Rescue of a lysosomal storage disorder caused by Grn loss of function with a brain penetrant progranulin biologic.

GRN mutations cause frontotemporal dementia (GRN-FTD) due to deficiency in progranulin (PGRN), a lysosomal and secreted protein with unclear function. Here, we found that Grn-/- mice exhibit a global deficiency in bis(monoacylglycero)phosphate (BMP), an endolysosomal phospholipid we identified as a pH-dependent PGRN interactor as well as a redox-sensitive enhancer of lysosomal proteolysis and lipolysis. Grn-/- brains also showed an age-dependent, secondary storage of glucocerebrosidase substrate glucosylsphingosine. We investigated a protein replacement strategy by engineering protein transport vehicle (PTV):PGRN-a recombinant protein linking PGRN to a modified Fc domain that binds human transferrin receptor for enhanced CNS biodistribution. PTV:PGRN rescued various Grn-/- phenotypes in primary murine macrophages and human iPSC-derived microglia, including oxidative stress, lysosomal dysfunction, and endomembrane damage. Peripherally delivered PTV:PGRN corrected levels of BMP, glucosylsphingosine, and disease pathology in Grn-/- CNS, including microgliosis, lipofuscinosis, and neuronal damage. PTV:PGRN thus represents a potential biotherapeutic for GRN-FTD.

Does dual-energy abdominal computed tomography increase the radiation dose to patients: a prospective observational study.

PURPOSE: The aim of our study was to compare single-energy (SECT) and dual-energy (DECT) abdominal computed tomography (CT) examinations in matched patient cohorts regarding the differences in effective radiation dose (ERD) and image quality performed in a third-generation dual-source computed tomography (DSCT) scanner. MATERIAL AND METHODS: Our study included 100 patients, who were divided randomly into 2 groups. The patients included in Group A were scanned by SECT, and Group B members were scanned by DECT. Volume CT dose index (CTDIvol), dose length product (DLP), and ERD for venous phase acquisition were recorded in each patient and were normalised for 40 cm. Analyses were performed by using statistical software (SPSS version 20.0 for windows), and Bonferroni correction for multiple comparisons was applied for p-values and confidence intervals. RESULTS: Average ERD based on DLP values normalised for 40 cm acquisition were obtained for both Group A and Group B. The mean ERD for Group A was 11.89 mSv, and for group B it was 6.87 mSv. There was a significant difference in these values between Group A and Group B as shown by a p-value of < 0.001. On subjective and objective analysis, there was no statistically significant difference in image quality between the 2 groups. CONCLUSIONS: The protocols in third-generation DSCT using dual-energy mode resulted in significant reductions in the effective radiation dose (by approximately 58%) compared to SECT in routine abdominal examination in matched cohorts. Therefore, the quantitative imaging potential of DECT can be utilised in needed patients with decreased radiation dose in third-generation DSCT.

High-Throughput Liquid Chromatography-Tandem Mass Spectrometry Quantification of Glycosaminoglycans as Biomarkers of Mucopolysaccharidosis II.

We recently developed a blood-brain barrier (BBB)-penetrating enzyme transport vehicle (ETV) fused to the lysosomal enzyme iduronate 2-sulfatase (ETV:IDS) and demonstrated its ability to reduce glycosaminoglycan (GAG) accumulation in the brains of a mouse model of mucopolysaccharidosis (MPS) II. To accurately quantify GAGs, we developed a plate-based high-throughput enzymatic digestion assay coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to simultaneously measure heparan sulfate and dermatan sulfate derived disaccharides in tissue, cerebrospinal fluid (CSF) and individual cell populations isolated from mouse brain. The method offers ultra-high sensitivity enabling quantitation of specific GAG species in as low as 100,000 isolated neurons and a low volume of CSF. With an LOD at 3 ng/mL and LLOQs at 5-10 ng/mL, this method is at least five times more sensitive than previously reported approaches. Our analysis demonstrated that the accumulation of CSF and brain GAGs are in good correlation, supporting the potential use of CSF GAGs as a surrogate biomarker for brain GAGs. The bioanalytical method was qualified through the generation of standard curves in matrix for preclinical studies of CSF, demonstrating the feasibility of this assay for evaluating therapeutic effects of ETV:IDS in future studies and applications in a wide variety of MPS disorders.

