Monoclonal antibody passive experimental autoimmune myasthenia gravis: current progress and future directions

Abstract

Experimental Autoimmune Myasthenia Gravis (EAMG) remains a cornerstone platform for mechanistic studies of pathogenic antibodies in myasthenia gravis (MG). In particular, monoclonal antibody passive-transfer EAMG (mAb-PTMG) has been instrumental in establishing causal links between defined antibody specificities and downstream neuromuscular junction (NMJ) dysfunction, as well as in dissecting key mechanisms underlying AChR antibody–mediated disease. More recently, PTMG studies using patient-derived MuSK monoclonal antibodies have expanded the mechanistic landscape of MG and provided an in vivo framework for evaluating the efficacy and mechanisms of action of agonistic anti-MuSK therapeutic antibodies. In this review, we summarize practical considerations for monoclonal antibody sourcing and preparation, standardized experimental workflows, major readouts and their interpretation, and representative applications across MG subtypes. We also highlight emerging opportunities for model refinement and translational application, with the aim of supporting mechanism-guided interpretation and the development of targeted antibody therapies.

Introduction

Myasthenia gravis (MG) is an organ-specific autoimmune disorder of the neuromuscular junction (NMJ), clinically characterized by fatigable muscle weakness [1]. Identification of pathogenic autoantibodies and the establishment of corresponding experimental autoimmune myasthenia gravis (EAMG) models are fundamental for elucidating disease mechanisms and advancing subtype-based diagnosis and therapy. To date, at least nine classes of MG-associated autoantibodies with potential pathogenic relevance have been reported, and additional candidate antibodies continue to emerge [2]. Accordingly, expanding the autoantibody repertoire and defining the functional significance of these candidates remain central directions in MG research. Using EAMG paradigms, antibodies against acetylcholine receptors (AChR) [3, 4], Muscle-specific receptor tyrosine kinase (MuSK) [5, 6], and Low-density lipoprotein receptor-related protein (LRP4) [7, 8] have been validated as directly pathogenic. Active EAMG studies further support the possibility that anti-Agrin antibodies may contribute to MG pathogenesis [9], whereas the pathogenic roles of other reported MG-associated antibodies require additional validation.

Animal models are essential tools for dissecting MG pathophysiology and for preclinical evaluation of therapeutic interventions. The EAMG system is generally divided into two major approaches: active immunization models and passive-transfer models (Fig. 1) [10]. Active immunization recapitulates key features of breakage of tolerance and the development of an endogenous polyclonal immune response, whereas passive transfer enables more direct attribution of disease manifestations to defined antibody preparations. Passive-transfer EAMG (PTMG) models generally fall into two categories: those induced by administering polyclonal immunoglobulins (IgG) and those induced by administering monoclonal antibodies (mAb). Notably, mAb–based passive-transfer models provide a highly controllable platform in which antigen specificity, epitope targeting, Fc effector functions, and antibody valency can be precisely defined and systematically tuned, while minimizing batch-to-batch variability. Accordingly, the following sections focus on mAb-based PTMG, covering their establishment, major antibody sources and formats, and their utility for mechanistic dissection and subtype-oriented translational studies.

Fig. 1.

Complementary EAMG models. A Active immunization with AChR induces antigen presentation, T/B-cell activation, and endogenous autoantibody production, modeling immune initiation and systemic immunopathology. B Passive transfer of exogenous antibodies (patient polyclonal IgG or defined mAbs) isolates antibody effector mechanisms with controllable variables (e.g., epitope, subclass, valency) and high reproducibility. C Together, both approaches capture key steps leading to NMJ injury in myasthenia gravis

PTMG: from patient serum or IgG to mAbs

The basic idea of a passive transfer model is to inject patient serum, purified IgG, or experimentally prepared antibodies into recipient animals, and then observe whether muscle weakness appears, as well as whether there are electrophysiological or morphological abnormalities at the NMJ. In early PTMG studies, patient serum or purified IgG was administered to mice to induce MG-like changes, providing key evidence that antibodies can be pathogenic [4, 7, 11, 12]. An advantage of this approach is that it more closely reflects the antibody composition observed in patients. However, these reagents are inherently polyclonal, comprising antibodies that recognize multiple epitopes and vary in subclass, affinity, and glycosylation, and are further influenced by donor-to-donor variability, disease stage, and treatment history. As a result, batch-to-batch consistency is limited. Induction of a measurable phenotype often requires relatively large amounts of purified IgG and, in some settings, repeated administrations over several days, which can constrain feasibility when patient material is scarce. Moreover, the onset and severity of weakness are variable rather than temporally uniform, reflecting differences in antibody titer and composition, recipient strain, and dosing regimen. Importantly, this variability limits mechanistic interpretation, as it becomes difficult to attribute pathogenic effects to specific antibody features. In particular, key questions such as which antibody subpopulations or subclasses drive pathogenicity, whether dose–response relationships are reproducible, and how consistently a given preparation induces weakness cannot be readily resolved in polyclonal systems. mAb PTMG mitigate these issues: anti-AChR mAbs can induce MG-like phenotypes, yet pathogenicity differs across clones, underscoring the importance of epitope specificity and antibody features in shaping disease [13]. Therefore, polyclonal and monoclonal EAMG are best viewed as complementary. Polyclonal systems capture clinical complexity better, whereas monoclonal systems are better suited for single-variable comparisons and provide more consistent conditions across experiments.

Mechanistic advantages of mAb PTMG: epitope, subclass, and valency

A key advantage of mAb PTMG is their defined, uniform composition. This enables systematic dissection of how individual variables—such as epitope specificity, IgG subclass, or valency—shape pathogenic effects.

First, at the epitope level, studies of AChR-associated MG have long identified the main immunogenic region (MIR) as a key immunodominant epitope [14]. mAb–based work demonstrated that human muscle AChR contains the MIR [15, 16] and further mapped this region to discrete residues on the AChR α subunit (e.g., residues 67–76) [17]. Mechanistically, MIR-specific mAbs do not interfere with acetylcholine binding to AChRs or directly affect channel gating. Instead, they predominantly cause AChR loss from the postsynaptic membrane by crosslinking AChRs and accelerating receptor degradation via antigenic modulation [18]. Notably, in patients with MG, complement-mediated injury is a major driver of AChR loss at the NMJ, and complement inhibition helps preserve postsynaptic AChR levels. Although receptor internalization may also occur, it is generally insufficient by itself to reduce AChR density to a level that significantly impairs muscle membrane depolarization. Recent studies further demonstrate that MIR-specific mAbs can also activate complement and exert pathogenic effects, which can be ameliorated by complement-targeted inhibitors such as CRIg/FH [19]. Collectively, establishing and validating the MIR through PTMG has been critical for elucidating the pathogenicity and mechanisms of mAbs.

Second, experimental evidence that IgG isotypes differ in their capacity to activate complement provides a methodological basis for such comparisons [20]. At the same time, Fcγ receptor–mediated effects—and species differences between mouse and human Fc receptor systems—can influence how Fc-dependent mechanisms are interpreted and generalized [21]. In anti-AChR mAb models, complement-dependent injury is strongly influenced by IgG subclass (e.g., IgG2a) and is closely linked to complement activation [22], a mechanism repeatedly highlighted in both clinical and basic studies [23, 24]. In addition, MuSK-related MG often features a high proportion of IgG4. Because IgG4 undergoes Fab-arm exchange and has effector functions that differ from the classic complement-activating IgG1 and IgG3 subclasses [25, 26]. mechanistic studies of MuSK-MG often rely more heavily on defined and controllable antibody formats [27–29]. Monoclonal antibody models enable controlled comparisons of complement-related effects by selecting different IgG isotypes or by engineering Fc constant-region variants (e.g., isotype switching) while preserving identical antigen specificity. This strategy could help explain why antibodies directed against the same target can produce markedly different degrees of tissue injury. Accordingly, studies should explicitly report the Fc format/engineering of the antibodies used, as well as the Fc receptor background of the recipient animals.

Third, antibody-induced antigenic modulation—driven by receptor crosslinking—promotes AChR internalization and accelerates receptor degradation. Evidence that MG IgG accelerates AChR degradation, and that this effect depends on crosslinking, suggests that antibody valency is an important determinant of disease phenotype [30]. mAb systems enable the generation of Fab, F(ab’)₂, or engineered mono- versus bivalent formats, making them well suited for directly testing how valency influences phenotypic outcomes.

Together mAb PTMG provides uniform antibody structure and single-epitope specificity, enabling consistent experimental conditions, improved reproducibility, and suitability for mechanistic studies. mAb PTMG allow precise dissection of pathogenic mechanisms at the NMJ, such as complement activation, receptor blockade, or receptor internalization.

