David P. Richman - US grants

The aim of this research is to study the immunoregulatory network in experimental autoimmune myasthenia gravis (EAMG) and in myasthenia gravis (MG), diseases in which the antigen, nicotinic acetylcholine receptor (AChR), is well defined. Such information may be applicable to other immunologically-based diseases in which the principle reaction is less well-understood. The idiotypic repertoire of the anti-AChR antibodies active in EAMG will be studied making use of rat monoclonal anti-AChR antibodies that have been prepared by means of hybridization of antibody-producing cells with myeloma cell lines. The fact that many of these monoclonal antibodies (mcabs) are capable of inducing EAMG when individually injected into normal animals makes it likely that some of these idiotypes (Ids) will be important in EAMG. Syngeneic animals will be immunized with anti-AChR mcabs to produce anti-idiotypic (anti-Id) serum and anti-Id mcabs. These anti-Id preparations will be used to search for the presence of Ids, and anti-Ids, in the serum and on the lymphocytes of both EAMG animals and patients with MG. Attempts will be made to modify the severity of EAMG by manipulating levels of important Ids by injection of anti-Id, either alone or in combination with cytotoxic drugs.

(Adapted from the applicant's abstract) The goal of this project is to determine the pathogenic mechanisms by which the autoantibodies in MG induce abnormal neuromuscular transmission and to devise means of blocking such effects. It is suggested that these studies would provide a groundwork for the development of antigen-specific treatments of this and other antibody-mediated autoimmune disorders. pEAMG induced by administration of monoclonal antibodies (mAbs) directed against the AChR in normal rates provides the experimental rationale for these studies. The hypothesis to be tested is that the major pathogenic mechanism in MG is complement-mediated damage to the AChR-containing post-synaptic membrane initiated by complement activation by bound anti-AChR Abs. Abs, whose complement activating capacity has been modified (F(ab')2), fragments of mAbs, and genetically engineered hybrid Abs with little of no complement activating activity will be produced and used in these studies. The engineered Abs are to be produced by myeloma cells transfected with recombinant genes which encode V regions from disease-inducing anti-AChR mAbs and constant regions which lack complement activating activity. The ability of the modified or engineered Abs to induce pEAMG or block EAMG induced by intact Abs will be assessed clinically, morphologically, electrophysiologically and by chemical and autoradiographic analysis of AChR content at muscle end plates.

The central theme of this Program Project is to use monoclonal antibodies (mAbs) directed against defined oligopeptides within the subunits of the nicotinic acetylcholine receptor (AcChR) as site-specific probes of AcChR structure (both primary and tertiary structure) and AcChR function. Special attention will be paid to mAbs whose binding interferes with the function of this transmembrane glycoprotein neurotransmitter receptor. Projects 1-4 will assess the effects of the mAbs on particular aspects of AcChR function: Project 1 - ligand binding, Project 2 - ion fluxes by stopped flow technique; Project 3 - ion channel function by patch clamp technique; Project 4 - neuromuscular transmission in vitro and in vivo. Projects 5 and 6 will localize the epitopes of these mAbs within the AcChR structure: Project 5 - production of rationally chosen synthetic oligopeptides mimicking specific sites within AcChR subunits, assessment of the contribution of glycosyl moieties to mAb epitopes, Project 6 -identification of bound mAbs on the low resolution three-dimensional model of AcChR by both small angle X-ray diffraction and high resolution electron microscopy, as well as localization of fluorescent probes. The Scientific Core will produce new mAbs directed against the peptides produced in Project 5 and will provide purified, characterized mAbs for the various projects. In Project 4, the peptides will be used to possibly develop a new treatment modality for experimental myasthenia gravis. This Program Project represents a collaborative effort of immunologists, biochemists, electrophysiologists, peptide chemists, and physical/structural chemists in which the information from the various projects, taken together, will allow for mapping of various sites in the primary sequence of AcChR, especially functionally important sites, onto the three-dimensional model.

