Background: Measles, mumps, and rubella outbreaks continue to occur despite high vaccination coverage, raising concerns about long-term vaccine-induced immunity. This study assessed serological protection against measles, mumps, rubella, and varicella in fully vaccinated Russian adolescents to identify immunity gaps and inform revaccination strategies. Methods: Serum IgG was quantified by commercial ELISA. Analytical cut-offs were: measles ≥ 0.18 IU/mL, rubella ≥ 30 IU/mL, mumps index ≥ 1.0 (index = OD/0.467), and varicella ≥ 1.0 IU/mL; equivocal results were classified as negative in the primary analysis and as positive in a sensitivity analysis. Geometric mean concentrations (GMC) were computed on the natural-logarithm scale. Results: Seropositivity differed markedly between infections (χ2 55.1; p < 0.001): rubella 81.4% (95% CI 74.6–86.7), varicella 57.7% (95% CI 49.8–65.2), mumps 53.2% (95% CI 45.4–60.9) and measles 41.0% (95% CI 33.6–48.9). GMC was lowest for measles (0.132; 95% CI 0.111–0.158) and highest for rubella (61.9; 95% CI 55.3–69.2). The measles estimate was sensitive to the treatment of equivocal results, rising to 53.2% (95% CI 45.4–60.9) at the manufacturer’s cut-off of ≥0.12 IU/mL; rubella rose to 94.9% at ≥16 IU/mL. Conclusions: Lower seropositivity rates for measles and mumps were identified among fully vaccinated adolescents, which fall below standard epidemiological benchmarks. In contrast, rubella seropositivity was robust at 81.4%. Because the measles estimate depended strongly on the assay cut-off applied, and because enzyme immunoassays are known to underestimate seroprevalence relative to neutralization testing, these findings describe the distribution of circulating antibody rather than the prevalence of susceptibility and warrant confirmation in larger representative samples with neutralization testing.
Bispecific antibodies (bsAbs) and bispecific antibody–drug conjugates (bsADCs) represent promising classes of emerging targeted therapeutics with the potential to overcome tumor heterogeneity and resistance in colorectal cancer (CRC). BsAbs can simultaneously engage multiple tumor antigens or immune cells or bind two distinct epitopes within a single target, enabling mechanisms of action beyond the capabilities of monoclonal antibodies. Bispecific T cell engagers facilitate targeted destruction of tumors through immune cell recruitment, while dual immune checkpoint inhibitors enhance immune activation by blocking T cell inhibitory signals. Furthermore, bsAbs can mediate dual signaling pathway inhibition through binding multiple receptor tyrosine kinase receptors or other tumor cell surface proteins. BsADCs integrate the dual-antigen recognition of bsAbs with targeted payload delivery, utilizing receptor-mediated endocytosis to deliver potent cytotoxic payloads selectively to CRC cells, while minimizing systemic toxicity. Recent advances in bsAb engineering, linker chemistry, site-specific conjugation, and payload design have accelerated the development of bsADCs for solid tumors, including CRC. BsAbs and bsADCs provide opportunities to improve tumor selectivity, enhance internalization, overcome antigen escape, and expand the population of CRC patients eligible for targeted therapy. Emerging preclinical studies demonstrate encouraging anti-tumor activity for bispecific modalities in CRC, while early clinical trials are beginning to establish their translational potential. This review summarizes the current landscape of bsAbs and bsADCs in therapeutic development for CRC, highlighting key biological targets, engineering strategies, mechanisms of action, and clinical status. We also discuss the major challenges facing clinical translation and provide perspectives on future directions for bispecific therapies in CRC.
