Charge variants in IgG1: Isolation,characterization, in vitro binding properties and pharmacokinetics in rats

Antibody charge variants have gained considerable attention in the biotechnology industry due to their potential influence on stability and biological activity. Subtle differences in the relative proportions of charge variants are often observed during routine biomanufacture or process changes and pose a challenge to demonstrating product comparability. To gain further insights into the impact on biological activity and pharmacokinetics (PK) of monoclonal antibody (mAb) charge heterogeneity, we isolated the major charge forms of a recombinant humanized IgG1 and compared their in vitro properties and in vivo PK. The mAb starting material had a pI range of 8.7–9.1 and was composed of about 20% acidic variants, 12% basic variants and 68% main peak. Cation exchange displacement chromatography was used to isolate the acidic, basic and main peak fractions for animal studies. Detailed analyses were performed on the isolated fractions to identify specific chemical modification contributing to the charge differences and were also characterized for purity and in vitro potency prior to being administered either subcutaneously (SC) or intravenously (IV) in rats. All isolated materials had similar potency and rat FcRn binding relative to the starting material. Following IV or SC administration (10 mg/kg) in rats, no difference in serum PK was observed, indicating that physiochemical modifications and pI differences among charge variants were not sufficient to result in PK changes. Thus, these results provided meaningful information for the comparative evaluation of charge-related heterogeneity of mAbs and suggested that charge variants of IgGs do not affect the in vitro potency, FcRn binding affinity or the PK properties in rats.Key words: mAb IgG1, charge heterogeneity, isoelectric point, neonatal Fc receptor (FcRn), pharmacokinetics, potency     

Nodal Signaling Regulates the Bone Morphogenic Protein Pluripotency Pathway in Mouse Embryonic Stem Cells

Members of the transforming growth factor-β superfamily play essential roles in both the pluripotency and differentiation of embryonic stem (ES) cells. Although bone morphogenic proteins (BMPs) maintain pluripotency of undifferentiated mouse ES cells, the role of autocrine Nodal signaling is less clear. Pharmacological, molecular, and genetic methods were used to further understand the roles and potential interactions of these pathways. Treatment of undifferentiated ES cells with SB431542, a pharmacological inhibitor of Smad2 signaling, resulted in a rapid reduction of phosphorylated Smad2 and altered the expression of several putative downstream targets. Unexpectedly, inhibition of the Nodal signaling pathway resulted in enhanced BMP signaling, as assessed by Smad1/5 phosphorylation. SB431542-treated cells also demonstrated significant induction of the Id genes, which are known direct targets of BMP signaling and important factors in ES cell pluripotency. Inhibition of BMP signaling decreased the SB431542-mediated phosphorylation of Smad1/5 and induction of Id genes, suggesting that BMP signaling is necessary for some Smad2-mediated activity. Because Smad7, a known inhibitory factor to both Nodal and BMP signaling, was down-regulated following inhibition of Nodal-Smad2 signaling, the contribution of Smad7 to the cross-talk between the transforming growth factor-β pathways in ES cells was examined. Biochemical manipulation of Smad7 expression, through shRNA knockdown or inducible gene expression, significantly reduced the SB431542-mediated phosphorylation of Smad1/5 and induction of the Id genes. We conclude that autocrine Nodal signaling in undifferentiated mouse ES cells modulates the vital pluripotency pathway of BMP signaling.     

NKG2D initiates caspase-mediated CD3zeta degradation and lymphocyte receptor impairments associated with human cancer and autoimmune disease

Deficiencies of the T cell and NK cell CD3ζ signaling adapter protein in patients with cancer and autoimmune diseases are well documented, but mechanistic explanations are fragmentary. The stimulatory NKG2D receptor on T and NK cells mediates tumor immunity but can also promote local and systemic immune suppression in conditions of persistent NKG2D ligand induction that include cancer and certain autoimmune diseases. In this paper, we provide evidence that establishes a causative link between CD3ζ impairment and chronic NKG2D stimulation due to pathological ligand expression. We describe a mechanism whereby NKG2D signaling in human T and NK cells initiates Fas ligand/Fas-mediated caspase-3/-7 activation and resultant CD3ζ degradation. As a consequence, the functional capacities of the TCR, the low-affinity Fc receptor for IgG, and the NKp30 and NKp46 natural cytotoxicity receptors, which all signal through CD3ζ, are impaired. These findings are extended to ex vivo phenotypes of T and NK cells among tumor-infiltrating lymphocytes and in peripheral blood from patients with juvenile-onset lupus. Collectively, these results indicate that pathological NKG2D ligand expression leads to simultaneous impairment of multiple CD3ζ-dependent receptor functions, thus offering an explanation that may be applicable to CD3ζ deficiencies associated with diverse disease conditions.     

