Development of in silico models to predict viscosity and mouse clearance using a comprehensive analytical data set collected on 83 scaffold-consistent monoclonal antibodies.

Biologic drug discovery pipelines are designed to deliver protein therapeutics that have exquisite functional potency and selectivity while also manifesting biophysical characteristics suitable for manufacturing, storage, and convenient administration to patients. The ability to use computational methods to predict biophysical properties from protein sequence, potentially in combination with high throughput assays, could decrease timelines and increase the success rates for therapeutic developability engineering by eliminating lengthy and expensive cycles of recombinant protein production and testing. To support development of high-quality predictive models for antibody developability, we designed a sequence-diverse panel of 83 effector functionless IgG1 antibodies displaying a range of biophysical properties, produced and formulated each protein under standard platform conditions, and collected a comprehensive package of analytical data, including in vitro assays and in vivo mouse pharmacokinetics. We used this robust training data set to build machine learning classifier models that can predict complex protein behavior from these data and features derived from predicted and/or experimental structures. Our models predict with 87% accuracy whether viscosity at 150 mg/mL is above or below a threshold of 15 centipoise (cP) and with 75% accuracy whether the area under the plasma drug concentration-time curve (AUC0-672 h) in normal mouse is above or below a threshold of 3.9 × 106 h x ng/mL.

VERITAS: Harnessing the power of nomenclature in biologic discovery.

We are entering an era in which therapeutic proteins are assembled using building block-like strategies, with no standardized schema to discuss these formats. Existing nomenclatures, like AbML, sacrifice human readability for precision. Therefore, considering even a dozen such formats, in combination with hundreds of possible targets, can create confusion and increase the complexity of drug discovery. To address this challenge, we introduce Verified Taxonomy for Antibodies (VERITAS). This classification and nomenclature scheme is extensible to multispecific therapeutic formats and beyond. VERITAS names are easy to understand while drawing direct connections to the structure of a given format, with or without specific target information, making these names useful to adopt in scientific discourse and as inputs to machine learning algorithms for drug development.

Successful management of hypothermic cardiopulmonary bypass in a malignant hyperthermia susceptible patient.

Malignant hyperthermia (MH) is a potentially lethal reaction in those that are genetically predisposed, frequently triggered by inhaled anesthetics. MH is often difficult to diagnose because it is accompanied by signs and symptoms that are shared with other disorders. The diagnosis is further obscured in cardiac surgical patients, as the signs of MH can be masked by the cardiopulmonary bypass circuit (CPB) and the use of induced hypothermia. In this case-report, we describe the successful anesthetic management of a 65-year-old MH-susceptible female, confirmed via caffeine halothane contracture test, with aortic regurgitation and ascending aortic dilatation who underwent a Bentall procedure. We have also identified certain key measures for the safe anesthetic management of these patients.

Discovery, characterization, and remediation of a C-terminal Fc-extension in proteins expressed in CHO cells.

Protein-based biotherapeutics are produced in engineered cells through complex processes and may contain a wide variety of variants and post-translational modifications that must be monitored or controlled to ensure product quality. Recently, a low level (~1-5%) impurity was observed in a number of proteins derived from stably transfected Chinese hamster ovary (CHO) cells using mass spectrometry. These molecules include antibodies and Fc fusion proteins where Fc is on the C-terminus of the construct. By liquid chromatography-mass spectrometry (LC-MS), the impurity was found to be ~1177 Da larger than the expected mass. After tryptic digestion and analysis by LC-MS/MS, the impurity was localized to the C-terminus of Fc in the form of an Fc sequence extension. Targeted higher-energy collision dissociation was performed using various normalized collision energies (NCE) on two charge states of the extended peptide, resulting in nearly complete fragment ion coverage. The amino acid sequence, SLSLSPEAEAASASELFQ, obtained by the de novo sequencing effort matches a portion of the vector sequence used in the transfection of the CHO cells, specifically in the promoter region of the selection cassette downstream of the protein coding sequence. The modification was the result of an unexpected splicing event, caused by the resemblance of the commonly used GGU codon of the C-terminal glycine to a consensus splicing donor. Three alternative codons for glycine were tested to alleviate the modification, and all were found to completely eliminate the undesirable C-terminal extension, thus improving product quality.