Background/Objectives: Artificial intelligence (AI) has transformed computational antibody engineering by enabling accurate prediction of antibody structures, rational optimization of therapeutic properties, and de novo antibody design. Recent advances in deep learning, protein language models, and generative AI have fundamentally changed the way antibodies are discovered and engineered. This review aims to present the historical evolution of computational antibody engineering, from early structure-based design strategies to modern AI-driven approaches, while highlighting the major computational tools, publicly available databases, current limitations, and future directions of the field. Methods: A comprehensive narrative review of the literature was conducted using PubMed, Scopus, Web of Science, and Google Scholar. Original research articles, methodological studies, and review papers published between 1985 and 2026 were evaluated. Publications were selected according to their scientific relevance, methodological quality, and contribution to the historical development of computational antibody engineering. Results: The review describes the progression of antibody engineering from phage display and structure-based computational methods to machine learning, deep learning, protein language models, and generative artificial intelligence. It summarizes key public databases supporting antibody research, discusses advances in antibody structure prediction and developability assessment, and reviews recent generative models capable of designing antibody sequences and structures. Current challenges, including limited experimental validation, dataset bias, prediction of highly flexible regions, model interpretability, and clinical translation, are also discussed. Conclusions: Artificial intelligence has fundamentally reshaped computational antibody engineering by integrating sequence, structural, and functional information into increasingly accurate predictive and generative frameworks. Although important challenges remain, recent developments indicate that AI-driven approaches will play an increasingly central role in the discovery and optimization of next-generation therapeutic antibodies.
GNR-068 is a proposed biosimilar to the ustekinumab reference product (RP), which works through the antagonism of interleukin 12 and interleukin 23. Ustekinumab RP is used for the treatment of chronic inflammatory conditions, including certain forms of plaque psoriasis, psoriatic arthritis, Crohn's disease, and ulcerative colitis. Objectives: The purpose of the study is to study the safety, pharmacokinetics, and immunogenicity of the ustekinumab biosimilar GNR-068 and the reference drug Stelara® after a single subcutaneous administration in healthy male volunteers. Methods: This was an open-label, randomized, comparative, parallel-group clinical trial of the safety, pharmacokinetics (PK), and immunogenicity of GNR-068 (JSC GENERIUM) and Stelara® (Manufacturer: Silag AG, Switzerland; RU Holder: JOHNSON & JOHNSON) after a single subcutaneous administration of 45 mg in healthy volunteers. During the trial, 146 volunteers were screened, and 122 were randomized. Results: The results showed that PK similarity was established based on 90% confidence intervals (CIs) for the ratios of geometric means of the primary endpoints of area under the concentration-time curve from time 0 extrapolated to infinity (AUC0-∞) and maximum observed serum concentration (Cmax) being contained within the pre-specified margin of 80.00-125.00%. The incidence of total ADA was lower in the GNR-068 group compared with the reference product group. Adverse events were similar between treatment groups and consistent with the safety profile of the ustekinumab RP. Conclusions: These results indicate that GNR-068 and the ustekinumab RP share similar PK and safety profiles.
Background: Idiopathic pulmonary fibrosis (IPF) is closely associated with fibroblast proliferation, macrophage polarization, and the accumulation of extracellular matrix (ECM). Targeting CSF-1R can rebalance macrophages, reduce ECM deposition, and block pro-fibrotic signaling. Methods: A Fab fragment targeting CSF-1R was screened via phage display. After sequence optimization, it was constructed into an IgG1 antibody (BC006). In vitro activity, domain binding, and signaling blockade were investigated, and in vitro safety indicators were evaluated. Efficacy was assessed using an induced IPF organoid-on-a-chip and bleomycin-induced mouse model. In vivo safety evaluation was conducted in cynomolgus monkeys. Results: BC006 showed an EC50 value of 226 ± 57 nM and a KD of 30 ± 2 nM. It specifically bound to the Ig4-5 domain of CSF-1R by inhibiting receptor dimerization without blocking ligand binding. It could dose-dependently inhibit the differentiation of monocytes into M2 macrophages and exert anti-IPF effects. In the induced IPF organoid-on-a-chip model, BC006 maintained lung barrier function and decreased α-SMA and Collagen I. It also improved lung function and attenuated the degree of fibrosis in the mouse model. Moreover, BC006 had no ADCC, CDC, cytokine release, or hemagglutination, and demonstrated favorable safety profiles in cynomolgus monkeys. Conclusions: BC006 is a novel anti-fibrosis antibody specifically targeting the CSF-1R Ig4-5 domain and offers a new therapeutic strategy for IPF.
