Daniel B. Drachman - US grants

The projects contained in this proposal are all closely related to the central theme of interactions of motor nerves and skeletal muscles. Myasthenia gravis (MG) is by far the most common clinical disorder of the neuromuscular junction, affecting acetylcholine receptors. We will continue our analysis of the pathogenetic mechanisms in MG, and will undertake therapeutic trials in experimental animals. We will extend our studies of neurotrophic mechanisms, both at the level of the nerve's influence, and at the level of the muscle's response. We will study nerve sprouting and regeneration, with particular emphasis on the factors that evoke nerve growth and the axonal mechanisms by which it is sustained. We have begun an extensive study of Coxsackievirus myopathy. This viral myopathy relates closely to the theme of the proposal, because only denervated or immature muscles are susceptible to infection, and because surface receptors are essential for infection to take place. Thus, the virus will be used as a biological probe of the muscle membrane during development and denervation. Finally, we plan to continue our studies of axonal transport in normal and abnormal peripheral nerve regeneration.

During the past decade, rapid strides have been made in understanding the pathophysiology, immunology, and treatment of myasthenia gravis (MG). It is now clear that the basic abnormality in MG is a decrease of acetylcholine receptors (AChRs) at neuromuscular junctions, brought about by an antibody-mediated autoimmune attack. Much is presently known about the antigen (AChR), the effector mechanisms of the autoimmune response, and the target (AChRs at neuromuscular junctions). However, MG is far from "solved;" there is a great deal more to be learned about the origin, the cellular and humoral immune mechanisms, and the ultimate cure for MG. The purpose of the planned conference is to bring together scientists from the many fields that impinge on MG, including clinical neurology, physiology, pharmacology, immunology, and molecular biology. Areas in which striking advances relevant to MG have been made in the past several years include: 1) molecular biology of the AChR; 2) new knowledge of the physiology of neuromuscular transmission; 3) basic mechanisms of immune responses and autoimmunity; 4) immunopathogenesis of MG, and of analogous diseases; 5) immunogenetics as applied to MG; 6) new strategies of treatment of MG. This conference represents a unique opportunity to bring together individuals whose combined knowledge and collaboration can lead to accelerated advances not only in MG, but also in the parent disciplines as well. The scientific content of the meeting will be both timely and important in its own right, but the effect of cross fertilization can be expected to enhance its impact greatly.

Myasthenia gravis (MG), which is by far the most common clinical disorder of the neuromuscular junction, is due to an antibody-mediated autoimmune attack against acetylcholine receptors (AChRs). The major challenge in MG is to use the detailed knowledge of its pathogenesis to design immunotherapeutic strategies that will a) specifically eliminate the anti-AChR autoimmune responses; b) have long-lasting effects, and c) eventually be applicalbe to human MG: We will develop and test a series of novel strategies for specific immunotherapy in the experimental model of myasthenia gravis in the rat (EAMG). 1) We will induce and propagate AChR-specific suppressor T lymphocytes, using two approaches that have already been successful in preliminary experiments in our hands: a) incubation with lymphocytes from EAMG rats with Cyclosporin A (CsA) plus AChR in vitro, b) incubation of lymphocytes from EAMG rats syngeneic lymphocytes with covalently coupled AChR. The suppressor cells induced by these methods, or soluble suppressor factors produced by them, will first be tested in a cell culture system, and then will be used to treat rats with EAMG. 2) We will attempt to treat EAMG by immunizing rats with a pure population of AChR-sensitized lymphoblasts. This method induces a powerful and broad spectrum anti-idiotypic reaction that should down-regulate the anti-AChR immune response. 3) We will use the promising method of "total lymphoid irradiation" (TLI) to attempt to induce long lasting and specific suppression of the anti-AChR response in animals with EAMG. ULtimately, the mehtods developed in this model system should be applicable to the treatment of human MG, and other autoimmune neuromuscular disorders.

