Search Results (299)

Hepatitis C virus (HCV) still lacks a licensed vaccine. The envelope glycoprotein E2 is a key neutralizing target, but its dense N-glycan shield can hinder epitope exposure. In this study, we revisit E2 glycan editing and examine whether single-site deletion preserves antigen integrity while improving immune responses in mice under a DNA immunization setting. Using a secreted E2 ectodomain (sE2384–661), we generated five N to D mutants at conserved sites (N1, N2, N4, N6, and N11) and evaluated them in a unified DNA immunization model with identical CpG content and delivery conditions across groups. The N2 mutant (N423, sE2-N2) maintained expression, secretion, and ER localization; furthermore, in mice, it was associated with higher anti-E2 titers and greater inhibition of H77 (genotype 1a) HCVcc at the tested dilutions, with limited activity against Con1 (1b). Cellular analyses showed increased IFN-γ ELISPOT counts and higher frequencies of granzyme B⁺/perforin⁺ CD8⁺ T cells after N2 immunization, while IL-4 remained low. Functionally, N2 elicited stronger specific lysis of CT26-sE2 targets in vitro and slowed CT26-sE2 tumor growth in vivo. In HCV-infected ICR^4R+ mice, therapeutic vaccination with sE2-N2 reduced blood HCV RNA and hepatic readouts compared with sE2. A monoclonal antibody isolated from sE2-N2-immunized mice (1C1) neutralized HCVcc in vitro and, after passive transfer, lowered viremia and liver signals in infected mice. Collectively, these findings indicate that selective removal of the N2 glycan preserves antigen properties and is associated with improved humoral and cellular immunity and measurable in vivo activity, supporting targeted glycan editing as a practical strategy to refine E2-based HCV vaccines. Full article

This study investigates the glycosylation patterns of serum IgG antibodies in relation to COVID-19 infection and vaccination, highlighting the potential of specific glycan profiles as biomarkers for immune responses. Using Spearman correlation analysis, distinct associations among glycan levels and various clinical laboratory parameters were identified, revealing complex, non-linear interactions that influence immune dynamics. Significant differences were observed in sialylated glycan profiles across patient groups, indicating that vaccination and natural infection elicit unique immune mechanisms and suggesting that vaccination induces favorable glycosylation changes. Notably, high-mannose glycans were found to correlate with other glycan types, underscoring their critical role in the immune response and suggesting their potential as biomarkers to differentiate between infection- and vaccination-induced immunity. The findings suggest that understanding these glycosylation dynamics may enhance diagnostic and therapeutic strategies, providing valuable tools for differentiating between immune responses elicited by infection and vaccination. Overall, this study contributes to the understanding of glycosylation’s impact on immune function in the context of COVID-19, emphasizing the importance of specific glycan markers, such as sialylated and high-mannose structures, in clinical applications. Full article

The landscape of first-line treatment for metastatic non-small cell lung cancer (NSCLC) without actionable driver mutations is rapidly evolving, currently dominated by pembrolizumab-based regimens. This review discusses the unique molecular characteristics of cemiplimab, a newer anti-PD-1 antibody, and defines its optimal positioning against established standards. Cemiplimab is a fully human IgG4 monoclonal antibody distinguished by two key features: an engineered hinge-region mutation that prevents Fab-arm exchange, ensuring exceptional molecular stability which minimizes anti-drug antibody (ADA) risks associated with unstable molecules; and a unique interaction with PD-1 glycosylation sites, potentially enhancing binding efficacy. These structural advantages may be particularly relevant in histologies like squamous NSCLC, where accumulating somatic mutations drive high neoantigen loads and heightened immune responses, creating an environment historically prone to ADA formation. Based on data from the pivotal EMPOWER-Lung program, we highlight cemiplimab’s exceptional promise in specific populations. Firstly, in the EMPOWER-Lung 1 trial, cemiplimab monotherapy demonstrated extraordinary survival benefits in a pre-specified analysis of the distinct “ultra-high” PD-L1 expression subgroup (TPS ≥90%), potentially surpassing historical benchmarks. Secondly, cemiplimab displays consistent, robust efficacy in challenging-to-treat squamous histology, both as monotherapy for patients with high PD-L1 expression and in combination with chemotherapy for patients with PD-L1 < 50%. In conclusion, cemiplimab establishes a unique therapeutic niche for patients with squamous histology and ultra-high PD-L1 expression, likely driven by its distinct structural stability and reduced immunogenicity. Full article