Background: Sex-related differences in SARS-CoV-2 viral susceptibility, severity, and recovery are prevalent. Factors such as age and biological variation provide some explanation for immune response, yet the use of biological sex as a variable may limit understanding of underlying individual mechanisms related to endocrine-immune function involved in long-term immune memory. Endogenous sex hormone concentrations provide a means to assess the magnitude or durability of antibody persistence and disease recovery. Objective: This study examined salivary concentrations of 17β-estradiol and testosterone, as well as the number of weeks since prior SARS-CoV-2 viral infection. The study aimed to determine whether endogenous sex hormones contributed additional explanatory value to antibody persistence beyond biological sex. Methods: A cross-sectional study included 75 college students (63% female) who reported weeks since previous SARS-CoV-2 infection (M = 15.41, SD = 7.13), confirmed through a positive SARS-CoV-2 anti-N rapid antibody test. Participants provided saliva samples, which were tested for concentration of 17β-estradiol and testosterone. Hierarchical multiple regression analyses examined the effects of biological sex and endogenous sex hormones on the persistence of SARS-CoV-2 antibodies following natural infection. Results: 17β-estradiol was the strongest positive predictor of SARS-CoV-2 antibody persistence (β = 0.63, p < 0.001). Departing from previous findings, biological sex was not a significant predictor of antibodies targeting the SARS-CoV-2 N protein. Conclusions: Findings underscored the role of estradiol on immune recovery in a healthy young adult population. Individual hormone variation appears more informative than sex classification for understanding antibody persistence and immune response.
Human Papillomavirus (HPV) infection is the leading cause of cervical cancer, presenting a significant global health challenge. While natural infection is widespread, the resulting immune response is often characterized by weak, delayed, and type-specific antibody production, offering unreliable protection against reinfection. This review provides a comparative analysis of natural versus vaccine-induced immunity, focusing on antibody kinetics, duration of protection, and cancer prevention efficacy. A comprehensive search of the literature from the last decade was conducted using PubMed, ScienceDirect, and Web of Science. The findings demonstrate that, unlike natural immunity, which is dominated by cellular responses with often incomplete seroconversion, prophylactic vaccination induces high titers of neutralizing IgG antibodies against the L1 capsid protein. These responses are durable, with protection persisting for over a decade, and recent data support the high efficacy of single-dose regimens. Furthermore, vaccination has shown utility in reducing infection persistence in HPV-positive individuals and provides critical protection in immunocompromised groups. Consequently, vaccine-induced immunity is consistently superior to naturally acquired immunity, supporting World Health Organization recommendations for universal vaccination as the primary intervention for reducing the global burden of HPV-related malignancies.
Immune-mediated neutropenias comprise a heterogeneous group of disorders characterized by antibody-mediated destruction of neutrophils, in which the detection of anti-neutrophil antibodies remains a significant diagnostic challenge. Human neutrophil antigens (HNAs) are key targets in both autoimmune and alloimmune conditions, and their identification requires an integrated laboratory approach combining serological assays, HNA genotyping, and clinical evaluation. However, variability in assay sensitivity, the presence of low-titer or conformationally dependent antibodies, and interference from anti-HLA antibodies may lead to inconclusive or misleading results. This review summarizes the immunological mechanisms underlying anti-HNA antibody-mediated neutropenia and critically evaluates current laboratory methods, including cell-based and bead-based assays. The role of HNA genotyping in supporting antibody identification and improving diagnostic accuracy is also discussed. In addition, we highlight the importance of interpreting serological findings according to antibody specificity and clinical context. An integrated and multidisciplinary diagnostic approach is essential to ensure accurate diagnosis and appropriate clinical management, while emerging technologies may further improve antibody detection in the future.
