Last update:
24-Mar-2005

OBJECTIVE The pathology of the autoimmune disease myasthenia gravis is largely explained by autoantibodies produced against the human muscle acetylcholine receptor. Plasmapheresis offers a significant therapeutic option but it removes important plasma components. The specific extracorporeal removal of the autoantibodies using immunoadsorption on matrix-immobilized receptor offers a solution to the problem, which is however hindered by the limited available native receptor. The objective of this study was the production of extracellular recombinant domains of the receptor subunits, and their utilization as specific immunoadsorbents of autoantibodies from myasthenic sera, for future application in a specific therapy for myasthenia.

METHOD The extracellular domains of the α, β, γ and ε subunits of the human acetylcholine receptor were expressed in Pichia pastoris, the proteins were purified by Ni²⁺-NTA agarose and their solubility was tested by gel filtration. Each subunit was immobilized on CNBr-sepharose and used in immunoadsorption experiments with 64 randomly chosen myasthenic sera. The autoantibodies which remained in the serum after immunoadsorption were quantified by radioimmunoassay.

RESULTS The extracellular domains (amino acids ~1-210) of the α, β, γ and ε subunits of the human muscle acetylcholine receptor were expressed and purified. Each recombinant protein had a near-native structure resembling the structure found in the full-length subunit of the intact receptor. Protein α1-210, immobilized on CNBr-sepharose, was used for the immunoadsorption of autoantibodies from 64 myasthenic sera and was shown to remove, on average, 35% of the antibodies. Correspondingly, β1-221, γ1- 218 and ε1-219, which had been immobilized on CNBr-sepharose could remove, on average, 22%, 21% and 18% of the autoantibodies, respectively. The combined use of α1-210 and γ1-218 in immunoadsorption experiments showed that the pathogenic autoantibodies could be removed in an additive fashion in 10 tested sera. Incubation of the polypeptides with human plasma showed that the immobilized proteins remained stable and did not dissociate from their insoluble matrix (sepharose). Moreover, the titration of the immunoadsorbent column with patient serum showed that a small quantity of immobilized protein (1 μg) could remove at least 2 pmoles of anti-receptor antibodies.

CONCLUSIONS These results suggest that the combined use of all subunits would remove the large majority of autoantibodies from most of the myasthenic sera, thus offering an option for the design and application of a novel, effective and antigen-specific therapy for myasthenia gravis.

Key words: Acetylcholine receptor, Immunoadsorption, Myasthenia gravis, Recombinant polypeptides, Therapy.