The smallest functional antibody fragment: Ultralong CDR H3 antibody knob regions potently neutralize SARS-CoV-2.

Cows produce antibodies with a disulfide-bonded antigen-binding domain embedded within ultralong heavy chain third complementarity determining regions. This "knob" domain is analogous to natural cysteine-rich peptides such as knottins in that it is small and stable but can accommodate diverse loops and disulfide bonding patterns. We immunized cattle with SARS-CoV-2 spike and found ultralong CDR H3 antibodies that could neutralize several viral variants at picomolar IC50 potencies in vitro and could protect from disease in vivo. The independent CDR H3 peptide knobs were expressed and maintained the properties of the parent antibodies. The knob interaction with SARS-CoV-2 spike was revealed by electron microscopy, X-ray crystallography, NMR spectroscopy, and mass spectrometry and established ultralong CDR H3-derived knobs as the smallest known recombinant independent antigen-binding fragment. Unlike other vertebrate antibody fragments, these knobs are not reliant on the immunoglobulin domain and have potential as a new class of therapeutics.

Diversity of ACE2 and its interaction with SARS-CoV-2 receptor binding domain.

COVID-19, the clinical syndrome caused by the SARS-CoV-2 virus, has rapidly spread globally causing hundreds of millions of infections and over two million deaths. The potential animal reservoirs for SARS-CoV-2 are currently unknown, however sequence analysis has provided plausible potential candidate species. SARS-CoV-2 binds to the angiotensin I converting enzyme 2 (ACE2) to enable its entry into host cells and establish infection. We analyzed the binding surface of ACE2 from several important animal species to begin to understand the parameters for the ACE2 recognition by the SARS-CoV-2 spike protein receptor binding domain (RBD). We employed Shannon entropy analysis to determine the variability of ACE2 across its sequence and particularly in its RBD interacting region, and assessed differences between various species' ACE2 and human ACE2. Recombinant ACE2 from human, hamster, horseshoe bat, cat, ferret, and cow were evaluated for RBD binding. A gradient of binding affinities were seen where human and hamster ACE2 were similarly in the low nanomolar range, followed by cat and cow. Surprisingly, horseshoe bat (Rhinolophus sinicus) and ferret (Mustela putorius) ACE2s had poor binding activity compared with the other species' ACE2. The residue differences and binding properties between the species' variants provide a framework for understanding ACE2-RBD binding and virus tropism.

GRANADA consensus on analytical approaches to assess associations with accelerometer-determined physical behaviours (physical activity, sedentary behaviour and sleep) in epidemiological studies.

The inter-relationship between physical activity, sedentary behaviour and sleep (collectively defined as physical behaviours) is of interest to researchers from different fields. Each of these physical behaviours has been investigated in epidemiological studies, yet their codependency and interactions need to be further explored and accounted for in data analysis. Modern accelerometers capture continuous movement through the day, which presents the challenge of how to best use the richness of these data. In recent years, analytical approaches first applied in other scientific fields have been applied to physical behaviour epidemiology (eg, isotemporal substitution models, compositional data analysis, multivariate pattern analysis, functional data analysis and machine learning). A comprehensive description, discussion, and consensus on the strengths and limitations of these analytical approaches will help researchers decide which approach to use in different situations. In this context, a scientific workshop and meeting were held in Granada to discuss: (1) analytical approaches currently used in the scientific literature on physical behaviour, highlighting strengths and limitations, providing practical recommendations on their use and including a decision tree for assisting researchers' decision-making; and (2) current gaps and future research directions around the analysis and use of accelerometer data. Advances in analytical approaches to accelerometer-determined physical behaviours in epidemiological studies are expected to influence the interpretation of current and future evidence, and ultimately impact on future physical behaviour guidelines.

Association of daily composition of physical activity and sedentary behaviour with incidence of cardiovascular disease in older adults.

