JPH09227408A - Treating composition for insulin-dependent diabetes mellitus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject medicine, comprising a glutamate decarboxylase protein or peptide and effective in treating or preventing insulin-dependent diabetes mellitus. SOLUTION: This medicine comprises a glutamate decarboxylase protein or peptide and preferably further an immunological activating fragment. The fragment contains an amino acid sequence Pro-Glu-Val-Lys. For example, the protein has the homology with a pancreatic 64 K protein in sequence and the 64 K protein has immunological reactivities with the glutamate decarboxylase enzyme. An islet isolated from the human pancreas is preferably used as a material.

Description

Detailed Description of the Invention

[0001]

[Industrial application] Diabetes has become a major health problem for people. Six million people in the United States are known to have diabetes, and five million more diagnosed Over 500,000 new diabetics are found each year with this unmet disease. In 1984, diabetes was a direct cause of death in 35,000 Americans, and contributed to a further 95,000 deaths.

Eye complications of diabetes are the leading cause of new blind, legally defined patients in the United States in people aged 20-74. The risk of lower limb amputation is 15 times greater in individuals with diabetes than in individuals without diabetes. This assessment is performed annually in the United States by people with diabetes and is approximately 12
It suggests that they experience more than half of the amputations of 5,000 feet.

Kidney disease is a frequent complication of severe diabetes. Approximately 30% of all new patients in the United States treated for end-stage renal disease have diabetes. This percentage is increasing at a rate of approximately 2% each year, so that within the next decade, kidney disease associated with diabetes will exceed half of all enrollees in end-stage renal disease programs. Will be

Individuals with diabetes are also at increased risk for periodontal disease. Periodontal infection progresses rapidly, leading to compromised metabolic inhibition as well as tooth loss.
Women with diabetes are at risk for serious complications of pregnancy. Current statistics suggest that maternal fetuses with diabetes have a mortality rate of approximately 7%.

The economic burden on diabetes is also great. Every year, patients with diabetes or patients with its complications run out of 24 million patients-days in hospitals. A conservative estimate of the total annual cost attributable to diabetes is at least $ 24 billion (American Diabetes Association rating, 198
8) and the overall economic impact of this disease is greater because the cost of additional healthcare is often more specific to certain complications of diabetes than to diabetes itself. Diabetes is a chronic, complex metabolic disease in which the body is unable to properly maintain and use carbohydrates, fats and proteins. Diabetes results from the interaction of various genetic and environmental factors and is characterized by high blood sugar levels resulting from a failure to produce insulin or impaired utilization thereof. Many cases of diabetes fall into two clinical types. Insulin-dependent diabetes mellitus (IDDM or IDD) and non-insulin-dependent diabetes mellitus (NIDDM or NIDD). Each type has a different prognosis, treatment and cause.

Approximately 5-10% of diabetics have IDD, previously referred to as juvenile diabetes because it appeared early in life, usually in childhood or adolescence. IDD is characterized by the inability to make insulin partially or completely. Patients with IDD die without daily insulin injections to control their illness.

[0007]

2. Description of the Related Art Elucidation of the etiology of diabetes made little progress until the mid-1970s, but at that time ID
Evidence began to accumulate, suggesting that D has an autoimmune etiology. It is now generally accepted that IDD results from the chronic autoimmune destruction of the insulin-producing pancreatic β-cells. Lymphocytes and other inflammatory cells are observed within the islets of Langerhans in newly diagnosed IDD patients and are regenerating (Langerhans) composed of β cells more than in other cell forms.
More found on the island. This active immune process is associated with various autoantibodies to the cytoplasmic and membrane antigens of β-cells, insulin and insulin receptors.

[0008] Therefore, IDD is a disease filled with autoantibodies. These include cytosolic islet cell autoantibodies (ICA) (Bottazo, GF, A. florin-Christensen (Flo).
rin-Cristensen), and D. Doniach [1974]
Lance.2: 1279-1283), islet cell surface autoantibodies (ICSA) (Maclaren, N., SW Huang, and J. Fogh [1975] Lancet.i: 997-1000). , Insulin autoantibody (IAA) (Palmer, JP, CM aspurine (Asp
lin), and P. Cleamons [1983] Science 222:
1337-1339) and possible anti-genotype insulin receptor-autoantibodies (Ins.RA) (Maron, R., D. Elias, M. de J. Bartelt, GJ Bruining, JJ Van Rood, Y.
Shechter, and IR Cohen [198
3] Nature 303: 817-818). 6 in 1982
It was reported that antibodies against 4,000M _r islet cell antigen were detected in IDD patients (Bekkeskov, S. et al. [1
982] Nature 298: 167-169). In a later publication, Beckeskov et al. Reported that the 64,000 M _r antibody was associated with IDD in the BB rat model (Bkkeskov, S.
[1984] Science 224: 1348-1350). Patients associated with individuals with IDD, and therefore known to be at risk of developing disease, were subsequently investigated by follow-up studies
(Bekkeskov, S. et al. [1987] J. Clin. Inves
t. 79: 926-934). Results of subsequent studies suggested that the 64,000 M _r antibody may be present in some IDD patients prior to the onset of clinical symptoms. However, in the same study, Beckeskov et al. Found that the function of the 64,000 M _r protein was unknown and that this data could not be concluded as to whether the 64,000 M _r antibody was present before the onset of decreased β-cell function. Reporting.

