Utility of Cellular Measurements of Non-Specific Endocytosis to Assess the Target-Independent Clearance of Monoclonal Antibodies.

Past studies have demonstrated higher clearance for monoclonal antibodies possessing increased rates of non-specific endocytosis. However, this metric is oftentimes evaluated indirectly using biophysical techniques or cell surface binding studies that may not provide insight into the specific rates of cellular turnover. Furthermore, few examples evaluating non-specific endocytosis have been reported for a therapeutic antibody that reached clinical assessment. In the current report, we evaluated a therapeutic human immunoglobulin G2 monoclonal antibody targeted against the interleukin-4 receptor alpha chain (IL-4Rα) that exhibited elevated target independent clearance in previous Phase 1 and 2 studies. We confirmed high non-specific clearance of the anti-IL-4Rα antibody as compared to a reference antibody during pharmacokinetic assessments in wild type mice where target-mediated disposition was absent. We then developed a cell-based method capable of measuring cellular protein endocytosis and demonstrated the anti-IL-4Rα antibody exhibited marked non-specific uptake relative to the reference compound. Antibody homology modeling identified the anti-IL-4Rα antibody possessed positive charge patches whose removal via targeted mutations substantially reduced its non-specific endocytosis. We then expanded the scope of the study by evaluating panels of both preclinical and clinically relevant monoclonal antibodies and demonstrate those with the highest rates of non-specific uptake in vitro exhibit elevated target independent clearance, low subcutaneous bioavailability, or both. Our results support the observation that high non-specific endocytosis is a negative attribute in monoclonal antibody development and demonstrate the utility of a generic cell-based screen as a quantitative tool to measure non-specific endocytosis of protein therapeutics at the single-cell level.

Greater Neighborhood Disadvantage Is Associated with Alterations in Fetal Functional Brain Network Structure.

OBJECTIVE: To determine the association between neighborhood disadvantage and functional brain development of in utero fetuses. STUDY DESIGN: We conducted an observational study utilizing Social Vulnerability Index (SVI) scores to assess the impact of neighborhood disadvantage on a prospectively recruited sample of healthy pregnant women from Washington, DC. Utilizing 79 functional MRI scans from 68 healthy pregnancies at a mean gestational age of 33.12 weeks, we characterized overall functional brain network structure using a graph metric approach. We utilized linear mixed effects models to assess the relationship between SVI and gestational age on five graph metrics, adjusting for multiple scans. RESULTS: Exposure to greater neighborhood disadvantage was associated with less well integrated functional brain networks, as observed by longer characteristic path lengths (L) and diminished global efficiency (GE) as well as diminished small world propensity (SWP). Across gestational ages, however, the association between SVI and network integration diminished to a negligible relationship in the third trimester. Conversely, SWP was significant across pregnancy, but the relationship changed such that there was a negative association with SWP earlier in the second trimester that inverted around the transition to the third trimester to a positive association. CONCLUSIONS: These data directly connect neighborhood disadvantage and altered functional brain maturation in fetuses. Our results suggest that even prior to birth, proximity to environmental stressors in the wider neighborhood environment are associated with altered brain development.

Rapid depletion of "catch-and-release" anti-ASGR1 antibody in vivo.

Targeting antigens with antibodies exhibiting pH/Ca2+-dependent binding against an antigen is an attractive strategy to mitigate target-mediated disposition and antigen buffering. Studies have reported improved serum exposure of antibodies exhibiting pH/Ca2+-binding against membrane-bound receptors. Asialoglycoprotein receptor 1 (ASGR1) is a membrane-bound receptor primarily localized in hepatocytes. With a high expression level of approximately one million receptors per cell, high turnover, and rapid recycling, targeting this receptor with a conventional antibody is a challenge. In this study, we identified an antibody exhibiting pH/Ca2+-dependent binding to ASGR1 and generated antibody variants with increased binding to neonatal crystallizable fragment receptor (FcRn). Serum exposures of the generated anti-ASGR1 antibodies were analyzed in transgenic mice expressing human FcRn. Contrary to published reports of increased serum exposure of pH/Ca2+-dependent antibodies, the pH/Ca2+-dependent anti-ASGR1 antibody had rapid serum clearance in comparison to a conventional anti-ASGR1 antibody. We conducted sub-cellular trafficking studies of the anti-ASGR1 antibodies along with receptor quantification analysis for mechanistic understanding of the rapid serum clearance of pH/Ca2+-dependent anti-ASGR1 antibody. The findings from our study provide valuable insights in identifying the antigens, especially membrane bound, that may benefit from targeting with pH/Ca2+-dependent antibodies to obtain increased serum exposure.