Reactive oxygen species,cellular redox systems,and apoptosis

Reactive oxygen species (ROS) are products of normal metabolism and xenobiotic exposure, and depending on their concentration, ROS can be beneficial or harmful to cells and tissues. At physiological low levels, ROS function as “redox messengers” in intracellular signaling and regulation, whereas excess ROS induce oxidative modification of cellular macromolecules, inhibit protein function, and promote cell death. Additionally, various redox systems, such as the glutathione, thioredoxin, and pyridine nucleotide redox couples, participate in cell signaling and modulation of cell function, including apoptotic cell death. Cell apoptosis is initiated by extracellular and intracellular signals via two main pathways, the death receptor- and the mitochondria-mediated pathways. Various pathologies can result from oxidative stress-induced apoptotic signaling that is consequent to ROS increases and/or antioxidant decreases, disruption of intracellular redox homeostasis, and irreversible oxidative modifications of lipid, protein, or DNA. In this review, we focus on several key aspects of ROS and redox mechanisms in apoptotic signaling and highlight the gaps in knowledge and potential avenues for further investigation. A full understanding of the redox control of apoptotic initiation and execution could underpin the development of therapeutic interventions targeted at oxidative stress-associated disorders.     

Hematopoietic progenitor kinase 1 is a critical component of prostaglandin E2-mediated suppression of the anti-tumor immune response

Lung cancer is the leading cause of cancer-related mortality in the world, resulting in over a million deaths each year. Non-small cell lung cancers (NSCLCs) are characterized by a poor immunogenic response, which may be the result of immunosuppressive factors such as prostaglandin E2 (PGE₂) present in the tumor environment. The effect of PGE₂ in the suppression of anti-tumor immunity and its promotion of tumor survival has been established for over three decades, but with limited mechanistic understanding. We have previously reported that PGE₂ activates hematopoietic progenitor kinase 1 (HPK1), a hematopoietic-specific kinase known to negatively regulate T-cell receptor signaling. Here, we report that mice genetically lacking HPK1 resist the growth of PGE₂-producing Lewis lung carcinoma (LLC). The presence of tumor-infiltrating lymphocytes (TILs) and T-cell transfer into T cell-deficient mice revealed that tumor rejection is T cell mediated. Further analysis demonstrated that this may be significantly due to the ability of HPK1 ^−/− T cells to withstand PGE₂-mediated suppression of T-cell proliferation, IL-2 production, and apoptosis. We conclude that PGE₂ utilizes HPK1 to suppress T cell-mediated anti-tumor responses.     

Leukocyte common antigen-related (LAR) tyrosine phosphatase positively regulates osteoblast differentiation by modulating extracellular signal-regulated kinase (ERK) activation

Protein tyrosine phosphatases (PTPs) are pivotal regulators of key cellular functions, including cell growth, differentiation, and adhesion. Previously, we reported that leukocyte common antigen-related (LAR) tyrosine phosphatase promotes osteoblast differentiation in MC3T3-E1 preosteoblast cells. In the present study, the mechanism of the regulatory action of LAR on osteoblast differentiation was investigated. The mineralization of extracellular matrix and calcium accumulation in MC3T3-E1 cells were markedly enhanced by LAR overexpression, and these effects were further increased by treatment with a MEK inhibitor. In addition, LAR overexpression dramatically reduced extracellular signal-regulated kinase (Erk) activation during osteoblast differentiation. In contrast, a marginal effect of the inactive LAR mutant on Erk activation was detected. Expression of osteoblast-related genes such as ALP, BSP, DLX5, OCN, and RUNX2, was increased by LAR overexpression during osteoblast differentiation. On the basis of these results, we propose that LAR functions as a positive regulator of osteoblast differentiation by modulating ERK activation. Therefore, LAR phosphatase could be used as a novel regulatory target protein in many bone-associated diseases, including osteoporosis.     

