Predicting microbial water quality in on-site water reuse systems with online sensors.

Widespread implementation of on-site water reuse is hindered by the limited availability of monitoring approaches that ensure microbial quality during operation. In this study, we developed a methodology for monitoring microbial water quality in on-site water reuse systems using inexpensive and commercially available online sensors. An extensive dataset containing sensor and microbial water quality data for six of the most critical types of disruptions in membrane bioreactors with chlorination was collected. We then tested the ability of three typological machine learning algorithms - logistic regression, support-vector machine, and random forest - to predict the microbial water quality as "safe" or "unsafe" for reuse. The main criteria for model optimization was to ensure a low false positive rate (FPR) - the percentage of safe predictions when the actual condition is unsafe - which is essential to protect users health. This resulted in enforcing a fixed FPR ≤ 2%. Maximizing the true positive rate (TPR) - the percentage of safe predictions when the actual condition is safe - was given second priority. Our results show that logistic-regression-based models using only two out of the six sensors (free chlorine and oxidation-reduction potential) achieved the highest TPR. Including sensor slopes as engineered features allowed to reach similar TPRs using only one sensor instead of two. Analysis of the occurrence of false predictions showed that these were mostly early alarms, a characteristic that could be regarded as an asset in alarm management. In conclusion, the simplest algorithm in combination with only one or two sensors performed best at predicting the microbial water quality. This result provides useful insights for water quality modeling or for applications where small datasets are a common challenge and a general advantage might be gained by using simpler models that reduce the risk of overfitting, allow better interpretability, and require less computational power.

mWTX-330, an IL-12 INDUKINE Molecule, Activates and Reshapes Tumor-Infiltrating CD8+ T and NK Cells to Generate Antitumor Immunity.

IL-12 is a pleotropic inflammatory cytokine, which has broad stimulatory effects on various immune cell populations, making it an attractive target for cancer immunotherapy. However, despite generating robust antitumor activity in syngeneic murine tumor models, clinical administration of IL-12 has been limited by severe toxicity. mWTX-330 is a selectively inducible INDUKINE molecule comprised of a half-life extension domain and an inactivation domain linked to chimeric IL-12 by tumor protease-sensitive linkers. Systemic administration of mWTX-330 in mice was well tolerated, resulted in robust antitumor immunity in multiple tumor models, and preferentially activated tumor-infiltrating immune cells rather than immune cells present in peripheral tissues. Antitumor activity was dependent on in vivo processing of the protease cleavable linkers and required CD8+ T cells for full efficacy. Within the tumor, mWTX-330 increased the frequency of cross-presenting dendritic cells (DC), activated natural killer (NK) cells, skewed conventional CD4+ T cells toward a T helper 1 (TH1) phenotype, drove regulatory T cells (Treg) fragility, and increased the frequency of polyfunctional CD8+ T cells. mWTX-330 treatment also increased the clonality of tumor-infiltrating T cells by expanding underrepresented T-cell receptor (TCR) clones, drove CD8+ T and NK cells towards increased mitochondrial respiration and fitness, and decreased the frequency of TOX+ exhausted CD8+ T cells within the tumor. A fully human version of this INDUKINE molecule was stable in human serum, was reliably and selectively processed by human tumor samples, and is currently in clinical development.

Discovery of a Conditionally Activated IL-2 that Promotes Antitumor Immunity and Induces Tumor Regression.

IL-2 is a cytokine clinically approved for the treatment of melanoma and renal cell carcinoma. Unfortunately, its clinical utility is hindered by serious side effects driven by the systemic activity of the cytokine. Here, we describe the design and characterization of a conditionally activated IL-2 prodrug, WTX-124, that takes advantage of the dysregulated protease milieu of tumors. WTX-124 was engineered as a single molecule containing an inactivation domain and a half-life extension domain that are tethered to a fully active IL-2 by protease-cleavable linkers. We show that the inactivation domain prevented IL-2 from binding to its receptors in nontumor tissues, thereby minimizing the toxicity associated with systemic exposure to IL-2. The half-life extension element improves the pharmacokinetic profile of WTX-124 over free IL-2, allowing for greater exposure. WTX-124 was preferentially activated in tumor tissue by tumor-associated proteases, releasing active IL-2 in the tumor microenvironment. In vitro assays confirmed that the activity of WTX-124 was dependent on proteolytic activation, and in vivo WTX-124 treatment resulted in complete rejection of established tumors in a cleavage-dependent manner. Mechanistically, WTX-124 treatment triggered the activation of T cells and natural killer (NK) cells, and markedly shifted the immune activation profile of the tumor microenvironment, resulting in significant inhibition of tumor growth in syngeneic tumor models. Collectively, these data demonstrate that WTX-124 minimizes the toxicity of IL-2 treatment in the periphery while retaining the full pharmacology of IL-2 in the tumor microenvironment, supporting its further development as a cancer immunotherapy treatment. See related Spotlight by Silva, p. 544.

