Assessing the binding properties of the anti-PD-1 antibody landscape using label-free biosensors.

Here we describe an industry-wide collaboration aimed at assessing the binding properties of a comprehensive panel of monoclonal antibodies (mAbs) against programmed cell death protein 1 (PD-1), an important checkpoint protein in cancer immunotherapy and validated therapeutic target, with well over thirty unique mAbs either in clinical development or market-approved in the United States, the European Union or China. The binding kinetics of the PD-1/mAb interactions were measured by surface plasmon resonance (SPR) using a Carterra LSA instrument and the results were compared to data collected on a Biacore 8K. The effect of chip type on the SPR-derived binding rate constants and affinities were explored and the results compared with solution affinities from Meso Scale Discovery (MSD) and Kinetic Exclusion Assay (KinExA) experiments. When using flat chip types, the LSA and 8K platforms yielded near-identical kinetic rate and affinity constants that matched solution phase values more closely than those produced on 3D-hydrogels. Of the anti-PD-1 mAbs tested, which included a portion of those known to be in clinical development or approved, the affinities spanned from single digit picomolar to nearly 425 nM, challenging the dynamic range of our methods. The LSA instrument was also used to perform epitope binning and ligand competition studies which revealed over ten unique competitive binding profiles within this group of mAbs.

Database-Centric Method for Automated High-Throughput Deconvolution and Analysis of Kinetic Antibody Screening Data.

The state-of-the-art industrial drug discovery approach is the empirical interrogation of a library of drug candidates against a target molecule. The advantage of high-throughput kinetic measurements over equilibrium assessments is the ability to measure each of the kinetic components of binding affinity. Although high-throughput capabilities have improved with advances in instrument hardware, three bottlenecks in data processing remain: (1) intrinsic molecular properties that lead to poor biophysical quality in vitro are not accounted for in commercially available analysis models, (2) processing data through a user interface is time-consuming and not amenable to parallelized data collection, and (3) a commercial solution that includes historical kinetic data in the analysis of kinetic competition data does not exist. Herein, we describe a generally applicable method for the automated analysis, storage, and retrieval of kinetic binding data. This analysis can deconvolve poor quality data on-the-fly and store and organize historical data in a queryable format for use in future analyses. Such database-centric strategies afford greater insight into the molecular mechanisms of kinetic competition, allowing for the rapid identification of allosteric effectors and the presentation of kinetic competition data in absolute terms of percent bound to antigen on the biosensor.

Inactivation of a GAL4-like transcription factor improves cell fitness and product yield in glycoengineered Pichia pastoris strains.

With a completely reengineered and humanized glycosylation pathway, glycoengineered Pichia pastoris has emerged as a promising production host for the manufacture of therapeutic glycoproteins. However, the extensive genetic modifications have also negatively affected the overall fitness levels of the glycoengineered host cells. To make glycoengineered Pichia strains more compatible with a scalable industrial fermentation process, we sought to identify genetic solutions to broadly improve cell robustness during fermentation. In this study, we report that mutations within the Pichia pastoris ATT1 (PpATT1) gene (a homolog of the Saccharomyces cerevisiae GAL4 [ScGAL4] transcriptional activator) dramatically increased the cellular fitness levels of glycoengineered Pichia strains. We demonstrate that deletion of the PpATT1 gene enabled glycoengineered Pichia strains to improve their thermal tolerance levels, reduce their cell lysis defects, and greatly improve fermentation robustness. The extension of the duration of fermentation enabled the PpATT1-modified glycoengineered Pichia strains to increase their product yields significantly without any sacrifice in product quality. Because the ATT1 gene could be deleted from any Pichia strains, including empty hosts and protein-expressing production strains alike, we suggest that the findings described in this study are broadly applicable to any Pichia strains used for the production of therapeutic proteins, including monoclonal antibodies, Fc fusions, peptides, hormones, and growth factors.

Critical review of the epidemiologic literature regarding the association between congenital heart defects and exposure to trichloroethylene.