Background: This study assesses the robustness of a legacy N-glycan profiling method for the therapeutic antibody MAB1 with different Peptide-N-glycosidase F (PNGase F) enzyme sources, solid phase extraction (SPE) cartridges, and reagent stability, aligning with ICH Q14 lifecycle management principles. Glycosylation profiling is critical for therapeutic antibodies as it influences both function and pharmacokinetics. Method: The legacy N-glycan profiling method, 2-aminobenzoic acid (2-AA) labeling combined with normal-phase HPLC, was re-evaluated to confirm consistent analytical performance in the context of evolving regulatory expectations. The evaluation focused on three key factors: PNGase F enzyme sources, solid-phase extraction (SPE) cartridges, and reagent stability. Results: Commercial PNGase F enzymes showed various performances, with some sources yielding significant differences. Several SPE cartridges were also tested, with certain formats displaying poor recovery and high variability, particularly for sialylated glycans. In addition, reagent stability studies revealed rapid degradation of the labeling reagent within a few days. Conclusions: These results underscore the importance of risk control, continual improvement, and lifecycle management to ensure reliable glycosylation analysis and the sustained robustness of legacy methods. Full article

Autoantibodies have long been regarded as passive reflections of immune dysregulation in connective tissue diseases (CTDs). Recent advances in systems immunology and molecular pathology have fundamentally redefined them as active molecular fingerprints that delineate distinct disease endophenotypes with predictive power for clinical trajectories and therapeutic responses. Rather than mere epiphenomena, autoantibodies encode precise information about dominant immune pathways, organ tropism, and pathogenic mechanisms. This review synthesizes emerging evidence that autoantibody repertoires—defined by specificity, structural properties, and functional characteristics—stratify patients beyond traditional clinical taxonomy into discrete pathobiological subsets. Specific signatures such as anti-MDA5 in rapidly progressive interstitial lung disease, anti-RNA polymerase III in scleroderma renal crisis, and anti-Ro52/TRIM21 in systemic overlap syndromes illustrate how serological profiles predict outcomes with remarkable precision. Mechanistically, autoantibody pathogenicity is modulated by immunoglobulin isotype distribution, Fc glycosylation patterns, and tissue-specific receptor expression—variables that determine whether an antibody functions as a biomarker or pathogenic effector. The structural heterogeneity of autoantibodies, shaped by cytokine microenvironments and B-cell subset imprinting, creates a dynamic continuum between pro-inflammatory and regulatory states. The integration of serological, transcriptomic, and imaging data establishes a precision medicine framework: autoantibodies function simultaneously as disease classifiers and therapeutic guides. This endophenotype-driven approach is already influencing trial design and patient stratification in systemic lupus erythematosus, systemic sclerosis, and inflammatory myopathies, and is reshaping both clinical practice and scientific taxonomy in CTDs. Recognizing autoantibodies as endophenotypic determinants aligns disease classification with pathogenic mechanism and supports the transition towards immunologically informed therapeutic strategies. Full article

A key function of naturally occurring antibodies is to control pathologically altered cells, such as those with aberrant glycosylation. Age-related diminution in the pool of B cells producing these immunoglobulins is linked to impaired anti-tumor immunity. In this study, the levels of antibodies against tumor-associated carbohydrate antigens (TACAs)—common in gastric cancer (GC) and other malignancies—were analyzed in 235 treatment-naïve GC patients (stages I–IV) and 76 healthy donors using a printed glycan array (PGA). We found that anti-glycan IgM levels, but not IgG, reduced with age in both patients and donors. Crucially, IgM levels against most glycans were significantly lower in the GC cohort compared with healthy donors, a trend that remained after age adjustment. Furthermore, an immunohistochemical analysis revealed that human anti-GalNAcα (Tn) antibodies—a well-characterized TACA in gastrointestinal cancers—bound to tumor cells and exhibited perinuclear and membrane staining in non-tumor surface cells within the same organ. These data support the hypothesis that gastric cancer patients have reduced levels of anti-glycan IgMs, which are responsible for the early recognition of transformed cells. This specific immunodeficiency may contribute to a permissive environment for tumor development. Full article

Human respiratory syncytial virus (RSV) remains a leading cause of severe lower respiratory tract infections in infants and immunocompromised populations, causing approximately 160,000 annual deaths globally. Despite recent approvals of prefusion F (pre-F) protein-based vaccines (Arexvy, Abrysvo) for older adults and pregnant women, pediatric vaccine development faces unique challenges including enhanced respiratory disease (ERD) risks, maternal antibody interference, and immature infant immune responses. Meanwhile, G protein glycosylation variability and NS1/NS2-mediated interferon suppression remain the outstanding difficulties in structure-based vaccine design. Additionally, current animal models demonstrate notable constraints in virus replication, host susceptibility, immune responses, clinical symptoms, and ERD phenomena. This review synthesizes current obstacles and innovative strategies, highlighting that the selection of multi-antigen strategies, appropriate adjuvants, and the development of more precise preclinical animal models are critical elements that will determine the efficacy and safety of future RSV vaccines. Full article

