Search Results (4,200)

Background: Neuropathic pain remains difficult to treat because current analgesics often provide insufficient efficacy or dose-limiting adverse effects. Nav1.7 is genetically validated as a key regulator of human pain sensation, but the development of selective small-molecule Nav1.7 inhibitors has been limited by the high similarity among voltage-gated sodium channel subtypes. Methods: We generated monoclonal antibodies selectively targeting Nav1.7, humanized them for therapeutic development, and evaluated their binding, selectivity, functional channel inhibition, systemic analgesic efficacy, and effects on neuronal activity in a rat model of partial sciatic nerve ligation-induced neuropathic pain. Results: The humanized antibodies showed high-affinity and selective binding to Nav1.7 and functionally inhibited the channel in cellular assays. After systemic administration to neuropathic pain model rats, the lead antibody produced robust analgesia lasting at least 96 h. Electrophysiological analyses demonstrated reduced mechanically evoked and spontaneous neuronal activity, and immunohistochemistry showed decreased mechanical stimulus-induced phosphorylation of extracellular signal-regulated kinase in dorsal root ganglion neurons. The antibodies did not impair physiological nociception or motor function under the tested conditions. Conclusions: These findings provide preclinical proof of concept that humanized anti-Nav1.7 antibodies can act as systemically administered, long-acting biologic analgesics for neuropathic pain while preserving normal nociceptive and motor functions. The clinical advancement of S-151128 further supports the translational potential of this modality. Full article

H9N2 avian influenza viruses inherently carry cross-species transmission potential, making continuous surveillance critical for pandemic prevention. This study focused on monitoring the 2024 H9N2 epidemic in Jiangsu Province’s external environment, analyzing its molecular evolution and receptor binding properties, assessing cross-species transmission and pandemic risks, and investigating serological antibody levels across different human populations. Environmental samples were collected from live poultry markets, farms, slaughterhouses, and bird habitats across Jiangsu, screened via quantitative PCR (qPCR), with positive samples used for virus isolation and whole-genome sequencing. Receptor binding properties were tested by hemagglutination assay, and H9N2 antibody levels were measured in 370 occupationally exposed individuals and 240 non-exposed individuals using hemagglutination inhibition (HI) assays. Among the 5779 collected samples, 6.89% tested H9N2-positive, and 12 strains belonging to the Eurasian lineage Y280-like clade G57 genotype were successfully isolated. All strains carried the HA-Q226L mutation, with 11 showing preferential binding to human α-2,6 receptors and one strain possessing dual receptor binding capability. Internal genes harbored mammalian adaptation mutations, and M2 proteins contained mutations conferring complete resistance to amantadine-class antiviral drugs. Serological tests revealed antibody positive rates of 4.05% in exposed populations and 2.5% in non-exposed populations, with no statistically significant difference between groups. These findings confirm that Jiangsu’s circulating H9N2 viruses have acquired human receptor preference and mammalian adaptation, posing silent infection and pandemic risks. Enhanced surveillance and the development of candidate vaccine stockpiles are strongly recommended. Full article

Proteins are the most common targets for antibody discovery and vaccine development, but their sequence variability can limit the breadth of resulting antigens. Lipids represent an alternative class of antigens due to their structural conservation and roles in host–pathogen interactions. Here, we describe the development and optimization of an in vitro antibody selection workflow using lipid-containing nanodiscs as antigen presentation platforms to enable phage and yeast display selections under conditions adapted for these non-protein targets. Lipopolysaccharide (LPS) nanodiscs were first used as a model system to evaluate selection strategies, including competitive and subtractive approaches to reduce non-specific binders, yielding peptide and single-chain variable fragment (scFv) binders that were affinity matured to improve binding signals. The same approach was subsequently used to select scFv antibodies that recognize lipid nanodiscs prepared from Yersinia pestis membrane lipid extracts. These antibodies show binding to lipid nanodiscs derived from Y. pestis, with evidence of selectivity relative to control nanodiscs. Overall, this work establishes a workflow for antibody selection against lipid-containing nanodisc antigens and highlights practical considerations associated with these targets. The approach may be useful for generating affinity reagents to membrane-associated lipids, although further characterization is required to define antigen specificity and functional activity. Full article