Daratumumab, a human IgG1 monoclonal antibody targeting CD38, is widely used in multiple myeloma and AL amyloidosis. Despite its clinical success, many patients fail to achieve durable responses or relapse, underscoring the importance of understanding resistance mechanisms. Drawing on experience from other better-studied monoclonal antibodies, resistance to daratumumab can be categorized into four main mechanisms: (1) reduced CD38 expression on plasma cells; (2) increased expression of complement inhibitory proteins (CD55/CD59), impairing complement-mediated cytotoxicity; (3) reduced drug bioavailability due to urinary loss in non-selective nephrotic syndrome; and (4) the development of neutralizing anti-daratumumab antibodies. Anti-drug antibodies (ADAs) may represent a potential mechanism of treatment failure through effects on pharmacokinetics, efficacy, and safety, even in patients on daratumumab therapy. Seven different trials have tested anti-daratumumab antibodies. Among them, anti-daratumumab antibodies were identified in only 0-2.4% of patients, and only in a small portion of these has it been proven to be neutralizing. Overall, ADAs appear rare, but these findings are likely underestimated due to short follow-up and suboptimal timing of assessment. In conclusion, standardized ADA monitoring, particularly months after treatment interruption or in cases of inadequate response or infusion-related reactions, may improve patient management and therapeutic outcomes.
BACKGROUND/OBJECTIVES:The autoimmune disorder Systemic Lupus Erythematosus (SLE) is characterized by increased titers of autoantibodies with different specificities against autoantigens, including the complement proteins C1q, C3 and Factor H. SLE is characterized by chronic inflammation and tissue damage due to the secretion of pro-inflammatory molecules and a tissue deposition of immune complexes formed by autoantibodies and their target antigens. The inflammatory process in SLE is maintained by the phospholipase A2 (PLA2) enzymes generating pro-inflammatory lipid mediators. Hereditary and environmental factors trigger SLE, with increased genetic heritability in first-degree relatives of SLE patients. METHODS:A cohort of 48 healthy FDRs of SLE patients was analyzed with the ELISA method for the presence of antibodies to complement proteins C1q, C3 and Factor H, with a focus on detecting autoepitopes both on immobilized and soluble C1q and its globular fragments ghA, ghB and ghC. The total serum PLA2 activity of FDRs was measured using the chromogenic substrate 4-nitro-3-octanoyloxy-benzoic acid (NOBA). RESULTS:Only C1q, and specifically its globular domains in both an immobilized and soluble state, was targeted by antibodies in the healthy FDRs similarly to the pattern established in SLE patients. In contrast, C3 and Factor H which are known autoantigens in SLE were not found as targets for the antibodies in the analyzed FDRs. Some of the FDRs showed increased serum PLA2 activity, which correlated weakly with anti-C1q antibodies. CONCLUSIONS:C1q and its globular domains are estimated as autoantigenic molecules for binding in the analyzed FDRs of SLE patients.
Background: The interaction between human immunoglobulin G (IgG)1 Fc and the Fc gamma receptor (FcγR) IIIa/CD16a elicits protective immune responses. Antibody N-glycosylation stabilizes the FcγR-binding interface and is thus essential for interaction with wildtype IgG1 Fc. Furthermore, the N-glycan introduces substantial compositional and functional heterogeneity, with distinct glycoforms providing different affinities and discrete responses in vivo. Accordingly, various engineering endeavors to improve antibody binding strive to boost the therapeutic efficacy of monoclonal antibodies but do not directly address compositional heterogeneity. Objective: Here, we describe a previously unexplored approach to engineer IgG1 Fc. We eliminated carbohydrate heterogeneity by removing the N-glycan but stabilizing the FcγR-binding interface with disulfide bonds. Conclusions: These newly generated Fc domains served as a starting point for protein engineering through yeast surface display to enhance receptor-binding affinity. We recovered Fc variants from this approach that demonstrated FcγRIIIa binding affinities comparable to the starting sequence and thus serve as a proof-of-principle for this strategy.