Antibodies are produced by cells of the adaptive immune response and recognize epitopes of microbial structures with high affinity and specificity. Antibodies are recognized by Fc fragment receptors (FcRs) found on the surface of phagocytic cells (neutrophils, monocytes, macrophages) and NK cells, among others. Hence, antibodies link the adaptive immune response with the innate immune response. The functions of antibodies are related to the N-glycosylation profile of these proteins. In this review, we describe how N-glycosylation of the Fc fragment of the different antibody classes is carried out, and which oligosaccharides are most commonly found in these antibodies. Subsequently, we summarize the biological effects of N-glycosylation of antibodies: on the binding of antibodies to FcRs (which affects various functions, such as antibody-dependent cellular cytotoxicity, antibody-dependent phagocytosis, and the production of pro- or anti-inflammatory chemokines and cytokines), on the ability of antibodies to activate complement and on the ability of some antibodies to directly neutralize the adhesion of bacteria and viruses to host cells (independently of Fab recognition). We describe how the N-glycosylation profile of antibodies is modified during certain infections (such as tuberculosis, COVID-19, influenza and dengue) and in response to vaccination, and the potential use of this profile to identify the stage and severity of an infection. Finally, we review the importance of N-glycosylation for the pharmacokinetic, pharmacodynamic and safety profiles of therapeutic monoclonal antibodies. Full article

Compared with classical Fc N-glycosylation, Fab N-glycosylation displays site heterogeneity and structural diversity. It contributes to immune regulation by modulating antibody stability, half-life, and antigen-binding activity, as well as by mediating blocking antibody effects. This review highlights the expression patterns and potential mechanisms of Fab N-glycosylated IgG in autoimmune diseases, pregnancy-induced immune tolerance, and tumor immune evasion, and discusses its structural and functional similarities to IgG4. Although Fab N-glycosylation plays an important role in both physiological and pathological conditions, the complexity of its glycan structures and variability in glycosylation sites hinder a precise understanding of its functional impacts. Clarifying these aspects is expected to emerge as a major focus in glycomics and antibody engineering research. Full article

Transgenic silkworms are promising host organisms for the production of therapeutic recombinant proteins due to their superior protein synthesis ability and human-like posttranslational modifications. In this study, we generated transgenic silkworms that secrete a recombinant human monoclonal antibody (mAb) against gp120 of human immunodeficiency virus (HIV) into their cocoons. Variations in the rearing temperature and humidity conditions had little effect on mAb properties, such as N-glycosylation. Next, we performed pilot-scale production of the mAb using three batches of transgenic silkworms. Rearing 22,000–45,000 silkworm larvae yielded 4–8 kg of cocoon shells per batch. Larval growth and development, as well as cocoon quality, were highly consistent across production batches. We extracted and purified the mAb from cocoon shells, yielding 6.1–7.6 g of purified mAb per kg of cocoons in each batch. Characterization of the purified mAb showed that the contents of oligomeric antibodies and host cocoon-derived proteins were less than 0.2% and 10 ppm, respectively, with high consistency among batches. From these results, we conclude that the transgenic silkworm system is sufficiently robust and reproducible for high-quality therapeutic mAb production. Full article

Background: Antibody-dependent cellular cytotoxicity (ADCC) is an immune response where antibodies bind to target cells and activate effector cells through Fcγ receptors, ultimately leading to the destruction of the target cells. Methods: This study examined the ADCC activities of charge variants of a therapeutic IgG₁, MAB1, using an internally developed reporter gene assay. In this assay, the proprietary target was expressed on DiFi cells, while FcγRIIIa was expressed on Jurkat effector cells. Results: The results revealed that different charge variants had varying levels of ADCC activity, with variants containing C-terminal lysine residues showing enhanced activity. The charge variants arose from modifications such as the presence of sialic acid at the glycan moiety, deamidation, and C-terminal lysine truncation, including K2 (two C-terminal lysine residues), K1 (one C-terminal lysine residue), and K0 (no C-terminal lysine residues) variants. Notably, the K1 and K2 variants demonstrated higher ADCC activity compared to the K0 and acidic variants. However, the observed increase was attributed not to the lysine residue itself, but rather to the MAN5 glycan associated with the lysine-containing variants. Conclusion: These findings challenge previous assumptions about the role of C-terminal lysine in ADCC, suggesting a shift in understanding the functional significance of charge variants and emphasizing the critical influence of glycan composition in therapeutic antibody efficacy. Full article

