Adapting Ferritin, a Naturally Occurring Protein Cage, to Modulate Intrinsic Agonism of OX40.

Ferritin is a multivalent, self-assembling protein scaffold found in most human cell types, in addition to being present in invertebrates, higher plants, fungi, and bacteria, that offers an attractive alternative to polymer-based drug delivery systems (DDS). In this study, the utility of the ferritin cage as a DDS was demonstrated within the context of T cell agonism for tumor killing. Members of the tumor necrosis factor receptor superfamily (TNFRSF) are attractive targets for the development of anticancer therapeutics. These receptors are endogenously activated by trimeric ligands that occur in transmembrane or soluble forms, and oligomerization and cell-surface anchoring have been shown to be essential aspects of the targeted agonism of this receptor class. Here, we demonstrated that the ferritin cage could be easily tailored for multivalent display of anti-OX40 antibody fragments on its surface and determined that these arrays are capable of pathway activation through cell-surface clustering. Together, these results confirm the utility, versatility, and developability of ferritin as a DDS.

Ubiquitin chain editing revealed by polyubiquitin linkage-specific antibodies.

Posttranslational modification of proteins with polyubiquitin occurs in diverse signaling pathways and is tightly regulated to ensure cellular homeostasis. Studies employing ubiquitin mutants suggest that the fate of polyubiquitinated proteins is determined by which lysine within ubiquitin is linked to the C terminus of an adjacent ubiquitin. We have developed linkage-specific antibodies that recognize polyubiquitin chains joined through lysine 63 (K63) or 48 (K48). A cocrystal structure of an anti-K63 linkage Fab bound to K63-linked diubiquitin provides insight into the molecular basis for specificity. We use these antibodies to demonstrate that RIP1, which is essential for tumor necrosis factor-induced NF-kappaB activation, and IRAK1, which participates in signaling by interleukin-1beta and Toll-like receptors, both undergo polyubiquitin editing in stimulated cells. Both kinase adaptors initially acquire K63-linked polyubiquitin, while at later times K48-linked polyubiquitin targets them for proteasomal degradation. Polyubiquitin editing may therefore be a general mechanism for attenuating innate immune signaling.

Generation of a Porcine Antibody Fab Fragment Using Protein Engineering to Facilitate the Evaluation of Ocular Sustained Delivery Technology.

Treatment of age-related macular degeneration (AMD) with anti-vascular endothelial growth factor (VEGF) biologic agents has been shown to restore and maintain visual acuity for many patients afflicted with wet AMD. These agents are usually administered via intravitreal injection at a dosing interval of 4-8 weeks. Employment of long-acting delivery (LAD) technologies could improve the therapeutic outcome, ensure timely treatment, and reduce burden on patients, caregivers, and the health care system. Development of LAD approaches requires thorough testing in pre-clinical species; however, therapeutic proteins of human origin may not be well tolerated during testing in non-human species due to immunogenicity. Here, we have engineered a surrogate porcine antibody Fab fragment (pigG6.31) from a human antibody for testing ocular LAD technologies in a porcine model. The engineered Fab retains the VEGF-A-binding and inhibition properties of the parental human Fab and has stability properties suitable for LAD evaluation. Upon intravitreal injection in minipigs, pigG6.31 showed first-order clearance from the ocular compartments with vitreal elimination rates consistent with other molecules of this size. Application of the surrogate molecule in an in vivo evaluation in minipigs of a prototype of the port delivery (PD) platform indicated continuous ocular delivery from the implant, with release kinetics consistent with both the results from in vitro release studies and the efficacy observed in human clinical studies of the PD system with ranibizumab (PDS). Anti-drug antibodies in the serum against pigG6.31 were not detected over exposure durations up to 16 weeks, suggesting that this molecule has low porcine immunogenicity.

Retinal and Lens Degeneration in New Zealand White Rabbits Administered Intravitreal TSG-6 Link Domain-Rabbit FAb Fusion Proteins.