Intermolecular interactions involving an acidic patch on immunoglobulin variable domain and the γ2 constant region mediate crystalline inclusion body formation in the endoplasmic reticulum.

Full-length immunoglobulins (Igs) are widely considered difficult to crystallize because of their large size, N-linked glycosylation, and flexible hinge region. However, numerous cases of intracellular Ig crystallization are reported in plasma cell dyscrasias. What makes some Ig clones more prone to crystallize during biosynthesis as well as the biochemical and cell biological requirements for this cryptic event are poorly understood. To investigate the underlying process of intracellular Ig crystallization we searched for model IgGs that can induce crystalline inclusions during recombinant overexpression. By testing various subunit combinations through mixing and matching of individual subunit chains derived from a panel of human IgG clones, we identified one secretion competent IgG2λ that induced needle-like crystalline inclusions in transfected HEK293 cells. Ig crystallization rarely occurred at steady-state cell growth conditions but was easily induced when ER-to-Golgi transport was pharmacologically blocked. Homology modeling revealed the presence of a prominent negatively-charged patch on the variable domain surface. The patch was composed of eight aspartic acids, of which five were in the heavy chain variable region and three were in the light chain. Crystallization occurred only when the two subunits were co-transfected and the intracellular crystals co-localized with ER resident proteins. Furthermore, subtype switching from IgG2 to IgG1 and stepwise neutralization of the acidic patch independently abrogated Ig crystallization events. The evidence supported that the formation of needle-like crystalline inclusions in the ER was underscored by multivalent intermolecular interactions between the acidic patch and undefined determinants present on the γ2 subunit constant region.

Impact of antibody subclass and disulfide isoform differences on the biological activity of CD200R and ßklotho agonist antibodies.

Agonism of cell surface receptors by monoclonal antibodies is dependent not only on its ability to bind the target, but also to deliver a biological signal through receptors to the cell. Immunoglobulin G2 antibodies (IgG2s) are made up of a mixture of distinct isoforms (IgG2-A, -B and A/B), which differ by the disulfide connectivity at the hinge region. When evaluating panels of agonistic antibodies against CD200 receptor (CD200R) or ßklotho receptor (ßklotho), we noticed striking activity differences of IgG1 or IgG2 antibodies with the same variable domains. For the CD200R antibody, the IgG2 antibody demonstrated higher activity than the IgG1 or IgG4 antibody. More significantly, for ßklotho, agonist antibodies with higher biological activity as either IgG2 or IgG1 were identified. In both cases, ion exchange chromatography was able to isolate the bioactivity to the IgG2-B isoform from the IgG2 parental mixture. The subclass-related increase in agonist activity was not correlated with antibody aggregation or binding affinity, but was driven by enhanced avidity for the CD200R antibody. These results add to the growing body of evidence that show that conformational differences in the antibody hinge region can have a dramatic impact on the antibody activity and must be considered when screening and engineering therapeutic antibody candidates. The results also demonstrate that the IgG1 (IgG2-A like) or the IgG2-B form may provide the most active form of agonist antibodies for different antibodies and targets.

Surgical site infection rates in dialysis patients undergoing endovascular procedures.