A therapeutic antibody, CSL305, has been developed, which combines inhibition of the complement classical and lectin pathways via complement C2 binding with an ability to act as an antagonist of the neonatal Fc receptor (FcRn). CSL305 binds to human C2 (huC2) but shows no binding or activity against mouse C2 precluding its use in mouse models of disease to fully assess in vivo efficacy. To circumvent this, a mouse strain was developed that replaced the expression of mouse C2 with huC2 by homologous recombination. These mice (huC2 “knock-in”; KI) were shown to express huC2 protein and to have complement activity. Interestingly, male huC2-KI mice showed much stronger complement activity compared to female mice and were also sensitive to inhibition by CSL305. Two models of disease using male huC2-KI mice were then used to assess the in vivo efficacy of CSL305. The first was an attenuated passive anti-glomerular basement membrane (GBM) glomerulonephritis model involving complement activation as its primary mechanism of action. CSL305 showed dose-dependent inhibition of disease as measured by urine albumin, with reductions in kidney cellular infiltration and plasma C3 cleaved fragments C3b/C3c/iC3b also observed. The second model was a collagen autoantibody-induced arthritis (CAIA) mouse model. Here, CSL305 showed a significant and dose-dependent inhibition of clinical score in both prophylactic and therapeutic settings, mediated exclusively via its FcRn mechanism of action. Although the animal models used in this study were found to preclude the demonstration of a synergistic effect on both mechanisms, CSL305 does act in vivo as both a complement inhibitor and as a FcRn antagonist to ameliorate disease. Full article

4-Chlorophenoxyacetic acid (4-CPA), a synthetic auxin analog, is employed in agriculture both as a plant growth regulator and as a constituent of herbicide formulations. Consequently, the establishment of simple and rapid detection methods is essential for effective environmental monitoring. This study reports the first development of a homogeneous fluorescence polarization immunoassay (FPIA) for the determination of 4-CPA. The monoclonal antibody (M1), raised against 4-CPA, was evaluated as a recognition element. Furthermore, two fluorescently labeled 4-CPA tracers—with ethylenediamine fluorescein thiocarbamate and aminohexylaminocarbonylfluorescein—were synthesized and purified, and their structures were unequivocally confirmed by high-performance liquid chromatography coupled with high-resolution mass spectrometric detection (HPLC-HRMS). Optimal concentrations of monoclonal antibodies and tracers were established, yielding a limit of detection of 1.2 ng/mL. The assay demonstrated a broad dynamic range of 2.3–300 ng/mL and a rapid analysis time of 15 min. Validation via the standard addition method in authentic open water samples resulted in recovery rates of 98–112%. To address the cross-reactivity with the prevalent herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), two novel strategies were devised and successfully implemented. The first approach involves the concurrent execution of two separate FPIAs—one for 2,4-D and one for 4-CPA—followed by the mathematical resolution of two analyte concentrations from the two measured binding values. The second strategy entails the preliminary selective removal of 2,4-D from sample matrices using affinity chromatography columns with immobilized anti-2,4-D antibodies prior to FPIA for 4-CPA. These proposed methodologies appear highly promising for overcoming the inherent limitations of traditional immunoassays when faced with significant cross-reactivity among structurally analogous compounds. Full article

Galectins are β-galactosyl-binding lectins with key roles in immune regulation and as pattern recognition receptors. To address their potential role(s) in viral infection of mucosal epithelia we currently investigate adhesion and entry mechanisms of the infectious hematopoietic necrosis virus (IHNV) using the zebrafish (Danio rerio) model system. We previously reported the recognition of IHNV envelope glycoprotein by the zebrafish galectin Drgal1-L2 and its inhibitory activity for viral adhesion to epithelial cells. Subsequently, we determined the structure of Drgal1-L2 and proposed a mechanism for Drgal1-mediated inhibition of IHNV spike fusion to the host epithelial cell. We now show that Drgal1 can also hinder viral adhesion and infection by binding to glycans on the host cell surface and epidermal mucus. We identified fibronectin, the reported IHNV receptor, as the cell surface glycoprotein recognized by Drgal1-L2. Surprisingly, IHNV also adhered in vitro to purified β1integrin, and pre-exposure of either IHNV or the immobilized β1integrin to Drgal1-L2 hindered IHNV adhesion. Binding of either anti-fibronectin or anti-β1integrin antibodies to the cell surface partially inhibited IHNV adherence. Drgal1-L2 also hindered IHNV adhesion by binding to mucus glycans. Taken together, our results suggest complementary mechanisms by which Drgal1-L2 may protect mucosal epithelial cells against IHNV infection and tentatively identify β1integrin as a novel receptor for IHNV. Full article