Fusion of biologic therapeutics to hyaluronic acid binding proteins, such as the link domain (LD) of Tumor necrosis factor (TNF)-Stimulated Gene-6 (TSG-6), is expected to increase vitreous residence time following intravitreal injection and provide for long-acting delivery. The toxicity of a single intravitreal dose of free TSG-6-LD and fusion proteins of TSG-6-LD and a nonbinding rabbit antibody fragment (RabFab) were assessed in New Zealand White rabbits. Animals administered free TSG-6-LD exhibited extensive lens opacities and variable retinal vascular attenuation, correlated with microscopic findings of lens and retinal degeneration. Similar but less severe findings were present in animals dosed with the RabFab-TSG-6-LD fusion proteins. In-life ocular inflammation was noted in all animals from 7-days postdose and was associated with high anti-RabFab antibody titers in animals administered fusion proteins. Inflammation and retinal degeneration were multifocally associated with evidence of retinal detachment, and hypertrophy and migration of vimentin, glial fibrillary acidic protein, and glutamine synthetase positive Müller cells to the outer nuclear layer. Further assessment of alternative hyaluronic acid binding protein fusions should consider the potential for retinal degeneration and enhanced immune responses early in development.

Experience using multielectrode cardiac catheters for detection of electrophysiologic activity of the human urinary bladder.

AIM: To determine the feasibility of commercially available multielectrode cardiac electrophysiology catheters to detect electrical activity in the human bladder. METHODS: Ten subjects requiring cystoscopy for the evaluation of lower urinary tract pathology were eligible for participation in our study. After routine rigid cystoscopy with a 70° cystoscope, various multielectrode cardiac electrophysiology catheters were introduced into the bladder. One of three catheters with different electrode configurations was used per subject. Electroanatomical images of the bladder were created and spontaneous electrical activity was recorded. Subjective response to electrical stimuli delivered across the electrodes (20 mA at 5 ms pulse width, rate 100 ms) was also recorded. The responses were qualitatively compared with that from a prior study. RESULTS: Electrical activity recorded at the dome of the bladder was less than 0.5 mV and low frequency. Myopotentials resembling smooth muscle were detected at electrodes near or within the trigone. A sensory response was reported with the use of pacing stimuli, with the sensation in the trigone being reported more often than the dome of the bladder. Stimulation in the trigone triggered sensory urgency and voiding in a patient with a history of overactive bladder. CONCLUSIONS: The use of multielectrode catheters to measure human bladder electrophysiologic activity is feasible. Issues with noise reduction still exist, though to a lesser extent with the multielectrode basket design than simple quadripolar one. Sensory responses to pacing stimuli may be useful for diagnostic and therapeutic purposes in the future.

Machine Learning in Epidemiology and Health Outcomes Research.

Machine learning approaches to modeling of epidemiologic data are becoming increasingly more prevalent in the literature. These methods have the potential to improve our understanding of health and opportunities for intervention, far beyond our past capabilities. This article provides a walkthrough for creating supervised machine learning models with current examples from the literature. From identifying an appropriate sample and selecting features through training, testing, and assessing performance, the end-to-end approach to machine learning can be a daunting task. We take the reader through each step in the process and discuss novel concepts in the area of machine learning, including identifying treatment effects and explaining the output from machine learning models.

Tailoring translational strength using Kozak sequence variants improves bispecific antibody assembly and reduces product-related impurities in CHO cells.

Optimal production of bispecific antibodies (bsAb) requires efficient and tailored co-expression and assembly of two distinct heavy and two distinct light chains. Here, we describe a novel technology to modulate the translational strength of antibody chains via Kozak sequence variants to produce bsAb in a single cell line. In this study, we designed and screened a large Kozak sequence library to identify 10 independent variants that can modulate protein expression levels from approximately 0.2 to 1.3-fold compared with the wild-type sequence in transient transfection. We used a combination of several of these variants, covering a wide range of translational strength, to develop stable single cell Chinese hamster ovary bispecific cell lines and compared the results with those obtained from the wild-type sequence. A significant increase in bispecific antibody assembly with a concomitant reduction in the level of product-related impurities was observed. Our findings suggest that for production of bsAb it can be advantageous to modify translational strength for selected protein chains to improve overall yield and product quality. By extension, tuning of translational strength can also be applied to improving the production of a wide variety of heterologous proteins.

Use of Stratified Cascade Learning to predict hospitalization risk with only socioeconomic factors.