Sources of antigen-specific mAbs

The generation of antigen-specific mAbs for PTMG can originate from immunized animals or directly from patients with MG. Both pathways offer distinct advantages for isolating pathogenesis antibodies (Fig. 2). Conceptually, these approaches differ not only in technical implementation but also in the biological information they capture. Animal immunization–based strategies tend to enrich for antigen-driven, high-affinity clones shaped by experimental conditions, making them well suited for controlled mechanistic studies. In contrast, patient-derived approaches provide access to naturally occurring autoreactive repertoires that reflect disease-relevant immune selection and thus offer greater translational relevance. Accordingly, these sources are complementary rather than interchangeable: animal-derived antibodies facilitate systematic mechanistic dissection, whereas patient-derived antibodies enable direct interrogation of human disease biology.

Fig. 2.

Sources and strategies for generating antigen-specific monoclonal antibodies. A Hybridoma and display-library–based antibody discovery from immunized mice. Splenic B cells isolated from antigen-immunized mice are fused with myeloma cells to generate hybridomas. Antigen-specific B cells can also be enriched by fluorescent autoantigen probes and single-cell sorting via flow cytometry. Antibody variable genes (VH and VL) are amplified by reverse transcription PCR and used to construct recombinant scFv/Fab libraries on phage, yeast, or mammalian display platforms. Antigen-binding clones are enriched and expanded for downstream characterization. B Single-cell cloning and display library construction from MG patient PBMCs. Peripheral blood mononuclear cells (PBMCs) from myasthenia gravis (MG) patients are stained to identify autoantigen-specific B cells, followed by antigen-specific single-cell sorting. VH and VL transcripts are recovered by single-cell RT-PCR to generate recombinant antibodies. Alternatively, total RNA can be extracted to construct scFv/Fab libraries, which are subjected to antigen panning for enrichment of specific binders. C Recombinant antibody reformatting and functional validation. Selected VH and VL sequences are cloned into expression vectors for full-length IgG reformatting. Recombinant antibodies are expressed, purified, and subjected to functional testing in vitro and in vivo to assess antigen binding, signaling interference, and pathogenic potential

Animal-derived mAb sources

Animal-derived mAbs are commonly generated by immunizing experimental animals with the relevant autoantigen or antigen fragment to elicit an adaptive B cell response. In PTMG studies, both mice and rats have been used, with Lewis rats being the most commonly employed model. Following repeated booster immunizations, splenic B cells are harvested as a rich source of antigen-experienced antibody-producing cells. Antibodies can then be isolated using one of the following approaches, each with distinct strengths and limitations.

Traditional hybridoma technology

Splenic B cells are fused with immortal myeloma cells to create hybridomas, which are screened for antigen specificity. Positive clones are subcloned to monoclonality, and the secreted mAb is purified. The variable regions can subsequently be sequenced and used to create recombinant mAbs for further engineering. This method is well established, robust, and yields stable antibody-producing cell lines with reproducible secretion. It remains a gold standard for generating murine mAbs and has been extensively validated in MG and EAMG research [22]. But hybridoma generation is time-consuming and labor-intensive, and fusion efficiency can be low. In addition, murine antibodies often require humanization for translational studies, and rare or low-frequency B-cell clones may be lost during fusion and subcloning [31, 32].

Single-cell sorting of immune B cells

Antigen-specific splenic B cells can be stained with fluorescently labeled autoantigen and isolated by single-cell flow cytometry. Sorted cells can be cultured or processed directly for variable-region sequencing, enabling reconstruction of paired heavy and light chains for recombinant antibody expression. By bypassing myeloma fusion, this strategy preserves native heavy–light chain pairing and provides rapid access to antigen-specific, often high-affinity clones. It is particularly advantageous for recovering rare B-cell populations and accelerating antibody sequence discovery. However, successful implementation depends on high-quality antigen probes and technical expertise in multicolor flow cytometry. Antigen labeling chemistry, probe valency, and gating strategies can bias which clones are captured, and downstream functional characterization relies entirely on recombinant expression and validation [33]. This strategy has been applied in other autoimmune models such as NMO to recover pathogenic recombinant antibodies and demonstrate disease-relevant pathology in vivo [34]. Application of similar single-cell approaches in MG/EAMG settings is conceptually feasible but has not yet been fully reported.

Antigen-specific library construction from B-cell repertoires

Total RNA from immune animal splenic B cells can be reverse transcribed to capture the heavy (VH) and light (VL) chain sequences. Recombinant single-chain variable fragment (scFv) or Fab libraries are then constructed and displayed on phage, yeast, or mammalian platforms. Iterative rounds of antigen panning and selection enrich antigen-binding clones, which can subsequently be reformatted into full-length recombinant monoclonal mAbs. Display-based approaches enable scalable screening of highly diverse libraries without reliance on individual cell culture and, when antigens are presented in a native-like conformation, can recover antibodies recognizing conformational epitopes and facilitate epitope-focused selection. However, selection outcomes are strongly influenced by antigen format, folding, and presentation during panning. Moreover, scFv/Fab formats may under-represent quaternary or membrane-proximal epitopes; this limitation can be partially mitigated by panning on antigen-stably expressing cell lines. Finally, enriched clones typically require reformatting and validation in a full-length IgG context [35]. Immune (immunization-derived) display libraries have been effectively used to recover antigen-specific clones from actively immunized animals in antibody-mediated autoimmune models [36], and such libraries can support antibody-transfer modeling—ranging from phage display–based autoantibody isolation to targeted autoantigen discovery—suggesting feasibility for active EAMG as a source for display-based mAb discovery, although practical constraints (e.g., native antigen presentation and membrane-proximal or quaternary epitopes) remain important.

Patient-derived mAb

Antibodies directly derived from patients with MG provide access to naturally occurring human autoreactive repertoires. When combined with experimental pathogenicity screening, they further offer the advantage of capturing bona fide disease-relevant “pathogenic” repertoires, including native somatic mutations and epitope specificities.

Single-cell sorting and RT-PCR cloning

Peripheral blood mononuclear cells (PBMCs) from MG patients can be stained for B-cell markers and autoantigen specificity, and single antigen-positive B cells are sorted. VH and VL are recovered from individual cells by single-cell RT-PCR, cloned into expression vectors, and recombinantly expressed as full-length human mAbs. This approach preserves authentic heavy–light chain pairing and enables the recovery of antibodies directly derived from human autoreactive B cell clones. When coupled with experimental pathogenicity screening, it provides a powerful platform for mechanistic studies and translationally relevant antibody discovery. Antigen-specific B cells are often rare in peripheral blood, limiting throughput. Sample availability, disease heterogeneity, and technical demands constrain large-scale antibody discovery [37, 38].

Patient autoantibody library construction

Total RNA from patient PBMCs or enriched B-cell subsets can be used to build high-diversity human scFv/Fab libraries for display. Following antigen panning (e.g., with AChR or MuSK extracellular domains), antigen-specific human antibody fragments are recovered and reformatted into full-length IgG for functional testing. This strategy can identify rare or low-frequency clones that may be missed by flow sorting alone. This strategy enables unbiased, high-throughput interrogation of patient antibody repertoire and can identify rare or low-frequency clones missed by single-cell approaches. Native heavy-light chain pairing is not preserved, and extensive downstream validation is required to confirm physiological relevance and pathogenic function [39]. Display-based antibody discovery (e.g., phage display) has been successfully used in autoimmune diseases to isolate monoclonal autoantibodies with demonstrated pathogenicity in vivo [40, 41]. These precedents support the feasibility of adopting analogous display-to-IgG-to-passive-transfer workflows for mechanistic studies, although published examples in MG remain relatively limited.

Although not specific to MG, each of these approaches has been successfully applied in autoantibody discovery and provides essential methodological context for the development and isolation of monoclonal antibodies using emerging technologies. Notably, several of these approaches, which have not been widely applied in PTMG previously, have now been incorporated into our PTMG-related research and can be further integrated with downstream recombinant engineering (e.g., humanization, Fc optimization, and valency modulation) to generate research tools or therapeutic candidates with defined properties.

Together, these strategies provide complementary access to different regions of the antibody repertoire, ranging from dominant antigen-driven responses to rare or low-frequency specificities, and should be selected based on the mechanistic or translational goals of the study.

Recombinant mAb engineering and modification

Beyond generating mAbs by hybridoma, single-cell cloning, or display technologies, recombinant engineering enables systematic “one-variable-at-a-time” experiments to distinguish epitope-driven binding from subclass-dependent biology: the same epitope-binding variable region can be paired with different constant regions, Fc variants, or multimerization formats, allowing causal attribution of clinical and pathological readouts (weakness, Compound muscle action potential (CMAP) decrement, NMJ morphology, complement deposition, and AChR/MuSK signaling changes) to defined antibody properties (Fig. 3).

Fig. 3.