A surgical site infection (SSI) is an infection related to surgery that develops within 30 days after an operation or within 1 year of implant placement. Postoperative SSIs are the most common health-care-associated infections, occurring in up to 5% of surgical patients. Endovascular surgical procedures related to vascular access are common in the dialysis population and may cause SSIs. A large outpatient vascular access system developed and implemented a surveillance program to measure and monitor SSIs in their population. The health-care surveillance system extended to 76 ambulatory care centers across the United States and Puerto Rico. Based on a recorded 92,880 patient encounters, the surveillance system tabulated 12,541 valid patient survey responses documenting self-reported symptoms of infection within a 30-day postoperative period. The SSI rate was tabulated based on the presence of two or more specified indicators of infection: antibiotics, pus, dehiscence, pain, warmth, and swelling. Patients undergoing interventional procedures received surveys at discharge. Data were collected and analyzed using SPSS software. Survey analysis indicated a less than 3% superficial incisional SSI rate in hemodialysis patients undergoing endovascular procedures. The SSI rate for clean wound procedures is generally 2% or less. These data indicate that dialysis patients undergoing interventional procedures in vascular access centers may have a slightly greater risk of developing SSIs due to the presence of additional risk factors including obesity, diabetes, and age. This study was limited by a set of loose diagnostic criteria self-reported by patients, which may have overestimated the prevalence of infection. SSIs are a serious medical problem associated with increased morbidity and mortality and increased medical care costs. All providers should consider an active surveillance program following endovascular procedures given the comorbidities associated with the dialysis population.

Integrated experimental setup for angle resolved photoemission spectroscopy of transuranic materials.

We have developed the Angle Resolved Photoemission Spectroscopy (ARPES) system for transuranic materials. The ARPES transuranic system is an endstation upgrade to the Laser Plasma Light Source (LPLS) at Los Alamos National Laboratory. The LPLS is a tunable light source for photoemission with a photon energy range covering the vacuum ultraviolet (VUV) and soft x-ray regions (27-140 eV). The LPLS was designed and developed for transuranic materials. Transuranic photoemission is currently not permitted at the public synchrotrons worldwide in the VUV energy range due to sample encapsulation requirements. With the addition of the ARPES capability to the LPLS system there is an excellent opportunity to explore new details centered on the electronic structure of actinide and transuranic materials.

A review of postoperative feeding regimens in infantile hypertrophic pyloric stenosis.

Infantile hypertrophic pyloric stenosis is a condition well known to pediatric surgeons. Postoperative length of hospital stay is a financial concern and remains a potential target for reduction in hospital costs. Ultimately, these costs are directly affected by the ability to effectively advance postoperative enteral nutrition. This review will serve to: 1) identify clinically relevant postoperative feeding patterns following pyloromyotomy, 2) review the relevant literature to determine an optimal feeding pattern, and 3) identify possible preoperative predictors that may determine the success of postoperative feeding regiments.

Sclerostin is expressed in articular cartilage but loss or inhibition does not affect cartilage remodeling during aging or following mechanical injury.

OBJECTIVE: Sclerostin plays a major role in regulating skeletal bone mass, but its effects in articular cartilage are not known. The purpose of this study was to determine whether genetic loss or pharmacologic inhibition of sclerostin has an impact on knee joint articular cartilage. METHODS: Expression of sclerostin was determined in articular cartilage and bone tissue obtained from mice, rats, and human subjects, including patients with knee osteoarthritis (OA). Mice with genetic knockout (KO) of sclerostin and pharmacologic inhibition of sclerostin with a sclerostin-neutralizing monoclonal antibody (Scl-Ab) in aged male rats and ovariectomized (OVX) female rats were used to study the effects of sclerostin on pathologic processes in the knee joint. The rat medial meniscus tear (MMT) model of OA was used to investigate the pharmacologic efficacy of systemic Scl-Ab or intraarticular (IA) delivery of a sclerostin antibody-Fab (Scl-Fab) fragment. RESULTS: Sclerostin expression was detected in rodent and human articular chondrocytes. No difference was observed in the magnitude or distribution of sclerostin expression between normal and OA cartilage or bone. Sclerostin-KO mice showed no difference in histopathologic features of the knee joint compared to age-matched wild-type mice. Pharmacologic treatment of intact aged male rats or OVX female rats with Scl-Ab had no effect on morphologic characteristics of the articular cartilage. In the rat MMT model, pharmacologic treatment of animals with either systemic Scl-Ab or IA injection of Scl-Fab had no effect on lesion development or severity. CONCLUSION: Genetic absence of sclerostin does not alter the normal development of age-dependent OA in mice, and pharmacologic inhibition of sclerostin with Scl-Ab has no impact on articular cartilage remodeling in rats with posttraumatic OA.