Background: Antinuclear antibodies (ANA) can be detected in patients with rheumatoid arthritis (RA) and pose many diagnostic challenges, especially when RA presents an atypical course and requires differentiation from other systemic connective tissue diseases (sCTDs). This study assessed ANA fluorescence patterns and immunoblot profiles, as well as the relationships between ANA titers, antibody expression intensity, and markers of disease activity in patients with RA. Methods: This single-center, cross-sectional, observational study included 81 RA patients (53 ANA-positive) meeting the 2010 ACR/EULAR classification criteria. ANA titers and fluorescence patterns were assessed using indirect immunofluorescence. Anti-extractable nuclear antigen (ENA) autoantibody profiles and expression intensity were assessed using immunoblot analysis. Demographic, clinical, and laboratory data were obtained. Spearman’s rank correlation coefficient was used to analyze the relationship between ANA titers and selected variables. Univariate and multivariate logistic regression analyses were performed to identify factors associated with ANA positivity. Results: The cohort consisted primarily of women (86.4%) with moderate disease activity. ANA fluorescence patterns were heterogeneous, with nucleolar and homogeneous patterns most frequently observed. Immunoblot analysis also revealed diverse autoantibody profiles without a clearly dominant specificity. Ro-52, SS-A, and Sm antibodies were detected more frequently, although their prevalence remained relatively low. No statistically significant correlations were found between ANA titers and inflammatory markers, serological parameters, or disease activity indices. Conclusions: RA patients with positive ANA demonstrated marked immunological heterogeneity, without concomitant symptoms of sCTD. A positive result in RA may reflect generalized immune dysregulation rather than a distinct clinical subtype. Further studies with larger cohorts are needed to clarify the clinical significance of ANAs in rheumatoid arthritis.
BACKGROUND:Viral attachment mediated by host cell surface receptors is the first step in viral infection. As a key cell surface receptor, heparan sulfate (HS) mediates the attachment and entry of numerous non-enveloped viruses in livestock, thereby serving as a crucial molecular target for studying virus-host interactions. METHODS:Based on the structural scaffold of a nanobody (Nb; PDB: 7TJC), we rationally designed and constructed a mutant Nb targeting HS, designated HS-Mut-Nb1, using molecular docking, site-directed mutagenesis, molecular dynamics (MD) simulations, and experimental characterization. RESULTS:Molecular docking indicated that the active site of wild-type Nb for HS binding was located within the cavity jointly formed by the complementarity-determining region 3 (CDR3) and the framework regions (FRs) of the wild-type Nb. A comprehensive analysis integrating virtual alanine scanning, site-directed mutagenesis, and MD simulations revealed that the combination of three point mutations (Phe47Arg, Asp99Tyr, and Tyr108Pro) significantly enhanced the binding affinity of Mut-Nb1 for HS, with a calculated binding free energy (ΔG) of -83.26 ± 3.06 kcal/mol. Enzyme-linked immunosorbent assay (ELISA) results further confirmed that Mut-Nb1 exhibited high affinity for HS (KD = 65.87 nM) and specificity (positive/negative ratio, P/N = 3.84; cross-reactivity, CR < 6.60%). CONCLUSIONS:This study not only provides novel candidate molecules for elucidating the mechanism of HS-virus interactions and developing related inhibitors but also offers a reference for the rapid construction of mutant Nbs.
BACKGROUND:The mucosal barrier presents a significant challenge for non-invasive delivery of macromolecular therapeutics, often requiring administration with poor bioavailability and increased toxicity risks. The polymeric immunoglobulin receptor (pIgR) contains an extracellular secretory component (SC) for immunoglobulin binding and a membrane-anchored stem domain capable of apical-to-basolateral transcytosis. We hypothesized that targeting the stem domain could enable active drug transport across mucosal barriers. METHODS:Using phage display, we identified four high-affinity nanobodies against human and murine pIgR. Two lead candidates (3LTHMP-4 and 3LTHMP-5) demonstrated efficient apical-to-basolateral transport in vitro (Transwell assays) and in vivo (fluorescence imaging). Engineered bispecific antibodies fusing these nanobodies with anti-IL-5 mAb reslizumab were administered via inhalation in a murine asthma model at one-tenth the intraperitoneal reslizumab dose. RESLUTS:The bispecific antibodies showed significant therapeutic efficacy, while reslizumab alone at equivalent concentrations failed to demonstrate efficacy. Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) revealed that both 3LTHMP-4 and 3LTHMP-5 specifically bind to the pIgR stem domain (residues 578-612), a region distinct from the dimeric IgA binding site. CONCLUSIONS:These findings suggest that stem domain-specific binding may facilitate transport across the mucosal barrier while preserving native receptor physiology, offering a potential strategy for effective transmucosal delivery of biologics.