Characterization of ASP8374, a fully-human, antagonistic anti-TIGIT monoclonal antibody.

The T-cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory motif (ITIM) domains (TIGIT) is a validated immune checkpoint protein expressed on memory CD4+T-cellls, Tregs, CD8+T-cell and natural killer (NK) cells. ASP8374 is a fully human monoclonal immunoglobulin (Ig) G4 antibody designed to block the interaction of TIGIT with its ligands and inhibit TIGIT signaling. ASP8374 exhibited high affinity binding to TIGIT and increased interferon (IFN)-γ production of cultured peripheral blood mononuclear cells (PBMCs) in a titratable manner. When used in combination with pembrolizumab, an anti-programmed death-1 (PD-1) antibody, ASP8374 induced higher T-cell activation in vitro than either treatment alone. An anti-mouse TIGIT antibody surrogate, mSEC1, displayed anti-tumor efficacy in an MC38 syngeneic mouse tumor model alone and in combination with an anti-programmed death-ligand 1 (PD-L1) antibody. In an additional syngeneic mouse tumor model (CT26), while mSEC1 alone did not demonstrate anti-tumor efficacy, mSEC1 combined with an anti-PD-1 antibody enhanced anti-tumor efficacy above that of the anti-PD-1 antibody alone. These data provide evidence that ASP8374 has therapeutic potential for advanced malignancies.

A Potent HER3 Monoclonal Antibody That Blocks Both Ligand-Dependent and -Independent Activities: Differential Impacts of PTEN Status on Tumor Response.

HER3/ERBB3 is a kinase-deficient member of the EGFR family receptor tyrosine kinases (RTK) that is broadly expressed and activated in human cancers. HER3 is a compelling cancer target due to its important role in activation of the oncogenic PI3K/AKT pathway. It has also been demonstrated to confer tumor resistance to a variety of cancer therapies, especially targeted drugs against EGFR and HER2. HER3 can be activated by its ligand (heregulin/HRG), which induces HER3 heterodimerization with EGFR, HER2, or other RTKs. Alternatively, HER3 can be activated in a ligand-independent manner through heterodimerization with HER2 in HER2-amplified cells. We developed a fully human mAb against HER3 (KTN3379) that efficiently suppressed HER3 activity in both ligand-dependent and independent settings. Correspondingly, KTN3379 inhibited tumor growth in divergent tumor models driven by either ligand-dependent or independent mechanisms in vitro and in vivo Most intriguingly, while investigating the mechanistic underpinnings of tumor response to KTN3379, we discovered an interesting dichotomy in that PTEN loss, a frequently occurring oncogenic lesion in a broad range of cancer types, substantially blunted the tumor response in HER2-amplified cancer, but not in the ligand-driven cancer. To our knowledge, this represents the first study ascertaining the impact of PTEN loss on the antitumor efficacy of a HER3 mAb. KTN3379 is currently undergoing a phase Ib clinical trial in patients with advanced solid tumors. Our current study may help us optimize patient selection schemes for KTN3379 to maximize its clinical benefits. Mol Cancer Ther; 15(4); 689-701. ©2016 AACR.

Methods for evaluating effects of an irinotecan + 5-fluorouracil/leucovorin (IFL) regimen in an orthotopic metastatic colorectal cancer model utilizing in vivo bioluminescence imaging.

