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Individual myasthenia gravis autoantibody clones can efficiently mediate multiple mechanisms of pathology

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Abstract

Serum autoantibodies targeting the nicotinic acetylcholine receptor (AChR) in patients with autoimmune myasthenia gravis (MG) can mediate pathology via three distinct molecular mechanisms: complement activation, receptor blockade, and antigenic modulation. However, it is unclear whether multi-pathogenicity is mediated by individual or multiple autoantibody clones. Using an unbiased B cell culture screening approach, we generated a library of 11 human-derived AChR-specific recombinant monoclonal autoantibodies (mAb) and assessed their binding properties and pathogenic profiles using specialized cell-based assays. Five mAbs activated complement, three blocked α-bungarotoxin binding to the receptor, and seven induced antigenic modulation. Furthermore, two clonally related mAbs derived from one patient were each highly efficient at more than one of these mechanisms, demonstrating that pathogenic mechanisms are not mutually exclusive at the monoclonal level. Using novel Jurkat cell lines that individually express each monomeric AChR subunit (α2βδε), these two mAbs with multi-pathogenic capacity were determined to exclusively bind the α-subunit of AChR, demonstrating an association between mAb specificity and pathogenic capacity. These findings provide new insight into the immunopathology of MG, demonstrating that single autoreactive clones can efficiently mediate multiple modes of pathology. Current therapeutic approaches targeting only one autoantibody-mediated pathogenic mechanism may be evaded by autoantibodies with multifaceted capacity.

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Acknowledgements

We thank Dr. Philip Coish for manuscript editing and Dr. Bhaskar Roy for advice on statistical analysis. The AQP4-specific human mAb58 was generously provided by Dr. Jeffrey L. Bennett of the Departments of Neurology and Ophthalmology, Programs in Neuroscience and Immunology, University of Colorado Anschutz Medical Campus, Aurora CO.

Funding

Minh C. Pham was supported by the NIH T32 predoctoral training grant (award number T32AI007019-46). Gianvito Masi is an MGNet Scholar Awardee. MGNet is a member of the Rare Disease Clinical Research Network Consortium (RDCRN) NIH U54 NS115054. All RDCRN consortia are supported by the network’s Data Management and Coordinating Center (DMCC) (U2CTR002818). Funding support for the DMCC is provided by the National Center for Advancing Translational Sciences (NCATS) and the National Institute of Neurological Disorders and Stroke (NINDS). Rosa Patzina was supported, in part, by the Biomedical Education Program (BMEP) funded by the German Academic Exchange Service (DAAD). Kevin C. O’Connor is supported by the National Institute of Allergy and Infectious Diseases of the NIH under award numbers R01-AI114780 and R21-AI142198, through an award provided through the Rare Diseases Clinical Research Consortia of the NIH and MGNet (award number U54-NS115054), and a pilot award from MGFA (Myasthenia Gravis Foundation of America). The funders had no role in the decision to publish or preparation of the manuscript.

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Authors and Affiliations

  1. Department of Immunobiology, Yale University School of Medicine, 300 George Street-Room 353J, New Haven, CT, 06511, USA

    Minh C. Pham, Gianvito Masi, Rosa Patzina, Abeer H. Obaid, Seneca R. Oxendine & Kevin C. O’Connor

  2. Department of Neurology, Yale University School of Medicine, New Haven, CT, 06511, USA

    Gianvito Masi, Rosa Patzina, Abeer H. Obaid, Seneca R. Oxendine, Richard J. Nowak & Kevin C. O’Connor

  3. Institute of Biomedical Studies, Baylor University, Waco, TX, 76706, USA

    Abeer H. Obaid

  4. Department of Dermatology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA

    Sangwook Oh & Aimee S. Payne

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  1. Minh C. Pham
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This study was originally conceived, initiated, and directed by KCO. MCP led the laboratory work at Yale, designed the study, optimized assays, performed experiments, interpreted data, and wrote the initial manuscript draft. All authors contributed to the acquisition, analysis, or interpretation of data. GM, RP, AHO, and SRO optimized assays, performed experiments, and interpreted data. RJN provided the clinical specimens and provided insight on the clinical and therapeutic relevance of the findings. SO and AP designed and optimized assays for subunit epitope mapping at the University of Pennsylvania. Figures were produced with GraphPad Prism, FlowJo, and BioRender by MCP. All authors contributed to the editing and revising of the manuscript.

Corresponding author

Correspondence to Kevin C. O’Connor.

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Conflict of interest

Dr. Aimee S. Payne has received equity, research support, patent licensing and other payments from Cabaletta Bio; patent licensing payments from Novartis; and consultant fees from Janssen. Dr. Sangwook Oh has received patent licensing payments from Cabaletta Bio. Dr. Richard J. Nowak has received research support from the NIH, Genentech, Alexion Pharmaceuticals, argenx, Annexon Biosciences, UCB Ra Pharmaceuticals, Myasthenia Gravis Foundation of America, Momenta, Immunovant, Grifols, and Viela Bio, now (Horizon Therapeutics). RJN has served as consultant/ advisor for Alexion Pharmaceuticals, argenx, Cabaletta Bio, CSL Behring, Grifols, Ra Pharmaceuticals, now a part of UCB Pharma, Immunovant, Momenta, and Viela Bio, now a part of Horizon Therapeutics; Dr. Kevin C. O’Connor has received research support from Ra Pharma, now (UCB Pharma), Alexion, now (AstraZeneca), Viela Bio, now (Horizon Therapeutics), and argenx. KCO is a consultant and equity shareholder of Cabaletta Bio. KCO has served as a consultant/advisor for Alexion Pharmaceuticals, now (AstraZeneca), and Roche. The authors have no additional financial interests. All other authors declare no competing financial interests.

Human participants

Deidentified human-derived specimens were retrieved from a biorepository established at the Yale University School of Medicine under the approval of Yale University’s Institutional Review Board.

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Pham, M.C., Masi, G., Patzina, R. et al. Individual myasthenia gravis autoantibody clones can efficiently mediate multiple mechanisms of pathology. Acta Neuropathol 146, 319–336 (2023). https://doi.org/10.1007/s00401-023-02603-y

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  • DOI: https://doi.org/10.1007/s00401-023-02603-y

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