Epitope-driven and Fc-dependent engineering of recombinant monoclonal antibodies. A Multivalency and format engineering. Antibody formats (IgG, F(ab’)₂, Fab) determine valency and receptor crosslinking capacity at the neuromuscular junction (NMJ). Bivalent IgG and F(ab’)₂ can crosslink AChR and alter receptor stability or clustering, whereas monovalent Fab lacks crosslinking ability and primarily blocks ligand binding. B Affinity and epitope mapping. Binding strength and kinetics (affinity/avidity) are characterized by competition and kinetic assays. Epitope definition is achieved using structural and proteomic approaches such as HDX-MS and cryo-EM to determine epitope location, orientation, and conformational changes upon antibody binding. C IgG subclass switching and D Fc-dependent effector functions. Different IgG subclasses (IgG1–IgG4) differentially engage complement (C1q) and Fcγ receptors (FcγR), modulating complement activation, immune cell recruitment, inflammatory signaling, and postsynaptic membrane damage at the NMJ

Epitope-driven binding biology

Multivalency and format engineering: bivalency, F(ab’)₂, Fab, and IgG fragments

Format engineering helps disentangle Fc-independent mechanisms from immune effector pathways. For AChR-related EAMG, comparing intact IgG with F(ab’)₂ and Fab fragments is informative because bivalency can drive receptor cross-linking and internalization, whereas Fab largely isolates direct blocking without cross-linking. In MuSK/LRP4-related models, format can be even more mechanistically revealing: some pathogenic antibodies may function by disrupting protein–protein interactions or by altering receptor clustering dynamics, which can be modulated by valency and geometry. Notably, a single antibody can engage multiple pathogenic mechanisms simultaneously and is strongly influenced by the local microenvironment as well as co-existing autoantibodies. However, by using matched fragments derived from the same mAb, investigators can dissect these mechanisms and determine the relative contribution of distinct effector pathways—including receptor modulation (cross-linking–driven internalization of AChR) and blockade of ligand–receptor interactions (e.g., the Agrin–LRP4–MuSK axis)—to the development and severity of muscle weakness in PTMG [1, 30, 42, 43].

Affinity, epitope mapping, and cross-competition

EAMG outcomes are highly sensitive to affinity/avidity and epitope location. Before in vivo experiments, antibodies should be characterized for binding affinity and kinetics (e.g., SPR/BLI), and epitope behavior should be defined using cross-competition and, when feasible, structural approaches (HDX-MS, cryo-EM, or mutagenesis scanning). These characterization data allow rational assembly of antibody panels in which only one feature is changed at a time—such as affinity, Fc effector function, or valency—so that functional differences can be interpreted without major confounding. Equally important, antibody activity in mAb PTMG depends on whether the antibody can actually bind the target antigen in the chosen species (mouse or rat). Because binding is epitope-dependent, even small sequence differences within the targeted epitope between human and rodent orthologs can markedly weaken or abolish binding. As a result, an antibody optimized for human relevance may fail to produce measurable effects in rodents not because it is non-pathogenic, but simply because its epitope is not sufficiently conserved. Therefore, confirming epitope mapping and cross-species reactivity should be considered a prerequisite for reliable mechanistic interpretation in passive-transfer models [44].

FC-dependent biology

IgG subclass switching

IgG subclass switching is a powerful recombinant strategy to interrogate how Fc-mediated effector functions shape disease expression in EAMG. By keeping the variable region (antigen specificity) constant while swapping the antibody Fc to different IgG subclasses, investigators can decouple epitope-driven binding from subclass-dependent biology—such as complement activation capacity, Fcγ receptor engagement, and downstream inflammatory amplification. This approach enables causal testing of whether a given pathogenic monoclonal antibody requires complement, FcγR signaling, or other Fc-dependent pathways to drive postsynaptic AChR loss, endplate injury, and impaired muscle membrane depolarization at the NMJ, ultimately leading to weakness and fatigability. It also provides a rational framework for mapping antibody “mechanism-of-action” across epitopes and targets.

Advantages: subclass switching offers a controlled, modular readout of Fc function, allowing direct comparison of disease phenotypes induced by the same paratope across Fc contexts; it can reveal effector-function thresholds (e.g., complement-dominant vs FcγR-dominant pathology), inform interpretation of patient subclass distributions, and guide selection of engineered Fc backbones for mechanistic or intervention studies.

Limitations: subclass reformatting may alter antibody valency/orientation, stability, or in vivo persistence, and interspecies differences in FcγR repertoires and complement pathways complicate direct translation between mouse and human subclasses. Therefore, studies should report Fc format, dosing/exposure, and tissue distribution, and ideally validate key conclusions using complementary genetic or pharmacologic perturbations (e.g., complement inhibition, FcγR blockade/knockout).

Future directions: systematic subclass panels—generated for matched epitope-defined mAbs—could be used to build “effector maps” linking antibody epitope, subclass, and NMJ lesion signatures (AChR loss, complement deposition, fragmentation, transmission failure). Coupling subclass-switch experiments with standardized electrophysiology (CMAP/EPP/mEPP), quantitative NMJ imaging, and intervention controls (e.g., complement inhibitors) would enable a more granular, pathway-resolved understanding of pathogenic mechanisms in EAMG.

Fc-silencing and Fc-enhancing variants: mapping complement vs FcγR contributions

Targeted Fc mutations can be used to selectively attenuate complement activation (e.g., disrupting C1q binding) and/or FcγR binding, generating Fc-silent antibodies that preserve binding but minimize inflammatory effector functions. In passive transfer paradigms, Fc-silent variants can address whether a given mAb causes disease predominantly by immune effector injury (MAC formation, inflammatory infiltration). Conversely, Fc-enhancing designs (increased FcγR binding or prolonged half-life) can be used to test threshold effects—whether increasing effector potency converts a “binding-only” antibody into a pathogenic one, or accelerates onset/severity in EAMG. Readouts to pair with Fc engineering: NMJ C3/C5b-9 deposition, macrophage/neutrophil markers, ultrastructural postsynaptic folds, and time-resolved electrophysiology (CMAP decrement) to distinguish early functional impairment from later structural loss [45–47].

FC-glycoengineering: tuning effector function without changing amino acid sequence

Fc glycosylation strongly influences FcγR binding and downstream inflammation. Glycoengineered antibodies (e.g., altered fucosylation or sialylation) provide a way to tune FcγR engagement while keeping the protein sequence unchanged, which can be attractive for mechanistic dissection in EAMG where subtle shifts in effector potency may influence phenotype severity or consistency. Such approaches can help determine whether a model is “effector-limited” (requires strong FcγR signaling) or “antigen-mechanism–limited” (primarily dependent on epitope-specific interference). Caveat: glycoforms can drift across production batches and expression systems; rigorous characterization of glycan profiles is important for reproducibility [48].

Together, these experimental and engineering strategies establish a multi-layered framework that links molecular antibody properties to in vivo pathogenic outcomes, thereby enabling mechanism-guided interpretation of MG and supporting the development of targeted therapeutic antibodies.

Procedure of mAbs PTMG

The workflow for establishing a mAb PTMG may appear straightforward, but experimental outcomes are in fact governed by three major classes of variables. First, the recipient context includes the animal strain, immune status (e.g., immunocompetent vs immunodeficient), and the complement and Fcγ receptor milieu. Second, the antibody reagent encompasses the monoclonal clone and epitope specificity, IgG subclass/Fc format, valency (e.g., IgG vs F(ab’)₂/Fab or engineered mono-/bivalent formats), and whether an Fc region is present. Third, the dosing strategy comprises the administered dose, route, frequency/number of injections, and overall exposure duration. These parameters differ substantially across studies. Without clear and standardized reporting, meaningful cross-study comparisons are difficult and experimental reproducibility is compromised.

Key model parameters for monoclonal antibodies

Based on Table 1, several conclusions emerge. First, antibodies directed MIR on the extracellular domain of the AChR α subunit—an epitope present in two copies per receptor—tend to induce disease more rapidly and at lower doses. Second, high-affinity binding alone is not sufficient for pathogenicity, as illustrated by antibodies that bind a single-copy extracellular epitope on the β subunit yet show limited or no in vivo effect. Third, including matched fragment controls (intact IgG, F(ab’)₂, and Fab) is critical for disentangling Fc- and complement-dependent injury from valency-dependent crosslinking and antigenic modulation. Finally, in MuSK-MG models, IgG4 pathogenicity primarily reflects blockade of MuSK signaling, whereas agonistic antibodies highlight a mechanistic basis for potential therapeutic strategies.

Table 1.