BACKGROUND AND OBJECTIVE: Published models predicting health related outcomes rely on clinical, claims and social determinants of health (SDH) data. Addressing the challenge of predicting with only SDH we developed a novel framework termed Stratified Cascade Learning (SCL) and used it for predicting the risk of hospitalization (ROH). MATERIALS AND METHODS: The variable set includes 27 SDH and "age" and "sex" for a cohort of diabetic patients. The SCL model uses three sub-models: SM1 (whole training set) stratifies training set into "predictable" and "unpredictable" subsets, SM2 (built on whole training set) classifies test set patients into "predictable" and "unpredictable", and SM3 (built on only the "predictable" subset) predicts the ROH for the patients classified as "predictable" by SM2. RESULTS: The SCL model does not improve either the AUC or the NPV of the basic classifier, but materially improves accuracy and specificity measures at the expense of lowering sensitivity for the "predictable" subset. Optimization of the risk thresholds of the sub-models does not noticeably change the AUC and NPV but further improves the accuracy and specificity at the expense of further lowering sensitivity. CONCLUSION: Since the SLC model yields low sensitivity it fails to predict high risk patients. But it yields high specificity that can be useful when the objective is to eliminate low-risk patients as candidates for further testing or treatment. The use of the SCL is not limited to healthcare, it can be applied to any predictive modeling problem when reliable predictions can only be made for a fraction of incoming data.

Trehalose Limits Fragment Antibody Aggregation and Influences Charge Variant Formation in Spray-Dried Formulations at Elevated Temperatures.

The preparation of PLGA rods for sustained release applications via a hot-melt extrusion process employs heat and mechanical shear. Understanding protein stability and degradation mechanisms at high temperature in the solid state is therefore important for the preparation of protein-loaded PLGA rods. The stability of a model protein, labeled Fab2, has been investigated in solid-state formulations containing trehalose at elevated temperatures. Spray-dried formulations containing varying levels of trehalose were exposed to temperatures ranging from 90 to 120 °C. Measurement of aggregation and chemical degradation rates suggests that trehalose limits Fab2 degradation in a concentration-dependent manner, but the effect tends to saturate when the mass ratio of trehalose to protein is around 1 in the solid formulation. The Fab2 secondary structure and spray-dried particle morphology were studied using circular dichroism and scanning electron microscopy techniques, respectively. On the basis of temperature and trehalose-dependent aggregation kinetics as well as changes in spray-dried particle morphology, a mechanism is proposed for the trehalose stabilization of proteins in solid state at elevated temperatures. The results reported here suggest that when fragment antibodies in the solid state are formulated with trehalose as excipient, a high temperature process such as hot-melt extrusion can be successfully accomplished with minimal degradation.

In Vivo Stability Profiles of Anti-factor D Molecules Support Long-Acting Delivery Approaches.

The collection of aqueous humor (phase 1 b/2 Mahalo study) from patients dosed intravitreally with anti-factor D (AFD; FCFD4514S, lampalizumab), a humanized antibody fragment previously under investigation to treat geographic atrophy (GA) secondary to age-related macular degeneration, presented a unique opportunity to examine AFD properties in clinical samples. We investigated AFD stability and target-binding characteristics to set up strategies for engineering and evaluating optimized molecules that enable less frequent dosing. Two variants, AFD.v8 and AFD.v14, were evaluated as alternatives to AFD for longer-acting treatments. Mass spectrometry, surface plasmon resonance, and immunoassay were used to assess AFD stability and binding activity in aqueous humor samples from Mahalo patients. In vitro stability and binding activity of AFD, AFD.v8, and AFD.v14 were assessed in human vitreous humor versus buffer at 37 °C over 16 weeks and in vivo in rabbits over 28 days along with pharmacokinetic determinations. In human aqueous humor, AFD specific binding was >85% through 30 days, and deamidation was <3% through 60 days, consistent with the AFD stability and binding activity in vitreous humor from humans in vitro and rabbits in vivo. Target binding, stability, and rabbit pharmacokinetic parameters of AFD.v8 and AFD.v14 were similar to those of AFD. Physiological stability and activity of AFD translated across in vitro and in vivo studies in humans and rabbits. The two variants AFD.v8 and AFD.v14 demonstrated comparable potency and pharmacokinetics. These findings, along with previously demonstrated improved solubility of AFD.v8 and AFD.v14, provide proof-of-concept for developing other similar long-acting therapeutic variants.

