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.

Stability Comparison Between Microglassification and Lyophilization Using a Monoclonal Antibody.

Producing solid-state formulations of biologics remains a daunting task despite the prevalent use of lyophilization and spray drying technologies in the biopharmaceutical industry. The challenges include protein stability (temperature stresses), high capital costs, particle design/controllability, shortened processing times and manufacturing considerations (scalability, yield improvements, aseptic operation, etc.). Thus, scientists/engineers are constantly working to improve existing methodologies and exploring novel dehydration/powder-forming technologies. Microglassification™ is a dehydration technology that uses solvent extraction to rapidly dehydrate protein formulations at ambient temperatures, eliminating the temperature stress experienced by biologics in traditional lyophilization and spray drying methods. The process results in microparticles that are spherical, dense, and chemically stable. In this study, we compared the molecular stability of a monoclonal antibody formulation processed by lyophilization to the same formulation processed using Microglassification™. Both powders were placed on stability for 3 months at 40 °C and 6 months at 25 °C. Both dehydration methods showed similar chemical stability, including percent monomer, charge variants, and antigen binding. These results show that Microglassification™ is viable for the production of stable solid-state monoclonal antibody formulations.

Insights into ultra-low affinity lipase-antibody noncovalent complex binding mechanisms.

Detection of host cell protein (HCP) impurities is critical to ensuring that recombinant drug products, including monoclonal antibodies (mAbs), are safe. Mechanistic characterization as to how HCPs persist in drug products is important to refining downstream processing. It has been hypothesized that weak lipase-mAb interactions enable HCP lipases to evade drug purification processes. Here, we apply state-of-the-art methods to establish lipase-mAb binding mechanisms. First, the mass spectrometry (MS) approach of fast photochemical oxidation of proteins was used to elucidate putative binding regions. The CH1 domain was identified as a conserved interaction site for IgG1 and IgG4 mAbs against the HCPs phospholipase B-like protein (PLBL2) and lysosomal phospholipase A2 (LPLA2). Rationally designed mutations in the CH1 domain of the IgG4 mAb caused a 3- to 70-fold KD reduction against PLBL2 by surface plasmon resonance (SPR). LPLA2-IgG4 mutant complexes, undetected by SPR and studied using native MS collisional dissociation experiments, also showed significant complex disruption, from 16% to 100%. Native MS and ion mobility (IM) determined complex stoichiometries for four lipase-IgG4 complexes and directly interrogated the enrichment of specific lipase glycoforms. Confirmed with time-course and exoglycosidase experiments, deglycosylated lipases prevented binding, and low-molecular-weight glycoforms promoted binding, to mAbs. This work demonstrates the value of integrated biophysical approaches to characterize micromolar affinity complexes. It is the first in-depth structural report of lipase-mAb binding, finding roles for the CH1 domain and lipase glycosylation in mediating binding. The structural insights gained offer new approaches for the bioengineering of cells or mAbs to reduce HCP impurity levels.Abbreviations: CAN, Acetonitrile; AMAC, Ammonium acetate; BFGS, Broyden-Fletcher-Goldfarb-Shanno; CHO, Chinese Hamster Ovary; KD, Dissociation constant; DTT, Dithiothreitol; ELISA, Enzyme-linked immunosorbent assay; FPOP, Fast photochemical oxidation of proteins; FA, Formic acid; F(ab'), Fragment antibodies; HCP, Host cell protein; IgG, Immunoglobulin; IM, Ion mobility; LOD, Lower limit of detection; LPLA2, Lysosomal phospholipase A2; Man, Mannose; MS, Mass spectrometry; MeOH, Methanol; MST, Microscale thermophoresis; mAbs, Monoclonal antibodies; PPT1, Palmitoyl protein thioesterase; ppm, Parts per million; PLBL2, Phospholipase B-like protein; PLD3, Phospholipase D3; PS-20, Polysorbate-20; SP, Sphingomyelin phosphodiesterase; SPR, Surface plasmon resonance; TFA, Trifluoroacetic acid.

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.

Uterine bioimpedance combined with artificial intelligence as a means of cancer detection.

This study hypothesised that benign and tumour-bearing uterine tissue could be differentiated by their unique electrical bioimpedance patterns, with the aid of artificial intelligence. Twenty whole, ex-vivo uterine specimens were obtained at the time of hysterectomy. A total of 11 benign and 9 malignant specimens were studied. A uterine bioimpedance probe was designed to measure the tissue between the endometrial and serosal layers of the uterus. The impedance data was then analysed with multiple instance learning and principal component analysis, forms of artificial intelligence. Final pathology results for the specimens included uterine sarcoma, adenocarcinoma, carcinosarcoma, and high-grade serous carcinoma. The analysis correctly identified 78% (7/9) of the malignant specimens and 82% (9/11) of the benign specimens. The overall accuracy of our analysis was 80%. Our results demonstrate distinction between electrical impedance properties of malignant and benign uterine specimens. Bioimpedance and artificial intelligence may have potential implications in risk assessment of patients and may subsequently guide surgical decision-making regarding route of organ removal.