Key parameters of representative monoclonal antibody passive transfer EAMG models

Name subclass	Source	Target, Epitope	Strain, sex, age	Administration scheme	Key phenotype (onset, severity)	Mechanism points	References
mAb 198 IgG1	Rat Hybridoma	Eel AChR, MIR, α subunit binding site	8–12-week-old female Lewis rat	Single intraperitoneal injection 80 µg	NR; Muscle weakness	Cross-links AChR and is associated with accelerated receptor degradation and impaired neuromuscular transmission; receptor modulation is implicated in this model	Lennon et al., [49]
371A IgG2b	Lewis Rat Hybridoma	Torpedo AChR, near cholinergic binding site	3–7-day-old chick	Single intraperitoneal or intravenous injection 28 mg/kg	Within 1 h; Complete paralysis after 1–2 h, EMG attenuation, severe motor defects	Binds to AChR cholinergic site, causing neuromuscular blockade and motor defects	Comez et al. [50]
370C IgG2a	Lewis Rat Hybridoma	Torpedo AChR, near cholinergic binding site	3–7-day-old chick	Single intraperitoneal or intravenous injection 38 mg/kg	Within 1 h; Complete paralysis after 1–2 h, EMG attenuation, severe motor defects	Binds to AChR cholinergic site, causing neuromuscular blockade and motor defects	Comez et al. [50]
383C IgG2b	Lewis Rat Hybridoma	Torpedo AChR, near cholinergic binding site	3–7-day-old chick	Single intraperitoneal or intravenous injection 31 mg/kg	Within 1 h; Complete paralysis after 1–2 h, EMG attenuation, severe motor defects	Binds to AChR cholinergic site, causing neuromuscular blockade and motor defects	Comez et al. [50]
132A IgG1	Lewis Rat Hybridoma	Torpedo AChR, Far from cholinergic binding site	3–7-day-old chick	Single intraperitoneal injection 30 mg/kg	No clinical symptoms or EMG abnormalities	Does not interfere with cholinergic site, no neuromuscular blockade	Comez et al. [50]
421H IgG2a	Lewis Rat Hybridoma	Torpedo AChR, Far from cholinergic binding site	3–7-day-old chick	Single intraperitoneal injection 31 mg/kg	No clinical symptoms or EMG abnormalities	Does not interfere with cholinergic site, no neuromuscular blockade	Comez et al. [50]
371A IgG2b	Lewis Rat Hybridoma	Torpedo AChR, Far from cholinergic binding site	3–7-day-old chick	Single intraperitoneal injection 37 mg/kg	No clinical symptoms or EMG abnormalities	Does not interfere with cholinergic site, no neuromuscular blockade	Comez et al. [50]
334C IgG2b	Lewis Rat Hybridoma	Torpedo AChR, Far from cholinergic binding site	3–7-day-old chick	Single intraperitoneal injection 30 mg/kg	No clinical symptoms or EMG abnormalities	Does not interfere with cholinergic site, no neuromuscular blockade	Comez et al. [50]
mAb 132A IgG2a	Lewis Rat Hybridoma	Torpedo AChR MIR, α subunit binding site	180–220 g female Lewis rat	Single intravenous injection 7.5–50 mg/kg	12–24 h; Mild clinical symptoms, moderate EMG attenuation	Associated with crosslinking-dependent AChR modulation; complement contribution was not specifically defined in this study	Christopher et al. [51]
mAb 561 IgG2b	Lewis Rat Hybridoma	Torpedo AChR MIR, α subunit binding site	180–220 g female Lewis rat	Single intravenous injection 5–10 mg/kg	12–24 h; Moderate clinical symptoms, moderate EMG attenuation	Likely low affinity, shorter binding time, causing mild immune response and complement activation	Christopher et al. [51]
mAb 265A IgG2a	Lewis Rat Hybridoma	Torpedo AChR MIR, α subunit binding site	180–220 g female Lewis rat	Single intravenous injection 5 mg/kg	12–24 h; Mild clinical symptoms, moderate EMG attenuation	Similar to mAb 132A, may cause medium EAMG severity via AChR cross-linking	Christopher et al. [51]
mAb 334C IgG2a	Lewis Rat Hybridoma	Torpedo AChR, MIR, α subunit binding site	180–220 g female Lewis rat	Single intravenous injection 10 mg/kg	12–24 h; Mild clinical symptoms, moderate EMG attenuation	Similar to mAb 132A and 265A, higher affinity, triggering immune activation and NMJ damage	Christopher M et al. [51]
mAb 153B IgG2a	Lewis Rat Hybridoma	Torpedo AChR, MIR, α subunit binding site	180–220 g female Lewis rat	Single intraperitoneal injection 10 mg/kg	24 h; Mild to moderate clinical symptoms, no significant electrophysiological changes	Low affinity, rapidly dissociating, minimal AChR degradation or dysfunction	Christopher et al. [51]
mAb 371A IgG2b	Lewis Rat Hybridoma	Torpedo AChR, MIR, α subunit binding site	180-220 g female Lewis rat	Single intravenous injection 0.25 mg/kg	12–24 h; Severe clinical symptoms	High-affinity MIR-binding mAb associated with severe EAMG, with evidence consistent with both complement activation and accelerated AChR degradation	Christopher et al. [51]
mAb 421H IgG2b	Lewis Rat Hybridoma	Torpedo AChR, MIR, α subunit binding site	180-220 g female Lewis rat	Single intravenous injection 50 mg/kg	12–24 h; Extremely severe clinical symptoms, rapid onset of myasthenia	Cross-links AChR and is associated with acute EAMG; complement activation likely contributes substantially	Christopher et al. [51]
mAb 63E IgG1	Lewis Rat Hybridoma	Torpedo AChR, MIR, α subunit binding site	180–220 g female Lewis rat	Single intravenous injection 1 mg/kg	48 h; Mild clinical symptoms, EMG attenuation is mild	Weak action, causes minor immune reaction by brief AChR cross-linking	Christopher et al. [51]
mAb 5.5 N/A	BALB/Bl Mouse Hybridoma	Torpedo AChR, MIR, α subunit binding site	2–7-day-old female chick	Single intraperitoneal injection 20–100 mg/kg	8–10 h; Severe respiratory distress at low dose (20–50 mg/kg), death within 20 h at higher dose (100 mg/kg), spontaneous recovery at low doses	Binds the AChR MIR and is associated with AChR downregulation in vivo	Miry C et al . [52]
mAb 5.14 N/A	BALB/Bl Mouse Hybridoma	Torpedo AChR, Non-binding site	2–7-day-old female chick	Single intraperitoneal injection	12 h; Mild clinical symptoms compared to mAb 5.5	Targets a conformational epitope and is associated with milder NMJ dysfunction, with less evidence of marked AChR loss	Miry C et al . [52]
mAb 5.34 N/A	BALB/Bl Mouse Hybridoma	Torpedo AChR, Non-binding site	2–7-day-old female chick	Single intraperitoneal injection	8 h; Milder clinical symptoms than mAb 5.5, less AChR damage	Targets a conformational epitope and is associated with milder NMJ dysfunction, with less evidence of marked AChR loss	Miry et al . [52]
mAb 42 IgG2a	Mouse Hybridoma	Torpedo and Electrophorus AChR, MIR, α subunit binding site	7–10-week-old female Lewis rat	Single intraperitoneal injection 3,300 pmol	Within 24 h; 72 h later AChR reduced by 50%	Cross-links AChR and is associated with AChR reduction; crosslinking-dependent receptor modulation likely contributes in this model	Tzartos et al., [42]
mAb 60 IgG1	Mouse Hybridoma	Torpedo and Electrophorus AChR, MIR, α subunit binding site	7–10-week-old female Lewis rat	Single intraperitoneal injection	No weakness	Does not bind Lewis rat AChR and therefore does not induce weakness or AChR loss; used as a specificity control	Tzartos et al., [42]
mAb 208 IgG2a	Mouse Hybridoma	Torpedo and Electrophorus AChR, MIR, α subunit binding site	4-week-old female Lewis rat	Single intraperitoneal injection 490–7,500 pmol	Within 2 days; significant symptoms, AChR reduced by 50%	Targets the AChR MIR and induces EAMG with AChR reduction; receptor modulation may contribute to the observed phenotype	Tzartos et al., [42]
mAb 210 IgG1	Mouse Hybridoma	Torpedo and Electrophorus AChR, MIR, α subunit binding site	4-week-old female Lewis rat	Single intraperitoneal injection 20–10,400 pmol	NR; Slow progression, mild effects, 5,190 pmol causes stronger symptoms	Associated with AChR reduction and relatively mild symptoms at low doses; receptor modulation may contribute in this model	Tzartos et al., [42]
mAb 198 IgG1	Rat Hybridoma	Torpedo AChR, MIR, α subunit binding site	4-week-old female Lewis rat	Single intraperitoneal injection 80 µg	NR; Muscle weakness	Cross-links AChR and is associated with accelerated receptor degradation and impaired neuromuscular transmission, supporting a role for receptor modulation in this model	Barchan et al., [53]
mAb 5.5 IgG1	Rat Hybridoma	AChR ligand binding site	4-week-old female Lewis rat	Single intraperitoneal injection N/A Combined with mAb 198	NR; Blocks AChR ligand binding site, reducing AChR loss	Blocks the ligand-binding site and attenuates mAb198-induced AChR loss, supporting a role for receptor modulation in this experimental setting	Barchan et al., [53]
mAb 35 IgG1	Rat Hybridoma	Torpedo AChR, MIR, α subunit binding site	4-week-old female Lewis rat	Single intraperitoneal injection N/A Combined with mAb 198	NR; Primarily binds to AChR's natural conformation, unable to effectively block MIR antigenic regulation	Shows conformation-dependent binding and is less effective than mAb198 at promoting receptor modulation, with correspondingly weaker AChR loss in this setting	Barchan et al., [53]
mAb 195 IgG1	Lewis Rat Hybridoma	Torpedo AChR, MIR, α subunit binding site	4-week-old female Lewis rat	Single intraperitoneal injection 0.1 mg	12 h after injection; Severe muscle weakness, mild to severe	Cross-links AChR and is associated with neuromuscular dysfunction;crosslinking-dependent receptor modulation likely contributes to the phenotype	Papanastasiou et al., [42]
mAb35 IgG1	Rat Hybridoma	Torpedo AChR, MIR, α subunit binding site	4-week-old female Lewis rat	Single intraperitoneal injection 0.15 mg	After 24–30 h; Weight loss, AChR content decrease	Induces EAMG with AChR reduction; crosslinking-dependent receptor modulation is implicated in this model	Poulas et al., [54]
mAb 35 IgG1	Lewis Rat Hybridoma	Torpedo AChR, MIR of AChR (similar to mAb 195)	4-week-old female Lewis rat	Single intraperitoneal injection 0.15 mg	12 h after injection; Significant muscle weakness and weight loss after 24 h	Similar to mAb 195, exacerbates muscle weakness and is consistent with a contribution from crosslinking-dependent AChR modulation	Papanastasiou et al., [42]
mAb198 IgG1	Rat Hybridoma	Torpedo AChR, MIR, α subunit binding site	6-week-old female Lewis rat	Single intraperitoneal injection 0.1 mg	24 h; Muscle weakness symptoms	Cross-links AChR and is associated with accelerated receptor degradation and impaired neuromuscular transmission, supporting a role for receptor modulation in this model	Hwang et al., [55]
mAb35 IgG1	Rat hybridoma	Torpedo AChR, MIR, α subunit binding site	6–8 weeks female Lewis rats	Single intraperitoneal injection 20 pmol/100 g	24–48 h; weight loss and clinical weakness, with NMJ MAC deposition	Induces PTMG with complement-mediated NMJ injury; suitable for evaluating complement-dependent pathogenic mechanisms	Song et al., [19]
aG101 IgG1	MG patients PBMCs Single-Cell Sorting	Homo AChR, MIR, α subunit binding site	4-week-old female Lewis rat	Single intraperitoneal injection 4 mg/kg	No significant muscle weakness, no severe clinical symptoms	Does not induce complement activation or significant antigenic regulation in AChR α subunit	Rose et al.,2022 [56]
bG402 IgG4	MG patients PBMCs Single-Cell Sorting	Homo AChR, MIR, β subunit binding site	4-week-old female Lewis rat	Single intraperitoneal injection 4 mg/kg	No significant muscle weakness, no severe clinical symptoms	Targets AChR β subunit, no complement activation or significant antigenic regulation	Rose et al., [56]
bG402-G1 IgG1	MG patients PBMCs Single-Cell Sorting	Homo AChR, MIR, β subunit binding site	4-week-old female Lewis rat	Single intraperitoneal injection 2 mg/kg (combined with aG101 2 mg/kg)	NR; Significantly reduces rod performance and AChR degradation	Strong complement activation, AChR cross-linking and aggregation causing NMJ damage	Rose et al., [56]
aG112 IgG1	MG patients PBMCs Single-Cell Sorting	Homo AChR, MIR, α subunit binding site	4-week-old female Lewis rat	Single intraperitoneal injection 2 mg/kg	No significant symptoms, monoclonal antibody did not trigger complement activation	Promotes AChR clustering without strong complement activation	Rose et al., [56]
mAb35 IgG1	Rat Hybridoma	Torpedo AChR, MIR, α subunit binding site	10–11-week-old LEW/Crl female rats	Intraperitoneal (I.P.) injection 20 pmol/100 g or subcutaneous (S.C.) injection 20 pmol/100 g	After 48 h; Weight loss, AChR content decrease	Induces EAMG with AChR reduction; crosslinking-dependent receptor modulation is implicated in this model	Arets et al., [57]
3F6C IgG4	Patients PBMCs; Single-Cell Sorting	Homo MuSK, MuSK Ig1	6-week-old female C57BL/6 mice	Single intraperitoneal injection 5 mg/kg	7–9 days;  > 50% O2 consumption reduction, 2–3 weeks before death	Inhibits MuSK phosphorylation, interfering with Lrp4-MuSK binding, leading to NMJ dysfunction	Oury et al., [28]
3B5 IgG4	Patients PBMCs; Single-Cell Sorting	Homo MuSK, MuSK Ig1	6-week-old female C57BL/6 mice	Single intraperitoneal injection 5 mg/kg	7–9 days;  > 50% O2 consumption reduction, 2–3 weeks before death	Inhibits MuSK phosphorylation, interfering with Lrp4-MuSK binding, leading to NMJ dysfunction	Oury et al., [28]
ARGX-119 IgG1	Patients PBMCs; Single-Cell Sorting	Homo MuSK, MuSK FZ/CRD	6-week-old female C57BL/6 mice	Single intraperitoneal injection 20 mg/kg after disease onset	Recovery after treatment; Significant improvement in survival rate, restored motor function, reduced muscle atrophy	Activates MuSK, restoring phosphorylation, promoting NMJ repair and counteracting MuSK MG mechanism	Oury et al., [28]

