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  Identification of self and non-self antigens implicated in autoimmune diseasesRelated Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Immunoglobulin, Antiserum, Antibody, Or Antibody Fragment, Except Conjugate Or Complex Of The Same With Nonimmunoglobulin Material, Anti-idiotypicIdentification of self and non-self antigens implicated in autoimmune diseases description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060159672, Identification of self and non-self antigens implicated in autoimmune diseases. Brief Patent Description - Full Patent Description - Patent Application Claims
FIELD OF THE INVENTION [0001] The present invention relates to the field of immunology and, in particular, to the identification of self and non-self antigens implicated in human autoimmune responses. The invention relates to methods of identifying such self antigens and provides examples of such antigens relating to Pemphigus vulgaris. The invention also relates to the use of such antigens for in vitro assays, animal models, therapeutic agents and vaccines. BACKGROUND OF THE INVENTION [0002] Human autoimmune diseases have a striking genetic association with particular alleles of major histocompatability complex ("MHC") class I or class II genes. The field was established by the seminal discovery of HLA-B27 linked susceptibility to ankylosing spondylitis, a chronic inflammatory joint disease (Brewerton (et al., 1973; Schlosstein et al., 1973). MHC associated susceptibility has now been documented for a variety of human autoimmune diseases, including insulin dependent diabetes mellitus (IDDM), rheumatoid arthritis (RA), Pemphigus vulgaris (PV), multiple sclerosis (MS) and myasthenia gravis (MG), just to name a few (Todd et al., 1987; Ahmed et al., 1990; Ahmed et al. 1991; Lanchbury & Panayi, 1991; Spielman & Nathenson, 1982; Protti et al., 1993). [0003] The MHC locus most commonly associated with autoimmune disease is the HLA-DRB locus (also known as DRB1), a highly polymorphic locus with over fifty known alleles. For example, a large body of epidemiological work has documented the association of rheumatoid arthritis with the DR4 (DRB1*0401, DRB1*0404) and DR1 (DRB1*0101) alleles, with the DR4 alleles conferring a higher risk than DR1 (Lanchbury & Panayi, 1991). The risk is dramatically increased when the subject is homozygous or heterozygous for DRB1*0401 and/or DRB1*0404. The observation that arthritis is associated with three DR alleles that are structurally similar led to the development of the `shared epitope` hypothesis as DRB1*0401, 0404 and 0101 share critical polymorphic residues in the DR.beta.67-71 cluster (Gregersen et al. 1987; Lanchbury & Panayi, 1991). These residues (in particular DR.beta.71) appear to be critical in defining the selectivity of peptide binding to the disease associated molecules. [0004] Pemphigus vulgaris (PV) is an autoimmune disease of the skin in which high titer auto-antibody production to an epidermal cell adhesion molecule (desmoglein 3) results in a loss of keratinocyte adhesion (acantholysis) and severe blister formation (Amagai et al., 1991). In different ethnic groups the disease is associated either with a DR4 allele (DRB1*0402) or with a rare DQ1 allele (DQB1*05032); only a small fraction of PV patients have neither susceptibility gene (Ahmed et al., 1991; Ahmed et al., 1990; Scharf et al., 1988). The DR4 subtype associated with pemphigus differs only at three positions in the DR.beta.67-71 cluster from the DR4 subtype associated with RA. The PV associated molecule has a negative charge (Glu) at the critical position (DR.beta.71); the neighboring position (DR.beta.70) is also negatively charged. The DR4 subtype associated with PV is the only one that carries a negative charge at DR.beta.71; a positive charge (Arg) is found at DR.beta.71 in the RA associated DR4 molecules. [0005] Efforts to identify sequence homologies between self-peptide epitopes that might be involved in autoimmunity and various bacterial and viral pathogens have therefore been made. These homology searches have focused on alignments with sequence identity. No success has been reported using such alignments in identifying epitopes from pathogens that could cross react with presumably pathogenic T cell lines from human patients with autoimmune disease (Oldstone, 1990). A sequence identity was recently found between an epitope in a Coxsackie virus protein and GAD65, suspected of being an autoantigen in diabetes. These peptides could reciprocally generate polyclonal T cell lines from mice that cross react with the other peptides (Tian, et al., 1994). No evidence, however, was provided that these peptides could stimulate clones from diabetic mice (or humans). [0006] Recent developments in the field, in particular the identification of allele specific peptide binding motifs have transformed the field (Madden et al., 1991; Rotschke & Falk, 1991). Based on this knowledge the structural basis for MHC linked susceptibility to autoimmune diseases can be reassessed at a level of detail sufficient for solving longstanding questions in the field. Motifs for peptide binding to several MHC class I and class II molecules have been defined by sequence analysis of naturally processed peptides and by mutational analysis of known epitopes. MHC class I bound peptides were found to be short (generally 8-10 amino acids long) and to possess two dominant MHC anchor residues; MHC class II bound peptides were found to be longer and more heterogeneous in size (Madden et al., 1991; Rotschke & Falk, 1991; Jardetzky et al. 1991, Chicz et al. 1993). Due to the size heterogeneity, however, it has proven more difficult to define MHC class II binding motifs based on sequence alignments. More recently, a crystal structure for HLA-DR1 demonstrated that there is a dominant hydrophobic anchor residue close to the N-terminus of the peptide and that secondary anchor residues are found at several other peptide positions (Brown et al., 1993). Even this work, however, could not provide a detailed description of the binding pockets of HLA-DR proteins, the particular residues involved in the formation of these pockets of the structural requirements or antigens for MHC binding. [0007] In the present disclosure, a detailed description of the HLA-DR antigen binding pockets is provided (Stern et al., 1994). With this information, together with functional information defining those amino acids of the self or non-self antigen that are needed for MHC binding and TCR contact (e.g., Wucherpfennig et al. 1994a,), binding motifs for the various HLA-DR allotypes may be developed, self epitopes involved in autoimmune disease may be identified and a method is provided for identifying bacterial and viral epitopes which may initiate a human autoimmune response. SUMMARY OF THE INVENTION [0008] The present invention provides, in one aspect, isolated polypeptides derived from the human desmoglein 3 protein and implicated as self epitopes in the autoimmune disease Pemphigus vulgaris (PV). These polypeptides consist essentially of the amino acid sequence disclosed herein and have been designated SEQ ID NO: 1. These polypeptides consist of SEQ ID NO: 1. In particular, the invention provides isolated polypeptides which consist of these sequences, the core MHC binding residues of these sequences, or the inner core MHC binding residues of these sequences. [0009] Compositions of the present invention comprise a pharmaceutically acceptable carrier and a polypeptide of SEQ ID NO: 1. The composition can also comprise a pharmaceutically acceptable salt of the polypeptide. A preferred pharmaceutically acceptable salt is an acetate. [0010] Compositions of the present invention can further comprising a pharmaceutically acceptable additive, such as a sugar or a surfactant. Acceptable sugars are those such as dextrose and mannitol. In one embodiment, the composition is formulated with about 5% sugar. The composition can further comprising buffers, such as dihydrate sodium citrate and monohydrate citric acid, and bulking agents, such as mannitol. In a further embodiment, a surfactant, such as can be polysorbate 20 or polysorbate 80, can be added to the composition in an amount of from about 0.01% to about 5%. One embodiment of the present invention comprises an immunogenic composition of the polypeptide. [0011] In one embodiment, the composition comprises a lyophilized polypeptide of SEQ ID NO: 1. In one embodiment, the lyophilized polypeptide has a reconstitution time of less than 15 minutes, more preferably, the reconstitution time is less than 10 minutes, more preferably, the reconstitution time is less than 5 minutes, and more preferably, the reconstitution time is less than 3 minutes. [0012] In one embodiment, the purity of the peptide is greater than 90%, more preferably, the purity is greater than 93%, more preferably, the purity is greater than 95%, and more preferably, the purity is greater than 96%. [0013] In one embodiment, the composition has bacterial endotoxin contamination of less than about 5 EU/mL, more preferably, the bacterial endotoxin contamination is less than about 3 EU/mL, more preferably, the bacterial endotoxin contamination is less than about 2 EU/mL, and more preferably, the bacterial endotoxin contamination is less than about 1.25 EU/mL. [0014] More preferably, the composition of the present invention has the formulation as set forth in Table 3. [0015] In another set of embodiments, the invention provides for pharmaceutical preparations for use in tolerizing individuals to auto-antigens. The preparations include a pharmaceutically acceptable carrier and an isolated human polypeptide which includes an amino acid sequence corresponding to a sequence motif for an HLA-DR protein which is associated with a human autoimmune disease. These polypeptides