Inhibition of WISE preserves renal allograft function.

Wnt-modulator in surface ectoderm (WISE) is a secreted modulator of Wnt signaling expressed in the adult kidney. Activation of Wnt signaling has been observed in renal transplants developing interstitial fibrosis and tubular atrophy; however, whether WISE contributes to chronic changes is not well understood. Here, we found moderate to high expression of WISE mRNA in a rat model of renal transplantation and in kidneys from normal rats. Treatment with a neutralizing antibody against WISE improved proteinuria and graft function, which correlated with higher levels of ß-catenin protein in kidney allografts. In addition, treatment with the anti-WISE antibody reduced infiltration of CD68(+) macrophages and CD8(+) T cells, attenuated glomerular and interstitial injury, and decreased biomarkers of renal injury. This treatment reduced expression of genes involved in immune responses and in fibrogenic pathways. In summary, WISE contributes to renal dysfunction by promoting tubular atrophy and interstitial fibrosis.

Underuse of cardiovascular preventive pharmacotherapy in patients presenting with ST-elevation myocardial infarction.

BACKGROUND: Multiple medications have proven efficacy for the primary prevention of coronary heart disease (CHD), but the appropriate patient population remains controversial. Even in the presence of multiple cardiovascular risk factors, many patients are not considered high risk and are not offered preventive medications despite proven efficacy. METHODS: We analyzed a prospective cohort of 1,710 consecutive ST-elevation myocardial infarction (STEMI) patients treated in a regional STEMI system from May 2007 to July 2010 and enrolled in a comprehensive database that includes preadmission medications. RESULTS: Of the 1,707 patients analyzed, 1,180 (69.1%) did not have known CHD before their event; and 482 (41.7%) of those patients had premature events (men <55 years old, women <65 years old). In patients without known CHD, cardiovascular risk factors were abundant (52.1% had hypertension, 43.6% had dyslipidemia, 41.4% had a family history of CHD, 58.5% were current or former smokers, and 14.9% were diabetic). Despite the high prevalence of risk factors, only 24.1% were on aspirin, 16.1% were on a statin, and only 7.8% were taking an aspirin and statin. Use of preventive medications was even less common in patients with premature events, including aspirin (15.2% vs 30.2%, P value < .001), statins (11.1% vs 19.5%, P value < .001), and the combination (5.6% vs 9.4%, P value < .001). CONCLUSIONS: Approximately 70% of a contemporary STEMI population did not have known CHD before their event, and >40% of those events would be considered premature. Despite the significant burden of cardiovascular risk factors, use of preventive therapy was alarmingly low in patients presenting with STEMI.

The Heart of New Ulm Project: using community-based cardiometabolic risk factor screenings in a rural population health improvement initiative.

Awareness of cardiovascular disease and diabetes risk factors can improve the health of individuals and populations. Community-based risk factor screening programs may be particularly useful for quantifying the burden of cardiometabolic risk in a given population, particularly in underserved areas. This study provided a description of a screening platform and how it has been used to monitor the cardiometabolic risk profile within the broader Heart of New Ulm Project, which is based in a rural Minnesota community. A cross-sectional, descriptive examination of baseline screening data indicated that 45% of the target population participated in the program over 8 months. Overall, 13% of the sample reported a personal history of diabetes or cardiovascular disease. Among the subset without active cardiometabolic disease, 35% were found to be at high risk for developing cardiovascular disease or type 2 diabetes over the next 8-10 years. A high prevalence of metabolic syndrome, high low-density lipoprotein cholesterol, obesity, and low fruit/vegetable consumption were of particular concern in this community. This article describes the use of screening results to inform the design of intervention programs that target these risk factors at both the community and individual levels. In addition, design considerations for future community-based cardiometabolic risk factor screening programs are discussed, with a focus on balancing program objectives related to health surveillance, research, and the delivery of preventive health care services.

