Mycobacterium abscessus (MABS), a rapidly growing non-tuberculous mycobacterium, causes disseminated infections in patients with adult-onset immunodeficiency due to anti-IFN-γ autoantibodies. We investigated whether cytokines produced by MABS-infected cells contribute to plasmablast survival and antibody production. To address this, we first analyzed the cytokine response in monocyte-derived macrophages infected with MABS and found that IL-6 was secreted at high levels. Since IL-6 is known to be produced by fibroblasts, we next characterized the intracellular survival of MABS and its ability to induce cytokine production in fibroblasts. We demonstrated that MABS invaded fibroblasts, replicated intracellularly, and induced high levels of IL-6 secretion. Given the established role of IL-6 in promoting plasmablast survival, we next developed an in vitro culture of B cell-derived plasmablasts to test the effect of IL-6. To do this, B cells from healthy donors were differentiated into early plasmablasts using Toll-like receptor (TLR) 7/8 agonist. After 4 days, plasmablasts were then cultured for an additional 3 days with either recombinant IL-6 or supernatants from infected macrophages. Our results demonstrated that both recombinant IL-6 and supernatants from infected macrophages promoted plasmablast survival and expansion. Blocking the IL-6 receptor with tocilizumab, but not inhibiting JAK1⁄2 with baricitinib, reduced plasmablast survival and IgG secretion in cultures treated with infected macrophage supernatants. Collectively, these findings suggest that infection-induced IL-6 promotes plasmablast survival and antibody production. Targeting IL-6 signaling could, therefore, represent a potential therapeutic strategy to modulate antibody responses.IMPORTANCEThe difficult-to-treat MABS infection is a major clinical problem in Asian patients with adult-onset primary immunodeficiency associated with anti-IFN-γ autoantibodies. Understanding the cytokine responses induced by MABS may support the development of cytokine-targeted therapies that control autoantibody production. In this study, we found that IL-6 was produced at high levels by both macrophages and fibroblasts following infection. Moreover, we demonstrated that IL-6 is a key cytokine promoting plasmablast survival, as IL-6 receptor blockade with tocilizumab significantly reduced plasmablast viability in cultures stimulated with infected macrophage supernatants. Together, these findings provide a rationale for future clinical investigation of cytokine-targeted therapeutic approaches.
Monoclonal antibodies (mAbs) are powerful tools for elucidating disease mechanisms. Capturing heterogeneity of patient responses-including clonality, somatic mutations, isotypes, and pathogenic potential-requires building large libraries of mAbs using high-throughput approaches. However, current techniques for identifying and isolating patient-derived mAbs targeting conformational epitopes on membrane proteins remain labor-intensive and inefficient. To address this challenge, we evaluated a cell-based optofluidic antibody discovery pipeline to generate patient-derived mAbs against myelin oligodendrocyte glycoprotein (MOG), a transmembrane autoantigen targeted in MOG antibody-associated disease (MOGAD). An optofluidic-based workflow incorporated mammalian display of human MOG (hMOG) in a live cell-based assay (CBA) format. B-cell receptor sequences from individual hMOG-binding B cells were cloned and expressed to generate mAbs from 3 patients. The hMOG binding specificity of these mAbs was validated in off-platform hMOG-CBAs. hMOG-specific antibody-secreting cells in one patient represented 0.02% of all single B cells screened. From these low-frequency populations, one patient-derived IgG mAb was successfully generated and validated. This IgG mAb, characterized by a high frequency of V-region somatic mutations (5%-12.2%), bound hMOG at concentrations as low as 1 ng/mL. This workflow enables rapid discovery of rare, patient-derived mAbs targeting conformational epitopes on membrane antigens, offering a scalable approach for dissecting autoantibody repertoires.