This table lists representative items to show differences in mechanisms and parameters. NR means not reported. MIR means main immunogenic region. MAC means membrane attack complex. RNS means repetitive nerve stimulation. CMAP means compound muscle action potential

Standardization of outcome measures

To improve cross-study comparability, mAb PTMG studies should adopt a harmonized outcome framework integrating: (1) a standardized clinical scoring system, (2) objective measurements of muscle strength and/or endurance, (3) prespecified electrophysiological readouts acquired using consistent protocols, and (4) quantitative analyses of NMJ pathology. The Methods section should explicitly define the parameters, scoring criteria, and prespecified interpretation rules for each endpoint to enhance reproducibility and enable meaningful comparisons across experiments [10, 30].

Clinical scoring scale

A 0–4 point clinical scale is recommended, with operational definitions that are practical and reproducible. Whenever feasible, scoring should be performed by blinded observers [10].

Score 0 (Normal): Normal spontaneous activity and grip, without clear fatigability.

Score 1 (Mild weakness): Reduced grip strength or easy fatigue; holding time shortens and/or grip weakens. Symptoms worsen with repeated activity, but the animal can still ambulate and feed normally.

Score 2 (Moderate weakness): Abnormal gait with instability, dragging, or reduced standing time. Clear fatigability is present and worsens after activity.

Score 3 (Severe weakness): Marked movement limitation and/or paralysis. Difficulty walking or standing for prolonged periods; impaired head lifting and a poor righting reflex may be observed. Supportive care may be required to maintain basic activity.

Score 4 (Humane endpoint): Moribund condition or predefined study endpoint, such as inability to eat/drink or signs consistent with impending respiratory failure. Animals reaching this score should meet prespecified ethical termination criteria and be sampled at endpoint or euthanized accordingly.

Objective measurements of muscle strength and behavior

To reduce the subjectivity inherent to clinical scoring, at least one objective, strength-related outcome should be included. Two commonly used measures are recommended.

Grip strength testing should be performed using a small-animal grip strength meter or a standardized force transducer. Mice grasp a metal bar with the forepaws while the operator applies a steady horizontal (or slightly downward) pull until release, and the peak force is recorded. Each testing day should include five sessions, with three trials per session; after excluding clear outliers, the mean value should be reported (either per session and/or averaged across sessions, as prespecified). To minimize variability, measurements should be conducted by the same operator within a consistent time window. Reports should specify the units (newtons or grams), calibration procedures, and whether values were normalized to body weight; reporting both raw and weight-adjusted values is recommended. A simple fatigue index can be calculated as: (mean of the last two sessions / mean of the first two sessions) × 100%, with all averaging and exclusion rules prespecified [7, 58].

Wire hang test: using a bar or grid apparatus, record the maximum hanging duration with a prespecified upper limit (e.g., 600 s). Because performance is influenced by body weight, habituation, and handling, this measure is best interpreted alongside grip strength and under controlled environmental conditions (temperature, noise, and testing time). Reports should specify the upper limit, stopping rule (release/fall), number of repeats, and the method used to summarize repeated measurements [58].

Minimum recommended outcome set: a clinical score plus grip strength (core endpoints), together with daily body weight monitoring. Wire hang test measures can be included as supportive secondary outcomes.

