InVivoMAb anti-human/rat/fish AChR

Targeted antibody blocking enables characterization of binding sites on immunoglobulin G (IgG), and can efficiently eliminate harmful antibodies from organisms. In this report, we present a novel peptide-denoted as a dual-functional conjugate of antigenic peptide and Fc-III mimetics (DCAF)-for targeted blocking of antibodies. Synthesis of DCAF was achieved by native chemical ligation, and the molecule consists of three functional parts: a specific antigenic peptide, a linker and the Fc-III mimetic peptide, which has a high affinity toward the Fc region of IgG molecules. We demonstrate that DCAF binds the cognate antibody with high selectivity by simultaneously binding to the Fab and Fc regions of IgG. Animal experiments revealed that DCAF molecules diminish the antibody-dependent enhancement effect in a dengue virus infection model, and rescue the acetylcholine receptor by inhibiting the complement cascade in a myasthenia gravis model. These results suggest that DCAFs could have utility in the development of new therapeutics against harmful antibodies.

Myasthenia gravis (MG) and experimental autoimmune myasthenia gravis (EAMG) are caused by Ab-mediated autoimmune responses to muscle nicotinic acetylcholine receptors (AChRs) that impair neuromuscular transmission, thereby causing muscle weakness. Previously, we discovered that i.p. injection of a therapeutic vaccine consisting of bacterially expressed cytoplasmic domains of human AChR subunits reduced the development of chronic EAMG in rats. In this article, we show that immunization with the therapeutic vaccine in adjuvants does not induce EAMG and, thus, is safe. The potency and efficacy of the therapeutic vaccine were greatly increased by s.c. administration of repeated low doses in IFA. Onset of chronic EAMG could be prevented. Established chronic EAMG could be rapidly reversed, modeling therapy of chronic MG. Therapy reduced pathological Abs assayed by immune precipitation of a main immunogenic region chimera. Successfully treated rats exhibited long-term resistance to reinduction of EAMG, suggesting a lasting cure of MG. A long-term effect of therapy was to change the isotype of the pathogenic Ab response from IgG2b, which fixes complement, to IgG1, which does not. Prevention and reversal of chronic EAMG was not caused by the isotype switch, but the isotype switch may contribute to resistance to reinduction of EAMG. Immunization with AChR cytoplasmic domains in adjuvant is promising as a safe, Ag-specific, potent, effective, rapidly acting, and long-lasting therapeutic approach to MG.

Mutations in AChR subunits, expressed as pentamers in neuromuscular junctions (NMJs), cause various types of congenital myasthenic syndromes. In AChR pentamers, the adult epsilon subunit gradually replaces the embryonic gamma subunit as the animal develops. Because of this switch in subunit composition, mutations in specific subunits result in synaptic phenotypes that change with developmental age. However, a mutation in any AChR subunit is considered to affect the NMJs of all muscle fibers equally. Here, we report a zebrafish mutant of the AChR delta subunit that exhibits two distinct NMJ phenotypes specific to two muscle fiber types: slow or fast. Homozygous fish harboring a point mutation in the delta subunit form functional AChRs in slow muscles, whereas receptors in fast muscles are nonfunctional. To test the hypothesis that different subunit compositions in slow and fast muscles underlie distinct phenotypes, we examined the presence of epsilon/gamma subunits in NMJs using specific antibodies. Both wild-type and mutant larvae lacked epsilon/gamma subunits in slow muscle synapses. These findings in zebrafish suggest that some mutations in human congenital myasthenic syndromes may affect slow and fast muscle fibers differently.

Myasthenia gravis (MG) is a debilitating and potentially fatal neuromuscular disease characterized by the generation of autoantibodies reactive with nicotinic acetylcholine receptors (AChR) that cause loss of AChR from the neuromuscular endplate with resultant failure of neuromuscular transmission. A role for complement (C) in the pathology of human MG has been suggested based upon identification of C activation products in plasma and deposited at the endplate in MG. In the rat model, experimental autoimmune MG (EAMG), C depletion or inhibition restricts clinical disease, further implicating C in pathology. The mechanisms by which C activation drives pathology in MG and EAMG are unclear. Here we provide further evidence implicating C and specifically the membrane attack complex (MAC) in the Lewis rat passive EAMG model of MG. Rats deficient in C6, an essential component of the MAC, were resistant to disease induction and endplate destruction was reduced markedly compared to C6-sufficient controls. After reconstitution with C6, disease severity and endplate destruction in the C6-deficient rats was equivalent to that in controls. The data confirm the essential role of the MAC in the destruction of the endplate in EAMG and raise the prospect of specific MAC inhibition as an alternative therapy in MG patients resistant to conventional treatments.