Rheumatoid arthritis-associated interstitial lung disease (RA-ILD) is one of the most severe extra-articular manifestations of rheumatoid arthritis (RA), requiring reliable biomarkers for early detection. This scoping review synthesized current evidence regarding the diagnostic performance and clinical associations of anti-mutated citrullinated vimentin (anti-MCV) antibodies in patients with RA-ILD. A comprehensive literature search was conducted across PubMed/MEDLINE, Embase, Scopus, and the Cochrane Library. Following systematic screening, two observational studies met the predefined inclusion criteria. Both included studies reported significantly higher anti-MCV positivity rates and/or serum levels in patients with RA-ILD compared with RA patients without pulmonary involvement. Specifically, one study identified an independent association between anti-MCV positivity and RA-ILD, while the other demonstrated significant correlations between anti-MCV titers and pulmonary function impairment, as well as disease activity markers. However, substantial heterogeneity was observed across the studies regarding assay platforms, positivity thresholds, and diagnostic cut-offs, which limits the direct comparability of results. While anti-MCV antibodies represent promising candidate biomarkers for RA-ILD, current evidence remains limited and is insufficient to establish definitive diagnostic, prognostic, or pathogenic significance. Consequently, larger, prospective, and multi-center studies utilizing standardized anti-MCV assay protocols are necessary to rigorously evaluate the clinical utility of these antibodies in the management of RA-ILD.
Although mAbs have great therapeutic potential, their use in medicine is currently limited by the high cost of their manufacturing. Significant developments in upstream processing technologies have caused downstream processing (DSP) to become the manufacturing cost-driver. DSP consists of a number of operations included in the capture, polishing, and formulation steps that contribute to excessive material and buffer consumption. This is particularly true for the capture and polishing steps, in which tedious and costly chromatographic operations are involved to ensure an adequate purity level of the medical product. The final formulation step also increases the burden of buffer consumption. This review focuses on those time- and material-consuming DSP operations and describes key issues and challenges related to their realization. In each of the steps, capture, polishing, and formulation, the platform processing approaches are presented as well as directions for their development. In addition, we present alternative nonchromatographic approaches that can potentially be used in the capture and polishing steps, such as precipitation or extraction. Furthermore, we describe mAb processing by crystallization, which can potentially serve as an alternative platform in both polishing and formulation steps.
BACKGROUND/OBJECTIVES:Cadherin-17 (CDH17, LI-cadherin) is a non-classical cadherin with an atypical structure and unique functions. CDH17 expression is restricted to normal intestinal epithelium. Furthermore, CDH17 functions as an oncoprotein that promotes tumor migration and invasion in colorectal, gastric, and pancreatic cancers. Therefore, CDH17 is an important diagnostic marker and therapeutic target. The CDH17-directed strategies, including monoclonal antibodies (mAbs), bispecific Abs, antibody-drug conjugates (ADCs), and chimeric antigen receptor (CAR) T cells, have been evaluated in preclinical and clinical studies. Therefore, developing mAbs that specifically recognize cell surface-expressing CDH17 is essential for advancing both tumor diagnosis and therapy. METHODS:Anti-human CDH17 mAbs (named Ca17Mabs) were developed by immunizing a mouse with CDH17-overexpressed cells and a high-throughput screening using flow cytometry. RESULTS:Among Ca17Mabs, a clone, Ca17Mab-5 (IgG1, κ) specifically recognized CDH17-overexpressed Chinese hamster ovary-K1 (CHO/CDH17) cells with no detectable cross-reactivity to 21 other CDHs by flow cytometry. Ca17Mab-5 also detected endogenous CDH17 in human colorectal cancer cell lines, COLO201 and COLO205. The apparent dissociation constant (KD) values of Ca17Mab-5 for CHO/CDH17 and COLO205 were estimated as 1.5 × 10-8 M and 1.3 × 10-8 M, respectively. Furthermore, Ca17Mab-5 detected endogenous CDH17 by Western blotting. In immunohistochemistry, Ca17Mab-5 exhibited clear membranous staining in normal colon epithelium, colorectal, gastric, and pancreatic cancers. CONCLUSIONS:Ca17Mab-5 is a versatile tool for detecting CDH17 and has potential for tumor diagnosis.