Contractile assessments

CMAPs during repetitive nerve stimulation (RNS) are recommended as an electrophysiological endpoint in EAMG studies. CMAP decrement (%) should be calculated as , where A₁ is the first and Aₙ the prespecified n-th response (commonly the 4th or 5th). Reports should specify the stimulated nerve and recorded muscle, stimulation frequency (typically 3 Hz, fixed within a study), number of stimuli per train, recording conditions (anesthesia, temperature control, electrode placement), and criteria for artifact exclusion. At least two trains per animal should be averaged. Both absolute CMAP amplitudes (mV) and decrement values (%) should be reported and aligned with clinical and strength assessments at matched time points. [8].

Force measurements

Evoked twitch or tetanic force measurements offer integrative functional correlates of neuromuscular transmission efficacy and muscle strength in vivo or ex vivo. twitch force measurements and related contractile responses evoked by nerve/muscle stimulation provide functional correlates of neuromuscular transmission efficacy and muscle strength in vivo or ex vivo, complementing NMJ-specific measures in EAMG models.

Electrophysiological measures

Electrophysiological assessments such as endplate potentials (EPPs) and miniature endplate potentials (mEPPs) recorded in ex vivo nerve-muscle preparations directly quantify synaptic transmission at the neuromuscular junction, with reductions in EPP amplitude and altered mEPP frequency/amplitude reflecting impaired acetylcholine release or receptor function.

NMJ morphology

For the evaluation of NMJ pathology, quantitative immunofluorescence analysis of pre- and postsynaptic NMJ morphology from confocal z-stack–derived images of individual junctions is recommended as a standardized and broadly applicable approach. At a minimum, three quantifiable features of postsynaptic AChR clusters should be reported, with prespecified sampling, imaging, and analysis criteria to ensure reproducibility and facilitate meaningful comparisons across studies.

Postsynaptic AChR clusters should be visualized using fluorescent α-bungarotoxin (α-BTX), a high-affinity and near-irreversible ligand of nicotinic AChRs. Optional co-staining with presynaptic markers (e.g., neurofilament and/or synaptic vesicle proteins) can aid NMJ localization and allow assessment of pre–post synaptic alignment. In studies addressing complement-mediated mechanisms, C3 or C5b-9 (membrane attack complex) may be included as additional mechanistic readouts. [37]

Sampling rules should be explicitly defined. Analyses should be restricted to one or two predefined muscles (e.g., diaphragm or soleus), with the rationale stated. A minimum of 30–50 NMJs per animal is recommended, sampled across multiple non-overlapping fields. Blinding is strongly recommended for both image acquisition and quantitative analysis. Reports should specify the image analysis software (e.g., ImageJ/Fiji) and thresholding strategy, including whether a single global threshold was applied or thresholds were adjusted based on background signal.

Quantitative endpoints should be prespecified and applied consistently within the same study. Commonly used metrics include: AChR cluster intensity: quantified from α-BTX fluorescence as background-corrected mean fluorescence intensity (or integrated density) within threshold-segmented AChR cluster ROIs acquired under identical imaging settings and, where appropriate, normalized to controls processed in parallel. AChR cluster (endplate) density, quantified as the number of α-BTX–positive endplates per unit muscle area (e.g., per mm2), provides an index of NMJ abundance and can capture NMJ loss or remodeling; however, because density is sensitive to sampling and sectioning, it is ideally interpreted together with innervation/occupancy metrics based on pre–post synaptic co-staining [59]. AChR cluster area, defined as the α-BTX–positive area per NMJ (µm2), reflecting changes in the size of postsynaptic specialization [59]. AChR cluster fragmentation, quantified using measures such as the number of fragments per cluster, a fragmentation index, or the proportion of discontinuous α-BTX–positive area [60].

Regardless of the specific metric employed, the segmentation algorithm, threshold criteria, and decision rules should be described in sufficient detail to enable replication and should remain consistent across all experimental groups.

Unified reporting template

It is recommended that each result table or summary table reports at least the following fields in a consistent format (Fig. 4).

1. Clinical score (0–4 scale): report the maximum clinical score reached during the study and the final score at the endpoint.

2. Strength and behavior (grip strength ± endurance): report raw grip strength and body weight–corrected grip strength (with units, device/model, and protocol details). If used, include a fatigue index (e.g., mean of last two sessions / mean of first two sessions × 100%) and wire hang test (upper limit, stopping rule, repeats, and summary metric).

3. CMAP: report baseline CMAP amplitude (mV), percentage decrement, stimulation frequency, and the selected n value (number of stimuli/trials) used for analysis.

4. Force measurements: report prespecified contractile outcomes, such as twitch force and tetanic force (and normalization to specific force if applicable), with stimulation and recording conditions defined.

5. Electrophysiological measures (NMJ transmission): report relevant synaptic readouts, including mEPP frequency and amplitude and EPP amplitude (and other prespecified parameters, such as quantal content if calculated), with acquisition conditions and analysis rules specified.

6. NMJ morphology (immunofluorescence): report the number of NMJs analyzed per animal, AChR intensity (background-corrected MFI or integrated density), AChR cluster/endplate density (NMJs per unit area), AChR cluster area (mean or median), and AChR fragmentation (with an explicit definition, e.g., fragments per endplate, fragmentation index, or discontinuous-area proportion).

Fig. 4.

Unified reporting template for outcome measures in EAMG studies

Using these standardized rules can greatly improve comparability across studies in later parameter summary tables, and it can reduce interpretation bias caused by differences in scales and parameters.

Collectively, these variables establish a structured framework in which defined antibody properties, host context, dosing strategies, and standardized outcome measures can be systematically integrated to achieve reproducible and mechanistically informative PTMG models.

Applications of mAb passive EAMG models in dissecting pathogenic mechanisms

mAb PTMG offers a precisely controlled yet highly informative framework to causally link defined autoantibody properties to NMJ dysfunction and injury. By isolating a single epitope- and paratope-defined antibody subclass, they enable clean dissection of how valency, subclass/Fc effector capacity, and target epitope placement drive complement activation, FcγR engagement, receptor cross-linking/internalization, and time-resolved electrophysiological impairment. This precision makes mAb EAMG particularly well suited for structure–function mapping and in vivo testing of engineered Fc variants (Fc-silent/enhanced or subclass-switched), allowing pathway-resolved attribution of complement- versus FcγR-dependent mechanisms across targets such as AChR, MuSK.

Pathogenic antibodies, non-pathogenic antibodies, neutral antibodies and protective antibodies

In mAbs studies, pathogenicity should be determined primarily by in vivo functional consequences, rather than by antigen binding alone. This distinction is important because many antibodies can recognize the target antigen without producing measurable functional impairment or structural injury. Indeed, PTMG with AChR-related mAbs have shown substantial heterogeneity in the ability of different anti-AChR clones to induce muscle weakness, indicating that binding and pathogenicity are not equivalent [13]. To summarize results across studies and improve comparability, this review uses the following clearer criteria for classification.

Pathogenic antibodies: after passive transfer, these antibodies are associated with detectable muscle weakness, often accompanied by evidence supporting NMJ dysfunction and/or injury. Reported supportive findings include electrophysiological abnormalities (e.g., reduced CMAP or RNS decrement) and structural changes at the NMJ (e.g., AChR reduction, complement deposition, or endplate ultrastructural changes) [4].

Neutral or weak antibodies: these antibodies bind the target antigen but do not consistently induce a phenotype under commonly used doses and observation windows, or they are associated only with mild or variable changes [13].

Protective antibodies: in principle, such antibodies could attenuate the effects of pathogenic antibodies through competitive binding or conformational modulation of the antigen. However, across the broader MG literature, well-validated and consistently reproducible “protective” monoclonal antibodies remain relatively uncommon [61]. Notably, an exception has been described in the context of MuSK, where a subset of agonistic monoclonal antibodies has been reported to exert beneficial effects. In particular, these agonistic antibodies were shown to ameliorate muscle weakness in a DOK7-CMS mouse model and to attenuate disease manifestations in experimental settings driven by pathogenic anti-MuSK antibodies. Mechanistically, their activity has been linked to promoting MuSK dimerization, thereby enhancing MuSK phosphorylation and downstream MuSK pathway activation, which in turn supports AChR clustering at the neuromuscular junction [28, 62]

AChR MG: the MIR epitope and multiple pathogenic mechanisms

In AChR MG, antibodies are not randomly distributed across the AChR surface but instead show a preferential targeting of several immunodominant regions. Early epitope-mapping studies using mAbs introduced the concept of the MIR, and subsequent work further localized critical segments to approximately residues 67–76 on the AChR α subunit [15, 17, 63]. This epitope-focused distribution provides a useful entry point for mAb–based disease models. Importantly, if mAbs directed against different epitopes of the same antigen exhibit distinct pathogenic effects, this observation suggests that epitope specificity itself may influence pathogenic potential, including the propensity to trigger complement-mediated injury or to promote receptor internalization and downstream processes. The pathogenic mechanisms of AChR antibodies are commonly summarized into three, partly overlapping, categories.