Eliminating untimely deaths of women from heart disease: highlights from the Minnesota Women's Heart Summit.

Despite national campaigns to increase awareness and reduce cardiovascular disease (CVD) mortality in women, CVD remains their leading cause of death, annually killing more women than men. Although some progress has been made in our understanding and treatment of CVD in women, the causes, extent, and demographic trends of observed sex differences and disparities remain uncertain, and the growing burden of CVD and its risk factors among younger women is concerning. The Minnesota Women's Heart Summit was convened to chart a course to eliminate premature deaths of women from heart disease. The multidisciplinary summit was hosted by the Minneapolis Heart Institute, Minneapolis Heart Institute Foundation, University of Minnesota, and Mayo Clinic. Presentations highlighted sex-based differences in symptoms, treatment, and outcomes, and panel experts provided commentary. Invited faculty and summit participants worked in small-group sessions to identify strategies to dissolve barriers, improve primary and secondary prevention, and enhance women's care and outcomes. This report summarizes strategies identified during the conference to serve as springboards for more substantive future initiatives. These include, for example, standardized data collection and use of existing data sets to inform perspectives on sex-related cardiovascular issues, mandatory reporting of sex-specific data, and increased attention to underserved/high-risk women. Participants acknowledged that implementing these ideas would be challenging and recommended key priorities/next action steps such as providing services close to "point-of-life" rather than "point-of-care" and creation of policies and regulations so that resources and environmental modifications encouraging healthier lifestyle choices are promoted. Additional research is needed to improve identification, treatment, and health behaviors and to address continued lack of awareness, symptom recognition delays, barriers to care, and outcome disparities-especially in diverse populations.

Therapeutic hypothermia after out-of-hospital cardiac arrest: evaluation of a regional system to increase access to cooling.

BACKGROUND: Therapeutic hypothermia (TH) improves survival and confers neuroprotection in out-of-hospital cardiac arrest (OHCA), but TH is underutilized, and regional systems of care for OHCA that include TH are needed. METHODS AND RESULTS: The Cool It protocol has established TH as the standard of care for OHCA across a regional network of hospitals transferring patients to a central TH-capable hospital. Between February 2006 and August 2009, 140 OHCA patients who remained unresponsive after return of spontaneous circulation were cooled and rewarmed with the use of an automated, noninvasive cooling device. Three quarters of the patients (n=107) were transferred to the TH-capable hospital from referring network hospitals. Positive neurological outcome was defined as Cerebral Performance Category 1 or 2 at discharge. Patients with non-ventricular fibrillation arrest or cardiogenic shock were included, and patients with concurrent ST-segment elevation myocardial infarction (n=68) received cardiac intervention and cooling simultaneously. Overall survival to hospital discharge was 56%, and 92% of survivors were discharged with a positive neurological outcome. Survival was similar in transferred and nontransferred patients. Non-ventricular fibrillation arrest and presence of cardiogenic shock were associated strongly with mortality, but survivors with these event characteristics had high rates of positive neurological recovery (100% and 89%, respectively). A 20% increase in the risk of death (95% confidence interval, 4% to 39%) was observed for every hour of delay to initiation of cooling. CONCLUSIONS: A comprehensive TH protocol can be integrated into a regional ST-segment elevation myocardial infarction network and achieves broad dispersion of this essential therapy for OHCA.

Two doses of sclerostin antibody in cynomolgus monkeys increases bone formation, bone mineral density, and bone strength.