In testing novel anticancer therapies, researchers strive to utilize models that reflect the human disease as much as feasible. In this regard, orthotopic models are frequently developed because cancer cells in these models form tumors in, and metastasize from, a tissue environment similar to the tissue of origin of the cancer cells. Here we adapted an orthotopic colorectal cancer model, in which HT-29 colorectal cancer cells form tumors in the rectal lining and metastasize to the para-aortic lymph nodes with high frequency. Firefly luciferase-expressing HT-29 cells were used in this model to realize the benefits of bioluminescence imaging (BLI). A combination of irinotecan, 5-fluorouracil (5-FU), and leucovorin (LV) (IFL) was used as a standard chemotherapeutic regimen positive control. BLI allowed for the demonstration of the effects of IFL on tumor growth in the rectal lining, with tumor weight measurements at the end of the study reflecting total tumor burden. BLI also allowed relatively easy demonstration of reduced tissue metastasis with IFL treatment, compared to more time-consuming histological techniques. It is concluded that the orthotopic colorectal cancer model approach described represents a valuable tool for validating treatment strategies in this indication.

Prioritization of EGFR/IGF-IR/VEGFR2 combination targeted therapies utilizing cancer models.

BACKGROUND: Rational strategies utilizing anticancer efficacy and biological principles are needed for the prioritization of specific combination targeted therapy approaches for clinical development, from among the many with experimental support. MATERIALS AND METHODS: Antibodies targeting epidermal growth factor receptor (EGFR) (cetuximab), insulin-like growth factor-1 receptor (IGF-IR) (IMC-A12) or vascular endothelial growth factor receptor 2 (VEGFR2) (DC101), were dosed alone or in combination, in 11 human tumor xenograft models established in mice. Efficacy readouts included the tumor burden and incidence of metastasis, as well as tumor active hypoxia inducible factor-1 (HIF-1), human VEGF and blood vessel density. RESULTS: Cetuximab and DC101 contributed potent and non-overlapping benefits to the combination approach. Moreover, DC101 prevented escape from IMC-A12 + cetuximab in a colorectal cancer model and cetuximab prevented escape from DC101 therapy in a pancreatic cancer model. CONCLUSION: Targeting VEGFR2 + EGFR was prioritized over other treatment strategies utilizing EGFR, IGF-IR and VEGFR2 antibodies. The criteria that proved to be valuable were a non-overlapping spectrum of anticancer activity and the prevention of resistance to another therapy in the combination.

Inhibitory activity of cetuximab on epidermal growth factor receptor mutations in non small cell lung cancers.

Mutations in the kinase domain of the epidermal growth factor receptor (EGFR) were identified in approximately 15% of all patients with non-small cell lung cancer (NSCLC). These mutations have been established as an indicator of superior response to gefitinib and erlotinib, small molecule inhibitors of the EGFR kinase domain. Whether these mutations would also render patients more susceptible to treatment with cetuximab (Erbitux), an EGFR-neutralizing antibody, is yet to be determined. In this study, we attempted to evaluate the effect of cetuximab on several NSCLC lines harboring some of the more common EGFR mutations (L858R and delL747-T753insS), as well as the recently identified kinase inhibitor-resistant mutation, T790M. We could show that the kinase activity of the abovementioned EGFR mutants was hindered by cetuximab, as detected by both cell-based phosphorylation and proliferation assays. Interestingly, cetuximab also induced enhanced degradation of the EGFR mutants as compared with the wild-type receptor. Most importantly, cetuximab successfully inhibited the growth of NSCLC lines in xenograft models. These results indicate the promising potential of cetuximab as a regimen for patients with NSCLC bearing these mutations.

Tumor growth inhibition with cetuximab and chemotherapy in non-small cell lung cancer xenografts expressing wild-type and mutated epidermal growth factor receptor.