The most recent Integrated Risk Information System review of trichloroethylene (TCE; CAS# 79-01-6) has suggested that congenital heart defects (CHD) are a critical endpoint associated with exposure to this solvent. This conclusion was drawn, at least partly, from epidemiologic data, including several relatively recent studies. The current article critically reviews this epidemiologic literature, focusing on study quality and consistency. Literature searches uncovered approximately a dozen studies that specifically addressed associations between TCE and congenital malformations in eight populations. Four of these reported positive associations between TCE and heart defects, with significant relative risks as high as 5-6 in some subgroups. However, each of the positive studies had substantial design or analytical flaws that could easily explain the results, thereby limiting the conclusions that can be drawn. Five studies found no positive association with TCE, and several of these reported substantially fewer cases than expected despite similar/higher exposures compared to positive studies, further detracting from causal conclusions. Overall, this epidemiologic literature provides no substantive or consistent evidence linking TCE to CHD.

shRNA screening identifies JMJD1C as being required for leukemia maintenance.

Epigenetic regulatory mechanisms are implicated in the pathogenesis of acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). Recent progress suggests that proteins involved in epigenetic control are amenable to drug intervention, but little is known about the cancer-specific dependency on epigenetic regulators for cell survival and proliferation. We used a mouse model of human AML induced by the MLL-AF9 fusion oncogene and an epigenetic short hairpin RNA (shRNA) library to screen for novel potential drug targets. As a counter-screen for general toxicity of shRNAs, we used normal mouse bone marrow cells. One of the best candidate drug targets identified in these screens was Jmjd1c. Depletion of Jmjd1c impairs growth and colony formation of mouse MLL-AF9 cells in vitro as well as establishment of leukemia after transplantation. Depletion of JMJD1C impairs expansion and colony formation of human leukemic cell lines, with the strongest effect observed in the MLL-rearranged ALL cell line SEM. In both mouse and human leukemic cells, the growth defect upon JMJD1C depletion appears to be primarily due to increased apoptosis, which implicates JMJD1C as a potential therapeutic target in leukemia.

Extreme sensitivity and the practical implications of risk assessment thresholds.

Traditional risk-assessment theory assumes the existence of a threshold for non-cancer health effects. However, a recent trend in environmental regulation rejects this assumption in favor of non-threshold linearity for these endpoints. This trend is driven largely by two related concepts: (1) a theoretical assumption of wide-ranging human sensitivity, and (2) inability to detect thresholds in epidemiologic models. Wide-ranging sensitivity assumes a subpopulation with extreme background vulnerability, so that even trivial environmental exposures are hazardous to someone somewhere. We use examples from the real world of clinical medicine to show that this theoretical assumption is inconsistent with the biology of mammalian systems and the realities of patient care. Using examples from particulate-matter air-pollution research, we further show that failure to reject linearity is usually driven by statistical rather than biological considerations, and that nonlinear/threshold models often have a similar or better fit than their linear counterparts. This evidence suggests the existence of practical, real-world thresholds for most chemical exposures.

Production of sialylated O-linked glycans in Pichia pastoris.

The methylotrophic yeast, Pichia pastoris, is an important organism used for the production of therapeutic proteins. Previously, we have reported the glycoengineering of this organism to produce human-like N-linked glycans but up to now no one has addressed engineering the O-linked glycosylation pathway. Typically, O-linked glycans produced by wild-type P. pastoris are linear chains of four to five α-linked mannose residues, which may be capped with ß- or phospho-mannose. Previous genetic engineering of the N-linked glycosylation pathway of P. pastoris has eliminated both of these two latter modifications, resulting in O-linked glycans which are linear α-linked mannose structures. Here, we describe a method for the co-expression of an α-1,2-mannosidase, which reduces these glycans to primarily a single O-linked mannose residue. In doing so, we have reduced the potential of these glycans to interact with carbohydrate-binding proteins, such as dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin. Furthermore, the introduction of the enzyme protein-O-linked-mannose ß-1,2-N-acetylglucosaminyltransferase 1, resulted in the capping of the single O-linked mannose residues with N-acetylglucosamine. Subsequently, this glycoform was extended into human-like sialylated glycans, similar in structure to α-dystroglycan-type glycoforms. As such, this represents the first example of sialylated O-linked glycans being produced in yeast and extends the utility of the P. pastoris production platform beyond N-linked glycosylated biotherapeutics to include molecules possessing O-linked glycans.

Characterization of the Pichia pastoris protein-O-mannosyltransferase gene family.