It has been reported that other antibodies against various non-β-cell specific molecules are present in IDD patients in increasing numbers. These include antibodies to tubulin, single and double stranded DNA, gastric parietal cells, intrinsic factor adrenal cortex cells, thyroid peroxidase enzyme and tiroglobulin. IDD patients have evidence of polyclonal activation of B-lymphocytes, which may be the result of increased antibody titers against various antigens.

[0010]

A further decade of research has shown that patients with IDD have a genetic marker called histocompatibility antigen that is associated with susceptibility to IDD. It shows that it is doing. Since these genetically susceptible markers are necessary but not sufficient for the development of IDD, some additional environmental factors, as yet unknown, may trigger the initiation of β-cell destruction and the development of diabetes. Seems necessary. Environmental factors, whether viral or chemical agents, initiate an immune response against β-cells, allowing the immune destruction of β-cells in genetically susceptible individuals (Atkinson, MA, N.
K. Maclaren. [1990] Scientific Amereica
n 7: 62-67). Therefore, finding diabetic patients in a pre-diabetic condition is essential in efforts to prevent the occurrence of this disease. Perhaps the single most important advance in diabetes research over the past two decades is the recognition that autoimmune destruction of beta cells can take months and years to complete. Currently, clinical diagnosis of diabetes is rarely made until the destruction process is almost complete, and until insulin injections are needed to prevent death.
Interfering with insulin-producing cells before they are irreversibly destroyed can provide a strategy to prevent the progression of diabetes and its complications.

Therefore, IDD before the disease progresses to the clinical stage
It is essential to find a means to accurately predict the onset of.

Although many biological markers are associated with IDD, to date, none of these markers have been uniformly present before the onset of clinical signs of disease. Not shown (Maclaren, NK [1988] Diabetes 3
7: 1591-1594). Such an early presence is essential for a compound to qualify as a useful predictive test of the disease. However, the initial presence alone is not sufficient for an accurate early detection method. Predictive tests based on the presence or absence of a particular marker are only valuable if the predictive marker is present only in those who will have the IDD and in others who do not. . Because the treatment of diabetes and the psychological impact of the disease can have a significant effect on the health of the person being diagnosed, it is important to develop a predictive test that is specific and therefore very unlikely to be falsely positive. is important.

It is also important to find a predictive test that can recognize the onset of the disease years before clinical symptoms appear. Early detection provides an opportunity to take action that can proactively or prevent serious health problems associated with clinical stage IDD. For example cyclosporin A,
Nonspecific immunosuppressive tests with drugs such as azathioprine and steroids have been performed in newly diagnosed patients. Although a limited number of disease remissions were gained over the first year of their use, these patients never returned to normal because beta cells had limited regenerative capacity and were Treatment that begins at times is often almost too late.

To date, no suitable predictive test for IDD has been found that meets these requirements. Although many autoantibodies are associated with the disease, studies that identify evenly predictive autoantibodies have not been successful. For example, 1976
Years ago, autoantibodies (ACA) that react with the cytoplasm of glucagon-secreting (α) cells in islet cells were described (Bottazo (Botta).
zo), GF, and R. Lendrum [1976] Lancet
2: 873-876). ACA may be a predictive marker for deficiency of the islet cell hormone glucagon, as autoantibodies to endocrine glands have been found to occur prior to or with the onset of clinical disease. So I thought. Unfortunately, later work revealed that ACA was not associated with defective alpha cell function, not associated with IDD, or with any identifiable pancreatic pathology (Winter). , WE, NK Maclaren, WJ
Riley, RH Unger, M. Neufold
(Neufeld), and PT Ozand (1984) Diabetes
33 (5): 435-437).

Studies into the predictive value of other antibodies associated with the clinical stage of the pancreas and IDD have created a tool for early detection of this disease. For example, both insulin autoantibodies (IAA) and islet cell autoantibodies (ICA) have been found to be present in many newly diagnosed patients. ICA has been shown to exist before the clinical stage of IDD, and has been found particularly in nondiabetic relatives at risk for IDD. But,
Neither ICA nor IAA provide predictive tests that confer almost absolute specificity and sensitivity to IDD.

Thus, it has been felt that there is a substantial and long-felt need for more accurate means of detecting and treating IDD in the early stages, before the onset of clinical symptoms and before the need for insulin therapy.