Background: The dynamics of humoral immune responses following primary and booster COVID-19 vaccinations are crucial to understand in order to optimize vaccination strategies. This study evaluates the magnitude and durability of SARS-CoV-2-specific IgG antibody responses across different vaccines in a large cohort of Bangladeshi adults. Methods: A total of 6300 adults from nine hospitals across eight divisions of Bangladesh were enrolled. Participants received two primary doses of either ChAdOx1 nCoV-19 (Covishield, Serum Institute of India, n = 2855), mRNA-1273 (Moderna, n = 578), BNT162b2 (Pfizer-BioNTech, n = 121), or Vero-cell-inactivated (Sinopharm, n = 2746) vaccines. Booster doses were administered at one-year intervals post-primary vaccination. SARS-CoV-2 spike receptor-binding domain (RBD)-specific IgG antibody responses were measured by ELISA using serum from vaccinees at multiple time points after two primary and two booster doses. Results: A total of 3745 individuals received booster 1 (third dose), with 59% receiving heterologous boosters (a different vaccine regimen than the primary doses). Only 5.5% (n = 347) of participants received a second booster one year after the first booster (among them, 99% received BNT162b2). Our results suggest that heterologous boosters with the mRNA vaccine induced higher IgG levels than homologous boosters for individuals who received primary vaccination with adenovirus vector-based ChAdOx1 nCoV-19 or a Vero-cell-inactivated vaccine. However, in those who initially received the mRNA-based vaccine, both homologous and heterologous boosters produced comparable IgG responses. Among all vaccine types, booster immunization with the Vero-cell-inactivated vaccine induced the lowest antibody responses. Longitudinal analysis demonstrated significantly high IgG levels over the 12 months following the first booster (p < 0.0001); however, IgG levels declined significantly after the second booster dose (fourth dose). Conclusions: Heterologous boosting strategies, particularly those involving mRNA vaccines, elicit stronger and more sustained IgG responses compared to a homologous booster. However, antibody waning after the second booster highlights the need for continued monitoring and potential additional vaccine strategies. Full article

Gastric cancer remains a major clinical challenge, underscoring the need for more effective drug delivery strategies. Approximately 10–20% of gastric cancers overexpress HER2, conferring aggressive tumor characteristics and poor survival, yet resistance to trastuzumab-based targeted therapy and limited intratumoral antibody penetration continue to restrict clinical outcomes. This study evaluated HER2-targeted exosomes as a delivery platform. Exosomes were engineered to express the p51 peptide, a high-affinity HER2-binding ligand, and loaded with 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), a potent HSP90 inhibitor. The cellular uptake and antitumor efficacy of p51-Exo^17-DMAG were assessed in vitro using NCI-N87 and AGS cells and in vivo using a mouse xenograft model. p51-modified exosomes exhibited superior HER2 specific uptake. Treatment with p51-Exo^17-DMAG significantly increased apoptosis, as demonstrated by elevated PARP and caspase3 cleavage, and downregulated oncogenic signaling molecules, including p-AKT, CDK2, VEGF, and c-Myc. Furthermore, p51-Exo^17-DMAG increased the number of TUNEL-positive cells. In the NCI-N87 xenograft model, systemic administration of p51-Exo^17-DMAG significantly inhibited tumor growth without toxicity or histological damage to major organs. Tumor analysis confirmed increased apoptosis and reduced proliferation in vivo. These findings demonstrate that p51-engineered exosomes provide an efficient, selective, and safe platform for HER2-targeted delivery of 17-DMAG, offering a promising precision medicine strategy for HER2-positive gastric cancer. Full article

Background: Although prolonged inflammatory symptoms are an infrequent and problematic adverse effect of vaccination that can occur in some people, the transient activation of acute phase reactants (APRs) is expected with adjuvanted vaccines and helps to potentiate immune responses. Methods: This experiment examined the association between vaccine reactogenicity and immunogenicity in monkeys immunized with an adjuvanted recombinant protein including a receptor binding domain–human IgG1-Fc fusion protein (RBD-Fc) sequenced from the ancestral Wuhan strain of SARS-CoV-2. The acute inflammatory reaction to immunization was assessed by determining the decline in serum iron levels at 24 h and the increase in the neutrophil-to-lymphocyte ratio (NLR) as the adherent neutrophil pool trafficked into circulation. Results: Robust primary and secondary antibody responses were elicited. Larger decreases in serum iron and higher NLRs were associated with a stronger inhibition of RBD binding with angiotensin-converting enzyme (ACE2) when five early viral variants of SARS-CoV-2 were tested, including Wuhan, Alpha, Beta, Gamma and Delta. Inhibition of ACE2-RBD binding was less evident when the Omicron variant was tested. Individual variation in the APR was also predictive of the persistence of cell-mediated immunity based on the number of interferon-expressing mononuclear cells activated by viral antigen in ELISpot assays. Conclusions: Rapid antibody responses to primary immunization and large secondary responses to booster immunizations were elicited by this adjuvanted recombinant RBD-Fc vaccine, and our analysis affirmed the view that a transient APR can enhance antibody binding with antigen proteins. Full article