The methylotrophic yeast, Pichiapastoris, is an important organism used for the production of therapeutic proteins. However, the presence of fungal-like glycans, either N-linked or O-linked, can elicit an immune response or enable the expressed protein to bind to mannose receptors, thus reducing their efficacy. Previously we have reported the elimination of ß-linked glycans in this organism. In the current report we have focused on reducing the O-linked mannose content of proteins produced in P. pastoris, thereby reducing the potential to bind to mannose receptors. The initial step in the synthesis of O-linked glycans in P. pastoris is the transfer of mannose from dolichol-phosphomannose to a target protein in the yeast secretory pathway by members of the protein-O-mannosyltransferase (PMT) family. In this report we identify and characterize the members of the P. pastoris PMT family. Like Candida albicans, P. pastoris has five PMT genes. Based on sequence homology, these PMTs can be grouped into three sub-families, with both PMT1 and PMT2 sub-families possessing two members each (PMT1 and PMT5, and PMT2 and PMT6, respectively). The remaining sub-family, PMT4, has only one member (PMT4). Through gene knockouts we show that PMT1 and PMT2 each play a significant role in O-glycosylation. Both, by gene knockouts and the use of Pmt inhibitors we were able to significantly reduce not only the degree of O-mannosylation, but also the chain-length of these glycans. Taken together, this reduction of O-glycosylation represents an important step forward in developing the P. pastoris platform as a suitable system for the production of therapeutic glycoproteins.

Review of the epidemiologic literature on residential exposure to perchloroethylene.

Perchloroethylene is a solvent that is widely used for dry cleaning. There has been considerable interest in the toxicity of this chemical because of the potential for low-level exposure among a large portion of the US population. Although substantial epidemiologic literature exists on high-level occupational exposure to perchloroethylene, there are relatively few studies dealing with lower-level residential exposure. In the current paper, the author reviews this limited residential literature, with special emphasis on strengths, limitations, and consistency. Reviewed studies primarily address neurobehavioral, cancer, and reproductive endpoints. Most studies used an ecological or cross-sectional design, with exposure defined by either drinking-water contamination or proximity to dry cleaning. In general, reviewed studies were highly exploratory, with inconsistencies and potential for bias that detract from interpretation of study findings. The magnitudes of reported effects are frequently incompatible with the effects reported from much higher occupational and human-chamber exposures. Overall, few reliable conclusions can be drawn from this sparse and highly limited body of literature.

Arabidopsis genes IRREGULAR XYLEM (IRX15) and IRX15L encode DUF579-containing proteins that are essential for normal xylan deposition in the secondary cell wall.

There are 10 genes in the Arabidopsis genome that contain a domain described in the Pfam database as domain of unknown function 579 (DUF579). Although DUF579 is widely distributed in eukaryotic species, there is no direct experimental evidence to assign a function to it. Five of the 10 Arabidopsis DUF579 family members are co-expressed with marker genes for secondary cell wall formation. Plants in which two closely related members of the DUF579 family have been disrupted by T-DNA insertions contain less xylose in the secondary cell wall as a result of decreased xylan content, and exhibit mildly distorted xylem vessels. Consequently we have named these genes IRREGULAR XYLEM 15 (IRX15) and IRX15L. These mutant plants exhibit many features of previously described xylan synthesis mutants, such as the replacement of glucuronic acid side chains with methylglucuronic acid side chains. By contrast, immunostaining of xylan and transmission electron microscopy (TEM) reveals that the walls of these irx15 irx15l double mutants are disorganized, compared with the wild type or other previously described xylan mutants, and exhibit dramatic increases in the quantity of sugar released in cell wall digestibility assays. Furthermore, localization studies using fluorescent fusion proteins label both the Golgi and also an unknown intracellular compartment. These data are consistent with irx15 and irx15l defining a new class of genes involved in xylan biosynthesis. How these genes function during xylan biosynthesis and deposition is discussed.

JARID2 regulates binding of the Polycomb repressive complex 2 to target genes in ES cells.