[0017]

The invention described in this specification is
Insulin-dependent diabetes mellitus (IDD) is accurately detected at an early stage, more specifically, IDD is detected before a significant loss of β-cell function, and it relates to a means for treating IDD at an early stage.

More specifically, it was found that autoantibodies against the islet cell 64,000 M _r protein existed several years before the clinical appearance of IDD was observed. This antibody is 64
Named KA. The early detection and treatment of this potentially debilitating disease facilitates ineffective interfering therapies at later stages of the disease, where irreversible destruction spreads.

64KA can be used alone to detect and predict the onset of IDD, or it can be used in combination with other autoantibodies such as ICA and IAA known to be associated with IDD. 64KA described herein is highly disease-specific for IDD. 64KA has been found to be present uniformly in children and young adults tested months and years prior to the commencement of IDD. The 64KA trial, despite the lack of other antibodies associated with this disease,
It was found to predict the onset of IDD.

It has also been found that protein antigens reactive with 64KA are useful in the treatment of IDD. ID as part of the invention
Described herein are novel means of treating D or delaying the onset of IDD. A novel means of treatment is to construct a novel hybrid β-cell 64K protein, that is, to construct a toxin product that can deprive immunological mediators involved in the pathogenesis of IDD .

Yet another aspect of the present invention is the imminence of islet cell transplantation of the pancreas by minimizing the immune-related destruction and rejection of the new pancreas by administering a 64K hybrid toxin molecule. The use of 64KA to diagnose and prevent destruction. The invention further relates to the use of anti-genotype antibodies against 64KA as a means of interference therapy.

The present invention further relates to the discovery that the pancreatic 64K protein has sequence homology with the glutamate decarboxylase (GAD) protein. Furthermore, 64K protein is GAD
It has immunological reactivity with antibodies to the enzyme. Thus, yet another element of the present invention relates to the use of GAD proteins or immunologically active fragments thereof for the diagnosis, prevention and treatment of IDD.

DETAILED DESCRIPTION OF THE INVENTION The invention described herein relates to agents for treating IDD discovered using 64K autoantibodies as an accurate and specific early indicator of the onset of IDD. The discovery of 64KA as a useful predictive marker for IDD stems from exhaustive biochemical studies focused on a large number of compounds associated with IDD. The high specificity of 64KA for IDD, as well as the frequent presence of 64KA in humans many years before the onset of IDD, is both unexpected. these
The nature of 64KA makes it useful as a means of early detection of IDD.

In order to ascertain whether certain substances are valid predictors of the onset of IDD, it is necessary to determine if these substances are present prior to the onset of clinical symptoms of diabetes. During this prediabetes period, circulating autoantibodies must be screened to identify susceptible individuals for many high-risk relatives of the affected proband, It is difficult to study because long-term observations are needed to document the natural history of β-cell failure. Therefore, animal models were used to increase and further confirm the data obtained for human testing.

Non-obese diabetic (NOD) mice are human IDD
Is a useful animal model for. Studies of NOD mice provide important insights into the sequence of pathogenic events and lead to an understanding of the nature of target island autoantigens involved in the autoimmune process. Another important model for IDD is the biobleeding (BB) rat. The present invention is
Discovered as a result of studies and tests involving BB rats and NOD mice.

It has been discovered that islet cell autoimmunity is associated with autoantibodies to β-cell proteins of relative molecular weight (M _r ) 64,000 (64K) in human and BB rat models. It was also determined that sera from newly diagnosed NOD mice also contained autoantibodies that immunoprecipitate the 64K antigen from detergent lysates of ³⁵ S-methionine labeled mouse islet cells (Atkins ( Atokinson, M., and NK McLaren (Mac
laren) [1988] Diabetes 38: 1587-1590). This autoantibody was detectable by the time of weaning in NOD mice, which disappeared within a few weeks of the onset of diabetes, with older non-diabetic mice and all three non-diabetic constitutional control lines tested. Did not exist in. In humans, 64KA was present prior to clinical diagnosis in 96% of those with a later onset of IDD below 35 years of age.
64KA was found to exist predictably well before the clinical appearance of IDD. The presence of 64KA was also found to be highly specific for IDD. 64 different from IAA or ICA
KA was not observed in individuals without a high or increased risk of developing IDD.

Materials and Methods Islet Cell Preparation Human pancreatic islets were isolated from cadaver pancreas as previously reported (Diabetes 37: 413-420, 1988). All islets in vitro in vitro in supplemented medium CMRL1066 (in vitro)
Held in. Average per batch 84,946 ± 11,061 (x ± SEM, n =
12) isolated islands of humans obtained, average island purity 91.2 ±
The insulin content per island was 2.3% and 0.4 ± 0.1 mU insulin. 4.68 ± 0.6 by Krebs (KREBS) perfusion test at alternating glucose concentrations of 60 mg / dl-300 mg / dl
A mean insulin stimulation index of 6 (G300 / G60) was given. All batches were labeled within 7 days.

