Search Results (1,688)

Background/Objectives: Multiple sclerosis (MS) constitutes a chronic autoimmune, inflammatory, and neurodegenerative disease, with dissemination in space and time, warranting diagnosis. Epstein–Barr virus (EBV) is increasingly recognized as a key contributor to MS pathogenesis. This review summarizes evidence on EBV-related mechanisms of currently approved disease-modifying therapies (DMTs) and emerging EBV-directed therapeutic strategies in MS. Methods: A systematic search of PubMed, Embase, Cochrane, and Web of Science was performed. Original English-language studies addressing EBV-related therapeutic mechanisms or EBV-targeted interventions in MS were included; 23 studies met the inclusion criteria. Results: Current DMTs may influence EBV-related immunity through diverse mechanisms, including modulation of B-cell subsets, altered lymphocyte trafficking, reduction in EBV-specific humoral responses, and restoration of T-cell surveillance. Monoclonal antibody-based therapies, particularly anti-CD20 agents and natalizumab, appear to affect the EBV–B-cell–immune axis through distinct but complementary mechanisms. Other interventions, including interferons, glatiramer acetate, dimethyl fumarate, autologous hematopoietic stem cell transplantation, and vitamin D supplementation, may also modulate EBV-specific cellular or humoral responses, although the magnitude and durability of these effects vary. Emerging EBV-directed approaches, including EBV-specific T-cell therapy, inhibition of specific proteins, modulation of autophagy, and cholesterol-dependent viral latency, provide additional support for targeting EBV-related pathways in MS. Conclusions: The therapeutic efficacy of DMTs in MS may extend beyond nonspecific immunomodulation and involve partial disruption of EBV-driven immune persistence. Further controlled studies are required to validate EBV-related biomarkers and determine whether direct EBV-targeted therapies can provide sustained clinical benefit. Full article

Currently, the most commonly used methods for detecting Mycoplasma are the culture method and the indicator cell culture method. However, both approaches exhibit low sensitivity and are incapable of detecting low-concentration contamination. In addition, the detection period may extend up to 28 days, which is unsuitable for rapid screening and may delay timely contamination control measures. To address these limitations, a Mycoplasma detection method based on nucleic acid amplification technology (NAT) was developed following a comparative analysis of gene sequences from various Mycoplasma species. The method was validated with respect to its detection performance and its applicability to biological product samples. DNA was extracted from Mycoplasma-contaminated samples using a magnetic bead-based nucleic acid extraction method. Universal primers were designed based on the highly conserved 16S rRNA gene sequence of Mycoplasma, and amplification was performed using multiplex quantitative PCR (qPCR) with fluorescent probes. The limit of detection (LOD) was established based on statistics of 24 replicates. Method specificity and robustness were evaluated according to the guidelines set by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH Q2), while sample applicability was assessed in accordance with the European Pharmacopoeia (EP) <2.6.7>. The NAT-based Mycoplasma detection method enabled rapid, qualitative identification of Mycoplasma contamination. The validated LOD was 10 CFU/mL, and the method met predefined requirements for sensitivity, specificity, and robustness. To assess applicability, real biological product samples, including monoclonal antibodies, antibody fusion proteins, bispecific antibodies, and trispecific antibodies, were spiked with 10 CFU/mL of standard Mycoplasma strains. All spiked samples tested positive. These findings confirm that the NAT-based Mycoplasma detection method is suitable for process control and product release testing in the production of biological products. Full article

