Discovery and Characterization of a Bicyclic Peptide (Bicycle) Binder to Thymic Stromal Lymphopoietin.

Thymic stromal lymphopoietin (TSLP) is an epithelial-derived pro-inflammatory cytokine involved in the development of asthma and other atopic diseases. We used Bicycle Therapeutics' proprietary phage display platform to identify bicyclic peptides (Bicycles) with high affinity for TSLP, a target that is difficult to drug with conventional small molecules due to the extended protein-protein interactions it forms with both receptors. The hit series was shown to bind to TSLP in a hotspot, that is also used by IL-7Rα. Guided by the first X-ray crystal structure of a small peptide binding to TSLP and the identification of key metabolites, we were able to improve the proteolytic stability of this series in lung S9 fractions without sacrificing binding affinity. This resulted in the potent Bicycle 46 with nanomolar affinity to TSLP (KD = 13 nM), low plasma clearance of 6.4 mL/min/kg, and an effective half-life of 46 min after intravenous dosing to rats.

BT8009; A Nectin-4 Targeting Bicycle Toxin Conjugate for Treatment of Solid Tumors.

Multiple tumor types overexpress Nectin-4 and the antibody-drug conjugate (ADC), enfortumab vedotin (EV) shows striking efficacy in clinical trials for metastatic urothelial cancer, which expresses high levels of Nectin-4, validating Nectin-4 as a clinical target for toxin delivery in this indication. Despite excellent data in urothelial cancer, little efficacy data are reported for EV in other Nectin-4 expressing tumors and EV therapy can produce significant toxicities in many patients, frequently leading to discontinuation of treatment. Thus, additional approaches to this target with the potential to extend utility and reduce toxicity are warranted. We describe the preclinical development of BT8009, a "Bicycle Toxin Conjugate" (BTC) consisting of a Nectin-4-binding bicyclic peptide, a cleavable linker system and the cell penetrant toxin mono-methylauristatin E (MMAE). BT8009 shows significant antitumor activity in preclinical tumor models, across a variety of cancer indications and is well tolerated in preclinical safety studies. In several models, it shows superior or equivalent antitumor activity to an EV analog. As a small hydrophilic peptide-based drug BT8009 rapidly diffuses from the systemic circulation, through tissues to penetrate the tumor and target tumor cells. It is renally eliminated from the circulation, with a half-life of 1-2 hours in rat and non-human primate. These physical and PK characteristics differentiate BT8009 from ADCs and may provide benefit in terms of tumor penetration and reduced systemic exposure. BT8009 is currently in a Phase 1/2 multicenter clinical trial across the US, Canada, and Europe, enrolling patients with advanced solid tumors associated with Nectin-4 expression.

"We know what they're struggling with": student peer mentors' embodied perceptions of teaching in a health professional education mentorship program.

This paper reports on a study of student peer mentorship in the context of nursing education in a higher education program in Canada. The study used an embodied hermeneutic phenomenological methodology to investigate student peer mentors' perceptions of teaching during peer mentorship. The data were collected over one calendar year (2019) and involved analysis of 10 participants' interview data and their 'body maps,' produced in response to guided questions. Through the data analysis a core theme of 'commitment to mentee growth' was identified, along with seven interrelated themes: sharing responsibility for learning, moderating stress, mediating power relations, navigating unknown processes, valuing creative approaches, offering generous acceptance, and facilitating confidence. Student peer mentorship has the potential to contribute to health professions education in a number of unique ways including through embodied attunement, trusting intersubjective relations, and dialogic education. This study is innovative in its purposeful design and aim to investigate both cognitive and embodied perceptions of student peer mentors. The findings point to the promise of student peer mentorship for advancing health sciences education. Implications for peer mentorship program development in health professions education are discussed.

Anticancer immunity induced by a synthetic tumor-targeted CD137 agonist.