are capable of binding to the HLA-DR protein to form a complex which activates autoreactive T cells in subjects having the autoimmune disease. The peptides are not derived from human collagen or human myelin basic protein. [0016] In particular embodiments, such pharmaceutical preparations are provided in which the HLA-DR protein is HLA-DR4 protein and the autoimmune disease is Pemphigus vulgaris. In addition, a particular sequence motif is provided for Pemphigus vulgaris and pharmaceuticals having peptides with this motif are provided. Specific embodiments of the pharmaceuticals include each of the polypeptides described above with respect to Pemphigus vulgaris. Thus, methods of tolerizing an individual to a Pemphigus vulgaris autoantigen are also provided. [0017] In another aspect of the invention, pharmaceuticals are provided for vaccination against a human pathogen implicated in the aetiology of autoimmune disease. These pharmaceutical preparations include a pharmaceutically acceptable carrier and an immunogenic preparation effective to immunize against a human pathogen. The human pathogen is one which in its native form includes a polypeptide having an amino acid sequence corresponding to a sequence motif for an HLA-DR protein which is associated with the autoimmune disease. These polypeptides are capable of binding to the HLA-DR protein to form a complex which activates T cells which become autoreactive and initiate the autoimmune disease. The preparations of the present invention specifically do not include such polypeptides but, rather, include other antigens from the pathogen. [0018] In particular embodiments, such pharmaceutical preparations are provided in which the HLA-DR protein is HLA-DR4 protein and the autoimmune disease is Pemphigus vulgaris. In addition, a particular sequence motif is provided for Pemphigus vulgaris and pharmaceuticals which lack peptides having this motif are provided. Specific embodiments of the pharmaceuticals include preparations lacking each of the polypeptides described above with respect to Pemphigus vulgaris. Thus, methods of immunizing an individual against pathogens which may cause Pemphigus vulgaris are also provided. [0019] The present invention also provides general methods for evaluating a peptide for an ability to induce an autoimmune response. These methods involve choosing an MHC HLA-DR molecule associated with the autoimmune response, selecting at least two major MHC binding pockets of the HLA-DR molecule, identifying sets of amino acid residues which bind within each of the selected pockets, developing a sequence motif for the HLA-DR molecule in which the sets of amino acids define the allowed amino acids at the corresponding positions of the motif, and then comparing the amino acid sequence of the peptide to the sequence motif. Peptides which match the motif have a much greater likelihood of inducing the autoimmune disease. In addition, if there is a known epitope implicated in the disease, the method may further include selecting at least one TCR contact residue of the epitope, identifying a set of amino acid residues which may serve as the TCR contact, and including this set in the motif at the appropriate position. In preferred embodiments, the motifs include restrictions on the residues at positions corresponding to at least the P1 MHC binding pocket and at least one of the P4 and P6 pockets. [0020] In another embodiment of the invention, methods are provided specifically for identifying foreign antigens implicated in human autoimmune response. These methods include the same steps as the previously described methods, but further include a comparison of the resulting sequence motif to sets of human pathogens. In preferred embodiments, peptide sequences from one or more species in the normal human intestinal flora are excluded from consideration. In another preferred embodiment, sequences from one or more species of pathogen which is negatively correlated with the incidence of the disease are excluded. In a most preferred embodiment, the human pathogen peptides are searched and evaluated on a computer database using the motif as a search criterion. [0021] The present invention provides, in one aspect, isolated peptides derived from the human desmoglein 3 (Dsg3) protein, and uses thereof. These peptides, for example, consist essentially of SEQ ID NO: 1, and bind to a HLA-DR4 protein to form a complex which activates autoreactive T cells in a subject having Pemphigus vulgaris. Continue reading about Identification of self and non-self antigens implicated in autoimmune diseases... Full patent description for Identification of self and non-self antigens implicated in autoimmune diseases Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Identification of self and non-self antigens implicated in autoimmune diseases patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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