Background/Objectives: Parkinson’s disease (PD) is a major neurodegenerative disorder with no cure. The goal is to develop an active immunotherapy targeting aggregated alpha synuclein (aSyn), the root cause of PD. TRB-001 is the lead candidate of a novel class of vaccines. It is a peptide/protein conjugate coupled to sugar residues, which is used to target and activate antigen-presenting cells, and addresses aSyn. Methods: A 33-year-old male, diagnosed with PD seven years previously, with a Hoehn & Yahr stage of 1, taking Levodopa/Benserazide (100/25 mg, 6× per day), Rotigotine (8 mg) and Rasagiline (1 mg), amounting to a Levodopa equivalent daily dose (LEDD) of 940 mg, also complicated by impulse control disorder, requested experimental therapy. He received a total of four TRB-001 administrations (weeks 0, 4, 8 and 34) following informed consent. The workup included safety, immunological and clinical parameters. Results: Vaccinations were well tolerated. They induced a high-titer aSyn-specific antibody (Ab) response. Titer increase was associated with a reduction in aSyn plasma levels, suggesting target engagement. The Ab titer and the reduction in aSyn plasma levels were both long-lived. The boost elicited a recall-type Ab titer increase and triggered avidity maturation (factor 7.8). Abs demonstrated a high degree of selectivity for aggregated aSyn (factor 30). Moreover, they were found to preferentially react with tissue from PD brain lysates. The Movement Disorder Society-Sponsored Unified Parkinson’s Disease Rating Scale (MDS-UPDRS) score for the patient remained essentially stable throughout the observation period of 53 weeks. At the time of the boost, the symptomatic PD therapy was simplified to Levodopa/Carbidopa/Entacapone 100/25/200 mg four times a day, amounting to an LEDD of 532 mg. This put an end to the symptoms of the impulse control disorder. Conclusions: Results obtained suggest that this new class of vaccines may yield Ab responses comparable in magnitude and target avidity to the therapeutic setting of monoclonal Abs. TRB-001 is currently being translated to a randomized, placebo-controlled Phase 1B study. Full article

Neuroinflammation is increasingly recognized as a key modulator of therapeutic response and adverse events in Alzheimer’s disease (AD), especially during anti-amyloid-β (Aβ) monoclonal antibody (Aβ-mAb) treatment. We applied longitudinal translocator protein (TSPO) positron emission tomography (PET) to evaluate TSPO-associated neuroinflammatory responses to chronic Aβ-mAb therapy and their modulation by the peroxisome proliferator-activated receptor γ (PPARγ) agonist pioglitazone. App^NL-G-F knock-in mice underwent TSPO-PET and Aβ-PET imaging at 5, 7.5, and 10 months of age across four treatment arms: placebo, Aβ-mAb, pioglitazone, and combination therapy. TSPO-PET detected early and progressive neuroinflammatory responses to Aβ-mAb that appeared lower with pioglitazone co-treatment. Both mono- and combination therapy were associated with altered temporal and spatial dynamics of the TSPO-PET signal. In addition, we applied a previously validated microglia desynchronization index based on TSPO-PET connectivity, which captured individual variation in regional TSPO-PET organization and correlated with cognitive performance. Together, TSPO-PET and its regional synchronicity can quantify longitudinal, region-specific treatment effects, which may help differentiate harmful from adaptive neuroinflammatory responses. These findings highlight the potential of TSPO-PET as a stratification biomarker to optimize therapeutic interventions. TSPO-PET therefore enables in vivo tracking of treatment-associated neuroinflammatory responses during anti-Aβ immunotherapy and provides a non-invasive framework for evaluating combination strategies targeting amyloid pathology and immune regulation in AD. Full article

Purpose: Lymph node (LN) excision is critical in oncologic surgery to provide important therapeutic and diagnostic information. LN evaluation helps in staging cancers, predicting prognosis and improving survival. The ultimate wish of a surgical oncologist would be to localize and dissect all pathologically positive LNs while avoiding the morbidity of removing true negative LNs. The goal of our study was to identify a reliable marker for clinical prediction of LNs with cancer cells from non-small cell lung cancer (NSCLC) versus LNs without. We identified epithelial cell adhesion molecule (EpCAM), a membrane protein normally expressed in epithelial tissues, including in lung. Patients and Methods: We used human specimens immunostained with anti-EpCAM monoclonal antibody. Results: EpCAM was expressed in NSCLC metastasis to LNs, as shown in 74 positive LNs from patients with resected primary NSCLC. Among pathologically negative LNs, regardless of PET avidity, EpCAM was absent; whereas among pathologically positive LNs, all PET uptake groups exhibited high EpCAM positivity. Together, these biomarkers had a 100% accuracy. There was no difference in expression between hilar and mediastinal LNs, nor between primary tumor histology. Conclusions: EpCAM may be useful for the surgical oncologist for preoperative or intraoperative detection of positive LNs from NSCLC. Full article

