Functional Profiling of Acetylcholine Receptor Antibodies Informs Therapeutic Outcome of FcRn Inhibitor Therapy in...

Myasthenia gravis (MG) is an autoimmune disorder of the neuromuscular junction most commonly associated with acetylcholine receptor (AChR) antibodies (AChR-Abs), detected in up to 80% of patients. The emergence of targeted therapies, such as neonatal Fc receptor (FcRn) inhibitors, highlights the need for standardized functional assays capable of dissecting AChR-Ab pathogenic mechanisms. By selectively reducing circulating IgG through FcRn blockade, these agents differ fundamentally from conventional immunosuppressants, emphasizing the need to characterize antibody-mediated AChR dysfunction and complement activation as potential markers of therapeutic response. However, current functional assays often rely on transiently transfected or heterogeneous cell systems, limiting reproducibility and scalability. A stably transfected human cell line expressing physiologically clustered adult AChRs could fill this methodological gap and enable standardized, quantitative evaluation of antibody effector functions.

We generated a stable rhabdomyosarcoma (RD) cell line expressing clustered human adult AChRs (hAChR-RDcl) using the PiggyBac transposon system for genomic integration of AChR subunit genes. The hAChR-RDcl line was applied to functional cell-based assays using serum samples from 30 patients with AChR-MG before and after one treatment cycle with efgartigimod. Clinical efficacy was assessed by Quantitative Myasthenia Gravis (QMG) and MG Activities of Daily Living (MG-ADL) scores.

The hAChR-RDcl cell line showed stable transcription and surface expression of adult AChR subunits, confirmed by RT-qPCR, α-bungarotoxin binding, and antibody-specific reactivity (mAb637 and patient serum samples). Baseline functional assays identified blocking antibodies in 15 of 30 (50%), internalizing antibodies in 20 of 30 (66.7%), and complement-activating antibodies in 14 of 30 (46.6%) samples. After efgartigimod treatment, all 3 antibody-mediated mechanisms were significantly reduced. Patients with persistent complement activity after treatment experienced more severe symptoms (higher QMG scores) compared with those showing other antibody-mediated pathogenetic mechanisms or complete suppression of antibody effector functions.

The hAChR-RDcl cell line represents a reliable and scalable platform for standardized functional profiling of AChR-Abs. Functional characterization of AChR-Abs provides a translational approach to monitor therapeutic responses and elucidate the mechanisms underlying FcRn inhibitor efficacy in MG.