BACKGROUND: In contrast to immune checkpoint inhibitors, the use of antibodies as agonists of immune costimulatory receptors as cancer therapeutics has largely failed. We sought to address this problem using a new class of modular synthetic drugs, termed tumor-targeted immune cell agonists (TICAs), based on constrained bicyclic peptides (Bicycles). METHODS: Phage libraries displaying Bicycles were panned for binders against tumor necrosis factor (TNF) superfamily receptors CD137 and OX40, and tumor antigens EphA2, Nectin-4 and programmed death ligand 1. The CD137 and OX40 Bicycles were chemically conjugated to tumor antigen Bicycles with different linkers and stoichiometric ratios of binders to obtain a library of low molecular weight TICAs (MW <8 kDa). The TICAs were evaluated in a suite of in vitro and in vivo assays to characterize their pharmacology and mechanism of action. RESULTS: Linking Bicycles against costimulatory receptors (e.g., CD137) to Bicycles against tumor antigens (e.g., EphA2) created potent agonists that activated the receptors selectively in the presence of tumor cells expressing these antigens. An EphA2/CD137 TICA (BCY12491) efficiently costimulated human peripheral blood mononuclear cells in vitro in the presence of EphA2 expressing tumor cell lines as measured by the increased secretion of interferon γ and interleukin-2. Treatment of C57/Bl6 mice transgenic for the human CD137 extracellular domain (huCD137) bearing EphA2-expressing MC38 tumors with BCY12491 resulted in the infiltration of CD8+ T cells, elimination of tumors and generation of immunological memory. BCY12491 was cleared quickly from the circulation (plasma t1/2 in mice of 1-2 hr), yet intermittent dosing proved effective. CONCLUSION: Tumor target-dependent CD137 agonism using a novel chemical approach (TICAs) afforded elimination of tumors with only intermittent dosing suggesting potential for a wide therapeutic index in humans. This work unlocks a new path to effective cancer immunotherapy via agonism of TNF superfamily receptors.

An Assay for Periplasm Entry Advances the Development of Chimeric Peptide Antibiotics.

The treatment of infection by Gram-negative bacteria is increasingly challenging as resistance to existing antibiotics spreads. Constrained peptides, selected for high target specificity and affinity via library display technologies, are an emerging therapeutic modality in many disease areas and may be a fertile source of new antibiotics. Currently, the utility of constrained peptides and other large molecules as antibiotics is limited by the outer membrane (OM) barrier of Gram-negative bacteria. However, the addition of certain moieties to large molecules can confer the ability to cross the OM; these moieties function as intramolecular trans-OM "vectors". Here, we present a method to systematically assess the carrying capacity of candidate trans-OM vectors using a real-time luminescence assay ("SLALOM", Split Luciferase Assay for Live monitoring of Outer Membrane transit), reporting on periplasmic entry. We demonstrate the usefulness of our tools by constructing a 3800 Da chimeric compound composed of a constrained bicyclic peptide (Bicycle) with a periplasmic target, linked to an intramolecular peptide vector; the resulting chimera is a broad-spectrum inhibitor of pathogenic Gram-negative bacterial growth.

Prevalence of iron deficiency in 62,685 women of seven race/ethnicity groups: The HEIRS Study.

BACKGROUND: Few cross-sectional studies report iron deficiency (ID) prevalence in women of different race/ethnicity and ages in US or Canada. MATERIALS AND METHODS: We evaluated screening observations on women who participated between 2001-2003 in a cross-sectional, primary care-based sample of adults ages ≥25 y whose observations were complete: race/ethnicity; age; transferrin saturation; serum ferritin; and HFE p.C282Y and p.H63D alleles. We defined ID using a stringent criterion: combined transferrin saturation <10% and serum ferritin <33.7 pmol/L (<15 µg/L). We compared ID prevalence in women of different race/ethnicity subgrouped by age and determined associations of p.C282Y and p.H63D to ID overall, and to ID in women ages 25-44 y with or without self-reported pregnancy. RESULTS: These 62,685 women included 27,079 whites, 17,272 blacks, 8,566 Hispanics, 7,615 Asians, 449 Pacific Islanders, 441 Native Americans, and 1,263 participants of other race/ethnicity. Proportions of women with ID were higher in Hispanics and blacks than whites and Asians. Prevalence of ID was significantly greater in women ages 25-54 y of all race/ethnicity groups than women ages ≥55 y of corresponding race/ethnicity. In women ages ≥55 y, ID prevalence did not differ significantly across race/ethnicity. p.C282Y and p.H63D prevalence did not differ significantly in women with or without ID, regardless of race/ethnicity, age subgroup, or pregnancy. CONCLUSIONS: ID prevalence was greater in Hispanic and black than white and Asian women ages 25-54 y. p.C282Y and p.H63D prevalence did not differ significantly in women with or without ID, regardless of race/ethnicity, age subgroup, or pregnancy.