Discovery and bio-optimization of human antibody therapeutics using the XenoMouse® transgenic mouse platform.

Since the late 1990s, the use of transgenic animal platforms has transformed the discovery of fully human therapeutic monoclonal antibodies. The first approved therapy derived from a transgenic platform--the epidermal growth factor receptor antagonist panitumumab to treat advanced colorectal cancer--was developed using XenoMouse(®) technology. Since its approval in 2006, the science of discovering and developing therapeutic monoclonal antibodies derived from the XenoMouse(®) platform has advanced considerably. The emerging array of antibody therapeutics developed using transgenic technologies is expected to include antibodies and antibody fragments with novel mechanisms of action and extreme potencies. In addition to these impressive functional properties, these antibodies will be designed to have superior biophysical properties that enable highly efficient large-scale manufacturing methods. Achieving these new heights in antibody drug discovery will ultimately bring better medicines to patients. Here, we review best practices for the discovery and bio-optimization of monoclonal antibodies that fit functional design goals and meet high manufacturing standards.

Engineering an IgG Scaffold Lacking Effector Function with Optimized Developability.

IgG isotypes can differentially bind to Fcγ receptors and complement, making the selection of which isotype to pursue for development of a particular therapeutic antibody important in determining the safety and efficacy of the drug. IgG2 and IgG4 isotypes have significantly lower binding affinity to Fcγ receptors. Recent evidence suggests that the IgG2 isotype is not completely devoid of effector function, whereas the IgG4 isotype can undergo in vivo Fab arm exchange leading to bispecific antibody and off-target effects. Here an attempt was made to engineer an IgG1-based scaffold lacking effector function but with stability equivalent to that of the parent IgG1. Care was taken to ensure that both stability and lack of effector function was achieved with a minimum number of mutations. Among the Asn297 mutants that result in lack of glycosylation and thus loss of effector function, we demonstrate that the N297G variant has better stability and developability compared with the N297Q or N297A variants. To further improve the stability of N297G, we introduced a novel engineered disulfide bond at a solvent inaccessible location in the CH2 domain. The resulting scaffold has stability greater than or equivalent to that of the parental IgG1 scaffold. Extensive biophysical analyses and pharmacokinetic (PK) studies in mouse, rat, and monkey further confirmed the developability of this unique scaffold, and suggest that it could be used for all Fc containing therapeutics (e.g. antibodies, bispecific antibodies, and Fc fusions) requiring lack of effector function or elimination of binding to Fcγ receptors.

Biological Characterization of a Stable Effector Functionless (SEFL) Monoclonal Antibody Scaffold in Vitro.

The stable effector functionLess (SEFL) antibody was designed as an IgG1 antibody with a constant region that lacks the ability to interact with Fcγ receptors. The engineering and stability and pharmacokinetic assessments of the SEFL scaffold is described in the accompanying article (Jacobsen, F. W., Stevenson, R., Li, C., Salimi-Moosavi, H., Liu, L., Wen, J., Luo, Q., Daris, K., Buck, L., Miller, S., Ho, S-Y., Wang, W., Chen, Q., Walker, K., Wypych, J., Narhi, L., and Gunasekaran, K. (2017) J. Biol. Chem 292). The biological properties of these SEFL antibodies were assessed in a variety of human and cynomolgus monkey in vitro assays. Binding of parent molecules and their SEFL variants to human and cynomolgus monkey FcγRs were evaluated using flow cytometry-based binding assays. The SEFL variants tested showed decreased binding affinity to human and cynomolgus FcγRs compared with the wild-type IgG1 antibody. In addition, SEFL variants demonstrated no antibody-dependent cell-mediated cytotoxicity in vitro against Daudi cells with cynomolgus monkey peripheral blood mononuclear cells, and had minimal complement-dependent cytotoxicity activity similar to that of the negative control IgG2 in a CD20+ human Raji lymphoma cell line. SEFL mutations eliminated off-target antibody-dependent monocyte phagocytosis of cynomolgus monkey platelets, and cynomolgus platelet activation in vitro These experiments demonstrate that the SEFL modifications successfully eliminated Fc-associated effector binding and functions.