Antibody Format and Serum Disposition Govern Ocular Pharmacokinetics of Intravenously Administered Protein Therapeutics.

Protein therapeutics have witnessed tremendous use and application in recent years in treatment of various diseases. Predicting efficacy and safety during drug discovery and translational development is a key factor for successful clinical development of these therapies. In general, drug related toxicities are predominantly driven by pharmacokinetic (PK) exposure at off-target sites. This work explores the ocular PK of intravenously administered protein therapeutics to understand impact of antibody format on off-site exposure. Species matched non-binding rabbit antibody proteins (rabFab and rabIgG) were intravenously administered to male New Zealand White rabbits at a single 1 mg bolus dose and exposure was measured up to 3 weeks. As anticipated based on absence of FcRn recycling, rabFab has relatively fast systemic PK (CL-943 mL/day and t1/2-1.93 days) compared to rabIgG (CL-18.5 mL/day and t1/2-8.93 days). Similarly, rabFab has lower absolute ocular exposure in ocular compartments (e.g., vitreous and aqueous humor) compared to rabIgG, despite higher relative exposures (measured as percent tissue partition in ocular tissues relative to serum, based on Cmax and AUC). In general, percent tissue partition based on AUC (in aqueous and vitreous humor) relative to serum exposure were 10.4 and 8.62 for rabFab respectively and 1.11 and 0.64 for rabIgG respectively. This work emphasizes size and format based ocular exposure of intravenously administered protein therapeutics. Findings from this work enable prediction of format based ocular exposure for systemically administered antibody based therapeutics and aid in selection of molecule format for clinical candidate to minimize ocular exposure.

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.

An Approach to Bioactivity Assessment for Critical Quality Attribute Identification Based on Antibody-Antigen Complex Structure.

Identification of critical quality attributes (CQAs) is an important step for development of biopharmaceuticals with intended performance. An accurate CQA assessment is needed to ensure product quality and focusing on development efforts where control is needed. The assignment of criticality is based on safety and efficacy. Efficacy is related to PK and bioactivity. Here, we developed a novel approach based on antibody-antigen complex structure and modeling as a complementary method for bioactivity assessment. To validate this approach, common product related quality attributes and mutagenesis data from several IgGs were assessed using available antibody-antigen complex structures, and results were compared with experimental data from bioactivity or binding affinity measurements. A stepwise evaluation scheme for structural based analysis is proposed; based on systematic assessment following the scheme, good correlation has been observed between structural analysis and experimental data. This demonstrates that such an approach can be applied as a complementary tool for bioactivity assessment. Main applications are 1) To decouple multiple attributes to achieve amino acid resolution for bioactivity assessment, 2) To assess bioactivity of attributes that cannot be experimentally generated, 3) To provide molecular mechanism for experimental observation and understand structure function relationship. Examples are provided to illustrate these applications.

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.

Production and analysis of a mammalian septin hetero-octamer complex.

The septins are filament-forming proteins found in diverse eukaryotes from fungi to vertebrates, with roles in cytokinesis, shaping of membranes and modifying cytoskeletal organization. These GTPases assemble into rod-shaped soluble hetero-hexamers and hetero-octamers in mammals, which polymerize into filaments and higher order structures. While the cell biology and pathobiology of septins are advancing rapidly, mechanistic study of the mammalian septins is limited by a lack of recombinant hetero-octamer materials. We describe here the production and characterization of a recombinant mammalian septin hetero-octamer of defined stoichiometry, the SEPT2/SEPT6/SEPT7/SEPT3 complex. Using a fluorescent protein fusion to the complex, we observed filaments assembled from this complex. In addition, we used this novel tool to resolve recent questions regarding the organization of the soluble septin complex. Biochemical characterization of a SEPT3 truncation that disrupts SEPT3-SEPT3 interactions is consistent with SEPT3 occupying a central position in the complex while the SEPT2 subunits are at the ends of the rod-shaped octameric complexes. Consistent with SEPT2 being on the complex ends, we find that our purified SEPT2/SEPT6/SEPT7/SEPT3 hetero-octamer copolymerizes into mixed filaments with separately purified SEPT2/SEPT6/SEPT7 hetero-hexamer. We expect this new recombinant production approach to lay essential groundwork for future studies into mammalian septin mechanism and function.

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.

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.

Influence of Charge, Hydrophobicity, and Size on Vitreous Pharmacokinetics of Large Molecules.

