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Supplementary Data from An Enzymatically Cleavable Tripeptide Linker for Maximizing the Therapeutic Index of Antibody–Drug Conjugates

Valine–citrulline is a protease-cleavable linker commonly used in many drug delivery systems, including antibody–drug conjugates (ADC) for cancer therapy. However, its suboptimal in vivo stability can cause various adverse effects such as neutropenia and hepatotoxicity, leading to dose delays or treatment discontinuation. Here, we report that glutamic acid–glycine–citrulline (EGCit) linkers have the potential to solve this clinical issue without compromising the ability of traceless drug release and ADC therapeutic efficacy. We demonstrate that our EGCit ADC resists neutrophil protease–mediated degradation and spares differentiating human neutrophils. Notably, our anti-HER2 ADC shows almost no sign of blood and liver toxicity in healthy mice at 80 mg kg−1. In contrast, at the same dose level, the FDA-approved anti-HER2 ADCs Kadcyla and Enhertu show increased levels of serum alanine aminotransferase and aspartate aminotransferase and morphologic changes in liver tissues. Our EGCit conjugates also exert greater antitumor efficacy in multiple xenograft tumor models compared with Kadcyla and Enhertu. This linker technology could substantially broaden the therapeutic windows of ADCs and other drug delivery agents, providing clinical options with improved efficacy and safety.

The Psychomotor Vigilance Test (PVT) is a widely used behavioral attention measure, with the 10-min (PVT-10) and 3-min (PVT-3) as two commonly used versions. The PVT-3 may be comparable to the PVT-10, though its convergent validity relative to the PVT-10 has not been explicitly assessed. For the first time, we utilized repeated measures correlation (rmcorr) to evaluate intra-individual associations between PVT-10 and PVT-3 versions across total sleep deprivation (TSD), chronic sleep restriction (SR) and multiple consecutive days of recovery. Eighty-three healthy adults (mean ± SD, 34.7 ± 8.9 years; 36 females) received two baseline nights (B1-B2), five SR nights (SR1-SR5), 36 h TSD, and four recovery nights (R1-R4) between sleep loss conditions. The PVT-10 and PVT-3 were completed every 2 h during wakefulness. Rmcorr compared responses on two frequently used, sensitive PVT metrics: reaction time (RT) via response speed (1/RT) and lapses (RT > 500 ms on the PVT-10 and > 355 ms on the PVT-3) by day (e.g., B2), by study phase (e.g., SR1-SR5), and by time point (1000–2000 h). PVT 1/RT correlations were generally stronger than those for lapses. The majority of correlations (48/50 [96%] for PVT lapses and 38/50 [76%] for PVT 1/RT) were values below 0.70, indicating validity issues. Overall, the PVT-3 demonstrated inadequate convergent validity with the “gold standard” PVT-10 across two different types of sleep loss and across extended recovery. Thus, the PVT-3 is not interchangeable with the PVT-10 for assessing behavioral attention performance during sleep loss based on the design of our study and the metrics we evaluated. Our results have substantial implications for design and measure selection in laboratory and applied settings, including those involving sleep deprivation.

Antibody-based therapy has shown great success in the treatment of many diseases, including cancers. While antibodies and antibody-drug conjugates (ADCs) have also been evaluated for central nervous system (CNS) disorders as well as brain tumors, their therapeutic efficacy can be substantially limited due to low permeability across the blood-brain barrier (BBB). Thus, improving BBB permeability of therapeutic antibodies is critical in establishing this drug class as a reliable clinical option for CNS diseases. Here, we report that, compared with a conventional heterogeneous conjugation, homogeneous conjugation of the synthetic BBB shuttle peptide angiopep-2 (Ang2) to a monoclonal antibody (mAb) provides improved binding affinity for brain microvascular endothelial cells