Lyme disease, caused by the spirochete, Borrelia burgdorferi sensu latu (Bbsl), is a tickborne infection of increasing incidence in North America, Europe and Asia. While vaccines based on Outer surface protein A (OspA) have proven highly efficacious at blocking Bbsl tick-to-human transmission, the high degree of antigenic variability among the major OspA serotypes (ST) has made the development of a broadly cross protective vaccine difficult. Recent profiling of protective human monoclonal antibodies (mAbs) has suggested the existence of conserved epitopes situated within OspA's central β-sheet (CBS), although direct comparisons of cross-serotype functionality has been hindered by biological differences among the major Bbsl genospecies. To address these issues, we developed a panel of isogenic B. burgdorferi reporter strains expressing the seven major OspA serotypes (ST1-7) and probed them with CBS-targeting mAbs to evaluate their complement-dependent borreliacidal activity. The mAbs segregated into three distinct classes: class 1 mAbs exhibited potent killing against all seven OspA serotypes, while classes 2 and 3 had restricted or no activity against two of the seven serotypes. Structural analysis of Fabs derived from each class of mAbs in complex with OspA ST1 showed that they target overlapping epitopes spanning β-strands 6-10 and involve contact with largely invariant residues. Further analysis of B. burgdorferi reporter strains expressing OspA variants from 17 additional Bbsl genospecies identified Lys-107 as a determinant of susceptibility for nearly all CBS mAbs. Taken together, these findings raise the prospect of structure-based design of a broadly protective monovalent Lyme disease vaccine.
Protein A resins are indispensable for monoclonal antibody (mAb) production, yet their condition and performance are traditionally assessed using indirect or qualitative methods. In this study, the multi-attribute method (MAM), previously applied to therapeutic protein characterization, is systematically adapted for the first time as a unified liquid chromatography-mass spectrometry (LC-MS) platform for Protein A resin analysis. Four Cytiva Protein A resins, MabSelect™, MabSelect SuRe™, MabSelect SuRe™ LX, and MabSelect™ PrismA, were evaluated by MAM for resin identity, Protein A ligand integrity, fouling by impurities, and cleaning performance. MAM enables resin-specific peptide fingerprinting and quantitative monitoring of Protein A ligand post-translational modifications (PTMs), including deamidation, isomerization, and fragmentation induced by repeated clean-in-place (CIP) cycles. Comparative analysis of virgin and used resins revealed ligand degradation and fouling despite engineered alkaline stability, with MabSelect™ showing the greatest susceptibility. Importantly, residual monoclonal antibodies (mAbs) and host cell proteins (HCPs) were directly detected and quantified from the resin matrix, providing a molecular-level assessment of resin cleaning effectiveness not achievable with conventional approaches. This work establishes MAM as a novel, sensitive, and comprehensive strategy for Protein A resin lifecycle management, delivering actionable insight for resin selection, cleaning optimization, and downstream process development.
Here, we report a simple and rapid extracellular vesicle (EV)-based membrane-type immunoglobulin-directed hybridoma screening (MIHS) platform that enables efficient isolation of monoclonal antibodies (mAbs) recognizing three-dimensional conformations of transmembrane proteins. Transmembrane proteins are essential membrane components involved in signal transduction, transport, and energy conversion. As they function in native conformations, isolating mAbs that recognize conformational epitopes is critical for both research and therapeutic use. However, traditional methods, such as the enzyme-linked immunosorbent assay (ELISA), can disrupt the antigen structure, making it challenging to screen for such mAbs. To overcome this limitation, we developed a screening strategy combining MIHS with EVs as antigen-presenting tools. EVs, which are naturally bounded by lipid bilayers, display transmembrane proteins in native conformations and can be easily isolated. As a model, we engineered cells co-expressing alkaline phosphatase (ALP) and green fluorescent protein, generating fluorescent EVs displaying ALP. Hybridomas were screened by MIHS using these EVs, followed by ELISA without direct antigen immobilization. All isolated mAbs recognized conformational ALP epitopes, demonstrating the structural selectivity of this method. These results demonstrate that the EV-based MIHS approach provides a convenient and structurally faithful strategy for isolating conformation-specific mAbs, offering broad utility in basic biology and antibody development.