Blood-brain barrier dysfunction in aging induces hyperactivation of TGFß signaling and chronic yet reversible neural dysfunction.

Aging involves a decline in neural function that contributes to cognitive impairment and disease. However, the mechanisms underlying the transition from a young-and-healthy to aged-and-dysfunctional brain are not well understood. Here, we report breakdown of the vascular blood-brain barrier (BBB) in aging humans and rodents, which begins as early as middle age and progresses to the end of the life span. Gain-of-function and loss-of-function manipulations show that this BBB dysfunction triggers hyperactivation of transforming growth factor-ß (TGFß) signaling in astrocytes, which is necessary and sufficient to cause neural dysfunction and age-related pathology in rodents. Specifically, infusion of the serum protein albumin into the young rodent brain (mimicking BBB leakiness) induced astrocytic TGFß signaling and an aged brain phenotype including aberrant electrocorticographic activity, vulnerability to seizures, and cognitive impairment. Furthermore, conditional genetic knockdown of astrocytic TGFß receptors or pharmacological inhibition of TGFß signaling reversed these symptomatic outcomes in aged mice. Last, we found that this same signaling pathway is activated in aging human subjects with BBB dysfunction. Our study identifies dysfunction in the neurovascular unit as one of the earliest triggers of neurological aging and demonstrates that the aging brain may retain considerable latent capacity, which can be revitalized by therapeutic inhibition of TGFß signaling.

Locating the lived body in client-nurse interactions: Embodiment, intersubjectivity and intercorporeality.

The practice of nursing involves ongoing interactions between nurses' and clients' lived bodies. Despite this, several scholars have suggested that the "lived body" (Merleau-Ponty, 1962) has not been given its due place in nursing practice, education or research (Draper, J Adv Nurs, 70, 2014, 2235). With the advent of electronic health records and increased use of technology, face-to-face assessment and embodied understanding of clients' lived bodies may be on the decline. Furthermore, staffing levels may not afford the time nurses need to be as "present" with their clients in embodied ways. The failure to attend to the lived body may contribute to missed opportunities for care and decreased quality of life for both clients and healthcare practitioners. In this paper, we undertake an analysis of selected aspects of the work of Maurice Merleau-Ponty. The aim is to advance understanding of the affordances this work may offer to enhancing client-nurse interactions within the practice of nursing. Merleau-Ponty's notions of embodiment, intersubjectivity and intercorporeality as articulated in his seminal texts The Phenomenology of Perception (New York, NY: Routledge, 2012) and The Visible and the Invisible (Evanston, IL: Northwestern University Press, 1968) are examined. These three constructs are discussed as they relate to the lived body in client-nurse interactions in nursing practice and education. Finally, implications of how attention to "the lived body" could shape interactions and have the potential to foster increased quality of life of clients and nurses are considered.

Bicyclic Peptides as a New Modality for Imaging and Targeting of Proteins Overexpressed by Tumors.

Molecular imaging of cancers using probes specific for tumor-associated target proteins offers a powerful solution for providing information regarding selection of targeted therapy, patient stratification, and response to therapy. Here we demonstrate the power of bicyclic peptides as targeting probes, exemplified with the tumor-overexpressed matrix metalloproteinase MT1-MMP as a target. A bicyclic peptide with subnanomolar affinity towards MT1-MMP was identified, and its radioconjugate showed selective tumor uptake in an HT1080 xenograft mouse model. Proteolytic stabilization of the peptide by chemical modification significantly enhanced the in vivo tumor signal [from 2.5%ID/g to 12%ID/g at 1 hour post injection (p.i.)]. Studies using mouse xenograft models with different cell lines show a robust correlation between tumor signals and in vivo MT1-MMP expression levels. Fatty acid modification of the bicyclic peptide extended its circulating half-life, resulting in increased tumor signals (36%ID/g at 6 hours p.i.). Comparative work with an equipotent radiolabeled MT1-MMP targeting antibody demonstrated starkly differential biodistribution and tumor accumulation properties, with the tumor signal slowly increasing to 6.2%ID/g within 48 hours. The rapid tumor penetration characteristics of bicyclic peptides, coupled with high potency and chemical versatility, thus offer high-contrast imaging probes for clinical diagnostics with compelling additional potential in targeted therapy.Significance: This work demonstrates the potential of bicyclic peptides as a platform for the development of high-contrast imaging probes for potential use in clinical cancer diagnostics and molecularly targeted therapeutics.