The pharmaceutical industry has seen significant growth in the development of antibody-based therapeutics, especially monoclonal antibodies (mAbs) and bispecific antibodies (bsAbs), used in the treatment of cancer and neurodegenerative diseases. However, their production and purification remain challenging. It is difficult to achieve both high product yield and the strict purity required for clinical use. Downstream processing is expensive and often involves trade-offs between efficiency and product quality. In addition, current purification methods do not fully remove contaminants, especially host cell proteins, residual DNA, and protein aggregates, affecting the safety and effectiveness of the final product. Recent advances in purification technologies, such as improved chromatography techniques and alternative separation methods, have shown promise in addressing some of these limitations. Process optimization and the integration of continuous manufacturing approaches are being explored to enhance efficiency and scalability. Furthermore, increased regulatory expectations are driving the need for more robust and reproducible purification strategies. As the antibody therapeutics market continues to expand, optimizing manufacturing and purification processes is crucial to achieve cost efficiency and large-scale production. This article discusses the main challenges in antibody production and downstream purification, focusing on monoclonal and bispecific antibodies, and compares current strategies to increase yield, improve purity, and reduce contaminants. Full article

Saposin-like protein 2 (SAP2) exhibits strong immunogenicity as an antigen for immunodiagnosis in ruminant and human fasciolosis. Most available immunodiagnostic test kits are based on polyclonal and monoclonal antibodies against antigens from Fasciola spp. Previous studies demonstrated that polyclonal and monoclonal antibodies against SAP2 showed high specificity and could effectively detect Fasciola spp. infections at an early stage. However, polyclonal antibodies are extremely difficult to produce, and quality control is not possible during production; the procedure also involves considerable financial investment. To overcome these problems, we developed a single-chain variable fragment (scFv) to control quality in each production cycle and reduce the cost of manufacturing immunodiagnostic kits. Our objectives were to produce and characterize an scFv that binds the SAP2 from the liver fluke Fasciola gigantica. We constructed the scFv by genetic engineering: we cloned immunoglobulin genes and linked them with flexible polypeptide linkers composed of repeating glycine and serine residues. We selected an scFv with high affinity for binding SAP2 using the phage-display technique and produced it using a prokaryotic expression system. The scFv was characterized via in silico and in vitro methods to confirm its specificity for SAP2, including IMGT/V-QUEST, IMGT/Collier-de-Perles, HADDOCK 2.4, ELISA, immunoblotting, and immunohistochemistry. The scFv was successfully produced and purified using Ni-NTA affinity chromatography. The purified scFvFgSAP2 was approximately 27 kDa, as confirmed by SDS-PAGE and immunoblot analysis. An indirect ELISA and immunoblotting indicated that scFvFgSAP2 had strong reactivity with F. gigantica compared to other parasite species. Moreover, immunolocalization of scFvFgSAP2 confirmed that it binds specifically to natural SAP2 in the cecal epithelium cells of F. gigantica. Therefore, this scFv targeting SAP2 is an effective material and can be used to develop immunodiagnostic procedures. Full article

