influence of Surface Coating on Metal Ion Release: Evaluation in Patients With Metal Allergy.

Nickel, chromium, and cobalt in stainless steel and Cobalt-chrome-molybdenum (CoCrMo) alloys may induce allergy. The objectives of this study were to evaluate surface coating regarding ion release, patch test reactivity, and arthroplasty performance. Materials and methods included patch test in 31 patients with metal allergy and 30 patients with no allergy to stainless steel and CoCrMo disks that are uncoated or coated by titanium nitride/zirconium nitride (TiN/ZrN). Assessment include atomic absorption spectrometry of released nickel, cobalt, and chromium from the disks after exposure to distilled water, artificial sweat and culture medium. Results showed that both coatings reduced the nickel and chromium release from stainless steel and CoCrMo disks and mostly the cobalt release from the disks (maximally 11.755 µg/cm(2)/5 d to 1.624 by Ti-N and to 0.442 by ZrN). Six of the 31 patients with metal allergy reacted to uncoated disks, but none reacted to the coated disks. The current authors report on exemplary patients with metal allergy who had symptom relief by revision with surface-coated arthroplasty. The authors concluded that the surface coating may prevent cutaneous and peri-implant allergic reactions. [Orthopedics. 2016; 39(3):S24-S30.].

762. A Quantitative Imaging Tool Box for the Functional Analysis of Chimeric Antigen Receptor - T Cell Immune Synapse Helps Identify Novel Structural and Functional Features of CAR T Cells

Introduction: The cytolytic immunological synapse (IS) in T cells is a discrete structural entity formed after the ligation of specific activation receptors and leading to the destruction of a cancerous cell through the targeted release of the contents of T cell lytic granules. Although the “native” T cell IS formation where the TCR binds to antigenic peptide-MHC complex has been intensely studied, the “engineered” CAR T cell synapse and its variations based on single chain variable fragment (scFv) design and co-stimulatory domain arrangements, still remains a black box. Our work focuses on the quantitative evaluation of the CAR T cell lytic IS using highly quantitative high and super resolution imaging techniques. Specifically, by combining quantitative interrogations of CAR synapse formation kinetics and dynamics at the cellular level, we are able to obtain an unprecedented level of insight into the effectiveness of therapy cells relative to the traditional measures of cytotoxicity and effector assays like chromium release, ELISPOT and flow cytometry. Super-resolution microscopy enables us to further “see” subtle variations in therapy cell quality at a near molecular level and can be used as a very precise functional readout of CAR T cell rational design. Methods: High and super resolution fluorescence microscopy platforms are used to image CAR T cell-target conjugates in the x, y and z dimensions. Imaging parameters include direct evaluation of CAR engagement at the IS by measuring mean fluorescence intensity (MFI) and volume of synaptic aggregates of CAR specific ligands as well as their relative distribution at the synapse. Results: We report here the quantitative measurement and comparison of single and dual CAR targeted ligand aggregation at the IS, lytic machinery re-arrangement and time to target cell killing. Our studies using this predictive imaging tool box establishes the superiority of T cells expressing a bispecific (tandem) CAR to those expressing one or two monospecific CARs by the presence of significantly increased accumulation of F-Actin (> five fold), greater convergence and polarization of the lytic granule machinery. Dual engagement of the targeted ligands by a tandem CAR also significantly offsets antigen escape variants in tumors predicting reduced tumor relapse. Furthermore, application of our imaging tool box reveals that tonic signaling of CARs containing a 4-1BB co-stimulatory domain can result in an almost three fold upregulation of Fas on plasma membrane and co-localization with Fas L leading to enhanced apoptosis and lower persistence of CAR T cells. Conclusions: Using our quantitative imaging tool box we are able to define a specific list of parameters to measure CAR engagement and lytic function at the IS. These imaging parameters can be directly applied to interrogate and predict functionality outcomes upon CAR design variations. By doing this we hope to provide crucial early insight in CAR T cell design that can guide improvements in the efficacy, safety and, ultimately, clinical effectiveness of CAR T cell therapies.