Complement-mediated endplate injury

Complement deposition at MG endplates, together with the presence of the membrane attack complex, has been consistently observed and supports the involvement of complement in disease pathology [64]. In EAMG models, ultrastructural localization of immune complexes and C3 at the endplate further supports this mechanism [65]. In PTMG, activation of the terminal complement pathway appears to contribute substantially to disease expression, and inhibition of terminal complement has been shown to attenuate clinical and pathological phenotypes [66]. In addition, experimental findings indicating that different IgG isotypes vary in their capacity to activate complement provide a plausible framework for understanding why antibodies recognizing the same antigen may produce differing degrees of tissue injury [20].

Antigenic modulation and accelerated receptor degradation

Early studies demonstrated that MG IgG can accelerate AChR degradation, leading to a reduction in receptor density at the endplate [67]. Subsequent work suggested that antibody-mediated cross-linking of AChRs represents an important prerequisite for this accelerated degradation process [14]. Together, these findings established the view that non-complement-mediated mechanisms can also substantially reduce AChR number. They further help explain why significant AChR loss can be observed in experimental settings in which complement involvement is limited or not emphasized.

Functional blocking

Certain antibodies are capable of directly interfering with acetylcholine binding to the receptor or of altering ion channel function. However, within most experimental frameworks, this mechanism appears to operate alongside complement-mediated injury and antigenic modulation, rather than acting as an isolated process [68].

In mAb PTMG studies, comparisons between full-length IgG and antibody fragments, including F(ab’)₂ and Fab, provide a more direct means of assessing the roles of valency and cross-linking in AChR reduction. In experiments emphasizing antigenic modulation, bivalent F(ab’)₂ fragments, which lack Fc regions, and monovalent Fab fragments differ in their ability to induce phenotypic changes and AChR loss. These observations suggest that bivalent cross-linking alone may promote receptor internalization and degradation [30]. A key advantage of this experimental design is that it allows Fc-dependent effects, including complement activation and Fcγ receptor engagement, to be separated from cross-linking–driven receptor modulation, enabling these mechanisms to be evaluated comparatively within the same model system.

Studies using mAbs have shown that, beyond differences in epitope specificity, mAbs recognizing the same epitope can simultaneously engage multiple pathogenic mechanisms. Specifically, a single mAb may contribute to disease through different combinations of pathogenic mechanisms, including complement activation, antigenic modulation with accelerated receptor internalization and degradation, and, in some cases, direct functional interference with receptor activity [27, 37]. These observations indicate that pathogenic mechanisms are not mutually exclusive and that epitope identity alone is insufficient to fully predict functional or pathological outcomes in PTMG. Notably, much of the supporting evidence has been derived from in vitro assays, and further validation in mAb PTMG models remains warranted.

MuSK MG: IgG4 features and signaling blockade

The discovery of MuSK antibodies provided a clear entry point for mechanistic studies of MG involving non-AChR targets [5]. A notable immunological feature of MuSK MG is the predominance of IgG4. IgG4 undergoes Fab-arm exchange and is often considered functionally monovalent, with relatively weak capacity to engage the classical complement pathway. These properties are thought to bias IgG4-mediated effects toward blocking signaling or disrupting protein–protein interactions, rather than efficiently cross-linking target antigens [69, 70]. Consequently, in MuSK-related experimental models, both antibody subclass and antibody format (e.g., full-length IgG versus fragments) can substantially influence the observed mode of action. Reported effects include interference with Agrin/LRP4/MuSK signaling, alterations in MuSK phosphorylation, and downstream destabilization of AChR clusters at the neuromuscular junction.

Passive transfer of anti-MuSK IgG1 monoclonal antibodies (e.g., 3B5 and 3F6C) has been reported to induce MG-like weakness in mice. Notably, these disease-like manifestations can be ameliorated by an agonistic anti-MuSK FZ mAb, consistent with the idea that distinct functional classes of MuSK antibodies may exert opposing effects on neuromuscular transmission [28]. In addition, a recently published PTMG study in mice showed that antibodies targeting specific MuSK domains can elicit weakness-related phenotypes accompanied by NMJ alterations, supporting the notion that domain-specific MuSK antibodies can carry pathogenic potential [71], Importantly, these findings also raise the possibility that mAbs directed against the same MuSK domain may differ in functional behavior, exhibiting either predominantly pathogenic or therapeutic-like properties depending on their epitope fine-specificity and/or mechanism of action. Collectively, these studies provide an experimental basis for examining whether epitope-level differences among MuSK antibodies shape both the magnitude of pathogenicity and the presence of therapeutic characteristics.

Other techniques to determine the pathogenic mechanisms of MG mAbs

In addition to passive-transfer models, complementary non-animal platforms provide critical functional and mechanistic insights into MG mAbs.

Cell-based assays (CBAs) are widely used to assess antigen binding, receptor clustering or internalization, complement deposition, and interference with signaling pathways such as the Agrin–LRP4–MuSK signalling. These assays can be performed in muscle cell models (e.g., C2C12 myotubes) [28], allowing quantitative evaluation of antibody-mediated effects on AChR clustering, MuSK phosphorylation, and downstream signaling events. In particular, CBAs have been instrumental in distinguishing between antibodies that activate complement and those that primarily disrupt receptor function or signaling [37].

Biophysical approaches, including surface plasmon resonance (SPR) [72] and biolayer interferometry (BLI) [73], further enable detailed characterization of antibody–antigen interactions, such as binding affinity, association/dissociation kinetics, epitope competition, and avidity or valency-dependent effects. These techniques are especially valuable for comparing monoclonal antibodies with subtle functional differences and for dissecting how epitope specificity and binding strength relate to pathogenic potential.

Additional methods, such as epitope mapping (e.g., peptide arrays or mutagenesis-based approaches) [74], glycosylation profiling [75], Fc effector function assays (including FcγR binding and complement activation assays) [76] and Cryo-EM [27], can further refine the functional characterization of MG autoantibodies. Together, these in vitro platforms provide a high-resolution framework for defining antibody properties, enabling mechanistic stratification of clones prior to in vivo testing.

Although these approaches cannot fully recapitulate the complexity of neuromuscular junction physiology or replace in vivo models for evaluating integrated disease expression, they are highly complementary. Importantly, they allow efficient prioritization of candidate antibodies and mechanistic hypotheses for downstream passive-transfer studies, thereby improving experimental throughput and interpretability.

Future perspectives

Identification of emerging pathogenic antibodies

At present, the pathogenic autoantibodies that are widely accepted in MG are directed against AChR, MuSK, and LRP4. However, additional antibodies have been detected in the sera of MG patients, including those targeting Agrin, titin, RyR2, cortactin, ColQ, and Kv1.4 [2]. The pathogenic relevance of these antibodies remains incompletely defined. Among these targets, Agrin is of particular interest.

Although Agrin is synthesized as a presynaptic protein, it is proteolytically cleaved and secreted as an active LG3 fragment, which binds to postsynaptic LRP4 and activates the Agrin/LRP4/MuSK signaling pathway. An active immunization EAMG model targeting Agrin LG3 has been successfully established, supporting its potential pathogenic relevance [9]. Nevertheless, further validation is required using passive-transfer EAMG models, particularly those based on mAbs, to more definitively determine its pathogenic role.

ColQ is a secreted extracellular matrix protein that anchors acetylcholinesterase (AChE) at the neuromuscular junction and is essential for maintaining AChE stability and function. To date, a pathogenic EAMG model for ColQ antibodies has not been reported, and its contribution to MG pathogenesis remains to be clarified.

In contrast, titin, RYR2, and cortactin are intracellular muscle proteins. Antibodies against these targets are generally considered secondary or accompanying antibodies that arise after disease onset, rather than primary pathogenic drivers. However, direct experimental evidence excluding their pathogenic potential is still limited, and further investigation is warranted.

The establishment of mAbs PTMG models provides a powerful approach to rigorously assess the pathogenicity of these candidate antibodies.

Moreover, a subset of MG patients remains seronegative for all currently recognized antibodies, suggesting that additional pathogenic autoantibodies may yet remain unidentified. Once discovered, systematic validation in EAMG models—particularly mAbs PTMG—will be essential for confirming their pathogenic relevance.

Further identification of pathogenic mechanism

Establishing that an autoantibody is pathogenic is often only a first step, and further work is typically required to define the underlying molecular mechanisms. Such mechanistic studies may include, but are not limited to, the following aspects: (1) which domain(s) of the target antigen are preferentially recognized, and whether immunodominant regions or “high-risk” epitopes can be identified [7, 15, 28, 71]; (2) whether antibodies directed against different epitopes produce distinct in vivo consequences—for example, showing a stronger propensity for complement-mediated injury versus a greater tendency to block signaling or disrupt protein–protein interactions [13]; and (3) whether these antibodies can reproducibly induce disease-relevant phenotypes in PTMG, together with a coherent chain of evidence linking NMJ structural alterations to functional deficits, and to further delineate the specific molecular pathways by which these antibodies ultimately cause NMJ injury [71, 77].