Metabolic labeling of islet cells Following in vitro culture for a minimum of 36 hours (6000 islets / 50 ml)
16 mM glucose, 20 mM HEPES, 100 u in RPMI 1640 medium
U / ml penicillin, 100 μg / ml streptomycin, 2% (v /
v) supplemented with normal human serum, 37 ° C, 95% air / 5% C
O ₂ ), these islets were replanted and incubated in supplemented RPMI1640 medium lacking methionine (1000 islets / ml) for 15 minutes. The cells were then (95% air / 5%) in methionine-free medium (1000 islands / ml) supplemented with ³⁵ S methionine (> 500 Ci / mmol) at a concentration of 0.5 mCi / 1x10 ³ islet cells. % CO ₂ ) It was cultured for 5 hours. Methionine-containing (0.5 mM) RPM supplemented with cells
Cultivated with I1640 medium (1000 islands / ml) for 30 minutes, twice (200xg, 5 minutes) 20 mM Tris (pH7.4), 150 mM Nacl, 100 KIE / ml aprotinin and 2 mM phenylmethylsulfonylfluoride (PM
SF) containing buffer and then snap frozen until extraction of those detergents
(-80 ° C).

Immunoprecipitation of 64K autoantigens Islet cells were lysed in the above buffer containing 2% TRITON ^™ X-114. Unnecessary substances were removed by ultracentrifugation (1
(00,000xg, 30 minutes, 4 ° C). The lysate was then separated into an aqueous phase, a sucrose phase and a detergent phase (Bordia (Bordia) prior to immunoprecipitation.
ier), C. [1981] J. Biol. Chem 256: 1064-1067). The lysate was treated with normal control human serum (10 μl per 100 μl supernatant, 1 h, 4
C) and then Protein A Sepharose C
L-4B was adsorbed in excess (100 μl of swollen protein A sepharose used for 25 μl of serum). An aliquot of unbound (precleared) lysate (100 μl containing 5-10 × 10 ⁶ cpm) was incubated with 25 μl of test serum (either IDD or control) for 18 hours (4 ° C.). Detergent extracts from 1000 islets for each serum sample tested were used. Pre-swollen Protein A Sepharose CL-4B was added to each assay tube and the reaction mixture was incubated for 45 minutes (4
° C). Protein A cephalometric - scan CL-4B was washed 5 times by centrifugation at then 0.5% Triton ^TM X-114 buffer (200 × g, 10 seconds), ice cold double distilled water (200 × g, 30 seconds) in In 1
Washed once. Bound protein was eluted and 80 mM Tris (p
H6.8), 3.0% (w / v) sodium dodecyl sulfate (SDS), 15% (v
/ v) sucrose, 0.001% (v / v) bromphenyl blue, and 5.0% (v / v) β-mercaptoethanol in a sample buffer for 3 minutes. Denatured by. The Sepharose beads were removed by centrifugation (200 x
g, 1 min), and the supernatant was electrophoresed through a discontinuous SDS 10% polyacrylamide separation gel, followed by Coomassie
Brilliant Bull-Staining and Enhance (New England Nuclear-Massachusetts,
Fluorography was performed using Boston.

ICA analysis (Neufeld, MN Maclaren,
W. Riley, D. Lezotte, H. McLaughlin, J. Silverstein, and A. Rosenbroom [1980] Diabetes 2
9: 589-592) and ICA was assayed by the indirect fluorescent antibody method on blood group O type cryocut pancreas sections. All results were read on the coded samples and control negative and positive sera were used in each batch. Serum was considered positive when fluorescent antibodies of the tissue were observed in the islets of the pancreas. Positive results are from the Immunology of Diabetes Workshops
Defined by (IDW), Noel K. McLaren (M
10 Juvenile Diabetes Fund (J) standardized by the global ICA Expert Examination Program managed by
It is positive when the concentration (or titer) of DF) is exceeded by the autoantibodies.

Insulin Autoantibody Binding Procedure The RIA method used to detect IAA was modified to maximize sensitivity without loss of specificity for IDD. Mean serum insulin binding has no family history of IDD9
Calculated from sera obtained from four non-diabetic Caucasian children and laboratory personnel (mean age 13.7 ± 7.7 years, 51 men / 43 women). None of these control individuals were positive for ICA. At the position of A14 at a concentration of 0.15 ng / ml
Optimal results have been found using monoiodinated human insulin radiolabeled with ¹²⁵ I. More recently, improved specificity of the assay could be achieved by measuring specific binding, which can be displaced through the addition of excess unlabeled insulin.