A novel antibody engineering strategy for making monovalent bispecific heterodimeric IgG antibodies by electrostatic steering mechanism.

Producing pure and well behaved bispecific antibodies (bsAbs) on a large scale for preclinical and clinical testing is a challenging task. Here, we describe a new strategy for making monovalent bispecific heterodimeric IgG antibodies in mammalian cells. We applied an electrostatic steering mechanism to engineer antibody light chain-heavy chain (LC-HC) interface residues in such a way that each LC strongly favors its cognate HC when two different HCs and two different LCs are co-expressed in the same cell to assemble a functional bispecific antibody. We produced heterodimeric IgGs from transiently and stably transfected mammalian cells. The engineered heterodimeric IgG molecules maintain the overall IgG structure with correct LC-HC pairings, bind to two different antigens with comparable affinity when compared with their parental antibodies, and retain the functionality of parental antibodies in biological assays. In addition, the bispecific heterodimeric IgG derived from anti-HER2 and anti-EGF receptor (EGFR) antibody was shown to induce a higher level of receptor internalization than the combination of two parental antibodies. Mouse xenograft BxPC-3, Panc-1, and Calu-3 human tumor models showed that the heterodimeric IgGs strongly inhibited tumor growth. The described approach can be used to generate tools from two pre-existent antibodies and explore the potential of bispecific antibodies. The asymmetrically engineered Fc variants for antibody-dependent cellular cytotoxicity enhancement could be embedded in monovalent bispecific heterodimeric IgG to make best-in-class therapeutic antibodies.

Asymmetrical Fc engineering greatly enhances antibody-dependent cellular cytotoxicity (ADCC) effector function and stability of the modified antibodies.

Antibody-dependent cellular cytotoxicity (ADCC) is mediated through the engagement of the Fc segment of antibodies with Fcγ receptors (FcγRs) on immune cells upon binding of tumor or viral antigen. The co-crystal structure of FcγRIII in complex with Fc revealed that Fc binds to FcγRIII asymmetrically with two Fc chains contacting separate regions of the FcγRIII by utilizing different residues. To fully explore this asymmetrical nature of the Fc-FcγR interaction, we screened more than 9,000 individual clones in Fc heterodimer format in which different mutations were introduced at the same position of two Fc chains using a high throughput competition AlphaLISA® assay. To this end, we have identified a panel of novel Fc variants with significant binding improvement to FcγRIIIA (both Phe-158 and Val-158 allotypes), increased ADCC activity in vitro, and strong tumor growth inhibition in mice xenograft human tumor models. Compared with previously identified Fc variants in conventional IgG format, Fc heterodimers with asymmetrical mutations can achieve similar or superior potency in ADCC-mediated tumor cell killing and demonstrate improved stability in the CH2 domain. Fc heterodimers also allow more selectivity toward activating FcγRIIA than inhibitory FcγRIIB. Afucosylation of Fc variants further increases the affinity of Fc to FcγRIIIA, leading to much higher ADCC activity. The discovery of these Fc variants will potentially open up new opportunities of building the next generation of therapeutic antibodies with enhanced ADCC effector function for the treatment of cancers and infectious diseases.

First record of plastiglomerates, pyroplastics and plasticrusts along the beaches of Tamilnadu, Southeast coast of India.

Plastic litter affects coastal and marine ecosystems globally. This study represents the first record of pyroplastics and plasticrust in the beaches of Tamil Nadu, India. All samples were FTIR spectroscopically examined to confirm the polymer composition of the suspected plastics. The 16 plastic formations were found in TamilNadu, including six plastiglomerates nine pyroplastics and one plasticrust. Five types of polymers (PET, PP, PVC, PA, and PE) were found on the plastic matrices. The study also revealed that pyroplastics and plasticrust formed by degradation of plastics through weathering in the coastal environment. The present study also found that four types of marine fouling organisms such as oyster larvae, bryozoan, barnacle and polychaete worm were encrusted on the two pyroplastics. The emergence of these new forms of plastic raises concerns about their interactions with the environment and biota.

Cigarette butt pollution in popular beaches of Morocco: Abundance, distribution, and mitigation measures.