Transdermal light neuromodulation: Optogenetics in the murine urinary tract.

BACKGROUND: Optogenetics is a biologic technique that uses light to control living neurons, which have been genetically modified to express light-sensitive ion-channels. OBJECTIVES: Using an adenovirus to modify the sciatic nerves of mice, we aim to demonstrate peripheral neuromodulation of bladder pain using transdermal light. STUDY DESIGN: This pilot study is divided into: A) Confirmation and Application and B) Behavioral Step. A) Six mice were injected with AAV6-hSyn-ChR2(H134R)-eYFP virus into their sciatic nerves. This encoded an excitatory opsin, enabling light-inducible stimulation. At 4-6 weeks after injection, we induced foot pain responses with an activating blue 475 nm wavelength of light. B) Two optogenetically primed mice and two control mice underwent anesthesia and capsaicin was instilled into their bladders via catheter. The catheters were removed and the mice awoke in a chamber that exposed them to either blue 475 nm light or no light. Groin licking was scored in a binary fashion by two blinded observers. RESULTS: A) All six mice exhibited pain response to 475 nm blue light either by licking of foot or avoidance of light. B) The optogenetically primed mice had a 48% reduction in bladder pain behavior when exposed to blue 475 nm light whereas the control mice had a 18% reduction. CONCLUSION: To our knowledge this is the first demonstration of the application of optogenetics to modulate sensation in the lower urinary tract. It suggests that the process of priming peripheral nerves for optogenetic modulation is possible and can be used to study bladder pain response in mice.

K11-linked polyubiquitination in cell cycle control revealed by a K11 linkage-specific antibody.

Polyubiquitination is a posttranslational modification where ubiquitin chains containing isopeptide bonds linking one of seven ubiquitin lysines with the C terminus of an adjoining ubiquitin are covalently attached to proteins. While functions of K48- and K63-linked polyubiquitin are understood, the role(s) of noncanonical K11-linked chains is less clear. A crystal structure of K11-linked diubiquitin demonstrates a distinct conformation from K48- or K63-linked diubiquitin. We engineered a K11 linkage-specific antibody and use it to demonstrate that K11 chains are highly upregulated in mitotic human cells precisely when substrates of the ubiquitin ligase anaphase-promoting complex (APC/C) are degraded. These chains increased with proteasomal inhibition, suggesting they act as degradation signals in vivo. Inhibition of the APC/C strongly impeded the formation of K11-linked chains, suggesting that a single ubiquitin ligase is the major source of mitotic K11-linked chains. Our results underscore the importance of K11-linked ubiquitin chains as critical regulators of mitotic protein degradation.

Enhancing the antitumor efficacy of a cell-surface death ligand by covalent membrane display.

TNF superfamily death ligands are expressed on the surface of immune cells and can trigger apoptosis in susceptible cancer cells by engaging cognate death receptors. A recombinant soluble protein comprising the ectodomain of Apo2 ligand/TNF-related apoptosis-inducing ligand (Apo2L/TRAIL) has shown remarkable preclinical anticancer activity but lacked broad efficacy in patients, possibly owing to insufficient exposure or potency. We observed that antibody cross-linking substantially enhanced cytotoxicity of soluble Apo2L/TRAIL against diverse cancer cell lines. Presentation of the ligand on glass-supported lipid bilayers enhanced its ability to drive receptor microclustering and apoptotic signaling. Furthermore, covalent surface attachment of Apo2L/TRAIL onto liposomes--synthetic lipid-bilayer nanospheres--similarly augmented activity. In vivo, liposome-displayed Apo2L/TRAIL achieved markedly better exposure and antitumor activity. Thus, covalent synthetic-membrane attachment of a cell-surface ligand enhances efficacy, increasing therapeutic potential. These findings have translational implications for liposomal approaches as well as for Apo2L/TRAIL and other clinically relevant TNF ligands.

Adults Hospitalized With Pneumonia in the United States: Incidence, Epidemiology, and Mortality.