Objective: This retrospective, intention-to-treat real-world study, designed by the Greek Research Alliance for the Study of headache and Pain (GRASP) sought to compare the effectiveness and safety of anti-CGRP monoclonal antibodies (anti-CGRP Mabs) to topiramate in preventing migraine. Patients and methods: Patients received either fremanezumab, erenumab, galcanezumab, eptinezumab, or topiramate for at least six months. Outcomes included reductions in monthly headache days (MHDs), ≥50% and ≥75% responder rates, monthly acute medication intake (MAI), MHDs with peak headache intensity ≥5 on VAS, migraine-related disability (MIDAS, HIT-6), quality of life (EQ-VAS), discontinuation rates and safety. Results: We included 409 migraine patients (median age 45.2 years), predominantly female (80%) and mostly with long-standing disease and high baseline burden. After six months, all treatments reduced MHDs. Mean MHDs decreased by -7.8 days with anti-CGRP Mabs versus -3.8 days with topiramate (p < 0.001). Higher ≥50% and ≥75% responder rates were observed across all anti-CGRP agents, compared to topiramate. Anti-CGRP Mabs also achieved greater reductions in moderate/severe MHDs, MAI, disability metrics, and superior QOL gains. Among the CGRP-targeted therapies, slight differences in effectiveness outcomes were present, though failing to demonstrate any specific superiority. Safety was favorable for anti-CGRP Mabs, whereas topiramate showed substantially higher adverse events and discontinuations. Conclusions: Anti-CGRP Mabs were more effective, produced greater reductions in disability and higher improvements quality-of-life metrics and were better tolerated than topiramate. Differences among individual anti-CGRP agents were modest and unlikely to represent a clinically meaningful superiority, supporting a class-wide benefit vs. topiramate in migraine prevention both in terms of effectiveness and safety.
Double-stranded nucleic acids can undergo transitions from canonical B/A-forms to alternate left-handed Z-DNA/Z-RNA (Z-NAs). Z-NAs are implicated in processes such as neuroinflammation in Alzheimer's disease, Lupus Erythematosus, microbial biofilms, and type I interferon-mediated human pathologies. Since endogenous Z-NA sensors like the Zα domain can induce B-to-Z transitions, monoclonal antibodies (mAbs) Z-D11 and Z22 have been regarded as conformation-specific tools to confirm Z-NA in situ, although high-resolution structural information remain unavailable. Here, we employed single-particle cryo-electron microscopy to determine structures of Z-D11 and Z22 bound to synthetic d(CG)6 12mer Z-DNA duplex. Both mAbs form filamentous trimers around the Z-DNA axis, further stabilized by Fab-Fab interactions. The mAbs achieve specificity through multiple backbone-dominated contacts to both Z-form backbone strands and the exposed guanine/cytosine bases in the major groove. This mode of recognition is dictated by shape complementarity rather than sequence specificity, sensing the alternating syn/anti backbone torsions and the phosphate zig-zag geometry unique to Z-DNA. Our data also suggest that these mAbs do not induce B-to-Z transitions under normal physiological conditions. Finally, comparison to other double-stranded NA-binding mAbs defines a similar structural logic adapted to different helical geometry recognition patterns, thus providing a framework for engineering highly specific nucleic acid probes.