PURPOSE: Targeting the epidermal growth factor receptor (EGFR) is a validated approach to treat cancer. In non-small cell lung cancer (NSCLC), EGFR contains somatic mutations in 10% of patients, which correlates with increased response rates to small molecule inhibitors of EGFR. We analyzed the effects of the monoclonal IgG1 antibody Erbitux (cetuximab) in NSCLC xenografts with wild-type (wt) or mutated EGFR. EXPERIMENTAL DESIGN: NSCLC cell lines were grown s.c. in nude mice. Dose-dependent efficacy was established for cetuximab. To determine whether combination therapy produces tumor regressions, cetuximab was dosed at half-maximal efficacy with chemotherapy used at maximum tolerated dose. RESULTS: Cetuximab showed antitumor activity in wt (A549, NCI-H358, NCI-H292) and mutated [HCC-827 (delE746-A750), NCI-H1975 (L858R, T790M)] EGFR-expressing xenografts. In the H292 model, cetuximab and docetaxel combination therapy was more potent to inhibit tumor growth than cetuximab or docetaxel alone. Cisplatin augmented efficacy of cetuximab to produce 6 of 10 regressions, whereas 1 of 10 regressions was found with cetuximab and no regression was found with cisplatin. Using H1975 xenografts, gemcitabine increased efficacy of cetuximab resulting in 12 of 12 regressions. Docetaxel with cetuximab was more efficacious with seven of nine regressions compared with single treatments. Cetuximab inhibited autophosphorylation of EGFR in both H292 and H1975 tumor lysates. Exploring the underlying mechanism for combination effects in the H1975 xenograft model, docetaxel in combination with cetuximab added to the antiproliferative effects of cetuximab but was the main component in this drug combination to induce apoptosis. CONCLUSIONS: Cetuximab showed antitumor activity in NSCLC models expressing wt and mutated EGFR. Combination treatments increased the efficacy of cetuximab, which may be important for the management of patients with chemorefractory NSCLC.

Vascular endothelial growth factor receptor 3 is involved in tumor angiogenesis and growth.

Vascular endothelial growth factor receptor 3 (VEGFR-3) binds VEGF-C and VEGF-D and is essential for the development of the lymphatic vasculature. Experimental tumors that overexpress VEGFR-3 ligands induce lymphatic vessel sprouting and enlargement and show enhanced metastasis to regional lymph nodes and beyond, whereas a soluble form of VEGFR-3 that blocks receptor signaling inhibits these changes and metastasis. Because VEGFR-3 is also essential for the early blood vessel development in embryos and is up-regulated in tumor angiogenesis, we wanted to determine if an antibody targeting the receptor that interferes with VEGFR-3 ligand binding can inhibit primary tumor growth. Our results show that antibody interference with VEGFR-3 function can inhibit the growth of several human tumor xenografts in immunocompromised mice. Immunohistochemical analysis showed that the blood vessel density of anti-VEGFR-3-treated tumors was significantly decreased and hypoxic and necrotic tumor tissue was increased when compared with tumors treated with control antibody, indicating that blocking of the VEGFR-3 pathway inhibits angiogenesis in these tumors. As expected, the anti-VEGFR-3-treated tumors also lacked lymphatic vessels. These results suggest that the VEGFR-3 pathway contributes to tumor angiogenesis and that effective inhibition of tumor progression may require the inhibition of multiple angiogenic targets.

Differential regulation of noxa in normal melanocytes and melanoma cells by proteasome inhibition: therapeutic implications.

Melanoma is the most aggressive form of skin cancer and advanced stages are invariably resistant to conventional therapeutic agents. Using bortezomib as a prototypic proteasome inhibitor, we have identified a novel and critical role of the proteasome in the maintenance of the malignant phenotype of melanoma cells that could have direct translational implications. Thus, melanoma cells from early, intermediate, and late stages of the disease could not sustain proteasome inhibition and underwent an effective activation of caspase-dependent and -independent death programs. This effect was tumor cell selective, because under similar conditions, normal melanocytes remained viable. Intriguingly, and despite of interfering with a cellular machinery in charge of controlling the half-life of the vast majority of cellular proteins, bortezomib did not promote a generalized disruption of melanoma-associated survival factors (including NF-kappaB, Bcl-2, Bcl-x(L), XIAP, TRAF-2, or FLIP). Instead, we identified a dramatic induction in vitro and in vivo of the BH3-only protein Noxa in melanoma cells (but not in normal melanocytes) in response to proteasome inhibition. RNA interference validated a critical role of Noxa for the cytotoxic effect of bortezomib. Notably, the proteasome-dependent regulation of Noxa was found to extend to other tumor types, and it could not be recapitulated by standard chemotherapeutic drugs. In summary, our results revealed Noxa as a new biomarker to gauge the efficacy of bortezomib specifically in tumor cells, and provide a new strategy to overcome tumor chemoresistance.

Targeting the platelet-derived growth factor receptor alpha with a neutralizing human monoclonal antibody inhibits the growth of tumor xenografts: implications as a potential therapeutic target.