Stable and Long-Lasting, Novel Bicyclic Peptide Plasma Kallikrein Inhibitors for the Treatment of Diabetic Macular Edema.

Plasma kallikrein, a member of the kallikrein-kinin system, catalyzes the release of the bioactive peptide bradykinin, which induces inflammation, vasodilation, vessel permeability, and pain. Preclinical evidence implicates the activity of plasma kallikrein in diabetic retinopathy, which is a leading cause of visual loss in patients suffering from diabetes mellitus. Employing a technology based on phage-display combined with chemical cyclization, we have identified highly selective bicyclic peptide inhibitors with nano- and picomolar potencies toward plasma kallikrein. Stability in biological matrices was either intrinsic to the peptide or engineered via the introduction of non-natural amino acids and nonpeptidic bonds. The peptides prevented bradykinin release in vitro, and in vivo efficacy was demonstrated in both a rat paw edema model and in rodent models of diabetes-induced retinal permeability. With a highly extended half-life of ∼40 h in rabbit eyes following intravitreal administration, the bicyclic peptides are promising novel agents for the treatment of diabetic retinopathy and diabetic macular edema.

The effect of an e-learning module on health sciences students' venipuncture skill development.

INTRODUCTION: Venipuncture is a psychomotor skill required in many healthcare professions. E-learning could be used to overcome current barriers in face-to-face learning in healthcare education such as insufficient classroom space or qualified instructors. We sought to evaluate the effectiveness of an e-learning module on students' performance when used in addition to in-class training. METHODS: Overall, 224 health sciences students were approached to participate in this pilot study. Recruited students were divided into control and study groups. The control group received only in-class training, whereas the study group had access to the e-learning module in addition to in-class learning. Both groups were evaluated on their self-confidence using a Likert scale, academic competence using a multiple-choice questionnaire, and psychomotor competence from video skill recordings using an in-house rubric. Nonparametric, independent sample Mann-Whitney tests were performed to evaluate differences between groups. RESULTS: Overall, 114 students provided written informed consent; 84 students (control: n = 50, study: n = 34) participated in at least one component of the study. Significantly higher (p = 0.017) academic competence scores were observed in the study group. Significantly higher confidence levels were also observed postintervention for both the control (p = 0.0025) and study (p = 0.0011) groups; however, no significant differences were found between the study and control groups before (p = 0.441) or after (p = 0.883) intervention. Finally, no significant differences (p = 0.428) were observed for psychomotor skills between the study arms. CONCLUSION: Our results suggest that there is potential for e-learning to increase the academic competence of students when used in conjunction with traditional learning; however, further research is needed to determine its efficacy on psychomotor skills.

Assessment of the Target Engagement and D-Serine Biomarker Profiles of the D-Amino Acid Oxidase Inhibitors Sodium Benzoate and PGM030756.

Irregular N-methyl-D-aspartate receptor (NMDAR) function is one of the main hypotheses employed to facilitate understanding of the underlying disease state of schizophrenia. Although direct agonism of the NMDAR has not yielded promising therapeutics, advances have been made by modulating the NMDAR co-agonist site which is activated by glycine and D-serine. One approach to activate the co-agonist site is to increase synaptic D-serine levels through inhibition of D-amino acid oxidase (DAO), the major catabolic clearance pathway for this and other D-amino acids. A number of DAO inhibitors have been developed but most have not entered clinical trials. One exception to this is sodium benzoate which has demonstrated efficacy in small trials of schizophrenia and Alzheimer's disease. Herein we provide data on the effect of sodium benzoate and an optimised Takeda compound, PGM030756 on ex vivo DAO enzyme occupancy and cerebellar D-serine levels in mice. Both compounds achieve high levels of enzyme occupancy; although lower doses of PGM030756 (1, 3 and 10 mg/kg) were required to achieve this compared to sodium benzoate (300, 1000 mg/kg). Cerebellar D-serine levels were increased by both agents with a delay of approximately 6 h after dosing before the peak effect was achieved. Our data and methods may be useful in understanding the effects of sodium benzoate that have been seen in clinical trials of schizophrenia and Alzheimer's disease and to support the potential clinical assessment of other DAO inhibitors, such as PGM030756, which demonstrate good enzyme occupancy and D-serine increases following administration of low oral doses.