Corrigendum: Translatability of findings from cynomolgus monkey to human suggests a mechanistic role for IL-21 in promoting immunogenicity to an anti-PD-1/IL-21 mutein fusion protein.

[This corrects the article DOI: 10.3389/fimmu.2024.1345473.].

Cellular Neonatal Fc Receptor Recycling Efficiencies can Differentiate Target-Independent Clearance Mechanisms of Monoclonal Antibodies.

In vivo clearance mechanisms of therapeutic monoclonal antibodies (mAbs) encompass both target-mediated and target-independent processes. Two distinct determinants of overall mAb clearance largely separate of target-mediated influences are non-specific cellular endocytosis and subsequent pH-dependent mAb recycling mediated by the neonatal Fc receptor (FcRn), where inter-mAb variability in the efficiency of both processes is observed. Here, we implemented a functional cell-based FcRn recycling assay via Madin-Darby canine kidney type II cells stably co-transfected with human FcRn and its light chain ß2-microglobulin. Next, a series of pH-dependent internalization studies using a model antibody demonstrated proper function of the human FcRn complex. We then applied our cellular assays to assess the contribution of both FcRn and non-specific interactions in the cellular turnover for a panel of 8 clinically relevant mAbs exhibiting variable human pharmacokinetic behavior. Our results demonstrate that the interplay of non-specific endocytosis rates, pH-dependent non-specific interactions, and engagement with FcRn all contribute to the overall recycling efficiency of therapeutic monoclonal antibodies. The predictive capacity of our assay approach was highlighted by successful identification of all mAbs within our panel possessing clearance in humans greater than 5 mL/day/kg. These results demonstrate that a combination of cell-based in vitro assays can properly resolve individual mechanisms underlying the overall in vivo recycling efficiency and non-target mediated clearance of therapeutic mAbs.

Regional homogeneity as a marker of sensory cortex dysmaturity in preterm infants.

Atypical perinatal sensory experience in preterm infants is thought to increase their risk of neurodevelopmental disabilities by altering the development of the sensory cortices. Here, we used resting-state fMRI data from preterm and term-born infants scanned between 32 and 48 weeks post-menstrual age to assess the effect of early ex-utero exposure on sensory cortex development. Specifically, we utilized a measure of local correlated-ness called regional homogeneity (ReHo). First, we demonstrated that the brain-wide distribution of ReHo mirrors the known gradient of cortical maturation. Next, we showed that preterm birth differentially reduces ReHo across the primary sensory cortices. Finally, exploratory analyses showed that the reduction of ReHo in the primary auditory cortex of preterm infants is related to increased risk of autism at 18 months. In sum, we show that local connectivity within sensory cortices has different developmental trajectories, is differentially affected by preterm birth, and may be associated with later neurodevelopment.

Central Autonomic Network and heart rate variability in premature neonates.

INTRODUCTION: The Central Autonomic Network (CAN) is a hierarchy of brain structures that collectively influence cardiac autonomic input, mediating the majority of brain-heart interactions, but has never been studied in premature neonates. In this study, we use heart rate variability (HRV), which has been described as the "primary output" of the CAN, and resting state functional MRI to characterize brain-heart relationships in premature neonates. METHODS: We studied premature neonates who underwent resting state functional MRI (rsfMRI) at term, (37-weeks postmenstrual age [PMA] or above) and had HRV data recorded during the same week of their MRI. HRV was derived from continuous electrocardiogram data during the week of the rsfMRI scan. For rsfMRI, a seed-based approach was used to define regions of interest (ROI) pertinent to the CAN, and blood oxygen level-dependent signal was correlated between each ROI as a measure of functional connectivity. HRV was correlated with CAN connectivity (CANconn) for each region, and sub-group analysis was performed based on sex and clinical comorbidities. RESULTS: Forty-seven premature neonates were included in this study, with a mean gestational age at birth of 28.1 +/- 2.6 weeks. Term CANconn was found to be significantly correlated with HRV in approximately one-fifth of CAN connections. Two distinct patterns emerged among these HRV-CANconn relationships. In the first, increased HRV was associated with stronger CANconn of limbic regions. In the second pattern, stronger CANconn at the precuneus was associated with impaired HRV maturation. These patterns were especially pronounced in male premature neonates. CONCLUSION: We report for the first time evidence of brain-heart relationships in premature neonates and an emerging CAN, most striking in male neonates, suggesting that the brain-heart axis may be more vulnerable in male premature neonates. Signatures in the heart rate may eventually become an important non-invasive tool to identify premature males at highest risk for neurodevelopmental impairment.