Acute kidney injury (AKI) caused by ischemia–reperfusion injury (IRI) involves rapid activation of innate immune responses, in which myeloid-derived immune cells critically shape injury severity. Y-box binding protein 1 (YB-1) regulates pro-inflammatory gene expression intracellularly and can be secreted to function extracellularly, yet how these two compartments jointly influence early IRI pathology remains poorly understood. To dissect the roles of intracellular myeloid versus extracellular YB-1, we subjected myeloid-specific Ybx1 knockout, Ybx1^fl/fl × LysM^cre, mice and wild-type (WT) littermates to unilateral renal IRI following administration of either a neutralizing anti-YB-1 antibody or control IgG. Kidney injury, inflammation, immune cell recruitment, neutrophil extracellular trap (NET) formation, antibody localization, and Fcγ receptor expression were assessed by qRT-PCR, histology, immunostaining, Western blotting, and flow cytometry. Myeloid-specific knockout of Ybx1 markedly reduced renal inflammation, neutrophil infiltration, NET formation, and tubular injury. This protective phenotype was lost when extracellular YB-1 was simultaneously reduced: anti-YB-1 treatment in knockout mice restored pro-inflammatory cytokine expression, increased tubular damage markers such as NGAL and KIM-1, exacerbated neutrophil recruitment and NET formation, and led to luminar accumulation of YB-1/anti-YB-1 immune complexes in tubular cells. Mechanistically, Ybx1-deficient myeloid cells exhibited significantly reduced CD16 expression, pointing to impaired Fcγ receptor-mediated phagocytosis as the cause of defective immune complex clearance. In contrast, wild-type mice efficiently cleared extracellular YB-1 complexes and showed no injury aggravation upon antibody treatment. Our findings identify myeloid YB-1 as a central regulator of early inflammatory injury in renal IRI and reveal that its protective depletion becomes pathogenic when extracellular YB-1 is simultaneously neutralized, likely due to unmasked defects in immune complex clearance. Full article

Antibody-drug conjugates (ADCs) have transitioned from clinically marginal agents into a defining therapeutic class for solid tumor oncology. In DESTINY-Breast03, trastuzumab deruxtecan achieved a four-fold progression-free survival advantage over trastuzumab emtansine, attributable not to antibody engineering but to the linker-payload axis: a cleavable peptide linker and a topoisomerase I payload with bystander activity. Sacituzumab govitecan extends the same logic to Trop-2-positive disease via extracellular payload release, and the framework now spans breast, urothelial, gynecologic, lung, gastric, and colorectal cancers, with enfortumab vedotin plus pembrolizumab displacing platinum chemotherapy as first-line therapy for urothelial cancer in EV-302 (median overall survival 31.5 versus 16.1 months). This review synthesizes ADC biology along three analytical axes. The mechanistic axis links each linker-payload-DAR configuration to a specific tumor-biology barrier: vascular limitation, which delivers approximately 0.1% of the administered dose to tumor tissue; the binding-site barrier, which concentrates exposure at the perivascular margin; and antigen mosaicism, which defeats internalization-dependent killing. The translational axis examines resistance as a coordinated failure across antigen modulation, trafficking, efflux, apoptotic execution, and lysosomal processing. The clinical axis traces the platform’s migration toward earlier-line and curative-intent settings. We close by examining whether the ADC delivery architecture translates to precision immunosuppression in autoimmune disease, where the glucocorticoid receptor modulator ADC ABBV-154 met placebo-controlled efficacy endpoints in rheumatoid arthritis but was discontinued because its benefit-risk profile did not differentiate it from existing biologic therapies. Full article

Monoclonal antibodies (mAbs) have been the subject of extensive study in recent years due to their recognition as highly promising therapeutic molecules offering high specificity and a low risk of side effects. Monitoring the structure of these molecules is crucial for developing new therapeutics, characterizing interactions with antigens or receptors, and explaining potential changes in activity between antibody production batches. However, commonly used biophysical approaches provide only low-spatial-resolution information, and conventional structural biology techniques such as crystallography and cryo-electron microscopy (cryo-EM) are difficult to apply to these highly dynamic proteins. Solution nuclear magnetic resonance (NMR) spectroscopy is the method of choice for structural studies of flexible proteins at atomic resolution; however, it has traditionally been limited to low-molecular-weight biological systems. In this review, we present recent advances in NMR spectroscopy and advanced isotopic labeling methods that have enabled the atomic-resolution study of both the crystallizable (Fc) and antigen-binding (Fab) fragments of antibodies. We show how NMR is becoming a powerful tool for investigating full-length mAbs at an atomic level, opening up new possibilities for the characterization and in-depth quality control of therapeutic antibodies in solution. Full article