There are substantial individual differences (resilience and vulnerability) in performance resulting from sleep loss and psychosocial stress, but predictive potential biomarkers remain elusive. Similarly, marked changes in the cardiovascular system from sleep loss and stress include an increased risk for cardiovascular disease. It remains unknown whether key hemodynamic markers, including left ventricular ejection time (LVET), stroke volume (SV), heart rate (HR), cardiac index (CI), blood pressure (BP), and systemic vascular resistance index (SVRI), differ in resilient vs. vulnerable individuals and predict differential performance resilience with sleep loss and stress. We investigated for the first time whether the combination of total sleep deprivation (TSD) and psychological stress affected a comprehensive set of hemodynamic measures in healthy adults, and whether these measures differentiated neurobehavioral performance in resilient and vulnerable individuals. Thirty-two healthy adults (ages 27–53; 14 females) participated in a 5-day experiment in the Human Exploration Research Analog (HERA), a high-fidelity National Aeronautics and Space Administration (NASA) space analog isolation facility, consisting of two baseline nights, 39 h TSD, and two recovery nights. A modified Trier Social Stress Test induced psychological stress during TSD. Cardiovascular measure collection [SV, HR, CI, LVET, BP, and SVRI] and neurobehavioral performance testing (including a behavioral attention task and a rating of subjective sleepiness) occurred at six and 11 timepoints, respectively. Individuals with longer pre-study LVET (determined by a median split on pre-study LVET) tended to have poorer performance during TSD and stress. Resilient and vulnerable groups (determined by a median split on average TSD performance) showed significantly different profiles of SV, HR, CI, and LVET. Importantly, LVET at pre-study, but not other hemodynamic measures, reliably differentiated neurobehavioral performance during TSD and stress, and therefore may be a biomarker. Future studies should investigate whether the non-invasive marker, LVET, determines risk for adverse health outcomes.

Background Blood pressure (BP) variability has reportedly been a risk factor for various clinical events. To clarify the influence of BP visit‐to‐visit variability on adverse events in patients with nonvalvular atrial fibrillation, a post hoc analysis of the J‐RHYTHM Registry was performed. Methods and Results Of 7406 outpatients with nonvalvular atrial fibrillation from 158 institutions, 7226 (age, 69.7±9.9 years; men, 70.7%), in whom BP was measured 4 times or more (14.6±5.0 times) during the 2‐year follow‐up period or until occurrence of an event, constituted the study group. SD and coefficient of variation of BP values were calculated as BP variability. Thromboembolism, major hemorrhage, and all‐cause death occurred in 110 (1.5%), 121 (1.7%), and 168 (2.3%) patients, respectively. When patients were divided into quartiles of systolic BP‐SD (P =0.015 for thromboembolism; HR, 2.60, 95% CI, 1.36–4.97, P =0.004 for major hemorrhage; and HR, 1.85, 95% CI, 1.11–3.07, P =0.018 for all‐cause death) after adjusting for components of the CHA 2 DS 2 ‐VASc score, warfarin and antiplatelet use, atrial fibrillation type, BP measurement times, and others. These findings were consistent when BP‐coefficient of variation was used instead of BP‐SD. Conclusions Systolic BP visit‐to‐visit variability was significantly associated with all adverse events in patients with nonvalvular atrial fibrillation. Further studies are needed to clarify the causality between BP variability and adverse outcomes in patients with nonvalvular atrial fibrillation. Registration URL: https://www.umin.ac.jp/ctr/ ; Unique Identifier: UMIN000001569.

Breast tumors generally consist of a diverse population of cells with varying gene expression profiles. Breast tumor heterogeneity is a major factor contributing to drug resistance, recurrence, and metastasis after chemotherapy. Antibody-drug conjugates (ADCs) are emerging chemotherapeutic agents with striking clinical success, including T-DM1 for HER2-positive breast cancer. However, these ADCs often suffer from issues associated with intratumor heterogeneity. Here, we show that homogeneous ADCs containing two distinct payloads are a promising drug class for addressing this clinical challenge. Our conjugates show HER2-specific cell killing potency, desirable pharmacokinetic profiles, minimal inflammatory response, and marginal toxicity at therapeutic doses. Notably, a dual-drug ADC exerts greater treatment effect and survival benefit than does co-administration of two single-drug variants in xenograft mouse models representing intratumor HER2 heterogeneity and elevated drug resistance. Our findings highlight the therapeutic potential of the dual-drug ADC format for treating refractory breast cancer and perhaps other cancers., Intratumor heterogeneity in breast cancer can limit the clinical success of antibody-drug conjugates (ADCs). In this study, the authors develop dual payload Her2-ADCs that show potent anti-tumor activity against heterogeneous breast tumors in vivo.