BACKGROUND: Moderate-to-vigorous physical activity (MVPA) is proposed as key for cardiovascular diseases (CVD) prevention. At older ages, the role of sedentary behaviour (SB) and light intensity physical activity (LIPA) remains unclear. Evidence so far is based on studies examining movement behaviours as independent entities ignoring their co-dependency. This study examines the association between daily composition of objectively-assessed movement behaviours (MVPA, LIPA, SB) and incident CVD in older adults. METHODS: Whitehall II accelerometer sub-study participants free of CVD at baseline (N = 3319, 26.7% women, mean age = 68.9 years in 2012-2013) wore a wrist-accelerometer from which times in SB, LIPA, and MVPA during waking period were extracted over 7 days. Compositional Cox regression was used to estimate the hazard ratio (HR) for incident CVD for daily compositions of movement behaviours characterized by 10 (20 or 30) minutes greater duration in one movement behaviour accompanied by decrease in another behaviour, while keeping the third behaviour constant, compared to reference composition. Analyses were adjusted for sociodemographic, lifestyle, cardiometabolic risk factors and multimorbidity index. RESULTS: Of the 3319 participants, 299 had an incident CVD over a mean (SD) follow-up of 6.2 (1.3) years. Compared to daily movement behaviour composition with MVPA at recommended 21 min per day (150 min/week), composition with additional 10 min of MVPA and 10 min less SB was associated with smaller risk reduction - 8% (HR, 0.92; 95% CI, 0.87-0.99) - than the 14% increase in risk associated with a composition of similarly reduced time in MVPA and more time in SB (HR, 1.14; 95% CI, 1.02-1.27). For a given MVPA duration, the CVD risk did not differ as a function of LIPA and SB durations. CONCLUSIONS: Among older adults, an increase in MVPA duration at the expense of time in either SB or LIPA was found associated with lower incidence of CVD. This study lends support to public health guidelines encouraging increase in MVPA or at least maintain MVPA at current duration.

Joint association between accelerometry-measured daily combination of time spent in physical activity, sedentary behaviour and sleep and all-cause mortality: a pooled analysis of six prospective cohorts using compositional analysis.

OBJECTIVE: To examine the joint associations of daily time spent in different intensities of physical activity, sedentary behaviour and sleep with all-cause mortality. METHODS: Federated pooled analysis of six prospective cohorts with device-measured time spent in different intensities of physical activity, sedentary behaviour and sleep following a standardised compositional Cox regression analysis. PARTICIPANTS: 130 239 people from general population samples of adults (average age 54 years) from the UK, USA and Sweden. MAIN OUTCOME: All-cause mortality (follow-up 4.3-14.5 years). RESULTS: Studies using wrist and hip accelerometer provided statistically different results (I2=92.2%, Q-test p<0.001). There was no association between duration of sleep and all-cause mortality, HR=0.96 (95% CI 0.67 to 1.12). The proportion of time spent in moderate to vigorous physical activity was significantly associated with lower risk of all-cause mortality (HR=0.63 (95% CI 0.55 to 0.71) wrist; HR=0.93 (95% CI 0.87 to 0.98) hip). A significant association for the ratio of time spent in light physical activity and sedentary time was only found in hip accelerometer-based studies (HR=0.5, 95% CI 0.42 to 0.62). In studies based on hip accelerometer, the association between moderate to vigorous physical activity and mortality was modified by the balance of time spent in light physical activity and sedentary time. CONCLUSION: This federated analysis shows a joint dose-response association between the daily balance of time spent in physical activity of different intensities and sedentary behaviour with all-cause mortality, while sleep duration does not appear to be significant. The strongest association is with time spent in moderate to vigorous physical activity, but it is modified by the balance of time spent in light physical activity relative to sedentary behaviour.

Integrating Sleep, Physical Activity, and Diet Quality to Estimate All-Cause Mortality Risk: A Combined Compositional Clustering and Survival Analysis of the National Health and Nutrition Examination Survey 2005-2006 Cycle.