Translatability of findings from cynomolgus monkey to human suggests a mechanistic role for IL-21 in promoting immunogenicity to an anti-PD-1/IL-21 mutein fusion protein.

AMG 256 is a bi-specific, heteroimmunoglobulin molecule with an anti-PD-1 antibody domain and a single IL-21 mutein domain on the C-terminus. Nonclinical studies in cynomolgus monkeys revealed that AMG 256 administration led to the development of immunogenicity-mediated responses and indicated that the IL-21 mutein domain of AMG 256 could enhance the anti-drug antibody response directed toward the monoclonal antibody domain. Anti-AMG 256 IgE were also observed in cynomolgus monkeys. A first-in-human (FIH) study in patients with advanced solid tumors was designed with these risks in mind. AMG 256 elicited ADA in 28 of 33 subjects (84.8%). However, ADA responses were only robust and exposure-impacting at the 2 lowest doses. At mid to high doses, ADA responses remained low magnitude and all subjects maintained exposure, despite most subjects developing ADA. Limited drug-specific IgE were also observed during the FIH study. ADA responses were not associated with any type of adverse event. The AMG 256 program represents a unique case where nonclinical studies informed on the risk of immunogenicity in humans, due to the IL-21-driven nature of the response.

Correlation of In Vitro Kinetic Stability to Preclinical In Vivo Pharmacokinetics for a Panel of Anti-PD-1 Monoclonal Antibody Interleukin 21 Mutein Immunocytokines.

The development of therapeutic fusion protein drugs is often impeded by the unintended consequences that occur from fusing together domains from independent naturally occurring proteins, consequences such as altered biodistribution, tissue uptake, or rapid clearance and potential immunogenicity. For therapeutic fusion proteins containing globular domains, we hypothesized that aberrant in vivo behavior could be related to low kinetic stability of these domains leading to local unfolding and susceptibility to partial proteolysis and/or salvage and uptake. Herein we describe an assay to measure kinetic stability of therapeutic fusion proteins by way of their sensitivity to the protease thermolysin. The results indicate that in vivo pharmacokinetics of a panel of anti-programmed cell death protein 1 monocolonal antibody:interleukin 21 immunocytokines in both mice and nonhuman primates are highly correlated with their in vitro susceptibility to thermolysin-mediated proteolysis. This assay can be used as a tool to quickly identify in vivo liabilities of globular domains of therapeutic proteins, thus aiding in the optimization and development of new multispecific drug candidates. SIGNIFICANCE STATEMENT: This work describes a novel assay utilizing protein kinetic stability to identify preclinical in vivo pharmacokinetic liabilities of multispecific therapeutic fusion proteins. This provides an efficient, inexpensive method to ascertain inherent protein stability in vitro before conducting in vivo studies, which can rapidly increase the speed of preclinical drug development.

Experience of early-life pain in premature infants is associated with atypical cerebellar development and later neurodevelopmental deficits.

BACKGROUND: Infants born very and extremely premature (V/EPT) are at a significantly elevated risk for neurodevelopmental disorders and delays even in the absence of structural brain injuries. These risks may be due to earlier-than-typical exposure to the extrauterine environment, and its bright lights, loud noises, and exposures to painful procedures. Given the relative underdeveloped pain modulatory responses in these infants, frequent pain exposures may confer risk for later deficits. METHODS: Resting-state fMRI scans were collected at term equivalent age from 148 (45% male) infants born V/EPT and 99 infants (56% male) born at term age. Functional connectivity analyses were performed between functional regions correlating connectivity to the number of painful skin break procedures in the NICU, including heel lances, venipunctures, and IV placements. Subsequently, preterm infants returned at 18 months, for neurodevelopmental follow-up and completed assessments for autism risk and general neurodevelopment. RESULTS: We observed that V/EPT infants exhibit pronounced hyperconnectivity within the cerebellum and between the cerebellum and both limbic and paralimbic regions correlating with the number of skin break procedures. Moreover, skin breaks were strongly associated with autism risk, motor, and language scores at 18 months. Subsample analyses revealed that the same cerebellar connections strongly correlating with breaks at term age were associated with language dysfunction at 18 months. CONCLUSIONS: These results have significant implications for the clinical care of preterm infants undergoing painful exposures during routine NICU care, which typically occurs without anesthesia. Repeated pain exposures appear to have an increasingly detrimental effect on brain development during a critical period, and effects continue to be seen even 18 months later.

