Antigen-specific apheresis of pathogenic autoantibodies from myasthenia gravis sera.

Myasthenia gravis (MG) is usually caused by autoantibodies against muscle nicotinic acetylcholine receptor (AChR), which is composed of five subunits (alpha(2)betagammadelta or alpha(2)betaepsilondelta). Current treatments, including plasmapheresis, are nonspecific, causing several side effects. We aim to develop an antigen-specific alternative to plasmapheresis, since the latter removes indispensable plasma components in addition to anti-AChR antibodies. We are developing a method for the selective depletion of the anti-AChR autoantibodies from patients' plasma through the construction of "immunoadsorbent" columns carrying AChR domains. We have expressed the extracellular domains (ECDs, amino acids approximately 1-210/220) of all human muscle AChR subunits in Pichia pastoris and, in preliminary experiments, in E. coli. The ECDs were immobilized (individually or mixed) on Sepharose beads, producing Sepharose-ECD columns, which were tested for their immunoadsorbing capacity on MG sera and shown to specifically eliminate major autoantibody fractions from several MG sera. The immobilized ECDs remained stable and did not dissociate from their matrix after incubation with serum, whereas the procedure was neither toxic nor immunogenic in two experimental rabbits. Testing the intact or antibody-depleted MG sera and the affinity purified autoantibodies showed that both the intact sera and the purified autoantibodies, but not the antibody-depleted sera, could induce AChR loss in cell cultures and experimental MG in rats. This preliminary study suggests that the myasthenic potency of MG sera is entirely due to their anti-AChR antibodies and therefore their depletion should be of therapeutic value. We conclude that ECD-mediated immunoadsorption can be used as an efficient, antigen-specific therapy for MG.

Isolation and functional characterization of anti-acetylcholine receptor subunit-specific autoantibodies from myasthenic patients: receptor loss in cell culture.

The muscle nicotinic acetylcholine receptor (nAChR) is the major autoantigen in the autoimmune disease myasthenia gravis (MG), in which autoantibodies bind to, and cause loss of, nAChRs. Antibody-mediated nAChR loss is caused by the action of complement and by the acceleration of nAChR internalization caused by antibody-induced cross-linking of nAChR molecules (antigenic modulation). To obtain an insight into the role of the various anti-nAChR antibody specificities in MG, we have studied nAChR antigenic modulation caused by isolated anti-subunit autoantibodies. Autoantibodies against the nAChR alpha or beta subunits were isolated from four MG sera by affinity chromatography on columns carrying immobilized recombinant extracellular domains of human nAChR expressed in the yeast Pichia pastoris. The isolated anti-alpha and anti-beta autoantibodies, as well as untreated MG sera, induced nAChR antigenic modulation in TE671 cells. Partially antibody-depleted sera exhibited reduced modulating activity, whereas a serum completely depleted of anti-nAChR antibodies exhibited no nAChR modulation. Interestingly, the anti-alpha autoantibodies were, on average, approximately 4.3 times more effective than the anti-beta autoantibodies. The present work supports the notion that anti-nAChR autoantibodies may be the sole nAChR-reducing factor in anti-nAChR antibody-seropositive MG, and that anti-alpha-subunit autoantibodies are the dominant pathogenic autoantibody specificity.