Hemolytic Uremic Syndrome (HUS) is a severe clinical manifestation primarily triggered by Shiga toxin-producing Escherichia coli (STEC). While Shiga toxins (Stx) are central to the development of systemic endothelial damage, current recombinant antibody developments have overwhelmingly focused on neutralizing the Stx2 subtype. However, numerous STEC isolates produce Stx1 either independently or alongside Stx2, revealing a critical need to diversify the antibody repertoire for comprehensive antitoxin therapies. To address this, we characterized two novel, fully human recombinant Fabs targeting Stx1 (FabB6:Stx1 and FabC8:Stx1) selected from a synthetic library via phage display. We evaluated their binding specificity and neutralizing activity in Vero and human proximal tubular epithelial (HK-2) cells, as well as in primary human glomerular endothelial cells (HGEC exposed to HUS-derived STEC supernatants. Both Fabs exhibited high specificity and nanomolar affinity for Stx1. Notably, they displayed cell-type-dependent neutralization profiles, with FabC8:Stx1 demonstrating superior and more consistent neutralization in HK-2 cells. Crucially, when evaluated alongside previously characterized anti-Stx2 antibodies (FabC11:Stx1/Stx2 and FabF8:Stx2), the Stx1-specific Fabs conferred complementary protection against clinical STEC isolates. These findings support the inclusion of Stx1-targeting recombinant antibodies into broader multi-toxin neutralization strategies, thereby expanding the therapeutic potential against STEC-associated diseases. Full article

Background: Gynecological cancers include collections of cancers with diverse cellular and molecular characteristics that often develop drug resistance, making them treatment-resistant. Biomolecule–drug conjugates (BDCs), especially antibody–drug conjugates (ADCs), have revolutionized the targeted therapy of cancer; however, the creation of these entities has so far been achieved by empirical, resource-intensive design methods. Objective: The aim of this review is to critically analyze how AI can be used for the rational design and optimization of high-affinity BDCs for gynecological cancer treatment. Methods and discussion: Recent advances in machine learning (ML)- and deep learning (DL)-based methods to predict biomolecule-target binding affinity, structural compatibility, linker stability, payload selection, trafficking in the cell, and biomolecule resistance mechanisms are summarized. The review also explores the possibilities for incorporation of structural, chemical, biological, and multi-omics data to enhance specificity, efficacy, and safety of conjugates. Besides antibody-based systems, AI-assisted design approaches with peptides, aptamers, and hybrid biomolecular systems are also included. This review also highlights parameters and experimental/numerical validation restrictions related to data quality, interpretability of models, regulatory aspects, etc. Conclusions: AI-based conjugate engineering is increasingly moving BDC development from a largely ‘trial and error’ approach to a more predictive and data-driven approach. While there are still challenges to be addressed in terms of translations and validations, the potential of AI approaches in the field of precision oncology and the development of more personalized treatment is promising in the context of gynecological cancers. Full article

Single-domain antibodies (sdAbs) are the smallest antigen-binding antibody (Ab) fragments (12–15 kDa) and have emerged as a versatile therapeutic platform. Their compact size, high solubility, stability, and ability to access cryptic epitopes distinguish them from conventional monoclonal Abs (mAbs) and larger Ab fragments. These properties are particularly attractive in ophthalmology, where molecular size, tissue penetration, and formulation constraints critically influence therapeutic performance. This narrative review summarizes the structural features, engineering strategies, immunogenicity considerations, and production platforms of sdAbs, with a focus on ocular applications. Preclinical studies demonstrate promising efficacy in retinal vascular diseases through targeting of VEGFA, ANG2, TNFα, and complement components, as well as in inflammatory and anterior segment disorders. SdAbs can be formatted as multimeric or Fc-fused constructs to extend intraocular half-life or delivered via gene therapy vectors as a sustained intraocular “biofactory” approach. Notably, recent work demonstrates the feasibility of vector-encoded sdAbs targeting complement C3 in vivo. While challenges remain regarding immunogenicity, pharmacokinetics, and regulatory pathways, the approval of several sdAb-based drugs in other fields underscores their clinical potential. SdAbs represent a promising next-generation modality for ocular therapeutics, enabling innovative strategies beyond conventional antibody formats. Full article