Influence of organic acids on kinetic release of chromium in soil contaminated with leather factory waste in the presence of some adsorbents

In this study, batch experiments were conducted to investigate the effects of nanoparticles (NPs) (MgO, ZnO, TiO2) and clay minerals (bentonite, zeolite) on the release of chromium (Cr) from leather factory waste (LFW) and LFW treated soil using organic acids. Chromium release from all treatments was studied in the presence of citric acid, oxalic acid and CaCl2 solutions. The results showed that, in all treatments, organic acids released more Cr than inorganic salt (CaCl2). The release of Cr by citric acid was higher than that by oxalic acid. In LFW treated soil and LFW, the release of Cr from the all treatments with NPs was less than that from the clay mineral treatments. On the other hand, in the presence of organic acids, Cr release by NPs and clay minerals decreased. Two kinetic models including pseudo-first- and pseudo-second-order model were tested to describe the time dependent Cr release data. Among the kinetic models used, the pseudo-second-order model generally gave the best fits to experimental data. Before and after release experiments, Cr in LFW, treated LFW, control soil and LFW treated soils were fractionated. In all treatments, the greatest amounts of Cr were found in the residual fraction (RES). The organic acids were effective in reducing the exchangeable (EXC), bound to organic matter (OM) and bound to carbonate (CAR) fractions of Cr in all treatments, whereas, after release of Cr from treated soils, Cr remained mainly in the RES fraction. The application of NPs and clay minerals in soil led to a significant transformation of Cr from mobile fractions to the RES fraction. Therefore, organic ligands played a dominant role in mobility and bioavailability of Cr and the removal of Cr by adsorbents.

Stainless steel corrosion scale formed in reclaimed water: Characteristics, model for scale growth and metal element release

Stainless steels generally have extremely good corrosion resistance, but are still susceptible to pitting corrosion. As a result, corrosion scales can form on the surface of stainless steel after extended exposure to aggressive aqueous environments. Corrosion scales play an important role in affecting water quality. These research results showed that interior regions of stainless steel corrosion scales have a high percentage of chromium phases. We reveal the morphology, micro-structure and physicochemical characteristics of stainless steel corrosion scales. Stainless steel corrosion scale is identified as a podiform chromite deposit according to these characteristics, which is unlike deposit formed during iron corrosion. A conceptual model to explain the formation and growth of stainless steel corrosion scale is proposed based on its composition and structure. The scale growth process involves pitting corrosion on the stainless steel surface and the consecutive generation and homogeneous deposition of corrosion products, which is governed by a series of chemical and electrochemical reactions. This model shows the role of corrosion scales in the mechanism of iron and chromium release from pitting corroded stainless steel materials. The formation of corrosion scale is strongly related to water quality parameters. The presence of HClO results in higher ferric content inside the scales. Cl− and SO42− ions in reclaimed water play an important role in corrosion pitting of stainless steel and promote the formation of scales.

Abstract PD3-07: TEM8 specific CAR T cells serve as a novel targeted therapy for triple negative breast cancer and its supporting endothelium

Abstract Background: Triple Negative Breast Cancer (TNBC) refers to an aggressive subtype of breast cancer negative for HER2, estrogen and progesterone receptors. Lacking these receptors, individuals with TNBC do not benefit from many of the targeted therapies for breast cancer. Tumor endothelial marker 8 (TEM8), originally identified as a tumor endothelium associated antigen, has more recently been implicated in TNBC pathogenesis and as a marker of breast cancer stem like cells. Here we report that T cells expressing a TEM8-specific chimeric antigen receptor (CAR) serve as a novel approach to target both TNBC cells and its supporting endothelium. CARs combine the specificity of a monoclonal antibody with the signaling properties of a T cell. Methods: We designed two novel TEM8-specific CAR molecules. A CAR molecule containing an exodomain derived from the anti-TEM8 L2 antibody, followed by CD28 and CD3-zeta signaling domains (second generation CAR) and CD28, 41BB and CD3-zeta signaling domains (third generation CAR), respectively. Retroviral transduction was used to express the TEM8 CAR transgene constructs on HEK 293T cells, then on primary T cells. Results: Immunofluorescence staining revealed that in a panel of primary TNBC breast cancer samples, TEM8 was overexpressed in comparison to normal adjacent breast tissue (6/6). Costaining with the pan-endothelial cell marker CD31 revealed that this overexpression was not confined to the endothelial compartment, but also present on tumor parenchymal cells. The immortalized TNBC cell lines (MDA-MB-231, MDA-MB-436, MDA-MB-468 and Hs578T) expressed endogenous levels of TEM8 protein as revealed by western blot. Greater than 90% transduction of primary human T cells was achieved using both of our CAR constructs, as detected by flow cytometry. TEM8 specific T cells displayed significantly higher killing of TEM8 positive TNBC and tumor endothelial cells (2H11 and bEND.3) in standard four hour chromium release assays when compared to both non-transduced or irrelevant (CD19) CAR T cells and secreted immunomostimulatory cytokines upon encounter of TEM8 positive cells in coculture assays. In a vascularized xenograft model, MDA MB468 cells were injected subcutaneously with matrigel into athymic nude mice and followed via bioluminescence imaging over the course of two months. Established tumors were treated with either, second or third generation TEM8 specific T cells, HER2 specific T cells, non-transduced T cells or left untreated. Relative to non-transduced T cells, TEM8 specific second and third generation CAR T cells significantly delayed tumor growth by 36 days and 50 days, respectively. Conclusion: TEM8 specific CAR T cells could serve as a novel targeted therapy for TNBC and supporting endothelium. Citation Format: Byrd T, Fousek K, Pignata A, Szot C, Bielamowicz K, Wakefield A, Koch J, Landi D, Seaman S, Wels W, Fletcher B, Hegde M, St Croix B, Ahmed N. TEM8 specific CAR T cells serve as a novel targeted therapy for triple negative breast cancer and its supporting endothelium. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD3-07.