Cigarette butts pose a significant challenge in managing solid litter, with an estimated 4.5 trillion of them being discarded into the environment annually. This study investigated the pollution of eleven beaches along the Moroccan Mediterranean by cigarette butts compared to other types of marine litter between 2018 and 2023. Sampling was conducted using the visual survey technique according to a standardized protocol. A total of 50, 575 items were collected with cigarette butts (14.62 %) and plastic caps/lids drinks (10.93 %) being the most common. Our survey of eleven beaches revealed 7395 cigarette butts, giving an average density of 0.06/m2 comparable to other countries in the Mediterranean. The analysis of the results shows significant differences in the abundance cigarette butts according to the beach typology, seasonality, sediment type, and number of beach users. Moreover, the study discovered a decrease in beach pollution during the COVID-19 pandemic lockdown, resulting in less litter collected compared to pre- and post-pandemic periods. The Cigarette Butt Pollution Index was categorized as very low pollution in M'diq and Kaa Asrass, low pollution in Ksar Sghir, Fnideq, Amsa, Oued Laou, significant pollution in Martil and Nador and sever pollution in Saidia and Sababia. The CBPI was higher during summer and winter, significantly associated with the density levels of beach users. The study recommends immediate action by the local administration to prevent the potential pollution of groundwater and sand by toxic substances leached from cigarette butts.

Role of advanced (magnetic resonance) neuroimaging and clinical outcomes in neonatal strokes: Experience from tertiary care center.

Neonatal strokes constitute a major cause of pediatric mortality and morbidity. Neuroimaging helps in its diagnosis as well as prognostication. However, advanced imaging, including magnetic resonance imaging (MRI), carries multiple challenges. Limited data exists in the literature on imaging-based predictors of neurological outcomes in neonatal stroke in the Indian population. In this study, we reviewed our available data on neonatal stroke patients between 2015 and 2020 for clinico-radiological patterns. During this period, 17 neonatal strokes were admitted and the majority were term births with a slight male preponderance. Seizures and encephalopathy were the most common presentation. Multiple maternal risk factors such as gestational diabetes, meconium-stained liquor, APLA syndrome, fever, deranged coagulation profile, oligohydramnios, cord prolapse, and non-progressive labor were seen. Cardiac abnormalities were seen in only less than half of these patients with the most common finding being atrial septal defects (ASD). Transcranial ultrasound was performed in eight neonates and the pick-up rate of ultrasound was poor. MR imaging showed large infarcts in 11 patients. The MCA territory was most commonly involved. Interestingly, five neonates had venous thrombosis with three showing it in addition to arterial thrombosis. Associated ictal, as well as Wallerian changes, were noted in 10. Although large territorial infarcts were the most common pattern, non-contrast MR angiography did not show major vessel occlusion in these cases. Outcomes were fairly good and only three patients had a residual motor deficit at 1 year. No recurrence of stroke was seen in any of the neonates.

Personal protective equipment (PPE) pollution driven by the COVID-19 pandemic in coastal environment, Southeast Coast of India.

The rise in the use of single-use plastics and personal protective equipment (PPE) has increased plastic waste in the marine environment. In this study, we surveyed the presence of PPE (face masks and gloves) discharged in 6 beaches along the coast of India. A total of 496 PPE were counted with an average density of 1.08 × 10-3 PPE m-2. The PPE density found was comparable to previous studies. Face masks were the most recorded type of PPE (98.39%), with gloves accounting for only 1.61% of the total. However, a significant reduction in the appearance of PPE was recorded on all six beaches, likely due to the increase in vaccination rates. The most contaminated places were the beaches with recreational activities + fishing. It has been noticed that the lack of awareness of environmental pollution and the negligence of the population and the mismanagement of municipal waste are the main causes of beach pollution by PPE. This study confirms the potential threat of PPE to terrestrial and aquatic organisms of multiple taxa in India, but further studies are needed to quantify the impact of this type of waste on marine animals.

Molecular architecture determines brain delivery of a transferrin receptor-targeted lysosomal enzyme.