Effect of ambiguous hemochromatosis gene test results on physician utilization.

BACKGROUND: Genetic test results may be available to greater numbers of people through genetic screening projects and other means. The effects of widespread genetic testing and notification of genetic test results, particularly added costs through increased physician utilization, have not been clearly established. METHODS: A primary care-based cohort of 20,306 participants (Hemochromatosis and Iron Overload Study, Ontario site) were tested for the C282Y and H63D mutations of the HFE gene and for abnormal serum ferritin (SF) and transferrin saturation levels. The primary outcome variable was the total number of physician claims per patient after genetic test notification by mail. Multiple Poisson regression was used to adjust for age, sex, baseline SF, diagnoses of arthritis, diabetes, heart failure and impotence, self-rated health, and the number of claims during the 12 months before notification of results. The reference group had no HFE mutations (wild type) and normal transferrin saturation/SF values. RESULTS: Participants with an ambiguous hemochromatosis gene test and normal iron levels had statistically significantly higher average physician utilization of 3.0%. Participants with HFE mutations (excluding C282Y homozygotes) and elevated iron values showed a 6% increase in physician utilization. CONCLUSIONS: The health effects, if any, of increased utilization in heterozygotes or those with mild ferritin elevations are unknown but are unlikely to be large at the population level. Ambiguous genetic test results are associated with increased physician service use and should be considered when assessing the complete societal costs of widespread genetic testing.

Potential nonresponse bias in a clinical examination after initial screening using iron phenotyping and HFE genotyping in the hemochromatosis and iron overload screening study.

BACKGROUND: Little is known about the factors affecting participation in clinical assessments after HEmochromatosis and IRon Overload Screening. METHODS: Initial screening of 101,168 primary care patients in the HEmochromatosis and IRon Overload Screening study was performed using serum iron measures and hemochromatosis gene (HFE) genotyping. Using iron phenotypes and HFE genotypes, we identified 2256 cases and 1232 controls eligible to participate in a clinical examination. To assess the potential for nonresponse bias, we compared the sociodemographic, health status, and attitudinal characteristics of participants and nonparticipants using adjusted odds ratios (ORs) and 95% confidence interval (CI). RESULTS: Overall participation was 74% in cases and 52% in controls; in both groups, participation was highest at a health maintenance organization and lowest among those under 45 years of age (cases: OR = 0.68; 95% CI 0.53, 0.87; controls: OR = 0.59; 95% CI 0.44, 0.78). In controls only, participation was also lower among those over 65 years of age than the reference group aged 46-64 (OR = 0.64; 95% CI 0.47, 0.88). Among cases, participation was higher in HFE C282Y homozygotes (OR = 3.98; 95% CI 2.60, 6.09), H63D homozygotes (OR = 2.79; 95% CI 1.23, 6.32), and C282Y/H63D compound heterozygotes (OR = 1.82; 95% CI 1.03, 3.22) than in other genotypes, and lower among non-Caucasians and those who preferred a non-English language than in Caucasians and those who preferred English (p < 0.0001). CONCLUSIONS: Subjects with greatest risk to have iron overload (C282Y homozygotes; cases > or =45 years; Caucasians) were more likely to participate in a postscreening clinical examination than other subjects. We detected no evidence of strong selection bias.

Screening for hemochromatosis and iron overload: satisfaction with results notification and understanding of mailed results in unaffected participants of the HEIRS study.