Cancer stem cells (CSCs) are a distinct subpopulation within a tumor that play an important role in tumor initiation, metastasis, therapeutic resistance, and cancer relapse. Their persistence is strongly influenced by the tumor microenvironment (TME), which provides a range of biological signals that maintain stemness, promote immune evasion, and resistance to cancer treatment. Therefore, effective targeting of CSCs is essential to improve therapeutic efficacy. In this review, we summarize the key characteristics of CSCs and their niche within the TME, emphasizing their interactions with immune cells, stromal components, and secreted factors. We also discuss the major challenges in targeting CSCs, including immune evasion, metabolic constraints, and intratumoral heterogeneity. We further highlight current and emerging immunotherapeutic strategies targeting CSCs, including immune checkpoint inhibitors, cancer vaccines, monoclonal antibodies, nanobodies, bispecific antibodies, antibody-drug conjugates (ADCs), CAR-T and CAR-NK cell therapies, oncolytic viruses, as well as innovative approaches such as targeted protein degradation. Finally, we emphasize the importance of a combinatorial approach that integrates CSCs targeting with modulation of the TME. Together, these strategies may lead to more durable responses, enhance therapy efficacy and reduce the risk of tumor recurrence. Full article

Background/Objectives: The envelope (E) protein of orthoflaviviruses contains antigenic sites that are composed of one or more epitopes, which can vary in antigenic specificity, including between viral species, strains, and even substrains. Monoclonal antibodies (mAbs) that bind these epitopes vary in functionality based on their specificity. This makes mAbs useful to study the differences in phenotypes between strains of viruses, such as the wild type (WT) and live attenuated vaccine strains of yellow fever virus (YFV). mAb 2D12 was raised against the 17D-204 YFV vaccine substrain virus (YF VAX^®) by Schlesinger et al. in 1983. However, it only neutralizes Asibi WT virus, not the 17D-204 vaccine substrain virus. Results: We confirmed these results and demonstrated that mAb 2D12 fails to neutralize all 17D vaccine substrains (17D-204, 17DD, and 17D-213), indicating that the minor differences between these virus substrains do not affect the epitope or functionality of mAb 2D12. In addition, mAb 2D12 was found to neutralize WT strain of French viscerotropic virus (FVV), with statistically indistinguishable neutralization from the WT strain Asibi. All but one of the live attenuated French neurotropic vaccine (FNV) derivative viruses had significantly lower neutralization than WT strains Asibi and FVV. FVV, Asibi, 17D, and FNV have many amino acid differences in the membrane (M) and E proteins. It is unclear which of them contributes to this differential neutralization. However, FNV and 17D have common amino acid substitutions from WT FVV and Asibi at positions M-36 and E-331, suggesting that one or both of these residues may contribute to the 2D12 epitope. Conclusions: Overall, mAb 2D12 is a valuable tool to distinguish WT virulent strains of YFV from live attenuated vaccine strains. Full article

Reversion of amino acid mutations in structural proteins is common in viral evolution. SARS-CoV-2 provides an unprecedented opportunity for ecological studies, thanks to the abundance of available whole genome sequences. YoyoMut allows regular scanning of open SARS-CoV-2 data, reporting on all cyclic and reverting mutations within all proteins (including Spike), with fine-grained trend visualization distinguishing non-mutated from mutated positions (either fixated or cyclically reversed). In the whole CoVSpectrum database, order of 100 reverting and 50 fixated mutations were identified on Spike. Classification is determined using alternative algorithms (based on threshold or slope inversion); finally, a 3D-protein structure allows us to identify spatial clustering of adjacent mutated positions. Systematic, automated monitoring of these behaviors aids immunologists and structuralists in their manual curation. By generating informative reports, our tool supports daily activities that have practical implications for vaccine and therapeutic anti-Spike monoclonal antibody design: prioritizing analysis of cyclic mutation and reversion models could help avoid the recent failures in their development and inform future strategies. Full article

