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  Targeted drug-formaldehyde conjugates and methods of making and using the sameUSPTO Application #: 20070203059Title: Targeted drug-formaldehyde conjugates and methods of making and using the same Abstract: A method and compositions for detecting autoimmunity to islet glucose-6 phosphatase related protein (IGRP). Detection of IGRP autoantibodies alone, and in combination with other molecules such as the 65-kDa form of glutamate decarboxylase (GAD65), insulin and insulin granule membrane proteins ICA512 (IA-2) and phogrin (IA-2) auto-antigens, provides an effective and reliable chemical assay for the diagnosis of autoimmune (type 1) diabetes. Additionally, this invention provides therapeutic regimens based on IGRP and related molecules for the amelioration of the diabetic clinical condition. Therefore, IGRP alone or in combination with other autoimmune diabetes associated antigens such as insulin, IA-2 and GAD65, is useful in the prediction (diagnosis), treatment (therapy), and prevention (prophylaxis) of diabetes. (end of abstract) Agent: Sheridan Ross PC - Denver, CO, US Inventors: John C. Hutton, Richard O'Brien, Howard Davidson, Seija Hackl USPTO Applicaton #: 20070203059 - Class: 514012000 (USPTO) Related Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Designated Organic Active Ingredient Containing (doai), Peptide Containing (e.g., Protein, Peptones, Fibrinogen, Etc.) Doai, Cyclopeptides, 25 Or More Peptide Repeating Units In Known Peptide Chain Structure The Patent Description & Claims data below is from USPTO Patent Application 20070203059. Brief Patent Description - Full Patent Description - Patent Application Claims
FIELD OF THE INVENTION [0001] The method and compositions of this invention provide a method for detecting autoimmunity to islet glucose-6-phosphatase related protein (IGRP). Detection of IGRP autoimmunity alone, or in combination with other molecules such as the 65-kDa form of glutamate decarboxylase (GAD.sub.65), insulin and insulin granule membrane proteins ICA512 (IA-2) and phogrin (IA2.beta.) auto-antigens, provides an effective and reliable chemical assay for the diagnosis of autoimmune (type 1) diabetes. Additionally, this invention provides therapeutic regimens based on IGRP and related molecules for the amelioration of the diabetic clinical condition. BACKGROUND OF THE INVENTION [0002] Diabetes has been termed the epidemic of the 21st century and over the past 50 years in Western societies has been doubling in incidence every 15 years. It exacts a huge socioeconomic toll because of its devastating microvascular and macrovascular complications and the need of patients to maintain a lifetime daily therapeutic regimen. Diabetes currently affects around two percent of the population and is increasing in incidence. It is a major cause of blindness, kidney disease and premature death, has immense socio-economic impact and places a heavy burden on healthcare facilities worldwide. The childhood-onset form of diabetes, which accounts for 10% of all cases in humans (IDDM, autoimmune or type 1 diabetes) results from an autoimmune process in which T-lymphocytes specifically destroy the insulin-secreting .beta.-cells of the pancreas. This implies that there is one or more .beta.-cell specific target(s) for destructive T-lymphocytes. [0003] Perhaps the single most important advance of the past three decades in diabetes research has been the recognition that autoimmune destruction of .beta.-cells takes months or years to reach completion. Whereas currently the clinical diagnosis of diabetes is almost never made until the destructive process is nearly complete and insulin injections are required to prevent death, intervention before the insulin-producing cells have been irreversibly destroyed can provide a strategy to prevent progression of diabetes and its complications. It is crucial, therefore, to find a means of accurately predicting the onset of autoimmune (type-1) diabetes before the disease has progressed to the clinical stage. [0004] The autoimmune destruction of pancreatic .beta.-cells is mediated mainly by T-lymphocytes of the CD4 and CD8 lineages. Single CD4 and CD8 clones can either accelerate diabetes progression or induce disease after adoptive transfer into genetically susceptible animals. Normally however, both types of cells act in concert, in both regulatory and effector capacities, at all phases of the disease, and the emergence of autoimmune diabetes is generally viewed as the consequence of their dysregulation. Based on the restricted TCR V.alpha. chain repertoire of diabetes-conferring CD4 and CD8 clones in the well-established nonobese diabetic (NOD) mouse model of autoimmune diabetes, the existence of a restricted number of primary targets for T-cell mediated attack appears likely. It is hypothesized that such targets would be .beta.-cell specific, or at least highly expressed in the .beta.-cell relative to the pancreatic .alpha., PP and D-cells. [0005] The majority of diabetic autoantigens that are defined molecularly have been discovered either by a candidate gene approach, or by serological investigations in diabetic humans. Insulin, the 65 kD form of glutamate decarboxylase (GAD) and the insulin granule membrane proteins ICA512 (IA2) and phogrin (IA2.beta.) are major targets of circulating islet cell autoimmunity in man. Other specificities include carboxypeptidase E, ICA69 and sulphated glycolipids. Of these, only insulin appears to be .beta.-cell specific whereas the others are broadly distributed among neuroendocrine tissues such as the brain, pituitary and adrenal medulla. One possible connection between these molecules is their association with the regulated pathway of secretion in the .beta.