The Polycomb group (PcG) proteins have an important role in controlling the expression of genes essential for development, differentiation and maintenance of cell fates. The Polycomb repressive complex 2 (PRC2) is believed to regulate transcriptional repression by catalysing the di- and tri-methylation of lysine 27 on histone H3 (H3K27me2/3). At present, it is unknown how the PcG proteins are recruited to their target promoters in mammalian cells. Here we show that PRC2 forms a stable complex with the Jumonji- and ARID-domain-containing protein, JARID2 (ref. 4). Using genome-wide location analysis, we show that JARID2 binds to more than 90% of previously mapped PcG target genes. Notably, we show that JARID2 is sufficient to recruit PcG proteins to a heterologous promoter, and that inhibition of JARID2 expression leads to a major loss of PcG binding and to a reduction of H3K27me3 levels on target genes. Consistent with an essential role for PcG proteins in early development, we demonstrate that JARID2 is required for the differentiation of mouse embryonic stem cells. Thus, these results demonstrate that JARID2 is essential for the binding of PcG proteins to target genes and, consistent with this, for the proper differentiation of embryonic stem cells and normal development.

Epidemiologic evidence for chloroprene carcinogenicity: review of study quality and its application to risk assessment.

This article evaluates the quality and weight of evidence associated with epidemiologic studies of cancer among occupational cohorts exposed to chloroprene. The focus is on liver, lung, and lymphohematopoietic cancers, which had been increased in early studies. Literature searches identified eight morbidity/mortality studies covering seven chloroprene-exposed cohorts from six countries. These studies were summarized and their quality was assessed using the 10 criteria suggested by the U.S. Environmental Protection Agency. The limitations within this literature (primarily the early studies) included crude exposure assessment, incomplete follow-up, uncertain baseline rates, and uncontrolled confounding by factors such as smoking, drinking, and co-exposure to benzene and vinyl chloride. Four cohorts were studied by the same group of investigators, who reported no overall increased associations for any cancers. This four-cohort study was by far the most rigorous, having the most comprehensive exposure assessment and follow-up and the most detailed documentation. This study also contained the two largest cohorts, including an American cohort from Louisville, Kentucky, that ranked at or near the top for each of the 10 quality criteria. There was evidence of a strong healthy worker effect in the four-cohort study, which could have hidden small excess risks. Small increased risks were suggested by internal or company-specific analyses, but these were most likely caused by uncontrolled confounding and low baseline rates. Overall, the weight of evidence does not support any substantial link between chloroprene exposure and cancer, but inconsistencies and a lack of control for major confounders preclude drawing firmer conclusions.

Do pollution time-series studies contain uncontrolled or residual confounding by risk factors for acute health events?

Acute health effects from air pollution are based largely on weak associations identified in time-series studies comparing daily air pollution levels to daily mortality. Much of this mortality is due to cardiovascular disease. Time-series studies have many potential limitations, but are not thought to be confounded by traditional cardiovascular risk factors (e.g., smoking status or hypertension) because these chronic risk factors are not obviously associated with daily pollution levels. However, acute psychobehavioral variants of these risk factors (e.g., smoking patterns and episodes of stress on any given day) are plausible confounders for the associations observed in time-series studies, given that time-series studies attempt to predict acute rather than chronic health outcomes. There is a fairly compelling literature on the strong link between cardiovascular events and daily "triggers" such as stress. Stress-related triggers are plausibly associated with daily pollution levels through surrogate stressors such as ambient temperature, daily workload, local traffic congestion, or other correlates of air pollution. For example, variables such as traffic congestion and industrial activity increase both stress-related health events and air pollution, suggesting the potential for classical confounding. Support for this argument is illustrated through examples of the well-demonstrated relationship between emotional stress and heart attack/stroke.

Interactions between subunits of Saccharomyces cerevisiae RNase MRP support a conserved eukaryotic RNase P/MRP architecture.

Ribonuclease MRP is an endonuclease, related to RNase P, which functions in eukaryotic pre-rRNA processing. In Saccharomyces cerevisiae, RNase MRP comprises an RNA subunit and ten proteins. To improve our understanding of subunit roles and enzyme architecture, we have examined protein-protein and protein-RNA interactions in vitro, complementing existing yeast two-hybrid data. In total, 31 direct protein-protein interactions were identified, each protein interacting with at least three others. Furthermore, seven proteins self-interact, four strongly, pointing to subunit multiplicity in the holoenzyme. Six protein subunits interact directly with MRP RNA and four with pre-rRNA. A comparative analysis with existing data for the yeast and human RNase P/MRP systems enables confident identification of Pop1p, Pop4p and Rpp1p as subunits that lie at the enzyme core, with probable addition of Pop5p and Pop3p. Rmp1p is confirmed as an integral subunit, presumably associating preferentially with RNase MRP, rather than RNase P, via interactions with Snm1p and MRP RNA. Snm1p and Rmp1p may act together to assist enzyme specificity, though roles in substrate binding are also indicated for Pop4p and Pop6p. The results provide further evidence of a conserved eukaryotic RNase P/MRP architecture and provide a strong basis for studies of enzyme assembly and subunit function.