Lymphocyte activation gene-3 (LAG-3) is a pivotal immune checkpoint receptor that exerts a negative regulatory effect on T-cell function. Although LAG-3-blocking antibodies have shown promising clinical potential, the inherent limitations of conventional monoclonal antibodies necessitate the development of novel antibody formats with enhanced biological and pharmacological properties. In this study, a panel of single-domain antibodies (sdAbs) targeting human LAG-3 was generated via phage display technology. Among these candidates, 2H-G7 was identified as a high-affinity sdAb that binds to LAG-3 with an equilibrium dissociation constant (K_D) in the nanomolar range. Notably, 2H-G7 potently blocks the interactions of LAG-3 with both of its key ligands, fibrinogen-like protein 1 (FGL1) and major histocompatibility complex class II (MHC-II). Its capacity to restore impaired T-cell function was validated by quantifying interleukin-2 (IL-2) secretion and CD69 expression in stimulated primary human peripheral blood mononuclear cells (PBMCs). Epitope mapping studies localized the binding site of 2H-G7 to the D1D2 extracellular domains of LAG-3, distinct from relatlimab, a clinically approved LAG-3-blocking antibody serving as the benchmark. In a xenogeneic mouse model of non-small-cell lung cancer (NSCLC), 2H-G7-Fc exhibited superior tumor growth inhibition efficacy compared with relatlimab. These findings demonstrate that 2H-G7 is a promising lead candidate for the development of next-generation LAG-3-targeted tumor immunotherapies. Full article

Nowadays, optical fiber-based biosensors are widely used in various fields, particularly in medical diagnostics and the selection of appropriate treatments for certain diseases. One example is cerebral ischemic disease, where many biomarkers are released and provide valuable information about the severity and progression of the disease. In this study, a long-period fiber grating (LPFG) biosensor was developed using a standard single-mode optical fiber and monoclonal antibody (IL-6 mAb) as the biological recognition element to detect IL-6, which is a protein associated with the inflammatory process. The assembly of the LPFG biosensor was characterized through optical and electronic microscopy to observe morphological changes at different stages of fabrication and the detection process. Additionally, micro-infrared spectroscopy was employed to identify functional groups in the protein region linked to the presence of IL-6. Experimental data were analyzed using principal component analysis, confirming the biosensor’s ability to detect IL-6 and providing insights into its fabrication process. Full article

Background: Stromal remodeling in the tumor microenvironment contributes to multiple myeloma (MM) progression and drug resistance, but the extracellular mediators that drive these changes remain incompletely defined. Extracellular enolase-1 (ENO1), including membrane-associated and secreted forms, has been implicated in tumor progression; however, its role in cancer-associated fibroblast (CAF)-associated stromal reprogramming in MM is unclear. Methods: The effects of extracellular ENO1 on stromal activation and tumor-supportive functions were examined in MM using MM–bone marrow stromal cell (BMSC) co-cultures, lactate production and viability assays, immunoblotting, cytokine analyses, and a subcutaneous xenograft model of bortezomib (BTZ)-resistant MM in male 6–7-week-old NOD.Cg-Prkdc^scid Il2rg^tm1Vst/Vst (NPG) mice. HuL001, an anti-ENO1 monoclonal antibody, was used to evaluate the therapeutic relevance of extracellular ENO1 targeting. Results: Extracellular ENO1 promoted fibroblast activation protein expression through plasmin-mediated transforming growth factor-β (TGF-β) activation and induced a CAF-associated stromal phenotype marked by enhanced glycolytic activity and increased secretion of tumor-promoting cytokines in MM-BMSC co-cultures. HuL001 suppressed these ENO1-driven effects. HuL001-pretreated stromal cells also exhibited reduced tumor-supportive activity in a BTZ-resistant MM xenograft model. In addition, HuL001 combined with lenalidomide overcame BTZ resistance in MM. Conclusions: Extracellular ENO1 drives CAF-associated stromal reprogramming in the MM microenvironment through the ENO1/plasminogen/plasmin/TGF-β axis. Therapeutic targeting of extracellular ENO1 with HuL001 may disrupt these tumor-supportive stromal activities and help overcome drug resistance in MM. Full article