IVGTT Procedure To investigate the insulin response to glucose stimulation, an intravenous glucose tolerance test (IVGTT) was performed.
(Srikanta, S., OP Ganda, GS
Eisenbarth, and JS Soerdona (S
oeldner) [1983] N. Engl. J. Med. 308: 322-325), the amount of glucose administered is 0.5 g / Kg given exactly intravenously over 2-4 minutes. After glucose injection of serum sample-1
Collected at 0, 1, 3, 5, 10, 15, 30, and 60 minutes. Insulin deficiency was compared to a control population of non-IDD individuals with ICA-negative insulin levels at 1 and 3 minutes
Defined as being the 3rd percentile (n = 15
0, age 20.3 ± 10.2 years, range 2 to 59 years). 70 for this analysis
Insulin levels below μIU / ml (<3rd percentile vs. 150 controls)
c) thought.

HLA DR Blood Grouping Method The HLA DR blood grouping method is Van Rood and Van Leuwen (Van Rood, JJ, A.
Van Leuwen, and JSPloem [1976] Nat
ure 262: 795-797). Standard control antisera was used for blood typing.

Construction of human island cDNA library A cDNA library for human island cells was constructed in Lambda gt11 vector (Young, RA, and Davis (Dav).
is), RW [1983] Proc.Natl.Acad.Sci. USA 80: 1194-11
98). Messenger RNA was isolated and analyzed by Chirgwin.
isolated and purified from approximately 200,000 purified human islets according to a modification of the method of Win et al. (Chirgwin,
JM, AE Przybyla, RJ McDonald's (M
acDonald), WJ Ruta (Rutter) [1979] Biochemistry
18: 5294-5299). Island with 1M 2-mercaptoethanol and 0.
Dissolved in 4M guanidine thiocyanate containing 1M Tris-HCL (pH 7.4). The lysate was layered on a 5.7M cushion of CsCl and 3 hours in a Beckman SW50.1 rotor for 17 hours.
It was centrifuged at 7,000 rpm. The RNA pellet was removed and resuspended in sterile water, followed by precipitation with 3 volumes of anhydrous ethanol. This final step was repeated 3 times to ensure that a clean preparation of RNA was obtained. Human islet total RNA was used with the cDNA for the library as a template, and the enzyme was present in the presence of human placental ribonuclease inhibitor, oligo (dT) primer, and four deoxyribonucleotide triphosphates. Reverse transcriptor
It was synthesized using ze (reverse transcriptase). The mixture at 40 degrees
It was cultured for 2 hours. The second strand of the cDNA was synthesized using the reaction products of the first strand and the enzymes E. coli DNA polymerase I and E. coli ribonuclease H. The mixture was sequentially incubated at 12 ° C. for 60 minutes, then 22 ° C. for 60 minutes, followed by heat inactivation at 65 ° C. for 10 minutes. Mix the mixture to ensure that the ends of the cDNA are flash ends.
Incubated with T4 DNA polymerase at 10 ° C for 10 minutes. This mixture of double-stranded cDNA (ds-cDNA) was then purified using extraction with an equal volume of buffer saturated phenol / chloroform followed by extraction with an equal volume of chloroform.
Unincorporated nucleotides were removed by the addition of an equal volume of 4M ammonium acetate followed by the addition of 2 volumes of anhydrous ethanol. After cooling on dry ice for 15 minutes, the mixture was centrifuged at 12,000 rpm in a microcentrifuge for 10 minutes. This precipitation step was repeated twice to ensure complete removal of unincorporated nucleotides.

Enzyme E in the presence of s-adenosyl-methionine
The ds-cDNA was prepared for cloning into the vector by a series of modification steps, starting with incubation with coRI methylase for 1 hour at 37 ° C. This methylated c
Synthesize DNA overnight at 15 ℃ in the presence of T4 DNA ligase and ATP
It was ligated to the EcoRI linker. The cohesive EcoRI ends were generated on the linkered cDNA by digestion with EcoRI endonuclease at 37 ° C for 5 hours. Following this step, excess linker was removed from the mixture by column chromatography. The material eluted in the void volume (space volume) of this column represents a ds-cDNA that has been methylated and linked to an EcoRI linker and dimensioned.

This material was ligated into the lambda gt11 vector digested with EcoRI and treated with alkaline phosphatase. Ligation was performed in the presence of T4-DNA ligase and ATP 15
It proceeded at ℃ overnight. Each ligation mixture was packaged into the phage lambda coat protein using a commercially available extract (Strategy). The level of packaged phage was determined by titration on the E. coli host strain Y1088. The complexity of the library was determined to be approximately 2.0 × 10 ⁶ pfu and the bromo-chloro-indolyl-galactoside (BCIG)
81% of them contained the inserted cDNA, as detected by the absence of color on the indicator. Recombinant library is screened with an oligonucleotide probe
It was amplified prior to training.

The following describes the procedure, including the best mode, for carrying out the present invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise stated.

Statistical analysis includes both chi-square analysis after the Yate correction factor is applied and Fisher's exact test (Snedecor).
(Snedecor), TN and WG Cochran [1967] S
tatistical Methods. Iowa State University Press, Ames, Iowa).