Mycobacterium leprae-Infected Macrophages Preferentially Primed Regulatory T Cell Responses and Was Associated with Lepromatous Leprosy.

BackgroundThe persistence of Mycobacterium leprae (M. leprae) infection is largely dependent on the types of host immune responses being induced. Macrophage, a crucial modulator of innate and adaptive immune responses, could be directly infected by M. leprae. We therefore postulated that M. leprae-infected macrophages might have altered immune functions.Methodology/Principal FindingsHere, we treated monocyte-derived macrophages with live or killed M. leprae, and examined their activation status and antigen presentation. We found that macrophages treated with live M. leprae showed committed M2-like function, with decreased interleukin 1 beta (IL-1beta), IL-6, tumor necrosis factor alpha (TNF-alpha) and MHC class II molecule expression and elevated IL-10 and CD163 expression. When incubating with naive T cells, macrophages treated with live M. leprae preferentially primed regulatory T (Treg) cell responses with elevated FoxP3 and IL-10 expression, while interferon gamma (IFN-gamma) expression and CD8+ T cell cytotoxicity were reduced. Chromium release assay also found that live M. leprae-treated macrophages were more resistant to CD8+ T cell-mediated cytotoxicity than sonicated M. leprae-treated monocytes. Ex vivo studies showed that the phenotype and function of monocytes and macrophages had clear differences between L-lep and T-lep patients, consistent with the in vitro findings.Conclusions/SignificanceTogether, our data demonstrate that M. leprae could utilize infected macrophages by two mechanisms: firstly, M. leprae-infected macrophages preferentially primed Treg but not Th1 or cytotoxic T cell responses; secondly, M. leprae-infected macrophages were more effective at evading CD8+ T cell-mediated cytotoxicity.

Chromium-51 (51Cr) Release Assay to Assess Human T Cells for Functional Avidity and Tumor Cell Recognition

Cytotoxic CD8+ T cells are able to specifically recognize and kill target cells through specific interaction between their T cell receptors (TCRs) and small immunogenic peptides (antigens) presented by major histocompatibility complex (MHC) molecules. The antigen recognition capacity and in vitro lytic activity of antigen-specific cytotoxic T cells can be assessed functionally in the so-called chromium 51 (51Cr) release assay, which was developed almost 50 years ago in our institution (Brunner et al., 1968). Radioactively-labelled cells deficient for endogenous antigen presentation [e.g., transporter for antigen presentation (TAP)-deficient T2 cells] and stably transfected with the MHC of interest (e.g., HLA-A2+) are typically used as targets during this 4h assay. Alternatively, 51Cr-labelled virus-infected or tumor cell lines presenting immunogenic antigens endogenously can serve as target cells (e.g., for the assessment of tumor recognition).In a peptide titration assay (section A), radioactively labelled target cells are pulsed with a serial dilution of the antigenic peptide and incubated at an effector (e.g., a CD8+ T cell clone) to target (51Cr -T2 cells) ratio (E:T) of 10:1 in a 96-well V-bottom plate for 4 h at 37 °C. In a tumor killing assay (section B), cytotoxic CD8+ effector cells are incubated at different ratios with the 51Cr-labelled target cell line (typically at E:T ratios of 30:1, 10:1, 3:1 and 1:1) in the presence or absence of the specific antigenic peptide (1 μM) and incubated for 4 h at 37 °C. At the end of the test, the amount of radioactivity release from the lysed target cells is determined in the supernatant using a liquid scintillation counter. The percentage of specific lysis, as well as the EC50 (i.e., 50% of maximal killing) and EMax values are then calculated, providing quantitative information about the antigen-specific functional avidity (i.e., the relative efficiency of T cell function based on antigen recognition via a defined TCR and maximal killing capacity of the analyzed T cells).