Sialyl Lewis A (sLeA), or the CA 19-9 marker, is the best validated and only FDA-approved serologic marker clinically used to monitor recurrence, progression, and therapy efficiency in pancreatic ductal adenocarcinoma (PDAC) patients, making it an attractive target for antibody development. Recent clinical trials have demonstrated satisfying safety profiles and unique expression in a range of malignancies that highlight CA 19-9 as an attractive TACA to target using a stand-alone drug or an adjuvant therapy. Hence, we set out to explore the use of synthetic sLeA in the development of additional monoclonal antibodies (mAbs) with enhanced sLeA recognition and improved efficacy. Two mAbs targeting sLeA were generated through mice immunization with synthetic sLeA glycoconjugates, synthetic glycan arrays, and hybridoma technology. We then compared the antigen-binding properties of the newly developed mAbs with those of the widely used mAb 1116-NS-19-9 and demonstrated improved affinity and specificity for native sLeA ectopically expressed in B16 melanoma cells, surpassing the performance of the established mAb 1116-NS-19-9. Of the two mAbs, GB11 was more promising. Therefore, to elucidate the structural origin of improved GB11's antigen binding, we conducted high-resolution mapping of the molecular recognition patterns between sLeA and the different antibodies using X-ray crystallography and STD NMR. These analyses revealed subtle yet critical differences in the glycan engagement and identified key structural features underlying enhanced GB11's recognition of sLeA. MD simulations further supported these observations, indicating distinct orientations of sLeA within the binding pockets of each mAb. Our results suggest improved recognition of the native sLeA antigen by the newly generated GB11 antibody, providing a detailed and high-resolution elucidation of the molecular interactions underlying this recognition. Our study provides a tool with improved theranostic properties against sLeA-overexpressing malignancies.
Developability assessment facilitates the selection of antibody drug candidates with desirable pharmaceutical properties. However, it remains uncertain whether agitation-induced aggregation can be predicted from standard developability parameters. Here, we investigated whether key biophysical parameters predict agitation-induced aggregation of monoclonal antibodies (mAbs). To this end, we generated a benchmark data set by characterizing the aggregation upon agitation in the presence of an air-liquid interface of ten approved mAbs reformulated in a common surfactant-free buffer. The extent of aggregation varied substantially among mAbs and was primarily dependent on antibody identity. Flow imaging microscopy combined with machine learning revealed micrometre-sized aggregates with distinct morphologies, consistent with aggregation at air-liquid interfaces. Examination of thin liquid films and foams confirmed the presence of aggregates directly at the air-liquid interface and, therefore, the critical role of this interface for antibody aggregation during agitation. We then applied fluorescence-based, light scattering, and chromatographic techniques to determine standard developability parameters for each mAb, including apparent melting temperature (Tm), nonreversibility onset temperature (Tnr), aggregation onset temperature (Tagg), diffusion self-interaction parameter (kD), hydrophobic interaction chromatography retention time, and relative monomer yield after isothermal refolding from chemical denaturants. Notably, none of these parameters correlated with agitation-induced aggregation. Finally, we assessed the surface properties of the mAbs via drop shape analysis and found that the combination of surface pressure and elastic modulus yields a good correlation with the concentration of micrometre-sized aggregates formed due to agitation. Overall, these findings highlight limitations in predicting mAb interfacial stability using standard developability assays and underscore the importance of studying antibody behavior at interfaces.
Favorable pharmacokinetic (PK) properties of antibody-drug conjugates (ADCs) enable higher systemic exposure and are associated with improved clinical efficacy, yet ADC PK optimization remains largely empirical and resource-intensive. We developed an in silico framework to predict half-lives of monoclonal antibodies (mAbs) and ADCs in C57BL/6 mice using multi-modal feature learning. To address structural complexity of ADCs, each compound was deconvoluted into three components: (1) antibody features represented using sequence descriptors, AlphaFold-based structural properties, or protein language model (PLM) embeddings; (2) conjugation-related features, including drug-to-antibody ratio and conjugation sites; and (3) physicochemical properties of linker-payloads. XGBoost models were trained using experimental half-life data from 118 mAbs and ADCs. Structure-based and PLM approaches achieved strong cross-validation (Pearson's r = 0.76), outperforming sequence-only models. In temporal validation of 32 mAbs and ADCs, the structure-based models demonstrated robust generalization (r = 0.79). PLM embeddings achieved comparable rank ordering (Spearman's ρ up to 0.81 for ESM-2), but lower quantitative accuracy relative to structure-based models. Overall, our study presents a scalable machine learning framework for early PK screening of mAbs and ADCs prior to synthesis and in vivo testing.