Background It is known that low diastolic blood pressure (DBP) is associated with long-term cardiovascular events after acute myocardial infarction (AMI). However, the impact of low diastolic blood pressure on short-term outcome has not yet been well investigated. Methods and results We included 15,208 patients who were hospitalized for AMI and registered in the Tokyo CCU network registry between 2013 and 2016. Thirty-day in-hospital mortality rate was 4.8% (728/15,208). To assess the relationship between DBP at the time of admission and 30-day mortality non-linearly, spline regression model was applied with the stratification of the cohort according to tercile of systolic blood pressure (SBP, low:≤122 mmHg, intermediate:123–148 mmHg, high:≥149 mmHg) and J-curve phenomenon was observed in the low and high SBP groups. In multivariate logistic regression analysis, adjusted odds ratio of the lowest quintile of DBP (≤64 mmHg) was 1.65 (95% CI:1.02–2.66) in low SBP group and 4.55 (95% CI:1.72–12.00) in high SBP group. Conclusion Low DBP was associated with increased 30-day in-hospital mortality rate after AMI even in patients with high SBP. Funding Acknowledgement Type of funding source: None

Acute myeloid leukemia (AML) is the most common and aggressive blood cancer in adults. In particular, significant unmet medical needs exist for effective treatment strategies for acute myelomonocytic leukemia (M4) and acute monocytic leukemia (M5) AML subtypes. Antibody–drug conjugates (ADC) are a promising drug class for AML therapy, as demonstrated by the FDA-approved anti-CD33 ADC, gemtuzumab ozogamicin (Mylotarg). However, CD33 is expressed in normal hematopoietic stem cells, highlighting the critical need to identify AML-specific targets to minimize the risk of potential adverse effects. We have demonstrated that the leukocyte immunoglobulin-like receptor subfamily B4 (LILRB4) is expressed at significantly higher levels on monocytic M4 and M5 AML cells than on normal counterparts. Here, we test whether LILRB4 is a promising ADC target to kill monocytic AML cells while sparing healthy counterparts. To this end, we generated ADCs from a humanized anti-LILRB4 mAb and the antimitotic payload, monomethyl auristatin F. The conjugates constructed were characterized and evaluated for LILRB4-specific cell killing potency, toxicity to progenitor cells, pharmacokinetics, and therapeutic efficacy. Our ADC linker technology platform efficiently generated homogeneous anti-LILRB4 ADCs with defined drug-to-antibody ratios. The homogeneous anti-LILRB4 ADCs demonstrated the capacity for LILRB4-mediated internalization, suitable physicochemical properties, and high cell killing potency against LILRB4-positive AML cells. Importantly, our data indicate that these ADCs spare normal progenitor cells. One of our homogeneous conjugates exerted a remarkable therapeutic effect and no significant toxicity in a xenograft mouse model of disseminated human AML. Our findings highlight the clinical potential of anti-LILRB4 ADCs in monocytic AML therapy.

Valine–citrulline linkers are commonly used as enzymatically cleavable linkers for antibody–drug conjugates. While stable in human plasma, these linkers are unstable in mouse plasma due to susceptibility to an extracellular carboxylesterase. This instability often triggers premature release of drugs in mouse circulation, presenting a molecular design challenge. Here, we report that an antibody–drug conjugate with glutamic acid–valine–citrulline linkers is responsive to enzymatic drug release but undergoes almost no premature cleavage in mice. We demonstrate that this construct exhibits greater treatment efficacy in mouse tumor models than does a valine–citrulline-based variant. Notably, our antibody–drug conjugate contains long spacers facilitating the protease access to the linker moiety, indicating that our linker assures high in vivo stability despite a high degree of exposure. This technology could add flexibility to antibody–drug conjugate design and help minimize failure rates in pre-clinical studies caused by linker instability., The valine-citrulline dipeptide, which is used as a cleavable linker for antibody-drug conjugates, is instable in mouse plasma. Here, the authors developed a glutamic acid–valine–citrulline tripeptide sequence as a stable alternative that still is susceptible to cathepsin-mediated cleavage.

The procedure followed by the Nuclear Metrology Laboratory (LMN) at the IPEN for the standardization of activity of 64Cu is described. The measurement was carried out in a 4π (PC) β-γ coincidence system. The activity per unit mass of the solution was determined by the extrapolation technique. The events were registered using a Time to Amplitude Converter (TAC) associated with a Multi-channel Analyzer. The gamma-ray emission probability per decay of the 1345.7keV transition of 64Cu determined with a calibrated REGe gamma-ray spectrometer was (0.472 ± 0.010) %, in agreement with the literature.