We aimed to compare all-cause mortality risk across clusters of adults ≥50 years of age (n = 1,035) with common lifestyle behaviors patterns, enrolled in the US National Health and Nutrition Examination Survey (2005-2006). Log-ratio coordinates of 24-hour movement pattern and z scores of diet quality were used as input into a model-based clustering analysis. A Cox regression model was fitted to ascertain the all-cause mortality risk associated with each cluster. Participants were clustered into 4 groups: 1) a group characterized by a better physical activity profile and longer sleep duration coupled with an average diet quality (cluster 1); 2) a group with the poorest activity profile and shortest sleep but also the best diet quality (cluster 2); 3) another group featuring lower levels of activity of either intensity and higher levels of sedentary behavior and also a poor diet quality score (cluster 3); and 4) a group with an average diet quality and the best activity profile in the sample (cluster 4). A combination of a poorer diet and activity profile increased the prospective risk of all-cause mortality. Our findings emphasize the importance of considering the combination of diet quality and 24-hour movement patterns when developing interventions to reduce the risk of premature mortality.

The physical activity paradox revisited: a prospective study on compositional accelerometer data and long-term sickness absence.

BACKGROUND: The 'physical activity paradox' advocates that leisure physical activity (PA) promotes health while high occupational PA impairs health. However, this paradox can be explained by methodological limitations of the previous studies-self-reported PA measures, insufficient adjustment for socioeconomic confounding or not addressing the compositional nature of PA. Therefore, this study investigated if we still observe the PA paradox in relation to long-term sick absence (LTSA) after adjusting for the abovementioned limitations. METHODS: Time spent on moderate-to-vigorous physical activity (MVPA) and remaining physical behaviors (sedentary behavior, standing, light PA and time in bed) at work and in leisure was measured for 929 workers using thigh accelerometry and expressed as isometric log-ratios (ilrs). LTSA was register-based first event of ≥6 consecutive weeks of sickness absence during 4-year follow-up. The association between ilrs and LTSA was analyzed using a Cox proportional hazards model adjusted for remaining physical behaviors and potential confounders, then separately adjusting for and stratifying by education and type of work. RESULTS: During the follow-up, 21% of the workers experienced LTSA. In leisure, more relative MVPA time was negatively associated with LTSA (20% lower risk with 20 min more MVPA, p = 0.02). At work, more relative MVPA time was positively associated with LTSA (15% higher risk with 20 min more MVPA, p = 0.02). Results remained unchanged when further adjusted for or stratified by education and type of work. CONCLUSION: These findings provide further support to the 'PA paradox'.

Plexiform schwannoma of the clitoris in a young girl: a case report.

BACKGROUND: Schwannoma (neurilemoma) is a benign, slow-growing tumor of the nerve sheath. These tumors are rarely found in the female genitalia and to date only 1 case of clitoral schwannoma has been reported in a young girl. We report here the second case of schwannoma of the clitoris. CASE: A 6-year-old girl presented with an enlarging clitoris. An alteration in her clitoral appearance had first been noted at 2 years of age. However, the size had further increased in the year prior to presentation. On examination her clitoris was normal in size but beneath the clitoral hood, predominantly on the left, there was a 3 x 2 cm irregular mobile mass. Her karyotype revealed normal 46XX female genotype. Magnetic resonance imaging of the abdomen and pelvis showed an isolated finding of diffuse enlargement of the clitoris with edema of the mons pubis. Surgical excision of the paraclitoral mass was performed. Intraoperatively the clitoral tip and shaft did not appear to be involved. However, the mass was found to be more diffuse, less well-defined, and more extensive than the clinical findings had suggested. On histology long spindle cells with nuclear palisading and focal Verocay body-like structures were found. There was mild to moderate pleomorphism. No mitotic figures were identified. There was diffuse staining of interweaving bundles for S100 protein and glial fibrillary acidic protein without staining for actin, desmin, or neurofilament. These features are consistent with a plexiform schwannoma. CONCLUSION: Although benign schwannomas rarely occur in the clitoris, we suggest that it should be considered as differential diagnosis for any young girl with clitoral or paraclitoral asymmetrical irregular mass.