BACKGROUND: Understanding the burden of community-acquired pneumonia (CAP) is critical to allocate resources for prevention, management, and research. The objectives of this study were to define incidence, epidemiology, and mortality of adult patients hospitalized with CAP in the city of Louisville, and to estimate burden of CAP in the US adult population. METHODS: This was a prospective population-based cohort study of adult residents in Louisville, Kentucky, from 1 June 2014 to 31 May 2016. Consecutive hospitalized patients with CAP were enrolled at all adult hospitals in Louisville. The annual population-based CAP incidence was calculated. Geospatial epidemiology was used to define ecological associations among CAP and income level, race, and age. Mortality was evaluated during hospitalization and at 30 days, 6 months, and 1 year after hospitalization. RESULTS: During the 2-year study, from a Louisville population of 587499 adults, 186384 hospitalizations occurred. A total of 7449 unique patients hospitalized with CAP were documented. The annual age-adjusted incidence was 649 patients hospitalized with CAP per 100000 adults (95% confidence interval, 628.2-669.8), corresponding to 1591825 annual adult CAP hospitalizations in the United States. Clusters of CAP cases were found in areas with low-income and black/African American populations. Mortality during hospitalization was 6.5%, corresponding to 102821 annual deaths in the United States. Mortality at 30 days, 6 months, and 1 year was 13.0%, 23.4%, and 30.6%, respectively. CONCLUSIONS: The estimated US burden of CAP is substantial, with >1.5 million unique adults being hospitalized annually, 100000 deaths occurring during hospitalization, and approximately 1 of 3 patients hospitalized with CAP dying within 1 year.

A pilot study of cardiac electrophysiology catheters to map and pace bladder electrical activity.

AIMS: This is a pilot study to evaluate the feasibility of using diagnostic cardiac electrophysiology catheters for recording intrinsic urinary bladder electrical activity and for electrical pacing capture of bladder tissue. METHODS: During cystoscopy, a curved quadripolar catheter was introduced and contact was made with the right and left halves of the dome and trigone in adult female patients undergoing cystoscopy. Electrical activity was recorded, using a commercially available cardiac electrophysiologic recording system, before and during pacing at 0.5-3.0 Hz. RESULTS: Apparent spontaneous electrical depolarizations were detected in both the trigone and the dome. The amplitude of these depolarizations was in the microVolt range. During pacing, local electrical capture was noted in the trigone, but not in the dome. CONCLUSIONS: Spontaneous low-amplitude electrical activity was detected in the bladder through the use of commercially available cardiac electrophysiology equipment. While these low-level signals could represent noise, the voltage, and morphology resemble detrusor muscle action potentials previously seen in animal studies. Pacing induced local electrical capture in the trigone but not the dome.

In silico selection of therapeutic antibodies for development: viscosity, clearance, and chemical stability.

For mAbs to be viable therapeutics, they must be formulated to have low viscosity, be chemically stable, and have normal in vivo clearance rates. We explored these properties by observing correlations of up to 60 different antibodies of the IgG1 isotype. Unexpectedly, we observe significant correlations with simple physical properties obtainable from antibody sequences and by molecular dynamics simulations of individual antibody molecules. mAbs viscosities increase strongly with hydrophobicity and charge dipole distribution and decrease with net charge. Fast clearance correlates with high hydrophobicities of certain complementarity determining regions and with high positive or high negative net charge. Chemical degradation from tryptophan oxidation correlates with the average solvent exposure time of tryptophan residues. Aspartic acid isomerization rates can be predicted from solvent exposure and flexibility as determined by molecular dynamics simulations. These studies should aid in more rapid screening and selection of mAb candidates during early discovery.

Evaluating the Use of Antibody Variable Region (Fv) Charge as a Risk Assessment Tool for Predicting Typical Cynomolgus Monkey Pharmacokinetics.