Advanced gastric (GAC) or gastroesophageal junction (GEJAC) adenocarcinoma continues to carry a poor prognosis. Understanding GAC/GEJAC at the molecular level has provided a new understanding and the basis for individualized approaches to treatment. The current biomarker-driven therapy focuses on four areas: microsatellite instability (MSI), human epidermal growth factor receptor-2 (HER2), programmed death ligand-1 (PD-L1) combined positive score, and claudin 18.2 (CLDN18.2). However, because of improving technology, the focus has shifted to cancer cell-surface proteins and peptides. Each of these GAC/GEJAC subgroups provides a different treatment pathway. The agents utilized to treat advanced GAC/GEJAC include immune checkpoint inhibitors (ICIs), chemotherapy, monoclonal antibodies (mAbs), and antibody–drug conjugate (ADC) therapy, as well as bispecific antibodies (BsAbs), but they are certainly not limited to the above. Drug development has shifted in recent years to establish different mechanisms that are attempting more sophisticated and targeted approaches, such as BsAbs and ADCs. Meanwhile, the development of cytotoxics has tapered off. Along with these developments in drug therapy, more therapies directed at CLDN18.2, HER2, MSI, EGFR, HER3 and trophoblast cell-surface antigen 2 (TROP2) are underway. Here we review future areas in advanced GAC, including zanidatamab’s potential role in HER2-positive advanced GAC and deciphering the abundance of anti-CLDN18.2, extending beyond investigative therapies. Full article

The continued evolution of SARS-CoV-2 has enabled an escape from most monoclonal antibodies, yet a subset of broadly neutralizing antibodies targeting three newly identified super-conserved RBD epitopes—SCORE-A, SCORE-B, and SCORE-C—retains remarkable activity against even the most recent JN.1-derived sublineages. Here, we employed an integrated computational framework combining conformational dynamics, mutational scanning, MM-GBSA binding energetics, and frustration profiling to dissect the molecular mechanisms by which XGI antibodies achieve broad neutralization and resistance to immune escape. Structural analysis revealed that all three SCORE epitopes share a common architecture: a highly conserved, minimally frustrated core that provides stable anchoring, flanked by peripheral regions that accommodate antibody-specific variations. Conformational dynamics showed that SCORE-A antibodies (XGI-183) rigidify the lateral epitope while leaving the RBM partially mobile; SCORE-B antibodies (XGI-198, XGI-203) clamp the RBM apex, directly blocking ACE2; and SCORE-C antibodies (XGI-171) allosterically loosen the RBM loop, impairing receptor engagement indirectly. Mutational scanning identified a hierarchical hotspot organization where primary hotspots (e.g., K356, T500, Y380, T385) are evolutionarily constrained and minimally frustrated, while secondary hotspots (e.g., V503, Y508, S383) are neutrally frustrated and represent the principal sites of immune-driven mutations. MM-GBSA decomposition revealed that van der Waals-driven hydrophobic packing dominates binding, with electrostatic interactions providing auxiliary stabilization. Critically, frustration analysis demonstrated that immune escape hotspots reside precisely in zones of neutral frustration—“energetic playgrounds” that permit mutational exploration without destabilizing the RBD—while minimally frustrated cores are evolutionarily locked. The comparative analysis of conformational versus mutational frustration distributions revealed a unifying principle: aligned neutral frustration yields permissive, escape-prone interfaces; decoupling enables the targeting of constrained cores; and the convergence of minimal frustration in both distributions creates invulnerable interfaces. These findings establish that broad neutralization arises not from ultra-high-affinity anchors but from strategic energy distribution across rigid, evolutionarily informed interfaces, providing a roadmap for designing next-generation therapeutics that target the invulnerable cores of viral surface proteins. Full article