Compositional analysis of the association between mortality and 24-hour movement behaviour from NHANES.

AIMS: Previous prospective studies of the association between mortality and physical activity have generally not fully accounted for the interplay between movement behaviours. A compositional data modelling approach accounts for relative scale and co-dependency in time-use data across physical activity behaviours of the 24-hour day. METHODS: A prospective analysis of the National Health and Nutrition Examination Survey 2005-2006 on N = 1468 adults (d = 135 deaths) in ages 50-79 years was undertaken using compositional Cox regression analysis. Daily time spent in sedentary behaviour, light intensity (LIPA) and moderate-to-vigorous physical activity (MVPA) was determined from waist-mounted accelerometer data (Actigraph 7164) and supplemented with self-reported sleep data to determine the daily time-use composition. RESULTS: The composition of time spent in sedentary behaviour, LIPA, MVPA and sleep was associated with mortality rate after allowing for age and sex effects (p < 0.001), and remained significant when other lifestyle factors were added (p < 0.001). This was driven primarily by the preponderance of MVPA; however, significant changes are attributable to LIPA relative to sedentary behaviour and sleep, and sedentary behaviour relative to sleep. The final ratio ceased to be statistically significant after incorporating lifestyle factors. The preponderance of MVPA ceased to be statistically significant after incorporating health at outset and physical limitations on movement. CONCLUSIONS: An association is inferred between survival rate and the physical activity composition of the day. The MVPA time share is important, but time spent in LIPA relative to sedentary behaviour and sleep is also a significant factor. Increased preponderance of MVPA may have detrimental associations at higher levels of MVPA.

Striking the Right Balance: Evidence to Inform Combined Physical Activity and Sedentary Behavior Recommendations.

BACKGROUND: Crucial evidence gaps regarding: (1) the joint association of physical activity and sedentary time with health outcomes and (2) the benefits of light-intensity physical activity were identified during the development of recommendations for the World Health Organization Guidelines on physical activity and sedentary behavior (SB). The authors present alternative ways to evidence the relationship between health outcomes and time spent in physical activity and SB and examine how this could be translated into a combined recommendation in future guidelines. METHODS: We used compositional data analysis to quantify the dose-response associations between the balance of time spent in physical activity and SB with all-cause mortality. The authors applied this approach using 2005-2006 National Health and Nutrition Examination Survey accelerometer data. RESULTS: Different combinations of time spent in moderate- to vigorous-intensity physical activity, light-intensity physical activity, and SB are associated with similar all-cause mortality risk level. A balance of more than 2.5 minutes of moderate- to vigorous-intensity physical activity per hour of daily sedentary time is associated with the same magnitude of risk reduction for all-cause mortality as obtained by being physically active according to the current recommendations. CONCLUSION: This method could be applied to provide evidence for more flexible recommendations in the future with options to act on different behaviors depending on individuals' circumstances and capacity.

Diversity of ACE2 and its interaction with SARS-CoV-2 receptor binding domain.

COVID-19, the clinical syndrome caused by the SARS-CoV-2 virus, has rapidly spread globally causing tens of millions of infections and over a million deaths. The potential animal reservoirs for SARS-CoV-2 are currently unknown, however sequence analysis has provided plausible potential candidate species. SARS-CoV-2 binds to the angiotensin I converting enzyme 2 (ACE2) to enable its entry into host cells and establish infection. We analyzed the binding surface of ACE2 from several important animal species to begin to understand the parameters for the ACE2 recognition by the SARS-CoV-2 spike protein receptor binding domain (RBD). We employed Shannon entropy analysis to determine the variability of ACE2 across its sequence and particularly in its RBD interacting region, and assessed differences between various species' ACE2 and human ACE2. As cattle are a known reservoir for coronaviruses with previous human zoonotic transfer, and has a relatively divergent ACE2 sequence, we compared the binding kinetics of bovine and human ACE2 to SARS-CoV-2 RBD. This revealed a nanomolar binding affinity for bovine ACE2 but an approximate ten-fold reduction of binding compared to human ACE2. Since cows have been experimentally infected by SARS-CoV-2, this lower affinity sets a threshold for sequences with lower homology to human ACE2 to be able to serve as a productive viral receptor for SARS-CoV-2.