DESCRIPTION: The pathogenesis of myasthenia gravis (MG) involves an antibody-mediated autoimmune response directed against acetylcholine receptors (AChR). Although current treatment of MG with immunosuppressive agents is reasonably effective, it has important drawbacks, including overall suppression of the immune system, and other adverse side effects. The goal of specific immunotherapy has remained elusive. Since the pathogenic AChR antibody response is T cell dependent, inactivation or elimination of AChR-specific T cells interrupts the immune response, with resulting clinical benefit. However, the fact that T cell responses to AChR are highly heterogeneous presents a strategic problem in designing specific immunotherapy. This proposal will explore and capitalize on a novel strategy of safe stimulation to target the entire spectrum of AChR-specific T cells, using denatured AChR (denAChR), in conjunction with genetically engineered agents that inactivate and/or eliminate the specifically stimulated T cells. The studies will be carried out in Lewis rats with experimental autoimmune MG (EAMG). DenAChR stimulates AChR-specific T cells as strongly as native AChR, but its reduced and non-pathogenic AChR antibody response avoids the risk of exacerbating MG. Stimulation is a necessary condition for the action of the therapeutic agents. CTLA4Ig is a soluble recombinant protein that blocks the B7 family of costimulatory molecules, and may induce anergy in antigen-stimulated T cells. Preliminary studies suggest that CTLA4Ig prevents primary EAMG and inhibits secondary EAMG, with interesting changes in the AChR antibody isotypes, suggestive of a switch in T helper cells from Th1 to Th2 type. DAB389IL2 is a fusion protein that binds to IL2 receptors on antigen-activated T cells, and kills them via a lethal diphtheria toxin moiety. Preliminary studies using DAB389IL2 in vitro and in vivo show marked inhibition of immune responses to AChR. Combined use of CTLA4Ig + DAB389IL2 appears to be even more potent, with a powerful effect on the difficult-to-treat secondary immune response to AChR. We will study the effects of this strategy on antibodies to Torpedo and autoantibodies to rat

DESCRIPTION (adapted from applicant's abstract): This is a proposal to develop and apply a unique and powerful new gene transfer strategy for specific immunotherapy of myasthenia gravis (MG). Ideally, therapy of MG should specifically inhibit the autoimmune response to the autoantigen, acetylcholine receptor (AChR), without otherwise interfering with the immune system. Because the antibody response to AChR is T cell dependant, elimination of the AChR-specific T cells interrupts the autoimmune disorder at a pivotal point, resulting in clinical benefit. This requires elimination of virtually all AChR-specific T cells. However, the marked heterogeneity of T cell responses to AChR in humans and in experimental animals presents a challenge in designing specific treatment capable of eliminating the entire repertoire of AChR-specific T cells. The investigator's targeting strategy is based on the fact that a myasthenic individual's own antigen presenting cells (APCs) can present AChR epitopes to that indivudual's entire repertoire of AChR specific T cells. They have adapted a method that induces the APCs to process and present AChR by inserting a cDNA construct that encodes the key immunogenic domain of the AChR flanked by signals that induce the APCs process and present it. These APCs target AChR-specific T cells highly effectively. In order to eliminate the targeted T cells, they utilize Fas ligands as a "warhead." When FasL interacts with Fas, which is abundantly expressed on activated T cells, it induces apoptosis and death of the T cells. Insertion of the gene for FasL in APCs induces them to express FasL, which effectively kills Fas-expressing target cells. It is essential to protect these APCs, which would die by FasL- induced "suicide," because they also express Fas. To protect these APCs, they insert a 3rd gene for a truncated form of FADD, which acts as a dominant negative inhibitor of Fas-mediated cell death, into the APCs. To insert all 3 genes simultaneously into APCs ex vivo, they have developed a vaccinia virus vector (vvv), which carries all 3 genes. This vaccinia vector can transfer multiple genes simultaneously, inducing high level production of the gene encoded proteins. When it is attenuated (by treatment with psoralen and UV light) the vvv does not replicate but does direct expression of the gene products. Finally, the expression of FasL directed by their vvv system should confer protection against an immune attack by the host's immune system on the virus vector itself. They now propose to optimize the system and develop it in two animal models: (1) experimental MG, including a new transgenic myasthenic mouse model; and (2) a transgenic mouse model of hemaglutinin (HA) sensitivity. The experiments described in the present proposal will develop this novel strategy in experimental animals, both for prevention and for treatment of EAMG. This method is designed to be suitable for treatment of human autoimmune diseases. When optimized in experimental animals, it will be adaptable for treatment of patients with MG and other autoimmune diseases.