Utility of physiologically based pharmacokinetic modeling to predict inter-antibody variability in monoclonal antibody pharmacokinetics in mice.

In this investigation, we tested the hypothesis that a physiologically based pharmacokinetic (PBPK) model incorporating measured in vitro metrics of off-target binding can largely explain the inter-antibody variability in monoclonal antibody (mAb) pharmacokinetics (PK). A diverse panel of 83 mAbs was evaluated for PK in wild-type mice and subjected to 10 in vitro assays to measure major physiochemical attributes. After excluding for target-mediated elimination and immunogenicity, 56 of the remaining mAbs with an eight-fold variability in the area under the curve (AUC0-672h: 1.74 × 106 -1.38 × 107 ng∙h/mL) and 10-fold difference in clearance (2.55-26.4 mL/day/kg) formed the training set for this investigation. Using a PBPK framework, mAb-dependent coefficients F1 and F2 modulating pinocytosis rate and convective transport, respectively, were estimated for each mAb with mostly good precision (coefficient of variation (CV%) <30%). F1 was estimated to be the mean and standard deviation of 0.961 ± 0.593, and F2 was estimated to be 2.13 ± 2.62. Using principal component analysis to correlate the regressed values of F1/F2 versus the multidimensional dataset composed of our panel of in vitro assays, we found that heparin chromatography retention time emerged as the predictive covariate to the mAb-specific F1, whereas F2 variability cannot be well explained by these assays. A sigmoidal relationship between F1 and the identified covariate was incorporated within the PBPK framework. A sensitivity analysis suggested plasma concentrations to be most sensitive to F1 when F1 > 1. The predictive utility of the developed PBPK model was evaluated against a separate panel of 14 mAbs biased toward high clearance, among which area under the curve of PK data of 12 mAbs was predicted within 2.5-fold error, and the positive and negative predictive values for clearance prediction were 85% and 100%, respectively. MAb heparin chromatography assay output allowed a priori identification of mAb candidates with unfavorable PK.

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.

Ex-utero third trimester developmental changes in functional brain network organization in infants born very and extremely preterm.

Introduction: The latter half of gestation is a period of rapid brain development, including the formation of fundamental functional brain network architecture. Unlike in-utero fetuses, infants born very and extremely preterm undergo these critical maturational changes in the extrauterine environment, with growing evidence suggesting this may result in altered brain networks. To date, however, the development of functional brain architecture has been unexplored. Methods: From a prospective cohort of preterm infants, graph parameters were calculated for fMRI scans acquired prior to reaching term equivalent age. Eight graph properties were calculated, Clustering Coefficient (C), Characteristic Path Length (L), Modularity (Q), Local Efficiency (LE), Global Efficiency (GE), Normalized Clustering (λ), Normalized Path Length (γ), and Small-Worldness (σ). Properties were first compared to values generated from random and lattice networks and cost efficiency was evaluated. Subsequently, linear mixed effect models were used to assess relationship with postmenstrual age and infant sex. Results: A total of 111 fMRI scans were acquired from 85 preterm infants born at a mean GA 28.93 ± 2.8. Infants displayed robust small world properties as well as both locally and globally efficient networks. Regression models found that GE increased while L, Q, λ, γ, and σ decreased with increasing postmenstrual age following multiple comparison correction (r2Adj range 0.143-0.401, p < 0048), with C and LE exhibited trending increases with age. Discussion: This is the first direct investigation on the extra-uterine formation of functional brain architecture in preterm infants. Importantly, our results suggest that changes in functional architecture with increasing age exhibit a different trajectory relative to in utero fetus. Instead, they exhibit developmental changes more similar to the early postnatal period in term born infants.

Robust sex differences in functional brain connectivity are present in utero.