Evaluating emerging issues in epidemiology.

Industry and government institutions need a credible approach for evaluating and responding to emerging public health issues. Representatives of industry, government, and academia met under the auspices of the International Life Sciences Institute's Health and Environmental Sciences Institute (HESI) to develop successful strategies for dealing with emerging issues based on historical case studies. The case studies chosen for evaluation were (1) tampon use and toxic shock syndrome; (2) hazardous waste and childhood cancer risk in Toms River, New Jersey; (3) fenfluramine and phentermine use and valvular heart disease; (4) silicone breast implants and cancer and auto-immune disease; and (5) progestational drugs and birth defects. We identified eight lessons from these case studies. Foremost, we recommend that public and private institutions not defer action until an issue is scientifically resolved and stress that cooperation among issue stakeholders is critical for effective issue resolution. We suggest establishing a research program as an effective way to assure that good science is included in resolution of the issue. We further recommend frequent and timely communication with all stakeholders, and the development of research approaches to fill gaps when the scientific data on an issue are limited.

GLP-2 receptor localizes to enteric neurons and endocrine cells expressing vasoactive peptides and mediates increased blood flow.

BACKGROUND & AIMS: Glucagon-like peptide-2 (GLP-2) is a nutrient-responsive hormone that exerts diverse actions in the gastrointestinal tract, including enhancing epithelial cell survival and proliferation, mucosal blood flow, and nutrient uptake and suppressing gastric motility and secretion. These actions are mediated by the G-protein-coupled receptor, GLP-2R. Cellular localization of the GLP-2R and the nature of its signaling network in the gut, however, are poorly defined. Thus, our aim was to establish cellular localization of GLP-2R and functional connection to vascular action of GLP-2 in the gut. METHODS: Intestinal cellular GLP-2R localization was determined with real-time, quantitative reverse-transcription polymerase chain reaction (qRT-PCR) of laser capture microdissected subtissue and fluorescence in situ hybridization and also with double and/or triple immunostaining of human and pig tissue using a validated GLP-2R polyclonal antibody. Superior mesenteric arterial blood flow and intestinal eNOS expression and phosphorylation were measured in TPN-fed pigs acutely (4 h) infused with GLP-2. RESULTS: We show that the porcine GLP-2R mRNA was expressed in the villus epithelium and myenteric plexus. GLP-2R protein was co-localized by confocal immunohistochemistry with serotonin in enteroendocrine cells and also with endothelial nitric oxide synthase (eNOS)-expressing and vasoactive intestinal polypeptide-positive enteric neurons. In neonatal pigs, GLP-2 infusion dose-dependently stimulated intestinal blood flow and coordinately upregulated the expression of intestinal eNOS mRNA, protein, and phosphorylation (eNOS-Ser1117). CONCLUSIONS: We conclude that the GLP-2-induced stimulation of blood flow is mediated by vasoactive neurotransmitters that are colocalized with GLP-2R in 2 functionally distinct cell types within the gastrointestinal tract.

Practical implications of nonlinear effects in risk-assessment harmonization.

Cancer and noncancer health effects have traditionally been handled differently in quantitative risk assessment. A threshold (i.e., safe exposure) has been assumed for noncancer health effects, and low-dose linearity without a threshold has been assumed for cancer. "Harmonization" attempts to reconcile these contrasting assumptions under one paradigm. Recent regulatory initiatives suggest that the U.S. Environmental Protection Agency may be leaning toward a harmonized, probabilistic/linear approach for noncancer health effects. Proponents of this approach cite variability in human susceptibility as an argument against thresholds (i.e., some individuals may be exquisitely sensitive at exposures well below threshold levels). They also cite the results of epidemiological models that suggest low-dose linearity for noncancer health effects. We will discuss the implications of these arguments and compare them to what is known about human biological variability in general. We will also touch on the regulatory implications of hormesis within this framework.