Cyclic amide bioisosterism: Strategic application to the design and synthesis of HCV NS5B polymerase inhibitors

Conformational modeling has been successfully applied to the design of cyclic bioisosteres used to replace a conformationally rigid amide bond in a series of thiophene carboxylate inhibitors of HCV NS5B polymerase. Select compounds were equipotent with the original amide series. Single-point mutant binding studies, in combination with inhibition structure–activity relationships, suggest this new series interacts at the Thumb-II domain of NS5B. Inhibitor binding at the Thumb-II site was ultimately confirmed by solving a crystal structure of 8b complexed with NS5B.     

The Catastrophic Decline of Tortoises at a Fenced Natural Area

Agassiz’s desert tortoise (Gopherus agassizii), a threatened species of the southwestern United States, has severely declined to the point where 76% of populations in critical habitat (Tortoise Conservation Areas) are below viability. The potential for rapid recovery of wild populations is low because females require 12–20 years to reach reproductive maturity and produce few eggs annually. We report on a 34-year mark-recapture study of tortoises initiated in 1979 at the Desert Tortoise Research Natural Area in the western Mojave Desert, California, USA, and provide substantive data on challenges faced by the species. In 1980, the United States Congress designated the Research Natural Area and protected the land from recreational vehicles, livestock grazing, and mining with a wildlife-permeable fence. The 7.77-km² study area, centered on interpretive facilities, included land both within the Natural Area and outside the fence. We expected greater benefits to accrue to the tortoises and habitat inside compared to outside. Our objectives were to conduct a demographic study, analyze and model changes in the tortoise population and habitat, and compare the effectiveness of fencing to protect populations and habitat inside the fence versus outside, where populations and habitat were unprotected. We conducted surveys in spring in each of 7 survey years from 1979, when the fence was under construction, through 2012. We compared populations inside to those outside the fence by survey year for changes in distribution, structure by size and relative age, sex ratios, death rates of adults, and causes of death for all sizes of tortoises. We used a Bayesian implementation of a Jolly Seber model for mark-recapture data. We modeled detection, density, growth and transition of tortoises to larger size-age classes, movements from inside the protective fence to outside and vice versa, and survival. After the second and subsequent survey years, we added surveys to monitor vegetation and habitat changes, conduct health assessments, and collect data on counts of predators and predator sign. At the beginning of the study, counts and densities for all sizes of tortoises were high, but densities were approximately 24% higher inside the fence than outside. By 2002, the low point in densities, densities had declined 90% inside the fence and 95% outside. Between 2002 and 2012, the population inside the fence showed signs of improving with a 54% increase in density. Outside the fence, densities remained low. At the end of the study, when we considered the initial differences in location, densities inside the fence were roughly 2.5 times higher than outside. The pattern of densities was similar for male and female adults. When evaluating survival by blocks of years, survivorship was higher in 1979–1989 than in 1989–2002 (the low point) and highest from 2002 to 2012. Recruitment and survival of adult females into the population was important for growing the population, but survival of all sizes, including juveniles, was also critical. Major events and activities driving the decline in populations both inside and outside the fence included illegal collecting, upper respiratory tract disease, and hyperpredation by the common raven (Corvus corax) on juvenile tortoises. Other sources of death were gunshots, vehicles, and predation by mammals. Outside the fence, fragmentation and deterioration of habitat was a critical driver. Between the first and last surveys, 2 different ecosystem processes were underway: recovery of vegetation and soils from grazing and vehicles inside the fence and continued deterioration outside the fence. Habitat outside the fence became increasingly denuded of shrubs and fragmented by roads and trails, and habitat fragments increased 50-fold. Outside the fence, biomass of non-native annual plants was higher and the cover of shrubs was lower, a reflection of ongoing deterioration. These changes and losses of habitat resulted in loss of shrub cover and sites for burrows, reduction in preferred food plants, and greater exposure to predators and extremes in temperature. Overall, the tortoise population and habitat inside the fence appeared to benefit from protection and showed signs of recovery at the end of the study. Fencing, control of vehicular access, and removal of livestock grazing were among several recommended management actions for critical habitat in the first recovery plan in 1994. At the end of the study, the Natural Area remained as 1 of 2 fenced, official protected areas for the species in the geographic range. We attribute fencing to continuing higher densities of adults inside the fence compared with outside the fence and promising signs of recovery. Densities of adults at the Natural Area also were 2.3 to 5.5 times higher than in 16 of the 17 Tortoise Conservation Areas (critical habitat units) within the geographic range. © 2020 The Authors. Wildlife Monographs published by Wiley Periodicals, LLC on behalf of The Wildlife Society.     