Sex-based differences in brain structure and function are observable throughout development and are thought to contribute to differences in behavior, cognition, and the presentation of neurodevelopmental disorders. Using multiple support vector machine (SVM) models as a data-driven approach to assess sex differences, we sought to identify regions exhibiting sex-dependent differences in functional connectivity and determine whether they were robust and sufficiently reliable to classify sex even prior to birth. To accomplish this, we used a sample of 110 human fetal resting state fMRI scans from 95 fetuses, performed between 19 and 40 gestational weeks. Functional brain connectivity patterns classified fetal sex with 73% accuracy. Across SVM models, we identified features (functional connections) that reliably differentiated fetal sex. Highly consistent predictors included connections in the somatomotor and frontal areas alongside the hippocampus, cerebellum, and basal ganglia. Moreover, high consistency features also implicated a greater magnitude of cross-region connections in females, while male weighted features were predominately within anatomically bounded regions. Our findings indicate that these differences, which have been observed later in childhood, are present and reliably detectable even before birth. These results show that sex differences arise before birth in a manner that is consistent and reliable enough to be highly identifiable.

Gestational age-related changes in the fetal functional connectome: in utero evidence for the global signal.

The human brain begins to develop in the third gestational week and rapidly grows and matures over the course of pregnancy. Compared to fetal structural neurodevelopment, less is known about emerging functional connectivity in utero. Here, we investigated gestational age (GA)-associated in vivo changes in functional brain connectivity during the second and third trimesters in a large dataset of 110 resting-state functional magnetic resonance imaging scans from a cohort of 95 healthy fetuses. Using representational similarity analysis, a multivariate analytical technique that reveals pair-wise similarity in high-order space, we showed that intersubject similarity of fetal functional connectome patterns was strongly related to between-subject GA differences (r = 0.28, P < 0.01) and that GA sensitivity of functional connectome was lateralized, especially at the frontal area. Our analysis also revealed a subnetwork of connections that were critical for predicting age (mean absolute error = 2.72 weeks); functional connectome patterns of individual fetuses reliably predicted their GA (r = 0.51, P < 0.001). Lastly, we identified the primary principal brain network that tracked fetal brain maturity. The main network showed a global synchronization pattern resembling global signal in the adult brain.

A Spontaneous Inversion of the X Chromosome Heterochromatin Provides a Tool for Studying the Structure and Activity of the Nucleolus in Drosophila melanogaster.

The pericentromeric heterochromatin is largely composed of repetitive sequences, making it difficult to analyze with standard molecular biological methods. At the same time, it carries many functional elements with poorly understood mechanisms of action. The search for new experimental models for the analysis of heterochromatin is an urgent task. In this work, we used the Rif1 mutation, which suppresses the underreplication of all types of repeated sequences, to analyze heterochromatin regions in polytene chromosomes of Drosophila melanogaster. In the Rif1 background, we discovered and described in detail a new inversion, In(1)19EHet, which arose on a chromosome already carrying the In(1)sc8 inversion and transferred a large part of X chromosome heterochromatin, including the nucleolar organizer to a new euchromatic environment. Using nanopore sequencing and FISH, we have identified the eu- and heterochromatin breakpoints of In(1)19EHet. The combination of the new inversion and the Rif1 mutation provides a promising tool for studies of X chromosome heterochromatin structure, nucleolar organization, and the nucleolar dominance phenomenon. In particular, we found that, with the complete polytenization of rDNA repeats, the nucleolus consists of a cloud-like structure corresponding to the classical nucleolus of polytene chromosomes, as well as an unusual intrachromosomal structure containing alternating transcriptionally active and inactive regions.

Enteroendocrine cell expression of split-GAL4 drivers bearing regulatory sequences associated with panneuronally expressed genes in Drosophila melanogaster.

In Drosophila melanogaster , hormone-secreting enteroendocrine cells are important for communication from the midgut to other tissues. Many lexA, GAL4, and split-GAL4 drivers that direct gene expression in enteroendocrine cells also confer expression in hormone-secreting cells of the central nervous system. This study examines the midgut expression of selected lexA, GAL4, and split-GAL4 transgenes carrying enhancer fragments previously associated with panneuronal gene expression to assess the experimental usefulness of these drivers for distinguishing the endocrine influences of CNS versus midgut cells on physiological processes.

Modulation of Ligand-Gated Glycine Receptors Via Functional Monoclonal Antibodies.