AIM: The purpose of this study was to assess the level of satisfaction and understanding of test results, by a sample of non-C282Y homozygous participants in the hemochromatosis and iron overload screening (HEIRS) study, who received serum ferritin (SF), transferrin saturation (TS), and HFE gene test results by mail. METHODS: Approximately 1 month after receiving test results by mail, participants were surveyed about understanding of and satisfaction with results notification. RESULTS: Overall, participants were satisfied with receiving test results by mail. Participants receiving results with one or two HFE mutations or TS and/or SF levels outside the normal range (an "alert value") were less likely to be satisfied with this method of notification. Participants with normal HFE test results understood their results and recommendations better than those with one or two mutations. Although all participants received results letters in their native language, English-speaking participants had higher mean understanding scores than Mandarin, Vietnamese, or Spanish-speaking participants. CONCLUSION: Participants were satisfied with receiving test results by mail. However, the level of understanding of the results was not sufficient for this mode of results notification to stand alone, especially for non-English speaking participants, and all participants with one or more test results outside the normal range.

The offer they can't refuse: parents and perinatal treatment decisions.

Despite a widespread myth of parental autonomy in decision-making for extremely preterm neonates, families in the United States are often not given access to accurate information about the consequences of preterm birth, resuscitation and treatment, or about their ethical options. Professional, philosophical, and financial incentives for hospitals and neonatologists to provide intensive treatment may trump parental wishes in delivery rooms and neonatal units. Parents may also be intimidated by the atmosphere of intensive care and by the behavior of committed staff. Prenatal advance directives allow parents to receive information on outcomes, treatments, and options, including palliative care, 'on their own turf' and as a part of routine prenatal counseling. The use of directives and other techniques for transparency in obstetric and neonatal care could improve the process of informed parental choice.

Health-related quality of life in a racially diverse population screened for hemochromatosis: results from the Hemochromatosis and Iron Overload Screening (HEIRS) study.

PURPOSE: The HEIRS Study screened 101,168 primary care participants for iron overload with serum transferrin saturation (TS), serum ferritin (SF), and C282Y and H63D mutations of the HFE gene. The objective of this study was to evaluate the impact of screening on participants' well-being. METHODS: All C282Y homozygotes, participants with an elevated TS and SF concentration, and a control group of phenotype-genotype negative persons, with neither C282Y nor H63D mutations in the HFE gene were recalled for a clinical evaluation. Health-related quality of life was assessed before screening and approximately 1 week after receipt of the results. Health worries were assessed only at follow-up. RESULTS: Participants (N = 1478) completed both initial and follow-up surveys. After adjusting for model covariates, phenotype and genotype combinations were statistically significant predictors of changes in psychological well-being (P = 0.0001) and general health (P = 0.0014). C282Y homozygotes with transient elevations in TS or SF were significantly more likely to worry about their health compared to study controls. Race, ethnicity, and preferred language subgroups differed on psychological well-being, general health, and health worry. CONCLUSION: Iron phenotype and HFE genotype are associated with health-related quality of life. Health worry was greatest among those considered genetically "at risk. " This may have important implications for multi-ethnic population-based screening studies in which genotype and phenotype are communicated.

Results communication and patient education after screening for possible hemochromatosis and iron overload: experience from the HEIRS Study of a large ethnically and linguistically diverse group.

PURPOSE: We assessed the effectiveness of educational interventions for conveying clinical findings and information about hereditary hemochromatosis (HH) and iron overload (IO) to individuals evaluated clinically after initial screening for HH/IO with serum ferritin (SF) concentration, transferrin saturation (TS), and HFE genotyping. METHODS: A questionnaire mailed to 2300 cases and controls 1 month after a letter summarizing clinical findings measured understanding of results and recommendations, knowledge of HH/IO, and satisfaction with information received. RESULTS: Of 1622 (70.5%) participants completing relevant items, 83.6% were satisfied with receiving initial screening results by mail, 93.4% found information clear and easy to understand, 89.2% generally felt they got enough information, but 47.5% still had questions. C282Y/C282Y homozygosity with normal TS/SF predicted the best understanding of genetic results. Many with no mutations thought relatives were at risk. Iron levels created most confusion, and a third incorrectly recalled treatment recommendations. Having any abnormal result, lower education, older age, and being non-white, and/or non-English speaking predicted lower understanding. CONCLUSIONS: Combining genotypic and phenotypic screening for HH/IO creates additional difficulties in communicating results-particularly to those with low health literacy. Explaining aberrant iron TS and SF levels and low-risk genotypes, follow-up recommendations, and risk to relatives will need creative, culturally appropriate strategies.