The Impact of Regeneration and Climate Adaptations of Urban Green-Blue Assets on All-Cause Mortality: A 17-Year Longitudinal Study.

Urban waterways are underutilised assets, which can provide benefits ranging from climate-change mitigation and adaptation (e.g., reducing flood risks) to promoting health and well-being in urban settings. Indeed, urban waterways provide green and blue spaces, which have increasingly been associated with health benefits. The present observational study used a unique 17-year longitudinal natural experiment of canal regeneration from complete closure and dereliction in North Glasgow in Scotland, U.K. to explore the impact of green and blue canal assets on all-cause mortality as a widely used indicator of general health and health inequalities. Official data on deaths and socioeconomic deprivation for small areas (data zones) for the period 2001-2017 were analysed. Distances between data zone population-weighted centroids to the canal were calculated to create three 500 m distance buffers. Spatiotemporal associations between proximity to the canal and mortality were estimated using linear mixed models, unadjusted and adjusted for small-area measures of deprivation. The results showed an overall decrease in mortality over time (ß = -0.032, 95% confidence interval (CI) [-0.046, -0.017]) with a closing of the gap in mortality between less and more affluent areas. The annual rate of decrease in mortality rates was largest in the 0-500 m buffer zone closest to the canal (-3.12%, 95% CI [-4.50, -1.73]), with smaller decreases found in buffer zones further removed from the canal (500-1000 m: -3.01%, 95% CI [-6.52, 0.62]), and 1000-1500 m: -1.23%, 95% CI [-5.01, 2.71]). A similar pattern of results was found following adjustment for deprivation. The findings support the notion that regeneration of disused blue and green assets and climate adaptions can have a positive impact on health and health inequalities. Future studies are now needed using larger samples of individual-level data, including environmental, socioeconomic, and health variables to ascertain which specific elements of regeneration are the most effective in promoting health and health equity.

Sedentary behaviour is associated with depression symptoms: Compositional data analysis from a representative sample of 3233 US adults and older adults assessed with accelerometers.

BACKGROUND: Evidence supporting the individual associations of sedentary behaviours with depression symptoms commonly ignores the inherent co-dependency between physical activity, sedentary behaviours and sleep in a given 24-hour period. Data analysis based on compositional methods effectively deals with this issue. AIM: To investigate the association between sedentary behaviour and depression symptoms synergistically using compositional analysis methods. METHODS: Participants were a representative sample of 3233 US adults and older adults from the 2005-2006 cycle of the NHANES with valid 24-hour lifestyle behaviours data (i.e., accelerometer-derived physical activity and sedentary behaviour and self-reported sleep) and available self-reported depression symptoms (PHQ-9). The association between sedentary behaviour and depression symptoms scoring was investigated using a compositional zero-inflated Poisson regression analysis. Subsequently, the model estimates were used to evaluate the effects on depression symptoms of replacing time spent in sitting activities with physical activity of different intensities and sleep. LIMITATIONS: The current study is limited by its cross-sectional design. Also, sleep time was self-reported, which could bias our estimations. RESULTS: Increased sedentary behaviour relative to other behaviours was statistically significantly associated with increased depression symptoms (p < 0.001). Reallocating 60 min time from sedentary behaviours to moderate-to-vigorous physical activity (MVPA) and sleep was associated with small reductions in depression symptoms. CONCLUSIONS: A synergistic compositional analysis of accelerometer data uncovered a detrimental association between sedentary behaviour and depression symptoms. These results add to evidence from previous studies. The observed association seems to be principally driven by corresponding reductions in MVPA and sleep duration.