Together, these considerations emphasize that robust mechanistic attribution requires an integrated framework that connects epitope/domain specificity, effector pathway engagement, and reproducible in vivo validation with matched structural and functional NMJ readouts. Applying such a framework can improve cross-study comparability and help move emerging antibody targets from association toward a more causal understanding of disease pathogenesis.

Upgrading the model system

A recurring limitation in current monoclonal antibody (mAb)–based EAMG studies is that murine and human immune effector systems are not fully aligned. In particular, differences in the structure and function of Fcγ receptor networks and complement pathways can influence the engagement and magnitude of Fc-dependent mechanisms, thereby complicating interpretation and cross-species generalization [21]. Therefore, model upgrades mainly focus on two directions, making the immune effector environment more similar to humans and making antigen structure more similar to humans.

Humanized recipient animals

Introducing key human Fcγ receptors or human FcRn into recipient animals can help render the in vivo behavior of human antibodies (including engineered formats) more comparable to clinical settings. When complement-dependent mechanisms are a focus, establishing a complement background that more closely resembles human complement activity may further improve translational relevance [21].

Humanized antigens

Transgenic expression of human extracellular domains/full-length humanized variants of AChR, MuSK, or LRP4 can reduce potential affinity or epitope biases arising from interspecies sequence differences [78–80]. This approach may allow binding properties and functional effects of patient-derived monoclonal antibodies in vivo to more closely reflect those observed in humans.

Overall, upgrading the model system is essential for improving the interpretability and translational value of mAb PTMG studies. Because Fc-dependent mechanisms are shaped by species-specific immune effector biology, and epitope engagement can be biased by interspecies antigen differences, model refinement should aim to better approximate both the human effector environment and the human antigen context. In practice, combining complementary strategies—such as recipient humanization (e.g., FcγR/FcRn and, where needed, complement background) together with antigen humanization—may provide a more robust framework for mechanistic dissection. Such upgraded platforms are expected to strengthen causal inference for emerging targets, improve cross-study comparability, and enhance the preclinical evaluation of epitope-defined therapeutic antibodies.

Mechanism-guided therapeutic stratification

MG treatment is gradually moving from mainly broad immunosuppression toward more mechanism guided targeted therapy. Clinical consensus statements and guidelines emphasize that treatment strategies should consider subtype, such as AChR positive and MuSK positive, severity, and crisis risk, and should be adjusted dynamically during long term management. From a mechanistic perspective, mAb PTMG has an important advantage. It can isolate one mechanism chain, such as the terminal complement pathway, pathogenic IgG level, or Fc effects, and observe intervention effects more directly. Therefore, it has strong explanatory value in early-stage evaluation of new therapies [81].

Complement-mediated endplate injury is supported by substantial experimental and pathological evidence in AChR MG [64, 82]. Clinically, the phase 3 randomized controlled trial REGAIN evaluated the terminal complement inhibitor eculizumab in anti AChR antibody positive, refractory generalized MG [82]. This trial is biologically consistent with passive EAMG results showing that the terminal complement pathway drives pathology and that inhibiting terminal complement can reduce phenotype [66]. In this context, the clinical use of eculizumab can be conceptually paralleled by mAb PTMG models applied across different antibody subgroups, which can serve as in vivo validation platforms for complement-dependent pathogenic mechanisms. Such models allow direct testing of whether complement activation meaningfully contributes to disease expression in distinct antibody types and subclasses, including newly identified patient-derived monoclonal antibodies, thereby strengthening mechanistic attribution at the level of individual antibodies.

The FcRn pathway regulates IgG recycling and serum half-life, making it a rational therapeutic target for reducing circulating pathogenic IgG. The phase 3 ADAPT trial evaluated the efficacy and safety of efgartigimod, an engineered Fc-fragment–based molecule, in generalized MG. The primary endpoint incorporated improvement in MG-ADL with maintenance for at least four weeks, supporting the concept that lowering total IgG can translate into clinical benefit within a relatively short time window [83]. This therapeutic logic is also broadly consistent with mAb PTMG: if exogenously administered mAbs can directly induce weakness, then reducing pathogenic mAbs burden would be expected to yield at least partially reversible symptomatic improvement. Notably, clinical responses to efgartigimod appear heterogeneous. In our unpublished observations, efgartigimod treatment reduced total IgG and, in most patients, was accompanied by an approximately proportional reduction in AChR antibody levels. However, a subset of patients showed clinical improvement despite no apparent reduction in AChR antibody titers during treatment. The mechanisms underlying this dissociation remain unclear, but these findings suggest that FcRn antagonism may exert therapeutic effects through more than one pathway, beyond a simple lowering of measured autoantibody concentration [84]. In this context, mAb PTMG could provide a useful in vivo platform to dissect potential mechanisms—such as changes in IgG distribution, Fc-dependent effector engagement, immune complex handling, or antibody functional properties—and to clarify which parameters best correlate with clinical benefit.

Therefore, an important application focus of mAb PTMG in this area is to systematically compare different antibody types and subclasses with respect to the extent and kinetics of reversibility in response to IgG-lowering strategies, complement inhibition, or Fcγ receptor–dependent modulation, thereby informing mechanism-guided therapeutic stratification. Beyond single-antibody testing, mAb PTMG provides a flexible in vivo platform to model clinically relevant antibody constellations—including the presence of a particular mAb, as well as different combinations of monoclonal antibodies that may act additively, synergistically, or even antagonistically. By linking defined antibody specificities, subclasses, and mixtures to differential responses to FcRn antagonists versus complement-targeted or Fc-modulating interventions, these models can help identify which antibody profiles are more likely to benefit from a given therapeutic mechanism. In this way, mAb PTMG may contribute mechanistic evidence to support treatment stratification and more rational therapy selection for patient subgroups defined by distinct monoclonal antibody signatures.

Although mAb-PTMG offers clear advantages—defined epitope specificity, precise control over antibody format and dosing, standardized experimental conditions, and high reproducibility for mechanistic studies—it cannot, on its own, capture the full complexity of MG. Accordingly, a coordinated, multi-model strategy is needed. Integrating active EAMG, polyclonal IgG PTMG, and mAb-PTMG in genetically or immunologically modified hosts can better encompass the pathogenic cascade of MG and improve the translation of mechanistic insights into therapeutic approaches.

Abbreviations

AChE

Acetylcholinesterase

AChR

Acetylcholine receptor

α-BTX

α-Bungarotoxin

BLI

Biolayer interferometry

C1q

Complement component 1q

C3

Complement component 3;

C5b-9

Complement component C5b-9

CMAP

Compound muscle action potential

CRD

Cysteine-rich domain

cryo-EM

Cryo-electron microscopy

DOK7-CMS

DOK7 congenital myasthenic syndrome

EAMG

Experimental autoimmune myasthenia gravis

EMG

Electromyography

EPP

Endplate potential

Fab

Fragment antigen-binding

F(ab')2

F(ab')2 fragment

Fc

Fragment crystallizable

FcγR

Fc gamma receptor

FcRn

Neonatal Fc receptor

FZ

Frizzled domain

HDX-MS

Hydrogen–deuterium exchange mass spectrometry

Ig1

Immunoglobulin-like domain 1

IgG

Immunoglobulin G

IgG1–IgG4

Immunoglobulin G subclasses 1–4

LG3

Laminin G-like domain 3

LRP4

Low-density lipoprotein receptor-related protein 4

MAC

Membrane attack complex

MG

Myasthenia gravis

MG-ADL

Myasthenia gravis activities of daily living

mAb

Monoclonal antibody

mAb-PTMG

Monoclonal antibody passive-transfer myasthenia gravis

mEPP

Miniature endplate potential

MIR

Main immunogenic region

MFI

Mean fluorescence intensity

MuSK

Muscle-specific receptor tyrosine kinase

NMJ

Neuromuscular junction

PBMC

Peripheral blood mononuclear cell

PTMG

Passive-transfer myasthenia gravis

RNS

Repetitive nerve stimulation

ROI

Region of interest

RT-PCR

Reverse transcription polymerase chain reaction

scFv

Single-chain variable fragment

SPR

Surface plasmon resonance

VH

Variable heavy chain

VL

Variable light chain

Author contributions

Z.Y. conceived and designed the review; L.T.Z., J.H.W.,S.Y.T. and Z.Y. contributed to make the figures; L.T.Z. and Z.Y. contributed to make the table; L.T.Z., M.Y.Z. and Z.Y. wrote the manuscript; all authors reviewed and approved the final version of the manuscript.

Funding

This work was supported by grants from the Sixth Round of Quanzhou City's High-Level Talent Team Recruitment Project (Grant No. 2023CT007), the National Natural Science Foundation of China (Grant No. W2441013). the Natural Science Foundation of Fujian Province (Grant No. 2021J01016), the Second Affiliated Hospital of Fujian Medical University Ph.D. Seedling Project (Grant No. BS202104).

Data availability

Not applicable. No new datasets were generated or analysed in this review.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.