Objectives To estimate the annual prevalence of different diagnostic categories by age, breed and sex in insured cats in Japan for which veterinary care claims had been made, and to identify if there is a pattern in these host factors. Materials and Methods Data from 48,187 cats insured for veterinary care in Japan in the period from April 2012 to March 2013 comprising 26,003 males and 22,184 females were analysed to calculate the annual prevalence of 18 diagnostic categories of disease by age, breed and sex. Results The prevalence was highest for urinary system disorders (12·2% for males and 10·0% for females), followed by digestive disorders (11·6% for males and 10·7% for females) and dermatological diseases (8·7% for males and 9·0% for females). The male cats had a higher prevalence than female cats for most diagnostic categories. The prevalence of cardiovascular, urinary, endocrine and neoplastic disorders increased with age; infectious and parasitic diseases had high prevalence at young ages, and the prevalence of respiratory, musculoskeletal disorders and injuries had bimodal peaks. Dermatological disorders had a high prevalence at all ages. A large variation in prevalence was observed between breeds for otic, dermatological, dental and cardiovascular disorders. Clinical Significance The findings can be used to increase awareness of patterns of health disorders in different categories of cat.

Background Although the patient's characteristics and outcome of acute myocardial infarction (AMI) have been sufficiently investigated and primary percutaneous coronary intervention (PCI) has been recognized as established treatment strategy, those of recent myocardial infarction (RMI) have not been fully evaluated. Purpose The purpose of the present study was to clarify clinical characteristics and in-hospital outcomes of RMI patients from the database of the Tokyo CCU network multicenter registry. Methods In Tokyo CCU network multicenter registry database from 2013 to 2016, 15788 consecutive patients were registered as AMI (within 24 hours from onset) and RMI (within 2–30 days from onset). However 1246 patients were excluded because of inadequate data. And we excluded 66 cases because of out of onset period and 129 cases that strongly suspected of involvement of vasospastic events. Therefore, remaining 14347 patients were categorized to RMI group (n=1853) and AMI group (n=12494), and analyzed. Results Compared with AMI group, average age was older (70.4±12.9 vs 68.0±13.4 years, p Kaplan-Meier analysis Conclusions RMI patients had distinctive clinical features in backgrounds and treatment strategies compared with AMI patients, and the major cause of death of RMI patients was different from that of AMI patients. Furthermore, even though treatment during hospitalization of RMI patients was well done, death due to mechanical complications continued to increase.

The procedure followed by the Nuclear Metrology Laboratory (LMN) at the IPEN-CNEN/SP, in Sao Paulo, for the primary standardization of 166Ho is described. The activity of 166Ho was determined by the efficiency extrapolation technique applied to a 4πβ(PC)-γ coincidence system using a gas flow proportional counter in 4π geometry coupled to a 76 × 76 mm NaI(Tl) crystal. The results for the γ-rays intensities at 80.57 and 1379.45 keV were 0.0651(11) and 0.00904(11), respectively.

The structural, magnetic, and electrical properties of InMnAs thin films were investigated to elucidate their ferromagnetism for potential applications in InP-based spintronics and magneto-optical integrated circuits. The films were prepared on InP substrates by molecular beam epitaxy at growth temperatures of 220–280 °C without further annealing. Except for the 220 °C-grown sample, the films showed ferromagnetism at room temperature. We found that the room-temperature ferromagnetism was predominantly caused by the existence of hexagonal MnAs clusters embedded in a homogeneous InMnAs matrix (InMnAs:MnAs system). High-resolution transmission electron microscope images revealed that the MnAs nanoclusters formed either elongated or round structures in the InMnAs host matrix. The experimental results indicate that the mixed systems of hexagonal MnAs embedded in InMnAs may provide potential ferromagnetic building blocks for InP-based spintronics.

In the present work, the determinations of k

A new method of non-destructive evaluation for residual tension load of ground anchors was developed. If the tendon tension part of a ground anchor is approximated by a “string”, the frequency of its free vibration is determined by line density and tension length of the PC steel and the operating tension. Although the free vibration of tendon tension part cannot be directly excited because it is in the ground, it is confirmed by a physical model experiment that the free vibration can be excited by applying a power at the extra length of anchor head. A series of field experiments was conducted by using a small vibrator and an accelerometer. A swept-frequency vibration was applied to the extra length of anchor head, and the vibration waveform was measured at the same position. The observed waveform was analyzed by running-spectrum analysis in order to find a resonance frequency which is used to calculate the residual tension load.