Eosinophilic myocarditis (EM) is a rare inflammatory heart disease often associated with eosinophilic granulomatosis with polyangiitis or hypereosinophilic syndrome. While anti-IL-5/5R and anti-IL-4/13 monoclonal antibodies (mAbs) efficacy in systemic eosinophilic diseases is established, data on EM are lacking. We aimed to: (1) characterize a single-center cohort of EM patients treated with mepolizumab, benralizumab, or dupilumab in combination with glucocorticoids and/or immunosuppressants; (2) systematically review published cases, comparing them with a contemporary cohort; (3) evaluate myocardial response and safety of mAbs in EM, in comparison with a historical cohort treated without mAbs at our center. Thirty-seven EM patients were included (19 from a contemporary cohort, 18 from the literature; 51% male; median age 47 years). Biologic treatments were mepolizumab (81%), benralizumab (14%), and dupilumab (5%). Median time to mAb initiation was 2.5 months; treatment duration 24 months. No EM relapses, deaths, or heart transplantations occurred. Glucocorticoids were tapered and withdrawn in 89% of cases, with no mAb discontinuations due to adverse events. In the contemporary cohort, mAb therapy was associated with improved LVEF (47% to 55%, p = 0.004), TnI normalization (95% to 12%, p < 0.001), and eosinophil reduction (95% to 11%, p < 0.001). Compared to EM patients managed with conventional immunosuppressants alone, the mAb group had no myocarditis relapses (0% vs. 25%) and lower follow-up eosinophil counts (0.04 ×109/L vs 0.85 ×109/L). In EM within eosinophilic disease, anti-IL-5/5R and anti-IL-4/13 mAbs showed steroid-sparing effects and favorable safety, suggesting potential benefit for disease control.
Treatment of Mycobacterium abscessus pulmonary disease (Mabs-PD) is a growing global health challenge. The lack of bactericidal antibiotics effective at therapeutically relevant concentrations underscores an urgent need for drug discovery. Targeting cell wall synthesis is a promising strategy for drug discovery, as exemplified by the clinical success of broad-spectrum β-lactam antibiotics. Here, we employ a bioluminescence-based whole-cell assay optimized to identify compounds that disrupt both cell wall synthesis and oxidative phosphorylation. A focused drug library screen against Mabs reveals a chemically tractable naphthalen-1-ylmethanamine scaffold with potent bactericidal activity. The optimized derivative GM47-1 targets MmpL3, compromises cell wall integrity, induces ATP leakage, and uncouples respiration. Further chemical optimization yields a derivative with nanomolar minimum inhibitory concentration, bactericidal activity against intracellular Mabs, and efficacy in a zebrafish infection model. Together, these findings identify a promising scaffold for therapeutic development and demonstrate the utility of this bioluminescence-based platform for discovering bactericidal agents against Mabs.
Therapeutic monoclonal antibodies (mAbs) are central to cancer treatment but often show incomplete efficacy. We show that transient pharmacologic inhibition of complex N-glycans in host cells ("glycoengineering") enhances the in vivo activity of multiple depleting mAbs, including mAbs already engineered for heightened potency. In preclinical models, glycoengineering improved α-CD20-mediated tumor clearance and survival through FcγRIIIa- and natural killer (NK) cell-dependent pathways. In B16-F10 melanoma, glycoengineering similarly enhanced anti-CD25 depletion of intratumoral regulatory T cells (Tregs). Notably, glycoengineering produced minimal changes in equilibrium binding affinity but markedly increased the mechanical durability of IgG-FcγRIIIa interactions under physiological shear stress. These results establish antibody effector function as a mechano-immunological process in which IgG-FcγR interactions can be tuned for resilience to physiological forces, thereby moving beyond the current affinity-centric paradigm in mAb engineering. Integrating mechanobiology into therapeutic development may enable mAbs optimized for the dynamic forces of human physiology, which provides a route to enhance next-generation immunotherapies.