-cell. [0006] Although B-lymphocytes may play a role in antigen presentation in type 1 diabetes and circulating autoantibodies provide useful pre-clinical markers for diabetic autoimmunity, it is clear that the humoral response per se contributes little to the pathogenesis of the disease. Given that the production of high affinity antibodies is a T-dependent process, it is reasonable to assume that autoreactive diabetogenic T-cells bind to peptides derived from the same molecules recognized by autoantibodies. Consistent with this assumption, insulin, phogrin and GAD have all been shown to be the target of spontaneous T-cell responses in NOD mice. [0007] However the converse is not necessarily true, and attempts to define the cognate antigen of pathogenic T-cell clones in NOD mice that have been selected solely on the basis of islet reactivity have been largely unsuccessful, suggesting that these do not correspond to known serological markers. Thus, much remains to be learned about the specificity and diversity of CD4 and CD8 T-cell responses and their cognate antigens. Such studies in man are impeded by the general inability to access or image the affected organ and the questionable relevance of T-cell responses detected in the peripheral circulation to immunological events occurring within the islet. [0008] Islet-specific CD4 and CD8 T cell clones have been isolated from spleen, lymph nodes or islet infiltrates of pre- or newly diabetic NOD mice and many accelerate disease in naive recipients. A subset of these transfer diabetes susceptibility to NOD scid and NOD rag2 .sup.(-/-) mice (which have no endogenous T or B cells) thus demonstrating that individual clones with a restricted, if not unique, antigenic specificity can cause disease. The extent to which natural disease is the result of clonotypic or antigen-restricted responses remains unclear. The restricted usage of V.alpha. chains in the TCRs of many of these clones points to the possibility that a limited number of antigenic epitopes and antigens are involved. [0009] Therefore, there exists a substantial and long-felt need for a more accurate means of detecting autoimmune diabetes in its early stages prior to the onset of clinical symptoms and the requirement of insulin therapy. This need would be met by identification of the molecular target(s) of the CD8 T-cell population that infiltrate the pancreatic islet and selectively destroy .beta.-cells while sparing the adjacent endocrine and exocrine tissue. Identification of such a target would make possible assays for the detection of diabetes-related autoimmunity based on: levels of circulating autoantibodies, lymphocyte proliferative responses to the protein and peptides derived from the protein, detection of lymphocytes in the circulation and tissues that react with MHC class I and MHC class II tetramer molecules that incorporate target IGRP peptides, and detection of lymphocytes in the circulation and tissue using ELISPOT assays that incorporate the protein or derived peptides to stimulate reactive cells. Further, the identification of such a protein target would allow the use of the target molecule as a recombinant protein to alter the response of the immune system in a way that is protective rather than destructive. This would involve the use of recombinant protein, chemically or physically modified forms of the protein, peptide sequences derived from the protein, chemically or physically modified and peptide homologues which could be used as a vaccine. BRIEF DESCRIPTION OF THE SEQUENCES [0010] SEQ ID NO.: 1 is an mRNA sequence of human IGRP. NM.sub.--021176. Homo sapiens islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), mRNA. [0011] SEQ ID NO.: 2 is an amino acid sequence of IGRP. NM.sub.--021176. Homo sapiens islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), amino acid. [0012] SEQ ID NO.: 3 is an mRNA sequence of mouse IGRP. NM.sub.--021331. Mus Musculus islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), mRNA. [0013] SEQ ID NO.: 4 is an amino acid sequence of mouse IGRP. NM.sub.--021331. Mus Musculus islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), amino acid. [0014] SEQ ID NO.: 5 is an amino acid sequence of the purported catalytic phosphatase motif of islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), amino acid. [0015] SEQ ID NO.: 6 is an amino acid sequence of a fragment of mouse IGRP. NM.sub.--021331. Mus Musculus islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), amino acid. [0016] SEQ ID NO.: 7 is an amino acid sequence of the NRP-V7 mimeotope. BRIEF DESCRIPTION OF THE DRAWINGS [0017] FIG. 1 shows immunofluorescence microscopic localization of IGRP. Rabbit polyclonal antibodies (1:250) raised to recombinant full-length IGRP were incubated with frozen normal mouse pancreas sections monoclonal to insulin or glucagon as indicated. IGRP co-localizes with insulin positive cells only. [0018] FIG. 2 shows the predicted structure and topology of IGRP. The protein is predicted to span the membrane 7 times. The model shown places the catalytic phosphatase motif KX.sub.nRPX.sub.nPSGHX.sub.nSRX.sub.nH (SEQ ID NO.: 5) in a lumenal position together with known sites of N-linked glycosylation. This orientation places the ER retention motif KKXX correctly on the cytosolic domain and the CD8 epitope in the lumen. [0019] FIG. 3 shows the spontaneous T-cell proliferative responses to IGRP in the NOD mouse. Lymphocytes prepared from pancreatic draining lymph nodes were mixed with splenocytes from the same animal and incubated for 3 days (500.times.10.sup.3 cells) in Click's medium with 50 .mu.M NOD mouse serum 50 .mu.M mercaptoethanol and the indicated recombinant antigens that were generated as fusion proteins with .beta.-galactosidase in the pUEX vector. Cultures were pulsed for 16 h with .sup.3H thymidine prior to harvest. [0020] FIG. 4 shows an analysis of pancreatic mononuclear cells using MHC Class I tetramers. Cells were isolated from collagenase digested mouse pancreas by gradient centrifugation and subject to 3 color FACS analysis using antibodies to CD45, CD8 and PE-streptavidin conjugated tetramers carrying peptides derived from IGRP. The gated (R1) population were 95% CD45 positive and composed of 12.5% CD8 cells. Of these, a significant proportion bound tetramer bearing the cognate peptide of the NY8.3 clone (Pep C). A control construct with mismatched class I (Pep A) and a second IGRP K.sup.d binding peptide (Pep B) gave only background staining. Continue reading... Full patent description for Targeted drug-formaldehyde conjugates and methods of making and using the same Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Targeted drug-formaldehyde conjugates and methods of making and using the same patent application. 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