The overall goal of this project is to analyze the autoimmune response to the acetylcholine receptor (ACHR) in myasthenia gravis (MG) and its experimental model, experimental autoimmune myasthenia gravis (EAMG), and to manipulate the expression of the autoantibodies. This analysis will provide the groundwork for the development of antigen-specific, or idiotope (Id)-specific, treatments of MG (and other autoimmune diseases as well) that will be more effective and less toxic than presently available therapies. A systematic survey of both the T cell and B cell immunogenic epitopes of the entire AChR in EAMG-susceptible and EAMG-resistant strains of mice in order to determine the role of the T cell repertoire in the diversity of the antibody response will be carried out. The T cell receptor (TcR) V region gene segment usage from T cell clones directed against the immunogenic T cell epitopes will be determined serologically and by sequencing cDNA or genomic DNA from these cell clones. The latter studies will determine whether the restricted TcR gene segment usage observed in the T-cell-mediated autoimmune disease, experimental allergic encephalomyelitis, also occurs in the antibody-mediated, T-cell-controlled autoimmune response in EAMG. Moreover, the effectiveness in blocking EAMG of anti-clonotypic responses against important TcRs will be assessed. Also, the role of T cells in the protection from EAMG induced by injection of, previously developed, anti-Id monoclonal antibodies will be analyzed. These studies will involve adoptive transfer of T cells from protected animals to naive animals and determination of TcR gene segment usage in the protected animals. The proposed experiments will primarily make use of the pepscanning procedure to survey the AChR epitopes for T cell and B cell immunogenicity, development of T cell clones and hybridomas, amplification of cDNA and genomic DNA by polymerase chain reaction, and direct sequencing of amplified products to determine TcR gene segment usage.

Myasthenia gravis (MG) and its animal model, experimental autoimmune myasthenia gravis (EAMG) are T-cell-dependent antibody (Ab)-mediated autoimmune disease directed at the acetylcholine receptor (AChR) in the endplate membrane of the neuromuscular junction (NMJ). Three pathogenic mechanisms by which the Abs induce the characteristic abnormalities at the NMJ have been proposed from studies of EAMG and MG: complement-mediated endplate membrane destruction increased AChR turnover by Ab crosslinking of adjacent AChRs, and functional blockage of intact AChRs. Our previous studies and those from other laboratories have suggested the hypothesis that the major mechanism is complement-mediated endplate membrane destruction. To test this hypothesis-syngeneic recombinant/chimeric (rec/chi) Abs will be produced from our library of rat anti-AChR monoclonal Abs, which have been engineered to have reduced or absent complement activating activity, but to retain the functions associated with the other two possible pathogenic mechanisms. These rec/chi Abs will be analyzed in vitro for their antigen binding and their effector functions and in vivo for their ability to induce the passively-transferred form of EAMG in syngeneic rats. Rec/chi Abs that are incapable of inducing EAMG will be tested, singly or in combination, for their ability to bock both passively- and actively-induced EAMG. Abs of this type will potentially represent a new antigen-specific treatment of MG. Therefore, similar human autologous rec/chi Abs will developed from anti-AChR B cells obtained from patients with MG, and their effector functions analyzed in vitro.

DESCRIPTION: This proposal seeks partial funding for the Ninth International Conference on Myasthenia Gravis, to be held in Santa Monica, CA. Speakers will be drawn from both the basic and clinical sciences and will present the current state of knowledge regarding the normal and abnormal function of the mammalian neuromuscular junction. A major goal is to integrate information from diverse disciplines, including biochemistry, structural biology, cellular and molecular neuroscience, pharmacology, immunology, and clinical neurology. Topics highlighted in the proposed conference will be molecular events involved in transmitter release, new postsynaptic antigens in autoimmune MG, and immune mechanisms underlying the Lambert-Eaton myasthenia syndrome. The conference is organized as a series of 35-minute oral presentations with five minutes of discussion following each presentation, in the morning and afternoon of each of the three days. The first half-day is devoted to basic features of the nmj, while the remainder is focused on various aspects of MG and LE. Poster presentations are selected from the abstracts submitted by all participants and will be shown in the early evening of the first two days. The conference will be advertised in the New York Academy's magazine, Science, and in other unstated related professional journals and newsletters. Women will be targeted with advertisements in publications of the Association of University Women, the Women's American Medical Association, the National Medical Association, and other unspecified organizations. Junior investigators and graduate students will pay a reduced registration fee.