Powassan virus (POWV) is a tick-borne orthoflavivirus that can cause severe neuroinvasive disease. There are no approved vaccines or therapeutics, and the incidence of POWV infection in humans is rising. Here, we isolated and characterized a panel of human monoclonal antibodies (mAbs) from a convalescent donor using single B-cell sorting with POWV EDIII and full-length E ectodomain as antigen bait. Binding and neutralization assays with both POWV reporter virus particles and an authentic strain identified multiple neutralizing mAbs that target distinct epitopes across the E glycoprotein, including EDIII lateral ridge/C-C' loop and non-EDIII regions. Four neutralizing mAbs were evaluated in a lethal mouse challenge, two of which conferred substantial protection. These findings define key targets of the human antibody response to POWV and highlight candidates for the development of human monoclonal antibody therapy for this emerging virus. Powassan virus is an emerging tick-borne orthoflavivirus with steadily increasing case numbers. With an approximately 10% case-fatality rate and no approved therapeutics or vaccines available, POWV represents a potential public health threat. Our results showing that human monoclonal antibodies can protect mice against POWV in a lethal challenge model provide a foundation for developing future effective immunotherapies against this virus.
Optical coherence tomography velocimetry (OCTV) was demonstrated with in-line processing of biologics for the first time. OCTV allowed the velocity of concentrated monoclonal antibodies (mAbs at 39.5-84.7 mg mL-1) to be probed in 3.4 pL volumes over distances 0-5 mm from the pipe walls. The large penetration depth is facilitated by the relatively low turbidity of mAbs at near-infrared wavelengths (1300 nm). The mAb solutions could be concentrated in situ and the changes to the viscoelasticity measured. Higher concentration mAb solutions became shear thinning (following the power law fluid model) and the amplitude of their velocity fluctuations decreased. Furthermore, dropping the pH of the mAb solutions induced a gelation phase transition and complex changes to the mAb rheology could be observed with OCTV e.g. thixotropy and the formation of a stationary boundary layer. Thus, in situ formulation of mAbs could be explored with OCTV under industrially relevant conditions.
To prepare monoclonal antibodies (mAbs) with high specificity and affinity against enhanced green fluorescent protein (EGFP), we used purified EGFP emulsified with Freund{L-End} 's adjuvant to immunize Balb/c mice. Mouse splenocytes were then fused with SP2/0 myeloma cells. Hybridoma cell lines stably secreting antibodies were obtained through indirect ELISA screening and subcloning with the limited dilution method. The epitope specificity, reactivity, and affinity of the antibodies were systematically analyzed via an additivity assay, isotype identification, Western blotting, and surface plasmon resonance (SPR). The results demonstrated the successful screening of two hybridoma cell lines, 1F11 and 3C11, which stably secreted antibodies specifically targeting EGFP, with isotypes of IgG2a and IgG2b, respectively. The additivity assay showed an additivity index (AI)>40%, indicating recognition of distinct antigenic epitopes. Antibody titers reached 1:512 000. Western blotting confirmed that the prepared antibodies could specifically recognize EGFP with a size of about 27 kDa. SPR analysis revealed affinity constants of 3.806×10-10 mol/L and 2.631×10-10 mol/L, respectively, both within the high-affinity range. These findings indicate that the prepared mAbs possess excellent specificity, reactivity, and high affinity, providing a specific and important biological tool for the immunological detection of EGFP-tagged proteins and the in vivo monitoring of vaccine strains. 为制备高特异性和亲和力的EGFP单克隆抗体(monoclonal antibody, mAb)，本研究以纯化的EGFP蛋白为抗原，经弗氏佐剂乳化后免疫Balb/c小鼠，将小鼠脾细胞与SP2/0骨髓瘤细胞进行融合，应用间接ELISA方法筛选阳性孔并利用有限稀释法进行亚克隆，获得稳定分泌抗体的杂交瘤细胞株。采用叠加试验、亚型鉴定、Western blotting及表面等离子体共振(surface plasmon resonance, SPR)技术，系统分析抗体的表位特异性、反应性及亲和力。结果发现，本研究成功筛选出2株能稳定分泌特异性针对EGFP蛋白单抗的杂交瘤细胞株1F11和3C11，其亚型分别为IgG2a和IgG2b;叠加试验显示叠加系数(additivity index, AI)大于40%，说明两者识别不同抗原表位;抗体效价检测发现效价均可达1:512 000;Western blotting分析证实其可特异性识别大小约为27 kDa的EGFP蛋白;SPR分析测得亲和力常数分别为3.806×10-10 mol/L和2.631×10-10 mol/L，均属于高亲和力水平。由此可见，本研究制备的mAbs具有良好的特异性、反应性及高亲和力，为EGFP标记蛋白的免疫学检测及疫苗株体内监测提供了特异性的重要生物工具。.