Delivery of biotherapeutics across the blood-brain barrier (BBB) is a challenge. Many approaches fuse biotherapeutics to platforms that bind the transferrin receptor (TfR), a brain endothelial cell target, to facilitate receptor-mediated transcytosis across the BBB. Here, we characterized the pharmacological behavior of two distinct TfR-targeted platforms fused to iduronate 2-sulfatase (IDS), a lysosomal enzyme deficient in mucopolysaccharidosis type II (MPS II), and compared the relative brain exposures and functional activities of both approaches in mouse models. IDS fused to a moderate-affinity, monovalent TfR-binding enzyme transport vehicle (ETV:IDS) resulted in widespread brain exposure, internalization by parenchymal cells, and significant substrate reduction in the CNS of an MPS II mouse model. In contrast, IDS fused to a standard high-affinity bivalent antibody (IgG:IDS) resulted in lower brain uptake, limited biodistribution beyond brain endothelial cells, and reduced brain substrate reduction. These results highlight important features likely to impact the clinical development of TfR-targeting platforms in MPS II and potentially other CNS diseases.

Histidine residue mediates radical-induced hinge cleavage of human IgG1.

Hydroxyl radicals induce hinge cleavage in a human IgG1 molecule via initial radical formation at the first hinge Cys(231) followed by electron transfer to the upper hinge residues. To enable engineering of a stable monoclonal antibody hinge, we investigated the role of the hinge His(229) residue using structure modeling and site-directed mutagenesis. Direct involvement of His(229) in the reaction mechanism is suggested by a 75-85% reduction of the hinge cleavage for variants in which His(229) was substituted with either Gln, Ser, or Ala. In contrast, mutation of Lys(227) to Gln, Ser, or Ala increased hinge cleavage. However, the H229S/K227S double mutant shows hinge cleavage levels similar to that of the single H229S variant, further revealing the importance of His(229). Examination of the hinge structure shows that His(229) is capable of forming hydrogen bonds with surrounding residues. These observations led us to hypothesize that the imidazole ring of His(229) may function to facilitate the cleavage by forming a transient radical center that is capable of extracting a proton from neighboring residues. The work presented here suggests the feasibility of engineering a new generation of monoclonal antibodies capable of resisting hinge cleavage to improve product stability and efficacy.

Enhancing antibody Fc heterodimer formation through electrostatic steering effects: applications to bispecific molecules and monovalent IgG.

Naturally occurring IgG antibodies are bivalent and monospecific. Bispecific antibodies having binding specificities for two different antigens can be produced using recombinant technologies and are projected to have broad clinical applications. However, co-expression of multiple light and heavy chains often leads to contaminants and pose purification challenges. In this work, we have modified the CH3 domain interface of the antibody Fc region with selected mutations so that the engineered Fc proteins preferentially form heterodimers. These novel mutations create altered charge polarity across the Fc dimer interface such that coexpression of electrostatically matched Fc chains support favorable attractive interactions thereby promoting desired Fc heterodimer formation, whereas unfavorable repulsive charge interactions suppress unwanted Fc homodimer formation. This new Fc heterodimer format was used to produce bispecific single chain antibody fusions and monovalent IgGs with minimal homodimer contaminants. The strategy proposed here demonstrates the feasibility of robust production of novel Fc-based heterodimeric molecules and hence broadens the scope of bispecific molecules for therapeutic applications.

Brain delivery and activity of a lysosomal enzyme using a blood-brain barrier transport vehicle in mice.

Most lysosomal storage diseases (LSDs) involve progressive central nervous system (CNS) impairment, resulting from deficiency of a lysosomal enzyme. Treatment of neuronopathic LSDs remains a considerable challenge, as approved intravenously administered enzyme therapies are ineffective in modifying CNS disease because they do not effectively cross the blood-brain barrier (BBB). We describe a therapeutic platform for increasing the brain exposure of enzyme replacement therapies. The enzyme transport vehicle (ETV) is a lysosomal enzyme fused to an Fc domain that has been engineered to bind to the transferrin receptor, which facilitates receptor-mediated transcytosis across the BBB. We demonstrate that ETV fusions containing iduronate 2-sulfatase (ETV:IDS), the lysosomal enzyme deficient in mucopolysaccharidosis type II, exhibited high intrinsic activity and degraded accumulated substrates in both IDS-deficient cell and in vivo models. ETV substantially improved brain delivery of IDS in a preclinical model of disease, enabling enhanced cellular distribution to neurons, astrocytes, and microglia throughout the brain. Improved brain exposure for ETV:IDS translated to a reduction in accumulated substrates in these CNS cell types and peripheral tissues and resulted in a complete correction of downstream disease-relevant pathologies in the brain, including secondary accumulation of lysosomal lipids, perturbed gene expression, neuroinflammation, and neuroaxonal damage. These data highlight the therapeutic potential of the ETV platform for LSDs and provide preclinical proof of concept for TV-enabled therapeutics to treat CNS diseases more broadly.