Poly(glycolic acid) (PGA) is an aliphatic polyester with the simplest chemical structure. Since PGA is susceptible to hydrolysis, various researchers studied the factors affecting the hydrolytic degradation behavior of PGA. The factors include the higher-order structure, pH, ionic electrolyte etc. In this study, to clarify the mechanism for the hydrolysis of PGA fibers, effect of the carboxylic acid content on the hydrolytic degradation behavior was investigated through the quantitative evaluation of the amounts of the carboxylic acid end group and the PGA monomer (glycolide, GL) in the fibers. Evaluation of the hydrolysis of PGA fibers through the analyses of the molecular weight retention and tensile strength retention after immersing in the phosphate buffer solution at 37 °C revealed that the hydrolysis is governed by the carboxylic acid content in the PGA fibers. It was also found that the amount of GL generated during the melt spinning process is affected by the amount and type of polymerization catalyst. In addition, the experimental results of fundamental analysis suggested that the polymerization catalyst also decomposes the GL into glycolic acid or glycolic acid dimers to generate carboxylic acid, and the rate of decomposition varies depending on the type and amount of catalyst. Based on these results, it was concluded that there is an indirect effect of the type and amount of polymerizing catalyst on the rate of hydrolysis of PGA fibers.

This functional magnetic resonance imaging (fMRI) study investigated the brain regions underlying language task performance in adult second language (L2) learners. Specifically, we identified brain regions where the level of activation was associated with L2 fluency levels. Thirty Japanese-speaking adults participated in the study. All participants were L2 learners of English and had achieved varying levels of fluency, as determined by a standardized L2 English proficiency test, the Versant English Test (Pearson Education Inc., 2011). When participants performed the oral sentence building task from the production tasks administered, the dorsal part of the left inferior frontal gyrus (dIFG) showed activation patterns that differed depending on the L2 fluency levels: The more fluent the participants were, the more dIFG activation decreased. This decreased activation of the dIFG might reflect the increased automaticity of a syntactic building process. In contrast, when participants performed an oral story comprehension task, the left posterior superior temporal gyrus (pSTG) showed increased activation with higher fluency levels. This suggests that the learners with higher L2 fluency were actively engaged in post-syntactic integration processing supported by the left pSTG. These data imply that L2 fluency predicts neural resource allocation during language comprehension tasks as well as in production tasks. This study sheds light on the neural underpinnings of L2 learning by identifying the brain regions recruited during different language tasks across different modalities (production vs. comprehension).

Peritoneal carcinomatosis is a major source of morbidity and mortality in patients with advanced abdominal neoplasms. Intraperitoneal chemotherapy (IPC) is an area of intense interest given its efficacy in ovarian cancer. However, IPC suffers from poor drug penetration into peritoneal tumors. As such, extensive cytoreductive surgery is required prior to IPC. Here, we explore the utility of iRGD, a tumor-penetrating peptide, for improved tumor-specific penetration of intraperitoneal compounds and enhanced IPC in mice. Intraperitoneally administered iRGD significantly enhanced penetration of an attached fluorescein into disseminated peritoneal tumor nodules. The penetration was tumor-specific, circulation-independent, and mediated by the neuropilin-binding RXXK tissue-penetration peptide motif of iRGD. Q-iRGD, which fluoresces upon cleavage, including the one that leads to RXXK activation, specifically labeled peritoneal metastases displaying different growth patterns in mice. Importantly, iRGD enhanced intratumoral entry of intraperitoneally co-injected dextran to approximately 300% and doxorubicin to 250%. Intraperitoneal iRGD/doxorubicin combination therapy inhibited the growth of bulky peritoneal tumors and reduced systemic drug toxicity. iRGD delivered attached fluorescein and co-applied nanoparticles deep into fresh human peritoneal metastasis explants. These results indicate that intraperitoneal iRGD co-administration serves as a simple and effective strategy to facilitate tumor detection and improve the therapeutic index of IPC for peritoneal carcinomatosis.

Time-gated interrogation of phosphorescent labels allows high sensitivity detection of analytes by excluding background fluorescence and stray excitation light. Here we report a method for preparing phosphorescent polymer nanospheres with optical properties specifically designed to enable time-gated detection with both organic and inorganic light-emitting diodes and photodiodes. The nanospheres were formed by co-precipitating the phosphorescent dye ruthenium diphenyl phenanthroline with the amphiphilic statistical co-polymer poly(styrene-co-maleic acid). To minimize aggregation and ensure effective encapsulation against water and oxygen ingress, the polymer chains were cross-linked with the small-molecule cross-linker bisphenol A diglycidyl ether, resulting in near-monodisperse nanospheres with excellent resilience against aggregation and quenching. The nanospheres exhibited long phosphorescence lifetimes of 5.3 ± 0.1 μs, high photoluminescence quantum efficiencies of 33 ± 5%, and excellent stability under UV irradiation. Using a blue light-emitting diode as a light-source and a silicon photodiode as a detector, a low detection limit of 4 μg ml−1 was achieved. The suitability of the nanospheres for bioanalysis was demonstrated by surface functionalising them with biotin to yield a simple biolabel for streptavidin.