Background/Objectives: The use of cross-linking enzymes for site-selective and efficient antibody modification has attracted considerable attention. Microbial transglutaminase (MTG)-mediated labeling of IgG at Gln295 has emerged as a promising strategy for preparing antibody-drug conjugates (ADCs). By contrast, selective modification of a specific Lys residue on native antibody surfaces using MTG remains challenging because most Lys residues exhibit low intrinsic reactivity. Here, we address this challenge by exploiting enzyme-antibody proximity together with screening for highly reactive Gln-donor substrates from a random peptide library. Methods: Reactive Gln-donor peptide substrates were first identified from a seven-amino-acid phage-displayed peptide library using a reactive Lys-containing peptide as bait. Based on the obtained sequence, an azide-functionalized Gln-donor peptide suitable for click chemistry was designed. Results: The designed substrate enabled efficient Lys65-selective modification of Fab fragments using a fusion of an engineered MTG zymogen and protein G (EzMTG-pG), followed by functionalization through click chemistry to yield fluorescent Fab conjugates. Conclusions: These results provide practical guidelines for substrate design in MTG-mediated site-selective protein modification.
Type I interferons (IFN-I), including IFN-α, IFN-β, and IFN-ω, are central to antiviral defence and immune regulation. Autoantibodies targeting IFN-I (anti-IFN-I AAbs) have emerged as key pathogenic factors in severe coronavirus disease 2019 (COVID-19) and are detectable in systemic lupus erythematosus (SLE), a prototypic IFN-driven autoimmune disease. Here we compare the prevalence and clinical impact of anti-IFN-I autoantibodies (Aabs) in COVID-19 and SLE based on a structured review of 53 studies from 2014 to 2025 and highlight the clinical associations and therapeutic opportunities presented by these autoantibodies. In COVID-19, neutralising anti-IFN-α and/or anti-IFN-ω AAbs were consistently associated with severe disease and impaired antiviral responses, particularly in older male populations. In SLE, anti-IFN-α AAbs were variably detected; neutralising antibodies were associated with reduced interferon gene signatures in some cohorts but inconsistent correlations with disease activity. Therapeutically, anti-IFN-I AAbs in COVID-19 may inform risk stratification and early antiviral strategies, whereas in SLE, IFN-α blockade, including IFN-α kinoid vaccination, demonstrates modulation of IFN signatures but variable clinical benefit. Notably, these findings reveal an immunological paradox: the same neutralising mechanism that impairs antiviral defence in COVID-19 may attenuate chronic IFN-driven inflammation in SLE. Taken together, anti-IFN-I AAbs exert context-dependent effects: pathogenic in acute viral infection yet potentially modulatory in chronic IFN-driven autoimmunity. Prospective longitudinal studies are required to further clarify their translational utility and long-term clinical impact.
Monoclonal antibody (mAb) discovery has been transformed by advances in single-cell technologies, microfluidics, high-throughput sequencing, and computational design. Modern platforms enable the interrogation of large numbers of individual B cells, directly linking antibody sequence with antigen specificity and functional activity. Microfluidic and optofluidic systems now support high-throughput compartmentalisation and functional screening of antibody-secreting cells, while sequencing-based approaches allow parallel recovery of paired heavy- and light-chain sequences. These developments have shifted antibody discovery from binding-based selection toward function-first paradigms, enabling the rapid identification of diagnostic and therapeutically relevant antibodies. Integration with computational tools, including machine learning and structure-based modelling, has further enabled the emergence of closed-loop discovery pipelines, in which experimental and in silico methods iteratively refine candidates. This review summarises key advances in single-cell microtools over the last decade and highlights how the convergence of experimental and computational technologies is reshaping antibody discovery toward scalable, data-driven, and increasingly automated platforms.