Examples 1-64KA Association with IDD Five groups of people were selected for the 64KA study. There were 31 newly diagnosed patients with IDD defined according to the established National Diabetes Data Group (NDDG) criteria (National Diabetes Data Group [1979] Classification and Diagnosis and Diabetes of Diabetes Mellitus). -Other categories of glucose tolerance-, "Diabetes 28: 1039
-1057). 26 non-diabetic control persons who also lack ICA and have no known family history of autoimmune disease;
Nondiabetic ICA and IAA in the newly diagnosed patient
40 relatives lacking illness; 36 nondiabetic but IC
A-positive people, 28 of whom are first-degree relatives of a proband with IDD, 4 are clearly healthy normal controls, and 4 have autoimmune Addison's disease or thyroiditis Patients; five unaffected relatives with IDD, ICA-negative but high-titer IAA-positive children; and sera before their documented initiation of IDD Twenty-eight additional people gathered in were also tested.

The above patients were from two future ICA screening population studies of more than 5,000 first-degree relatives of probands with IDD, and 8200 normal control individuals. Approximately 50 of the normal control people obtained through
00 were school students. All sera were stored at 20 ° C for 1 week to 7 years prior to autoantibody analysis.

64KA was extremely ill in this disease in that it was present in 84% of newly diagnosed IDD patients and was completely absent from the population of subjects tested (p <0.0001). It turned out to be specific.

Example 2-64KA as an Indicator of IDD To analyze the ability of 64KA to actually predict the onset of IDD, serum samples from 28 individuals were analyzed for 64KA.
Serum samples were collected prior to the onset of any clinical appearance of IDD. At later points in their lives, each of the 28 individuals developed clinical symptoms of IDD. These 28
Twenty-five of those had clinical symptoms before age 35.

It was found that in 24 out of 25 (96%) people under the age of 35 at the onset of clinical IDD, 64KA was present. Tested serum samples from these individuals have been taken somewhere between 3 months and 7 years prior to the clinical onset of IDD (27 months on average). The presence of 64KA occurs 7 years before the onset of clinical symptoms.

The same serum samples were tested for the presence of ICA and IAA. ICA was present in 22 of 28 samples (78%) compared to 64KA, which is present in 82% of all cases, and
Was present in only 13 of the 27 samples (48%). These results are shown in Tables 1-4. Clearly ID
The predictive value of 64KA for D is the best of all three autoantibodies.

In another analysis of 9 IDD patients with disease onset, retention of response SUSTACAL ^™ versus loss of conserved islet cell insulin showed a better correlation with 64KA than ICA. Therefore, 64KA also provides an indication of the amount of β cells remaining.

Table 1 64,000 M _r autoantibody frequency group n 64KA ICA control 26 0 (0) 0 (0) newly diagnosed IDD 31 26 (84) 25 (81) ICA- / IAA-relatives 40 1 (2 ) 0 (0) ICA + Relatives 28 23 (82) 28 (100) ICA + Others 8 7 (87) 8 (100) ICA- / IAA + <5 years old 5 4 (80) 0 (0) (ICA ... Island cell autoantibodies, IAA ... Insulin autoantibodies)

Table 2 Summary of discovery of non-diabetic patients at risk of IDD due to positive ICA IDD-related autoantibodies Plasma insulin IVGTT Age of age HLA-DR confirmed ICA ^* 64K IAA (1 + 3 min ( percentile )) M 6 3,-IDD family survey 80 +-144 (40) M 9 3,1 IDD family survey 40 +-57 (<1) F 11 3,4 IDD family survey 80 + + ND M 11 3,4 IDD family survey 80 + + 105 (20) F 12 4,3 IDD Family Survey 40 +-194 (60) F 13 2,4 IDD Family Survey 40 + + 62 (<3) M 17 3,4 IDD Family Survey 20 +-67 ( <3) M 20 2,4 IDD Family Survey 40 + + 58 (<1) F 29 3,4 IDD Family Survey 40 +-57 (<1) F 30 3,-IDD Family Survey 160 +-82 (10) F 33 3,8 IDD Family Survey 20 +-125 (30) F 34 3,4 IDD Family Survey 40 +-167 (50) M 44 4,1 IDD Family Survey 20--ND F 10 4,8 IDD Family Survey 360 +-98 (20) F 38 4,-IDD Family Survey 20 + + 71 (5) M 7 4,8 IDD Family Survey 40 + + 114 (25) M 7 3,4 IDD Family Survey 20 + + 116 ( 25) M 10 4,-IDD Family Survey 160 + + 52 (<1) F 10 5.9 IDD Family Survey 20 + + 128 (30) F 43 3,1 IDD Family Survey 80--293 (70) F 13 1, 3 IDD Tribe Survey 80--100 (20) M 5 2,4 IDD Family Survey 40 +-125 (30) F 39 4,-IDD Family Survey 80 + + 112 (25) M 11 3,4 IDD Family Survey 40 + + 56 (<1) F 35 4,7 IDD Family Survey 40 +-420 (90) M 43 3,7 IDD Family Survey 160--ND F 38 3,4 IDD Family Survey 20--184 (60) F 26 ND IDD Family Survey 40 + + ND M 8 3,4 School Survey 160 + + 67 (<3) M 10 3,4 School Survey 160--268 (75) F 13 4,6 School Survey 40 +-67 (<3 ) M 11 3,4 School survey 320 + + 124 (30) M 16 3,4 Addison's disease / thyroiditis 160 +-ND M 32 3,4 Addison's disease / thyroiditis 80 +-197 (60) F 41 3, 4 Addison's disease / thyroiditis 320 + + 69 (<3) M 12 3,4 Addison's disease / thyroiditis 160 +-149 (40) ^* JDF units