To compare the longitudinal cognitive changes of AChEIs and mAbs separately across three different cognitive measurements. We conducted a systematic review and meta-analysis of randomized controlled trials (RCTs) in individuals with mild cognitive impairment (MCI) or mild Alzheimer's disease (AD). Major databases (PubMed, Embase, CENTRAL, PsycINFO, and ClinicalTrials.gov) were searched from inception to April 6, 2025. The primary outcomes were changes in the Alzheimer's Disease Assessment Scale-Cognitive Subscale-14 Items (ADAS-Cog), Mini-Mental State Examination (MMSE), and Clinical Dementia Rating-Sum of Boxes (CDR-SB). Overall, monoclonal antibodies (7 trials, n = 8010) exhibited comparatively homogeneous and an increasing separation from placebo over time within trials, whereas acetylcholinesterase inhibitors (9 trials, n = 4993) showed greater heterogeneity and an apparent attenuation of effect with longer follow-up. On ADAS-Cog, acetylcholinesterase inhibitors demonstrated a nonsignificant pooled effect with substantial heterogeneity (MD = -0.24, 95% CI -1.22 to 0.73; I2 = 76%), whereas monoclonal antibodies were associated with a statistically significant and relatively consistent improvements within trials (MD = -1.27, 95% CI -1.69 to -0.84; I2 = 0%). On MMSE and CDR-SB, acetylcholinesterase inhibitors yielded modest and borderline effects, respectively (MMSE MD = 0.10, 95% CI 0.07 to 0.13; CDR-SB MD = -0.11, 95% CI -0.22 to 0), whereas monoclonal antibodies showed statistically significant benefits (MMSE MD = 0.42, 95% CI 0.07 to 0.77; CDR-SB MD = -0.41, 95% CI -0.62 to -0.20). For CDR-SB, AChEIs showed minimal statistical heterogeneity (I2 = 0%) despite variable individual trajectories, whereas mAbs showed moderate heterogeneity (I2 = 56%). In patients with MCI or mild AD, mAbs showed consistent efficacy on MMSE and ADAS-Cog but demonstrated heterogeneous effects on CDR-SB. In contrast, AChEIs showed heterogeneous and modest efficacy across all three measurements. These findings should be interpreted with caution due to the study's limitations.
Host cell proteins (HCPs) are important process-related safety concern factors during drug development, clinical trials and manufacturing, and their removal becomes particularly challenging when associated with antibody aggregates. Residual HCPs in drug products could trigger adverse effects or compromise drug efficacy and stability. This study aims to enhance the HCP removal specifically in monoclonal antibodies (mAbs) with high aggregate content before protein A chromatography by introducing reduction and denaturation steps. A model mAb was purified by different processes, and the impact on HCP removal was evaluated by enzyme-linked immunosorbent assay (ELISA) and Liquid chromatography-mass spectrometry (LC-MS). The HCP profiles in high aggregate, low aggregate and monomeric mAbs were analyzed by LC-MS. The additional reduction and denaturation steps improved HCP removal rate by 1.08 log10 reduction value (LRV) compared to the control process. For the relatively abundant high-risk HCPs, 45% were completely removed. HCP removal in the model mAb was significantly enhanced, especially in high aggregate forms. This method can reduce HCP carry-over from mAbs aggregates in subsequent downstream processing and can provide an efficient HCP removal strategy for manufacturing to improve patient safety and drug stability.