The development of bone-rebuilding anabolic agents for treating bone-related conditions has been a long-standing goal. Genetic studies in humans and mice have shown that the secreted protein sclerostin is a key negative regulator of bone formation. More recently, administration of sclerostin-neutralizing monoclonal antibodies in rodent studies has shown that pharmacologic inhibition of sclerostin results in increased bone formation, bone mass, and bone strength. To explore the effects of sclerostin inhibition in primates, we administered a humanized sclerostin-neutralizing monoclonal antibody (Scl-AbIV) to gonad-intact female cynomolgus monkeys. Two once-monthly subcutaneous injections of Scl-AbIV were administered at three dose levels (3, 10, and 30 mg/kg), with study termination at 2 months. Scl-AbIV treatment had clear anabolic effects, with marked dose-dependent increases in bone formation on trabecular, periosteal, endocortical, and intracortical surfaces. Bone densitometry showed that the increases in bone formation with Scl-AbIV treatment resulted in significant increases in bone mineral content (BMC) and/or bone mineral density (BMD) at several skeletal sites (ie, femoral neck, radial metaphysis, and tibial metaphysis). These increases, expressed as percent changes from baseline were 11 to 29 percentage points higher than those found in the vehicle-treated group. Additionally, significant increases in trabecular thickness and bone strength were found at the lumbar vertebrae in the highest-dose group. Taken together, the marked bone-building effects achieved in this short-term monkey study suggest that sclerostin inhibition represents a promising new therapeutic approach for medical conditions where increases in bone formation might be desirable, such as in fracture healing and osteoporosis.

Multipotent CD15+ cancer stem cells in patched-1-deficient mouse medulloblastoma.

Subpopulations of tumorigenic cells have been identified in many human tumors, although these cells may not be very rare in some types of cancer. Here, we report that medulloblastomas arising from Patched-1-deficient mice contain a subpopulation of cells that show a neural precursor phenotype, clonogenic and multilineage differentiation capacity, activated Hedgehog signaling, wild-type Patched-1 expression, and the ability to initiate tumors following allogeneic orthotopic transplantation. The normal neural stem cell surface antigen CD15 enriches for the in vitro proliferative and in vivo tumorigenic potential from uncultured medulloblastomas, supporting the existence of a cancer stem cell hierarchy in this clinically relevant mouse model of cancer.

Effect of anti-fibrinolytic therapy on experimental melanoma metastasis.

Anti-fibrinolytic agents such as aprotinin and epsilon-aminocaproic acid (EACA) are used clinically to decrease peri-operative bleeding. Use of these treatments during cancer-related surgeries has led to investigation of the effect of fibrinolysis inhibition on cancer cell spread. The ability of aprotinin to reduce proteolytic activity of proteases required for metastasis suggests that it could have an anti-metastatic effect in patients undergoing tumor resection. However, many metastatic cells in the vasculature of a secondary tissue are associated with a micro-thrombus. The association of tumor cells with thrombi has been shown to increase their survival; therefore inhibition of plasmin-mediated fibrinolysis might instead increase metastatic cell survival by enhancing the association between thrombi and tumor cells. The goal of this work was to determine the effect of anti-fibrinolytic treatment on experimental metastasis and to establish the role of coagulation factors in this effect. The metastatic ability of B16F10 melanoma cells was evaluated in vivo following cell or animal pre-treatment with aprotinin or EACA. Additionally, a novel in vivo technique was developed, to permit analysis of tumor cell association with thrombi in the lung microvasculature using confocal microscopy. Aprotinin and EACA treatment of mice resulted in a significant increase in lung metastasis. Aprotinin treatment increased the size of thrombi in association with cells arrested in lung capillaries. This study suggests that clinical use of anti-fibrinolytic agents for cancer-related surgeries could result in increased metastatic ability of those cells shed immediately prior to and during surgery, and that this approach thus requires further study.