Platelet-derived growth factor receptor alpha (PDGFRalpha) is a type III receptor tyrosine kinase that is expressed on a variety of tumor types. A neutralizing monoclonal antibody to human PDGFRalpha, which did not cross-react with the beta form of the receptor, was generated. The fully human antibody, termed 3G3, has a Kd of 40 pmol/L and blocks both PDGF-AA and PDGF-BB ligands from binding to PDGFRalpha. In addition to blocking ligand-induced cell mitogenesis and receptor autophosphorylation, 3G3 inhibited phosphorylation of the downstream signaling molecules Akt and mitogen-activated protein kinase. This inhibition was seen in both transfected and tumor cell lines expressing PDGFRalpha. The in vivo antitumor activity of 3G3 was tested in human glioblastoma (U118) and leiomyosarcoma (SKLMS-1) xenograft tumor models in athymic nude mice. Antibody 3G3 significantly inhibited the growth of U118 (P=0.0004) and SKLMS-1 (P <0.0001) tumors relative to control. These data suggest that 3G3 may be useful for the treatment of tumors that express PDGFRalpha.

Neuroendocrine tumors and second primary malignancy--a relationship with clinical impact?

BACKGROUND: Neuroendocrine tumors (NET) are frequently associated with synchronous or metachronous secondary primary malignancies (SPM). The aim of this study was to report on 14 patients with NET and SPM from a series of 96 patients with NET. PATIENTS AND METHODS: Fourteen patients with NET and synchronous or metachronous SPM were reviewed for primary site and characteristics of NET and associated SPMs as well as the outcome of these combined malignancies. RESULTS: From 1987 to 2002, 14 (14.6%) out of 96 patients with NET were identified with SPM. The median age of the patients at diagnosis of NET was 69 years (range: 56-86 yrs). There were nine female and five male patients. The localization of NET was: four in appendix, three ileum, two duodenum, one stomach, one jejunum, one pancreatic tail, one rectum and one lung. Five patients had synchronous SPM (two colon cancers with one double colon cancer, one gastric cancer, one bladder cancer, one ovarian cancer) and nine metachronous SPM (two basal cell carcinomas, one colon cancer, two breast cancer, one gastric MALT-lymphoma, one ductal pancreatic adenocarcinoma, one bladder cancer, one hepatocellular carcinoma), three months to five years after diagnosis of NET. Five patients died of metastatic tumor (three SPM: 1, 7, 10 yrs; two NET: 1, 9 yrs), two patients died of other causes (1, 7 yrs), three patients are alive with metastatic tumor (two NET: 5, 6 yrs; one SPM: 10 yrs) while four patients are tumor-free (6 ms, 2, 9, 10 yrs). CONCLUSION: NET is associated to a high degree with gastrointestinal and genitourinary SPM. In 5/14 (36%) patients SPM was diagnosed synchronously, while in 8/14 (57%) patients SPM was diagnosed metachronously. In 8/14 patients (57%) primary symptoms were caused by SPM. As a consequence, every NET should be regarded as an index tumor and risk-adapted follow-up with thorough investigation, mainly of the GI and genitourinary tracts, is to be recommended.

Differential apoptotic response to the proteasome inhibitor Bortezomib [VELCADE, PS-341] in Bax-deficient and p21-deficient colon cancer cells.

Targeting the ubiquitin-proteasome pathway has emerged as a promising approach for treating cancer. Bortezomib (VELCADE, formerly known as PS-341), a potent and reversible proteasome inhibitor, is being evaluated in clinical trials for treating multiple myeloma, and various other types of hematologic and solid tumors. Proteasome inhibitors are known to induce apoptosis in human cancer cells. Nevertheless, the mechanisms of apoptosis induced by proteasome inhibitors remain unclear. In this study, we investigated the role of p53 and its downstream targets in bortezomib-induced apoptosis in HCT116 human colon cancer cells. We demonstrated that bortezomib induced p53, and activated its downstream genes p21, PUMA and Bax in a p53-dependent fashion. However, apoptotic response to bortezomib was not affected by the deletion of p53. Surprisingly, we found that bortezomib-induced apoptosis was markedly enhanced in the p21-knockout cells, while significantly decreased in the BAX-knockout cells. Furthermore, in the cells deficient for both Bax and p21, apoptosis was restored to the level in the parental or the p53-deficient cells. The opposite effects of Bax and p21 were unrelated to the extent of proteasome inhibition, and were also observed in cells treated with different proteasome inhibitors. These results indicate that p53 downstream targets can collectively modulate apoptotic response to bortezomib and other proteasome inhibitors.