Habitat Features Predict Carrying Capacity of a Recovering Marine Carnivore

The recovery of large carnivore species from over-exploitation can have socioecological effects; thus, reliable estimates of potential abundance and distribution represent a valuable tool for developing management objectives and recovery criteria. For sea otters (Enhydra lutris), as with many apex predators, equilibrium abundance is not constant across space but rather varies as a function of local habitat quality and resource dynamics, thereby complicating the extrapolation of carrying capacity (K) from one location to another. To overcome this challenge, we developed a state-space model of density-dependent population dynamics in southern sea otters (E. l. nereis), in which K is estimated as a continuously varying function of a suite of physical, biotic, and oceanographic variables, all described at fine spatial scales. We used a theta-logistic process model that included environmental stochasticity and allowed for density-independent mortality associated with shark bites. We used Bayesian methods to fit the model to time series of survey data, augmented by auxiliary data on cause of death in stranded otters. Our model results showed that the expected density at K for a given area can be predicted based on local bathymetry (depth and distance from shore), benthic substrate composition (rocky vs. soft sediments), presence of kelp canopy, net primary productivity, and whether or not the area is inside an estuary. In addition to density-dependent reductions in growth, increased levels of shark-bite mortality over the last decade have also acted to limit population expansion. We used the functional relationships between habitat variables and equilibrium density to project estimated values of K for the entire historical range of southern sea otters in California, USA, accounting for spatial variation in habitat quality. Our results suggest that California could eventually support 17,226 otters (95% CrI = 9,739–30,087). We also used the fitted model to compute candidate values of optimal sustainable population abundance (OSP) for all of California and for regions within California. We employed a simulation-based approach to determine the abundance associated with the maximum net productivity level (MNPL) and propose that the upper quartile of the distribution of MNPL estimates (accounting for parameter uncertainty) represents an appropriate threshold value for OSP. Based on this analysis, we suggest a candidate value for OSP (for all of California) of 10,236, which represents 59.4% of projected K. © 2021 The Authors. The Journal of Wildlife Management published by Wiley Periodicals LLC on behalf of The Wildlife Society.     

Body Condition of Wintering Pacific Greater White-Fronted Geese

Extreme changes to key waterfowl habitats in the Klamath Basin (KB) on the Oregon–California border and the Sacramento Valley (SV) in California, USA, have occurred since 1980. The spatial distribution of Pacific greater white-fronted geese (Anser albifrons sponsa; geese) has likewise changed among these areas and population size has grown from 79,000 to >600,000 geese during the same period. To assess the effects of landscape changes and spatial-temporal distribution of geese, we collected Pacific greater white-fronted geese during winters of 2009–2010 and 2010–2011 in the KB and SV and compared their body condition to geese collected during 1979–1980 and 1980–1981. We modeled body and lipid mass to assess body condition for each sex independently and examined the influence of collection day, year, and region. Body condition of geese varied throughout the winter and within years in a nonlinear fashion. We detected an increase in body condition in both sexes during December and January in the SV, which corresponds with improved habitat conditions and increases seen in other species in the region. Body condition upon arrival in fall migration varied by year for females and by year and region for males. Males and females arrived in poorer body condition during 2010–2011 than all other study years and males in the KB during 2010–2011 had extremely low lipid mass, reflecting poor regional habitat conditions induced by drought. Body condition of females varied over spring, by year, and by region and regional effects were evident for males. Body condition was significantly higher for geese in the SV than in the KB during spring. Our results suggest that Pacific greater white-fronted geese have adapted to a changing landscape and have adjusted historical spatial use patterns to take advantage of more favorable conditions in the SV between 1979 and 2010. © 2021 The Wildlife Society.