Nerve growth factor (NGF) is a member of the neurotrophin family, essential for neuronal survival and phenotypic maintenance. However, in vitro, its function can be disrupted by oxidative posttranslational modifications such as tyrosine nitration. Nitrated NGF (NO2NGF) has been shown to have a gain-of-toxic, pro-apoptotic, activity in motoneuron cultures. Herein, we report the generation and characterization of monoclonal antibodies (mAbs) that specifically recognize NO2NGF to unravel its formation in vivo. Using hybridoma technology, we produced mAbs with high affinity and selectivity for NO2NGF, as demonstrated immunochemically and by surface plasmon resonance. The antibodies elicit neutralizing capacity to NO2NGF in neuronal cells. Nitrated Tyr52 within the NGF48-58 sequence was identified as the primary antigenic determinant by crystallographic analysis of antibody:peptide complexes at atomic resolution, peptide-based epitope mapping and molecular dynamics simulations, whereas local sequence NGF motifs around the nitrated tyrosine are important for protein specificity. The antibodies revealed NO2NGF accumulation in amyotrophic lateral sclerosis (ALS) rodent models and human subjects. Indeed, immunofluorescence showed selective accumulation of NO2NGF in spinal cord regions undergoing motor neuron degeneration, as well as in sciatic nerves and neuromuscular junctions. Our findings establish NGF nitration as an oxidative hallmark in ALS and demonstrate that monoclonal antibodies targeting this chemical modification are powerful tools for both mechanistic studies and biomarkers development. This work proposes a link between neurotrophin nitration and neurodegenerative disease progression and opens avenues for therapeutic exploration along the peroxynitrite-tyrosine nitration pathway.
Macrophage phagocytosis is a major cytotoxic mechanism for therapeutic monoclonal antibodies (mAbs) that opsonize target cells. This antibody-dependent cellular phagocytosis (ADCP) can occur via the Fcγ or complement pathways, but the relative contribution of these pathways to mAb-mediated cell clearance is not known. Here, we analyzed the kinetics, functional cooperation, and phagocytic capacities of Fcγ receptor-dependent and complement-dependent ADCP, separately and concomitantly, in primary macrophages challenged with mAb-opsonized lymphocytes. Using quantitative live-cell imaging of primary mouse macrophages, genetic disruption of Fcγ receptor signaling, and controlled modulation of complement activity, we directly compared the kinetics, capacity, and exhaustion behavior of ADCP via the Fcγ (fADCP) and complement (cADCP) pathways. cADCP operates as a mechanistically independent phagocytic pathway with distinct temporal dynamics. Relative to fADCP, cADCP exhibits delayed onset but substantially greater cumulative target clearance. When both pathways are engaged simultaneously, their effects on target removal are additive, indicating functional non-redundancy. Notably, macrophages rendered refractory to further phagocytosis following fADCP retain full capacity for cADCP, demonstrating that complement receptor-mediated engulfment can bypass Fcγ receptor-associated hypophagia. However, despite its greater capacity, cADCP is also finite, as increasing target burden induces a dose-dependent state of complement-associated phagocytic exhaustion that is kinetically distinct from fADCP hypophagia and is largely reversible within 24 h. Our mechanistic understanding of mAb-mediated cytotoxicity via ADCP is disproportionately focused on Fcγ receptor engagement and signaling, with comparatively less emphasis on the role of complement activation. These findings establish complement-mediated ADCP as a quantitatively powerful macrophage effector pathway that can be leveraged to enhance the overall cytotoxic efficacy of mAbs. Further, our work provides a functional framework for understanding how Fcγ receptor and complement pathways differentially contribute to macrophage cytotoxic capacity and highlights effector exhaustion as a shared but mechanistically distinct constraint on sustained antibody-mediated cell clearance. Effector exhaustion therefore represents a fundamental bottleneck to durable responses to ADCP-inducing therapeutic antibodies, but one that can potentially be mitigated via utilization of both ADCP pathways.