[0048] Table 3 IDD occurs before the test has been Mato Me IDD-related that was seen in the first serum sample that can be obtained from a patient self-antibody HLA-DR start age verification diabetes before period (month) ICA ^* 64K IAA M 3,3 5 IDD Family Survey 9-+-M 4,6 10 IDD Family Survey 34 20 + + F 4,7 11 IDD Family Survey 34 40 + + M 3,3 12 IDD Family Survey 8 40 +-F 4, 6 15 IDD Family Survey 3 80 +-M 3,4 15 IDD Family Survey 19 80 + + M 3,4 17 IDD Family Survey 14-+-M 3,4 19 IDD Family Survey 3 40 +-M ND 21 IDD Family Survey Survey 32 80 +-F 4,-33 IDD Family Survey 14 10 + + F 4,5 38 IDD Family Survey 2 80--M 3,4 42 IDD Family Survey 42 80 + + M 4,6 5 IDD Family Survey 30 - ^** - ^** + M 3,4 15 IDD Family Survey 6-+ + M 3,4 30 IDD Family Survey 13 160 + ND M ND 4 IDD Family Survey 3 20--M 6,1 33 IDD Family Survey 14 - ^** +-F 4,6 37 IDD Family survey 3 80--F 3,4 7 Glucose intolerance 1 + (NT) + + M 4,4 15 Glucose intolerance 2 80 + + F 3,6 17 Glucose Intolerance 2 20 +-M 2,3 18 Glucose intolerance 12 40 +-M ND 27 Glucose intolerance 6 40 + + F 2,3 40 Glucose intolerance 19 + (NT)--F 1,4 9 School survey 3 40 + + F 3,4 12 School survey 44 320 + + F 3,3 24 Grés-Veise disease 72- ^** +-F 1,4 14 Thyroiditis 75 + (NT) + + 22/28 23/28 13/27 (78%) (82 %) (48%) NT = JDF titer not measured ND = not measured ^* = JDF unit ^** = converted to autoantibody positive in later sample data

Table 4 Discovery of non-diabetic patients at risk of IDD due to positive IAA Summary IDD-related autoantibodies Plasma insulin IVGTT Age of age HLA-DR confirmed ICA ^* 64K IAA (1 + 3 min ( percentile )) F 2 4,5 IDD Family Study-+ + 129 (40) M 3 2,1 IDD Family Study-+ + 27 (<1) F 5 ND IDD Family Study-+ + ND M 0.5 1,6 IDD Family Study-+ + 342 F 3 ND IDD Family Survey--+ ND

Example 3-64KA and IDD in NOD mice
Related Two recently two types of animals (BB rat and NOD mouse) have been developed that can serve as models for the study of human IDD. Using animal models to assess as an indicator of IDD is the number of high-risk relatives of ill probands who must screen for circulating autoantibodies to find susceptible individuals. Because of their prevalence, the pre-diabetes period is invaluable because it is difficult to study and because of the long-term observations that need to document the natural history of β-cell failure in the disease. NOD mouse and B
B rats provide important insights into the etiological sequence of events involved in human IDD. Analysis of these animals also leads to an understanding of the nature of target islet cell autoantigens involved in the autoimmune process. We therefore IDD NOD mice and BB rats
Were tested for their possible 64K autoantibodies both before and after the onset of.

Male and female NODs and subjects of various ages (BALB
Serum was obtained from mice of the / c, C57BL / 6, C2H / Hej) strain. NOD
The diagnosis of IDD in mice was characterized by their throat paw and weight loss and persistence of hyperglycemia above 240 mg / dl. Daily for all diabetic animals to maintain health
Received daily insulin doses of 0.5-2.0 units of protamine-zinc insulin (Eli Lilly, Indianapolis, Ind.).