Avian influenza A viruses pose ongoing threats to human and animal health, with H7 subtypes causing outbreaks globally. In Mexico, highly pathogenic H7N3 viruses have circulated in poultry since 2012, causing sporadic human infections. Here we analyzed genetic markers in hemagglutinin sequences from Mexican H7N3 isolates and conducted serological assays on human populations with poultry exposure. Our results show conserved avian-like receptor binding sites, thus limiting human adaptation, alongside antigenic drift and acquisition of glycosylation sites likely driven by vaccination. Serological testing of 1103 individuals revealed no detectable antibodies against H7N3, indicating a naïve population. Phylogenetic analyses revealed multiple virus clades circulating regionally. These findings suggest that while current H7N3 viruses have limited capacity for sustained human transmission, the lack of population immunity underscores the importance of continued surveillance and risk assessment to mitigate potential pandemic threats. Full article

Background/Objectives: This study comprehensively characterized the O- and N-glycosylation profiles of bispecific antibodies (BsAbs) via advanced analytical techniques to evaluate their structural and functional implications. Methods: High-resolution MS revealed O-xylosylation at Ser468 within the (G4S)4 linker peptide, which was identified as xylose with a molecular weight of 132.042 Da. HILIC-HPLC analysis of N-glycosylation revealed glycan species engineered to eliminate Fc effector functions. O-glycosylation analysis via β-elimination followed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) identified xylose as the predominant glycan. Results: O-xylosylation does not affect the binding of BsAbs to either antigen Programmed Death-1 (PD-1) or Vascular Endothelial Growth Factor (VEGF). Notably, O-xylosylation interactions with mannose receptor represent the first discovery highlighting potential immunomodulatory roles. Conclusions: This study highlights the critical importance of monitoring comprehensive glycosylation characterization during the development of BsAb with (G4S)n linkers to ensure optimal therapeutic efficacy, safety, and reduced immunogenic potential. Full article

Background: Recombinant monoclonal antibodies (mAbs) represent the fastest-growing sector of the biopharmaceutical industry, with their efficient expression being a key technological factor for scalability. Objectives: In this study we compared the performance of two bicistronic vectors, which alternate the positions of the light and heavy chain coding genes, employing a wild-type Encephalomyocarditis virus (EMCV) IRES functional element to drive expression of the second gene. Methods: Using two neutralizing anti-SARS-CoV-2 IgG1 antibodies as model molecules, we conducted transient transfections in the commercially available ExpiCHO^TM platform. Following protein A affinity purification and quantification, vectors positioning the light chain as the first cistron consistently yielded higher expression levels than those with the heavy chain upstream. To confirm the quality attributes of the mAbs, we applied a comprehensive analytical workflow, including SDS-PAGE and Western blot for molecular mass and purity, circular dichroism for secondary structure, intrinsic tryptophan fluorescence for tertiary structure, and SEC-HPLC for quaternary structure and aggregate detection. Additionally, we assessed binding affinity to the target using spot blot and surface plasmon resonance, analyzed N-glycosylation profiles by HILIC-HPLC and mass spectrometry, and examined molecular structure by transmission electron microscopy. Results and Conclusions: Together, these results provide insight into the impact of gene positioning within bicistronic vectors on mAb expression efficiency and quality, supporting optimization strategies for scalable recombinant antibody production. Full article

Rheumatoid arthritis (RA) is the most common inflammatory polyarthritis. In addition, 60–80% of patients express anti-citrullinated protein antibodies (ACPAs), which serve as a diagnostic marker for RA. The effector functions of these autoantibodies can be heavily affected by the N-glycosylation of their Fc region. Here we present a comparison of the Fc N-glycosylation of ACPA IgG to that of non-ACPA IgG from the same patients, and of healthy controls, in an IgG isoform-specific manner. We isolated ACPA and normal serum IgG, digested by trypsin, and separated the resulting peptide mixture by a reversed-phase nanoLC coupled to a Bruker Maxis II Q-TOF, and determined the relative abundance of glycoforms. The paired analysis of galactosylation and sialylation of the IgG subclasses of ACPA and non-ACPA IgG has shown a significant, moderate negative correlation with the inflammatory markers, the level of C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR), as well as with rheuma-factor (RF), but not with the disease activity score (DAS) or cyclic citrullinated peptide specific antibodies (anti-CCP). However, we detected a significant negative correlation between glycosylation and DAS in the non-ACPA IgG fractions. Furthermore, the isoform-specific analysis revealed additional insight into the changes of the glycosylation features of IgG in RA: changes in the frequencies of the bisecting GlcNAc unit between sample groups could be explained by only the IgG1 isoform; while invariance in fucosylation is the result of the superposition of two isoforms with opposite changes. These results highlight the importance of analyzing immunoglobulin glycosylation in an isoform-specific manner. Full article