Abstract B053: Designing chimeric antigen receptors for personalized immunotherapy: Rapid assembly of CARs from principal components using “EZ-CAR” platform

Abstract Clinical data demonstrates that one chimeric antigen receptor (CAR) design targeting T cells to a given tumor-associated antigen (TAA) will have varying therapeutic potential in different patients. For example, second generation CD19-specific CARs activated via chimeric CD28/CD3ζ or CD137/CD3ζ exhibit superior clinical responses when autologous genetically modified T cells are administered to patients with acute versus chronic B-lineage leukemias. Therefore, we investigated whether CAR species can be designed to improve the anti-tumor effect for a given tumor. This is particularly appealing as our clinical approach to gene therapy based on the electro-transfer of DNA plasmids from the Sleeping Beauty (SB) transposon system reduces the cost and complexity to manufacture individual CAR designs for small subsets of patients. Our approach to personalizing CAR+ T cells is based on the generation of a large number of CAR molecules that can be screened and assessed for their ability to benefit a given patient. We have developed an approach for high throughput assembly of CARs using triple site-specific recombination system termed “EZ-CAR platform”. This technology allows the rapid combination of 3 components of a prototypical CAR from (i) the single chain variable fragment (scFv) that defines specificity, (ii) the scaffold/hinge that appends the scFv from the cell surface, and (iii) one or more intracellular signaling domains. As proof-of-concept we generated a CD19-specific CAR using the EZ-CAR platform in parallel with CD19 CAR control CAR+ T cells (CG CAR). Both CD19 CAR sequences were inserted into Sleeping Beauty transposon vectors and electroporated into T cells. After electroporation the T cells were cultivated in presence of CD19+ Activating and Propagating Cells (AaPCs) for antigen specific expansion of the T cells. The expression of the CARs in the T cells surface was measured every week by flow cytometry (Fc+ expression), showing similar CAR expression in both groups. We also performed a Chromium Release Assay (CRA) to evaluate the killing function of CD19 CAR+ T cells generated by EZ-CAR platform against tumor cells. After 4 hours of incubation the percentage of specific cell lysis was 52% by the EZ- CAR T cells and 49% by the CG CAR T cells. These results showed that using this technology we were able to generate functional CAR+ T cells. Then we started a rapid production of CARs using a library of plasmids containing the three components of a CAR molecule: (i) anti-CD19 scFv (ii) 5 hinges with different sizes and (iii) different combinations of 7 signaling domains with the CD3ζ domain. The EZ-CAR platform is in a dynamic process where new CAR components are been added to our library. Transfection of HEK 293 cells with plasmid containing the CAR transgene were used to screen 30 different CAR constructs to ensure the expression of the CAR protein on the cell surface. The high throughput testing of individual CAR molecules has been undertaken using the iQue Screener (Intellicyt), a high throughput flow cytometer, where cytotoxic assays are performed using engineered target cells expressing a fluorescent granzyme B reporter. The release of IL-2, IFN-γ and TNF-α by the CAR+ T cells and the cytotoxic activity are concurrently evaluated. The CAR designs that result in superior killing will be further tested using tumor cells from patients. Citation Format: Ana Beatriz Korngold, Brian Rabinovich, Helen Huls, Laurence Cooper. Designing chimeric antigen receptors for personalized immunotherapy: Rapid assembly of CARs from principal components using “EZ-CAR” platform. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B053.