Discovering and improving novel peptide therapeutics.

Peptides have a number of advantages over small molecules in terms of specificity and affinity for targets, and over antibodies in terms of size. However, sensitivity to serum and tissue proteases coupled with short serum half-life has resulted in few recombinant library derived peptides, making the transition from lead to drug on the market. Recently, a series of technologies have been developed to address both these issues: selection methodologies addressing protease resistance have been developed that when combined with methods such as pegylation antibody Fc attachment and binding to serum albumin look likely to finally turn therapeutic peptides into a widely accepted drug class.

Compositional analyses of the associations between sedentary time, different intensities of physical activity, and cardiometabolic biomarkers among children and youth from the United States.

INTRODUCTION: Compositional data analysis is one appropriate method for co-dependent data, even when data are collected for a subdivision of the 24-hour period, such as the waking day. Objectives were to use compositional analyses to examine the combined and relative associations of sedentary time (ST), light-intensity physical activity (LPA), moderate-intensity physical activity (MPA), and vigorous-intensity physical activity (VPA) with cardiometabolic biomarkers in a representative sample of children and youth. METHODS: This cross-sectional study included 2544 participants aged 6-17 years from the 2003-2006 United States National Health and Nutrition Examination Survey. ST (<100 counts per minute), LPA (100 counts per minute to <4 METs; Freedson age-specific equation), MPA (4 to <7 METs), and VPA (≥7 METs) were accelerometer-derived. Cardiometabolic biomarkers included waist circumference, body mass index (BMI) z-score, HDL-cholesterol, C-reactive protein, and blood pressure. Triglycerides, glucose, insulin, and LDL-cholesterol were measured in a fasting sub-sample of adolescents (n = 670). Compositional linear regression models were conducted. RESULTS: The composition of ST, LPA, MPA, and VPA was significantly associated with BMI z-score, log waist circumference, systolic and diastolic blood pressure, HDL-cholesterol, and log plasma glucose (variance explained: 1-29%). Relative to the other three behaviors, VPA was negatively associated with BMI z-score (γVPA = -0.206, p = 0.005) and waist circumference (γVPA = -0.03, p = 0.001). Conversely, ST was positively associated with waist circumference (γST = 0.029, p = 0.013). ST and VPA were also positively associated with diastolic blood pressure (γST = 2.700, p = 0.018; γVPA = 1.246, p = 0.038), relative to the other behaviors, whereas negative associations were observed for LPA (γLPA = -2.892, p = 0.026). Finally, VPA was positively associated with HDL-cholesterol, relative to other behaviors (γVPA = 0.058, p<0.001). CONCLUSIONS: The ST and physical activity composition appears important for many aspects of cardiometabolic health in children and youth. Compositions with more time in higher-intensity activities may be better for some aspects of cardiometabolic health.

Compliance with 24-h Movement Behaviour Guidelines among Belgian Pre-School Children: The ToyBox-Study.

The 24-h day-containing physical activity, sedentary behaviour and sleep-in pre-school children has not yet been extensively investigated. The aim of the current study was to investigate pre-schoolers' compliance with the 24-h movement behaviour guidelines (i.e., three hours/day total physical activity, a maximum of one hour/day of screen time and 10⁻13 h sleep/night). In total, 595 pre-schoolers (53.3% boys, mean age: 4.2 years) provided complete data for the three behaviours. Physical activity was objectively measured with accelerometers, while screen time and sleep were parent-reported through questionnaires. The proportion of pre-schoolers complying with the 24-h movement behaviour guidelines was calculated on weekdays and on weekend days. Low compliance rates were found: 10.1% on weekdays and only 4.3% on weekend days. The majority of pre-schoolers complied with the sleep duration guidelines (>90% on weekdays and weekend days), followed by the screen time guidelines (61% on weekdays and 28% on weekend days). The lowest compliance rates were found for physical activity (<20% on weekdays and weekend days). Overall, low percentages of pre-schoolers complying with the 24-h movement behaviour guidelines were found, and the lowest compliance was found for physical activity.