DESCRIPTION (provided by applicant): Anti-muscle-specific kinase (MuSK) myasthenia (AMM) is a newly-described disease of neuromuscular transmission characterized by fatigable weakness, muscle wasting and circulating antibodies (Abs) to MuSK, a transmembrane receptor kinase crucial to the formation of this synapse. AMM differs from myasthenia gravis (MG) in its severity, the more focal nature of the weakness and the associated muscle wasting. Remarkably little is known concerning its pathogenesis or etiology, including whether there is an accompanying cellular immune response to MuSK and what mechanisms underlie the abnormal neuromuscular transmission and muscle wasting. The treatment of this disease is nearly completely unknown. Current clinical practice is to use the treatment protocols developed for (seropositive) MG. However, data have accumulated demonstrating that many of these treatments, e.g. cholinesterase inhibitors, thymectomy and immunoglobulin infusion, are minimally effective. Our overall goal is to develop more specific and effective treatments of AMM. To accomplish this, we have produced an animal model of AMM in Lewis rats, termed experimental AMM (EAMM), to serve as a platform for analysis of the pathogenic mechanisms underlying the human disease, including the mechanisms active at the neuromuscular junction (NMJ) and the mechanisms underlying the immune attack on this synapse. The model disease, which is induced by a single injection of the MuSK 60 isoform of the protein is quite severe, resulting in fatigable weakness and muscle wasting leading to death within 27 days, along with severe disruption of both the postsynaptic and presynaptic components of the NMJ. The proposed studies will address the hypothesis that AMM is an Ab-mediated disease and that the MuSK Abs alter the function of MuSK at the mature NMJ, thereby inducing the weakness and muscle wasting that are characteristic of this disease. A corollary of that hypothesis is that MuSK, in addition to its known role in the developing NMJ, plays an important role in the maintenance of the mature synapse. The first specific aim involves the analysis of the pathogenic mechanisms operative at the NMJ in EAMM through clinical, electrophysiologic and morphologic studies of the NMJ, focusing on the signal transduction pathways activated by MuSK. The second specific aim analyzes the nature of the autoimmune response in EAMM by assessing both the Ab and T cell responses to MuSK in these animals, along with studies of passive transfer of the disease with both immunoglobulin derived from EAMM serum and lymphocytes from EAMM spleens and lymph nodes. PUBLIC HEALTH RELEVANCE: Relevance: Remarkably little is known concerning the pathogenesis or etiology of anti-muscle-specific kinase (MuSK) myasthenia (AMM), including whether the MuSK Abs are, in fact, pathogenic, whether there is an accompanying cellular immune response and what mechanisms underlie the abnormal neuromuscular transmission and muscle wasting. The treatment of this disease is also nearly completely unknown. This project will provide the means for determining the underlying mechanisms in AMM in order to identify new treatments and provide an animal model for testing these treatments.

The long term goal of this developmental project is improve the treatment of myasthenia gravis (MG) and, possibly, other antibody (Ab)-mediated autoimmune diseases, by specifically targeting the autoimmune process, with little or no effect on the normal functioning of the immune system. We will make use of syngeneic chimeric autoantibody receptor (CAAR)- expressing T cells (CAART) to attack autoAb-producing B cells in the rat model of muscle- specific kinase (MuSK)-MG, experimental autoimmune MuSK myasthenia (EAMM). CAART are modeled after chimeric antigen receptor (CAR) T cells (CART) that express an engineered CAR to target tumor cells, by employing the single chain Fv of an anti-tumor Ab fused to the T cell receptor framework. For CAAR, the single chain anti-tumor Fv is replaced by the autoantigen, in this case, MuSK ectodomain, to target the anti-MuSK Ab displayed on the surface of the autoimmune B cells. The previous use of CAART has involved a chimeric human/mouse animal model of pemphigus vulgaris (PV) in immunodeficient mice. The current autoimmune model better approximates human autoimmune disease in that it is induced by active immunization of an immunocompetent host. The Specific Aims are: 1) assess, and optimize, in vitro the ability of rat MuSK CAART to reduce/eliminate syngeneic anti-MuSK B cells and MuSK Ab secretion; 2) assess the ability of injected syngeneic MuSK CAART to prevent and treat EAMM, as well as screening for off-target and other adverse effects. cDNA coding the autoantigen will be inserted into a lentiviral vector that has been effective in transfecting human T cells to produce human PV CAART. In preliminary EAMM studies, this vector has successfully transduced rat T cells as well. For the in vitro experiments, two types of autoimmune B cells will be used as targets for the CAART: anti-MuSK hybridomas and polyclonal anti-MuSK B cells isolated from spleens of MuSK-immunized rats. Assays for CAART efficacy will include measures of in vitro B cell killing: Cr release and reduced MuSK Ab-secreting B cells in an ELISPOT. In terms of in vivo treatment of EAMM, dose-finding experiments will be carried out by assessing disease severity clinically, electrophysiologically and by morphometric analysis of the neuromuscular synapse, the target structure in MG. Potential adverse effects of injected CAART will be monitored assessing neuromuscular junction structure and function, cytokine levels, blood chemistries and blood counts and by necropsy screen of various organs.