The pharmacokinetic (PK) behavior of monoclonal antibodies in cynomolgus monkeys (cynos) is generally translatable to that in humans. Unfortunately, about 39% of the antibodies evaluated for PKs in cynos have fast nonspecific (or non-target-mediated) clearance (in-house data). An empirical model relating variable region (Fv) charge and hydrophobicity to cyno nonspecific clearance was developed to gauge the risk an antibody would have for fast nonspecific clearance in the monkey. The purpose of this study was to evaluate the predictability of this empirical model on cyno nonspecific clearance with antibodies specifically engineered to have either high or low Fv charge. These amino acid changes were made in the Fv region of two test antibodies, humAb4D5-8 and anti-lymphotoxin α. The humAb4D5-8 has a typical nonspecific clearance in cynos, and by making it more positively charged, the antibody acquires fast nonspecific clearance, and making it less positively charged did not impact its clearance. Anti-lymphotoxin α has fast nonspecific clearance in cynos, and making it more positively charged caused it to clear even faster, whereas making it less positively charged caused it to clear slower and within the typical range. These trends in clearance were also observed in two other preclinical species, mice and rats. The effect of modifying Fv charge on subcutaneous bioavailability was also examined, and in general bioavailability was inversely related to the direction of the Fv charge change. Thus, modifying Fv charge appears to impact antibody PKs, and the changes tended to correlate with those predicted by the empirical model.

Contribution of Antibody Hydrodynamic Size to Vitreal Clearance Revealed through Rabbit Studies Using a Species-Matched Fab.

We have developed a tool Fab fragment of a rabbit monoclonal antibody that is useful for early evaluation in rabbit models of technologies for long acting delivery (LAD) of proteins to the eye. Using this Fab we show that vitreal clearance can be slowed through increased hydrodynamic size. Fab (G10rabFab) and Fab' (G10rabFab') fragments of a rabbit monoclonal antibody (G10rabIgG) were expressed in Chinese hamster ovary (CHO) cells and purified using antigen-based affinity chromatography. G10rabFab retains antigen-binding upon thermal stress (37 °C) for 8 weeks in phosphate-buffered saline (PBS) and can be detected in rabbit tissues using an antigen-based ELISA. Hydrodynamic radius, measured using quasi-elastic light scattering (QELS), was increased through site-specific modification of the G10rabFab' free cysteine with linear methoxy-polyethylene glycol(PEG)-maleimide of 20000 or 40000 molecular weight. Pharmacokinetic studies upon intravitreal dosing in New Zealand white rabbits were conducted on the G10rabFab and PEGylated G10rabFab'. Results of single and multidose pharmacokinetic experiments yield reproducible results and a vitreal half-life for G10rabFab of 3.2 days. Clearance from the eye is slowed through increased hydrodynamic size, with vitreal half-life showing a linear dependence on hydrodynamic radius (RH). A linear dependence of vitreal half-life on RH suggests that molecule diffusivity makes an important contribution to vitreal clearance. A method for prediction of vitreal half-life from RH measurements is proposed.

Macrolide Use and Neutrophil Function/Cytokine Levels in Hospitalized Patients with Community-Acquired Pneumonia: A Pilot Study.

PURPOSE: The objective of this study was to measure plasma cytokine levels and blood neutrophil functions as well as clinical outcomes in hospitalized patients with community-acquired pneumonia (CAP) treated with or without macrolide use--a known modulator of inflammatory response. METHODS: Subjects with CAP had peripheral blood analyzed for some neutrophil functions (degranulation of secretory vesicles and specific granules, respiratory burst response and phagocytosis) and ten cytokine levels measured in serum and sputum supernatants. Neutrophil function in healthy volunteers was also measured for reference. Values were measured on the day of enrollment, days 2-4 and 5-7, depending on a patient's length of stay. Early and late clinical outcomes were also evaluated. All values were compared between those treated with or without a macrolide. RESULTS: A total of 40 subjects were in this study; 14 received macrolide treatment, and 26 did not. Neutrophil function in the macrolide group was not significantly different compared to the non-macrolide group. None of the median cytokine levels or IQRs were statistically significant between the groups. However, a trend toward decreased IL-6, IL-8, and IFN-γ levels, and favorable clinical outcomes were present in the macrolide group. CONCLUSIONS: This pilot study showed no statistical difference between cytokine levels or neutrophil activity for CAP patients prescribed a macrolide containing regimen. Considering the trend of lower cytokine levels in the macrolide group when comparing the 5- to 7-day time period with the non-macrolide group, a full study with an appropriate sample size may be warranted.