ACP-196 Is a Second Generation Inhibitor of Bruton Tyrosine Kinase (BTK) with Enhanced Target Specificity

Background: Signaling through the B-cell receptor (BCR) results in cell survival and proliferation, and this pathway is critical for the progression of many B-cell malignancies, such a chronic lymphocytic leukemia (CLL). BTK, a TEC family kinase, is essential to the BCR signaling cascade. The recent approval of the first-in-class BTK inhibitor for the treatment certain B-cell malignancies, ibrutinib, has validated BTK as a clinical target. ACP-196 is a novel, second generation inhibitor of BTK with an IC50of 3 nM against purified BTK and EC50 of 8 nM in a human whole-blood CD69 B-cell activation assay. In vivo, oral administration of ACP-196 inhibits BCR-induced CD69 expression in murine splenic lymphocytes with an ED50 of 1.3 mg/kg compared with 2.9 mg/kg for ibrutinib. ACP-196 also has improved target specificity over ibrutinib with 323-,94-, 19- and 9-fold selectivity over the other TEC kinase familymembers (ITK, TXK, BMX, and TEC, respectively) and no activity against EGFR (Covey AACR, 2015). TXK and ITK are critical for T-cell receptor activation and development. ITK is also necessary for natural killer (NK)-cell mediated antibody dependent cell cytotoxicity (ADCC), which is important for the efficacy of monoclonal antibody therapy, eg, obinutuzumab. Additionally, therapeutic blockade of EGFR signaling is employed for treatment of epithelial cancers and is associated with adverse events, such as diarrhea and skin rash. Here, we describe results from signaling and functional assays to further elucidate the differential effects of ACP-196 on primary CLL cells compared with T-cells, NK-cells and epithelial cells.Methods: CLL patient samples were evaluated for pathway activation in the presence and absence of ACP-196 using phospho-protein immunoblotting. Helper T-cell (Th) skewing assays were performed using purified naive CD4+ T-cells from C57BL/6, Itk knock out (KO) and Itk/Txk double KO mice in the presence ACP-196 or ibrutinib. CD8+ T-cell activity was assessed by cytolytic activity (CTL) assays using T-cells isolated from C57BL/6 mice. The CTL response was initiated with various effector:T-cell ratios using C57BL/6 T-cells and murine BALB/c lymphoblasts. To evaluate NK-cell ADCC function, standard chromium release assays were done using purified NK-cells isolated from healthy volunteer peripheral blood mononuclear cells and cultured with obinutuzumab-coated (10 µg/mL) CLL cells in the presence and absence of ACP-196. Immunoblotting of H460 lung cancer cells for phospho-EGFR was used to evaluate the effect of ACP-196 on EGFR signaling.Results: ACP-196 treatment of primary human CLL cells showed a dose-dependent (0.01μM-1μM) decrease in p-BTK (Y223), p-ERK (T202/Y204) and p-AKT (T308) and p-IκB-α (S32), without impact on total protein levels. These findings demonstrate ACP-196 inhibits autophosphorylation of BTK as well as down-stream effectors of BCR signaling.Phospho-protein immunoblotting of primary human T-cells and Jurkat cells showed that ACP-196 does not inhibit T-cell receptor signaling molecules downstream of ITK and TXK such as IκBα, NFAT, or JunB. In in vitro helper T cell skewing assays, ACP-196, unlike ibrutinib, showed no effect on differentiation of Th subsets (Th1/Th2/Th17) or regulatory T cells (Tregs). The effect of ibrutinib on Th and Treg differentiation was similar to that observed in cells from Itk KO and Itk/Txk KO mice. In addition, ACP-196 had no effect on CD8+ T-cell viability or cytotoxicity as compared with ibrutinib-treated CD8+ T cells (46% and 36% reduction, respectively).In the in vitro ADCC assay, ACP-196 had no effect on NK cell function (chromium release of 34.8% vs 39.6% for the vehicle control). Lastly, at clinically relevant concentrations ACP-196 did not attenuate autophosphorylation of EGFR in H460 lung epithelial cells demonstrating no effect of ACP-196 on EGFR signaling.Conclusions: ACP-196 selectively inhibits BTK and BCR signaling at pharmacologic concentrations without off-target effects on T-cells, NK-cells and lung epithelial cells. ACP-196 is currently in Phase 3 trials for the treatment of CLL (ClinicalTrials.gov NCT0247568 and NCT02477696).This work was supported by the NIH NCI R01CA197870 and Acerta Pharma. DisclosuresGulrajani:Acerta Pharma: Employment. Covey:Acerta Pharma BV: Employment, Equity Ownership, Patents & Royalties. Kaptein:Acerta Pharma: Employment. Van Lith:Acerta Pharma: Employment. Izumi:Acerta Pharma: Employment. Hamdy:Acerta Pharma BV: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Ulrich:Acerta Pharma: Employment. Lannutti:Acerta Pharma: Employment. Johnson:Acerta Pharma: Research Funding.