PURPOSE: Development of therapeutics for retinal disease with improved durability is hampered by inadequate understanding of pharmacokinetic (PK) drivers following intravitreal injection. Previous work shows that hydrodynamic radius is correlated with vitreal half-life over the range of 3 to 7 nm, and that charge and hydrophobicity influence systemic clearance. Better understanding the molecular attributes affecting vitreal elimination half-life enables improved design of therapeutics and enhances clinical translatability. METHODS: Impacts of charge and hydrophobicity on vitreal PK in the rabbit were systematically assessed using antibody and antibody fragment (Fab) variant series, including ranibizumab, altered through amino acid changes in hypervariable regions of the light chain. The impact of molecule size on vitreal PK was assessed in the rabbit, nonhuman primate, and human for a range of molecules (1-45 nm, net charge -1324 to +22.9 in rabbit), including published and internal data. RESULTS: No correlation was observed between vitreal PK and charge or hydrophobicity. Equivalent rabbit vitreal PK was observed for ranibizumab and its variants with isoelectric points (pI) in the range of 6.8 to 10.2, and hydrophobicities of the variable domain unit (FvHI) between 1009 and 1296; additional variant series had vitreal PK similarly unaffected by pI (5.4-10.2) and FvHI (1004-1358). Strong correlations were observed between vitreal half-life and hydrodynamic radius for preclinical species (R 2 = 0.8794-0.9366). CONCLUSIONS: Diffusive properties of soluble large molecules, as quantified by hydrodynamic radius, make a key contribution to vitreal elimination, whereas differences in charge or hydrophobicity make minor or negligible contributions. TRANSLATIONAL RELEVANCE: These results support estimation of vitreal elimination rates based on molecular size in relevant preclinical species and humans.

Protein conjugates and fusion proteins as ocular therapeutics.

Long-acting delivery (LAD) of ocular therapeutics has potential to improve the standard of care for ocular diseases, such as age-related macular degeneration (AMD), by increasing patient compliance and reducing overall treatment burden on patients and healthcare providers. Although relatively few ocular LAD technologies are currently on the market, a variety of emergent and novel protein engineering-based technologies are being investigated in both the laboratory and clinical settings. Here, we review some of the key indications and treatments that would benefit from the development of LAD for the treatment of ocular diseases and examine the current state of LAD technologies that leverage protein-engineering approaches as well as nascent technologies with potential for future impact.

Identification and characterization of an octameric PEG-protein conjugate system for intravitreal long-acting delivery to the back of the eye.

Innovative protein engineering and chemical conjugation technologies have yielded an impressive number of drug candidates in clinical development including >80 antibody drug conjugates, >60 bispecific antibodies, >35 Fc-fusion proteins and >10 immuno-cytokines. Despite these innovations, technological advances are needed to address unmet medical needs with new pharmacological mechanisms. Age-related eye diseases are among the most common causes of blindness and poor vision in the world. Many such diseases affect the back of the eye, where the inaccessibility of the site of action necessitates therapeutic delivery via intravitreal (IVT) injection. Treatments administered via this route typically have vitreal half-lives <10 days in humans, requiring frequent administration. Since IVT injection is burdensome to patients, there exists a strong need to develop therapeutics with prolonged residence time in the eye. We report here a strategy to increase retention of a therapeutic fragment antibody (Fab) in the eye, using an anti-complement factor D Fab previously optimized for ocular delivery. Polyethylene glycol structures, varying in length, geometry and degree of branching, were coupled to the Fab via maleimide-activated termini. A screening strategy was developed to allow for key determinants of ocular half-life to be measured in vitro. After compound selection, a scalable process was established to enable tolerability and pharmacokinetic studies in cynomolgus monkeys, demonstrating an increase in vitreal half-life with no associated adverse events. Further, we show that the technique for compound selection, analytical characterization, and scalable production is general for a range of antibody fragments. The application of the technology has broad impact in across many therapeutic areas with the first major advancement in the treatment of an important ocular disease.

In Search of Mobile Applications for Urogynecology Providers.

INTRODUCTION: Thousands of medical applications (apps) are available for mobile devices. Finding accurate, health care provider-centered apps may be time consuming and frustrating for urogynecologists. The objective of this study was to identify and evaluate urogynecology (urogyn) apps using a modified APPLICATIONS scoring system. MATERIALS AND METHODS: Urogyn apps were identified from the Apple iTunes and Google Play Stores using the following 10 MeSH terms: urogynecology, incontinence, prolapse, urinary tract infection, pelvic surgery, fecal incontinence, defecation disorder, voiding disorder, urethral diverticulum, and fistula. Patient-centered and inaccurate apps were excluded. The remaining apps were evaluated with a modified APPLICATIONS scoring system, which included both objective and subjective criteria to determine each app's ability to aid in clinical decision making and to provide informational data. Objective rating components were price, paid subscription, literature referenced, in-app purchases, Internet connectivity, advertisements, text search field, interplatform compatibility and incorporated images, figures, videos, and special features. Subjective rating components were ease of navigation and presentation. RESULTS: Our search yielded 133 and 235 apps in the Apple iTunes and Google Play Stores, respectively. Only 8 apps (4 of which were in both stores) were determined to be accurate and useful; these were evaluated using the modified APPLICATIONS scoring system. The top-rated app was Practical Urology. CONCLUSION: Few accurate clinical decision-making and informational apps exist for urogynecologists. Apps varied by comprehensiveness and quality. This study highlights the importance of systematically reviewing and rating medical apps. It also emphasizes the need for developing accurate apps for urogynecologists that improve health care provider performance and patient outcomes.