Ion channels are targets of considerable therapeutic interest to address a wide variety of neurologic indications, including pain perception. Current pharmacological strategies have focused mostly on small molecule approaches that can be limited by selectivity requirements within members of a channel family or superfamily. Therapeutic antibodies have been proposed, designed, and characterized to alleviate this selectivity limitation; however, there are no Food and Drug Administration-approved therapeutic antibody-based drugs targeting ion channels on the market to date. Here, in an effort to identify novel classes of engineered ion channel modulators for potential neurologic therapeutic applications, we report the generation and characterization of six (EC50 < 25nM) Cys-loop receptor family monoclonal antibodies with modulatory function against rat and human glycine receptor alpha 1 (GlyRα1) and/or GlyRα3. These antibodies have activating (i.e., positive modulator) or inhibiting (i.e., negative modulator) profiles. Moreover, GlyRα3 selectivity was successfully achieved for two of the three positive modulators identified. When dosed intravenously, the antibodies achieved sufficient brain exposure to cover their calculated in vitro EC50 values. When compared head-to-head at identical exposures, the GlyRα3-selective antibody showed a more desirable safety profile over the nonselective antibody, thus demonstrating, for the first time, an advantage for GlyRα3-selectivity. Our data show that ligand-gated ion channels of the glycine receptor family within the central nervous system can be functionally modulated by engineered biologics in a dose-dependent manner and that, despite high protein homology between the alpha subunits, selectivity can be achieved within this receptor family, resulting in future therapeutic candidates with more desirable drug safety profiles. SIGNIFICANCE STATEMENT: This study presents immunization and multiplatform screening approaches to generate a diverse library of functional antibodies (agonist, potentiator, or inhibitory) raised against human glycine receptors (GlyRs). This study also demonstrates the feasibility of acquiring alpha subunit selectivity, a desirable therapeutic profile. When tested in vivo, these tool molecules demonstrated an increased safety profile in favor of GlyRα3-selectivity. These are the first reported functional GlyR antibodies that may open new avenues to treating central nervous system diseases with subunit selective biologics.

FetalGAN: Automated Segmentation of Fetal Functional Brain MRI Using Deep Generative Adversarial Learning and Multi-Scale 3D U-Net.

An important step in the preprocessing of resting state functional magnetic resonance images (rs-fMRI) is the separation of brain from non-brain voxels. Widely used imaging tools such as FSL's BET2 and AFNI's 3dSkullStrip accomplish this task effectively in children and adults. In fetal functional brain imaging, however, the presence of maternal tissue around the brain coupled with the non-standard position of the fetal head limit the usefulness of these tools. Accurate brain masks are thus generated manually, a time-consuming and tedious process that slows down preprocessing of fetal rs-fMRI. Recently, deep learning-based segmentation models such as convolutional neural networks (CNNs) have been increasingly used for automated segmentation of medical images, including the fetal brain. Here, we propose a computationally efficient end-to-end generative adversarial neural network (GAN) for segmenting the fetal brain. This method, which we call FetalGAN, yielded whole brain masks that closely approximated the manually labeled ground truth. FetalGAN performed better than 3D U-Net model and BET2: FetalGAN, Dice score = 0.973 ± 0.013, precision = 0.977 ± 0.015; 3D U-Net, Dice score = 0.954 ± 0.054, precision = 0.967 ± 0.037; BET2, Dice score = 0.856 ± 0.084, precision = 0.758 ± 0.113. FetalGAN was also faster than 3D U-Net and the manual method (7.35 s vs. 10.25 s vs. ∼5 min/volume). To the best of our knowledge, this is the first successful implementation of 3D CNN with GAN on fetal fMRI brain images and represents a significant advance in fully automating processing of rs-MRI images.

Development of an immunoassay for aglycosylated murine IgG1 in mouse serum via generation of a specific tool antibody.

Aim: To develop a method for the quantitation of effector functionless mouse surrogate IgG1 drug molecules in mouse matrices. Materials & methods: A panel of antibodies that bound specifically to N297G mutation-containing mouse IgG molecules was generated in rats. The panel was screened to identify an antibody that could be used as both the capture and detection reagent in an electrochemiluminescent immunoassay. Results & conclusion: The quantitative assay developed with the N297G-specific antibody passed acceptance criteria across multiple IgG1 fragment crystallizable (Fc)-containing protein formats and provides accurate quantitation of the total levels of mouse surrogate protein Fc present in in vivo mouse serum samples. These results are useful in understanding drug integrity and the development of precise pharmacokinetic/pharmacodynamic relationships.