Extended disordered proteins: targeting function with less scaffold.

It has been estimated that a large fraction of cellular proteins are natively disordered. Current opinion largely holds that natively disordered proteins are more 'adaptive', leading to advantages in regulation and in binding diverse ligands. Here, we argue for another, simple, physically based reason. Disordered proteins often have large intermolecular interfaces, the size of which is dictated by protein function. For proteins to be stable as monomers with extensive interfaces, protein size would need to be 2-3 times larger. This would either increase cellular crowding or enlarge the size of the cell by 15-30%, owing to the increase in the sequence length. Smaller sizes of cells, proteins, DNA and RNA conserve energy. Thus, disordered proteins provide a simple yet elegant solution to having large intermolecular interfaces, but with smaller protein, genome and cell sizes.

How different are structurally flexible and rigid binding sites? Sequence and structural features discriminating proteins that do and do not undergo conformational change upon ligand binding.

Proteins are dynamic molecules and often undergo conformational change upon ligand binding. It is widely accepted that flexible loop regions have a critical functional role in enzymes. Lack of consideration of binding site flexibility has led to failures in predicting protein functions and in successfully docking ligands with protein receptors. Here we address the question: which sequence and structural features distinguish the structurally flexible and rigid binding sites? We analyze high-resolution crystal structures of ligand bound (holo) and free (apo) forms of 41 proteins where no conformational change takes place upon ligand binding, 35 examples with moderate conformational change, and 22 cases where a large conformational change has been observed. We find that the number of residue-residue contacts observed per-residue (contact density) does not distinguish flexible and rigid binding sites, suggesting a role for specific interactions and amino acids in modulating the conformational changes. Examination of hydrogen bonding and hydrophobic interactions reveals that cases that do not undergo conformational change have high polar interactions constituting the binding pockets. Intriguingly, the large, aromatic amino acid tryptophan has a high propensity to occur at the binding sites of examples where a large conformational change has been noted. Further, in large conformational change examples, hydrophobic-hydrophobic, aromatic-aromatic, and hydrophobic-polar residue pair interactions are dominant. Further analysis of the Ramachandran dihedral angles (phi, psi) reveals that the residues adopting disallowed conformations are found in both rigid and flexible cases. More importantly, the binding site residues adopting disallowed conformations clustered narrowly into two specific regions of the L-Ala Ramachandran map. Examination of the dihedral angles changes upon ligand binding shows that the magnitude of phi, psi changes are in general minimal, although some large changes particularly between right-handed alpha-helical and extended conformations are seen. Our work further provides an account of conformational changes in the dihedral angles space. The findings reported here are expected to assist in providing a framework for predicting protein-ligand complexes and for template-based prediction of protein function.

Sequence and structure analysis of parallel beta helices: implication for constructing amyloid structural models.

Increasing evidence suggests that amyloids and parallel beta helices may share similar motifs. A systemic analysis of beta helices is performed to examine their sequence and structural characteristics. Ile prefers to occur in beta strands. In contrast, Pro is disfavored, compatible with the underlying assumption in Pro-scanning mutagenesis. Cys, Asn, and Phe form significant homostacking (identical amino acid interactions). Asn is highly conserved in the high-energy, left-handed alpha-helical conformation, where it frequently forms amide stacking. Based on the observed prominent stacking of chemically similar residues in parallel beta helices, we propose that within the "cross-beta" framework, amyloids with longer peptide chains may have common structural features of in-register, parallel alignment, with the side chains forming identical amino acid ladders. The requirement of ladder formation limits the combinations of side chain interactions. Such a limit combined with environmental conditions (e.g., pH, concentration) could be a major reason for the ability of most polypeptides to form amyloids.