Compositional Analysis of the Associations between 24-h Movement Behaviours and Health Indicators among Adults and Older Adults from the Canadian Health Measure Survey.

This study investigated the association between the allocation of time-use over the 24-h day between sleep, sedentary behaviour (SB), light-intensity physical activity (LPA) and moderate-to-vigorous-intensity physical activity (MVPA)) and health indicators. A cross-sectional analysis of Canadian Health Measures Survey data was undertaken using compositional data analysis. SB, LPA and MVPA were derived from Actical accelerometers, whilst sleep was self-reported by respondents. The analysis was stratified by age; adults (aged 18⁻64 years; n = 6322) and older adults (65⁻79 years; n = 1454). For adults, beneficial associations were observed between larger proportions of MVPA relative to time in other behaviours and body mass index (BMI), waist circumference, aerobic fitness, resting heart rate, high-density lipoprotein (HDL) cholesterol, triglycerides, blood glucose and insulin levels. More time spent in sleep relative to other movement behaviours was deleteriously associated with aerobic fitness, HDL cholesterol, insulin, C-reactive proteins and grip strength but beneficially with low-density lipoprotein cholesterol. Relative time spent in LPA was deleteriously associated with BMI and beneficially with triglycerides and grip strength. In older adults, these associations were blunted or disappeared but larger proportions of MVPA were associated with better mental health. The importance to health of MVPA when explicitly considered relative to other movement behaviours was confirmed.

Covalent antibody display--an in vitro antibody-DNA library selection system.

The endonuclease P2A initiates the DNA replication of the bacteriophage P2 by making a covalent bond with its own phosphate backbone. This enzyme has now been exploited as a new in vitro display tool for antibody fragments. We have constructed genetic fusions of P2A with single-chain antibodies (scFvs). Linear DNA of these fusion proteins were processed in an in vitro coupled transcription-translation mixture of Escherichia coli S30 lysate. Complexes of scFv-P2A fusion proteins covalently bound to their own DNA were isolated after panning on immobilized antigen, and the enriched DNAs were recovered by PCR and prepared for the subsequent cycles of panning. We have demonstrated the enrichment of scFvs from spiked libraries and the specific selection of different anti-tetanus toxoid scFvs from a V-gene library with 50 million different members prepared from human lymphocytes. This covalent antibody display technology offers a complete in vitro selection system based exclusively on DNA-protein complexes.

An in vitro selection strategy for conferring protease resistance to ligand binding peptides.

One drawback to the use of peptides as therapeutics has been their susceptibility to proteolysis. Here, we have used an in vitro display technology, CIS display, to enhance the proteolytic resistance of ligand-binding peptides by selection of protecting motifs from a large peptide library. The premise to this selection was that certain linear peptides within a library could form structures capable of preventing the access of proteases to defined cleavage sites without affecting ligand binding. A diverse 12-mer peptide library was inserted between a FLAG epitope motif and a thrombin cleavage site and this construct was fused to the bacterial initiator protein RepA for CIS display selection. After five rounds of selection, protection motifs were isolated that were capable of preventing proteolytic cleavage of the adjacent thrombin site. Some of the selected peptides were also resistant to more promiscuous proteases, such as chymotrypsin and trypsin, which were not used in the selection. The observed resistance to thrombin, trypsin and chymotrypsin translated into increased resistance to plasma proteases in vitro and to an increase in circulating half-lives in rats. This method can be applied to enhancing the in vivo stability of therapeutic peptides.