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  • The cytoplasmic domain of muscle AChR is

    2024-02-23

    The cytoplasmic domain of muscle AChR is not accessible to CAY10433 in vivo. Theoretically, therapy with the cytoplasmic domains should be safe. Safety is demonstrated by the facts that: (1) rats repeatedly immunized with the cytoplasmic domains in TiterMax adjuvant do not develop EAMG, although they produced antibodies which can bind to solubilized native AChRs; and (2) serum antibodies from these rats do not bind the MIR, passively transfer EAMG, or cause antigenic modulation of AChR [94]. Suppression of EAMG accompanied by high level of antibodies to AChR cytoplasmic domains implies the possibility of therapeutic effects of these antibodies. Adjuvants promote the formation of antibodies to antigens. Thus, adjuvants may enhance the potency and efficacy of therapy with the cytoplasmic domains. We found that specific immunosuppression of EAMG with a therapeutic vaccine consisting of human AChR cytoplasmic domains is more potent when it is administered s.c. in adjuvant immediately after the acute phase [94]. As a therapeutic adjuvant, the modest adjuvant incomplete Freund’s adjuvant (IFA) CAY10433 is more effective than the more potent adjuvant TiterMax that is used with Torpedo AChR to induce EAMG. IFA induces a predominantly Th2 biased response, while TiterMax induces a strong Th1 response [95]. Administration of the therapeutic vaccine s.c. in IFA is 8-fold more potent than i.p. administering the vaccine in saline. It causes a 5-fold reduction in pathological autoantibody level to the MIR and a 5-fold increase in autoantibody level to rat muscle AChR that is primarily directed at pathologically irrelevant cytoplasmic epitopes. The efficacy of specific immunosuppressive therapy of EAMG is also improved by optimizing the dose and schedule of therapeutic vaccine in IFA. One mg of therapeutic vaccine in IFA at weekly intervals between days 14 and 49 after induction of EAMG eliminates development of chronic EAMG almost completely, reducing the antibody level to the MIR 8-fold (Fig. 4A and B) [94]. Vaccination results in a 100-fold increase in antibody level to AChR cytoplasmic domains. Benefits of therapy are long lasting. The rats treated with any dose in Fig. 4A are resistant to re-induction of EAMG by re-immunization with Torpedo AChR in TiterMax 23 weeks after the last therapeutic dose (Fig. 4C and D). Their resistance to re-induction of EAMG does not result from the changes in AChR amount or density in their neuromuscular junctions, since these successfully treated rats are as susceptible to passive transfer of EAMG by a mAb to the MIR as are normal rats. Their resistance to re-induction of EAMG is not a result of having survived EAMG either, because untreated rats that survived EAMG are not resistant to re-induction of EAMG. Therapy does not result in deletion or unresponsiveness of autoreactive lymphocytes. Successfully treated rats develop rapid and strong immune responses to both Torpedo AChR and the MIR after re-immunization with Torpedo AChR [94]. Antibodies in these rats bind to the extracellular surface of rat muscle AChR, causing antigenic modulation of AChR. However, these serum antibodies are ineffective at either passively transferring EAMG or fixing complement in response to binding MIR/AChBP chimera. After re-immunization with Torpedo AChR, therapy shifts predominant isotypes of antibodies to the MIR in these rats from IgG2b (which strongly fixes complement) to IgG1 (which fixes complement poorly). Because of this isotype switching, serum antibodies from successfully treated rats are less effective at fixing complement, and thus are less pathogenic. This therapeutic regime started on day 35 or 92 during the chronic phase rapidly reverses existing chronic EAMG (Fig. 5A, B, C, and D). This closely mimics treatment of chronic MG. Therapy rapidly suppresses the production of antibodies to the MIR. The rapid onset of the therapy suggests that it must involve inhibition or apoptosis of autoantibody-producing plasma cells. All of these properties make this therapeutic vaccine an attractive candidate for antigen-specific therapy for human MG.