Brunnstedt (Brunstedt
ndstedt), J., Nielsen, JH, Lanmark
(Lernmark), A. and Hagedorn Study Group, Methods in Diabetes Research, J. Lana.
r), SL edition (Pohl) [John Willy & Sons (John
Willey & Sons), New York, 1987]) and isolated BALB / c islets and labeled with ³⁵ S methionine. RPMI 1640 (GIBCO, New York) medium supplemented with islet cells
The islet cells were washed twice (4 ° C.) in (2.0% fetal bovine serum, 100 units / ml penicillin, 100 μg / ml streptomycin), followed by 20 mM Tris (pH 7.4), 150 mM NaCl, 1000 KIE / ml tray scyllo-. And washed once in a buffer containing 2 mM PMSF. The cells were then placed in 1% Nonidet P-40 (NP-4
0) (v / v) in the above buffer. Unnecessary substances were removed by ultracentrifugation (100,000 xg, 30 minutes). The lysate was incubated with normal mouse serum (Balb / c) (10 μl per 100 μl supernatant, 1 hour, 4 ° C.) and subsequently adsorbed to excess protein A Sepharose CL-4B. This culture / adsorption procedure was repeated 3 times. Aliquots (100 μl) of unbound (precleared) lysate were incubated for 18 hours (4 ° C.) with either 25 μl of NOD serum or control serum. For each test tube
100 μl of preswollen protein A Sepharose CL-4B was added and the reaction mixture was incubated for 45 minutes (4 ° C.). Protein A Sepharose CL-4B was then washed 5 times by centrifugation in 0.5% NP-40 buffer (200xg, 10 seconds) and once in cold water (200xg, 30 seconds). The bound immune complex was 80 mM Tris (pH 6.8), 3.0% (w / v) sodium dodecyl sulfate (SDS), 15% sucrose, 0.001 bromophenyl blue and
Denaturation was achieved by boiling for 5 minutes in sample buffer containing (5.0% v / v) β-mercaptoethanol. The beads of cepharose were removed by centrifugation (200 xg, 1
Min), and the supernatant was electrophoresed through a SDS 12% polyacrylamide separation gel, followed by Coomassie staining and oat.
Tradiography was performed.

The first test was to see if 64KA occurred in NOD mice. Serum was newly diagnosed
8 female NOD mice with IDD (mean age at onset 104.5 ± 13.5 days, range 93-134 days) and BALB / c (n = 5), C
57 BL / 6 (n = 4), C3H / HeJ (n = 2) strains 11 healthy control mice (2 males and 9 females, mean age 97.7 ± 30.4 days, range 50-120 days) Serum was obtained from.

At the onset of their diabetes, 64KA was NO
It was detected in 87% of D but 64KA was not detected in any of the non-diabetic control lines (P <0.001). 64K at high percent in NOD mice with newly diagnosed IDD
The presence of autoantibodies against, combined with the absence of 64KA in age-matched non-diabetic control mice, indicates that 64KA is specific for IDD and its presymptomatic status. means. The presence of 64KA is also one of the first detectable events and is generally present at or even before weaning, the earliest age at which insulitis is found.

Example 4-As an index of IDD in NOD mice
To find out about the natural history of 64K autoantibodies for 64K AIDD of 5 mice (2 males and 3 females at weaning period,
Serum samples obtained from ages 23-25 days) were analyzed and their pancreas histologically examined for insulin. Insulin was detected as a clear intraislet infiltration of lymphocytes on hematoxylon- and eosin-stained pancreatic sections containing 5 or more islets. 64KA was already present in 80% (4/5) of these mice. At the same time, early-life insurance was also present in three of the four mice whose pancreatic tissues were analyzed. Two animals (both males) tested at ages of 190 days and above that did not produce any IDD symmetrically (both males) did not have 64KA, although both had insulinis.

Example 5-64 KA in BB rats In a study of IDD found in BB rats (n = 8), autoantibodies to the 64K protein appeared early in the natural history of disease,
Significant specificity is shown as the marker for IDD (not present in the control of 6 Lewis rats). Person, NOD
Mouse, and the discovery of 64KA in BB rats, M _r 64,000
Autoimmunity to islet cell proteins is of considerable etiological importance, suggesting that the 64K protein may have important physiological functions in normal β cells.

[0057] Example regard to biochemical characterization biochemical characterization of 6-64K antigen, higher resolution 64K protein gel, which has two components (63,500 and 64,000 M _r)
Suggesting that they can consist of doublets that differ by about 0.5 kilodaltons between. The 64K component migrates to the M _r 64,000 range under reducing conditions and can be found as a dimer (approximately 130K) under non-reducing conditions. 64
The K protein exhibits charge heterogeneity in that it has isoelectric points of 6.4 and 6.7. The 64K component most likely represents an integral islet cell membrane protein due to the hydrophobicity observed in the immunoprecipitation of the detergent phase extract. "64K antigen" used in this patent application
The term refers to a compound, compounds or complexes that behaves on a gel in the manner described herein. The 64K antigen can also be identified by immunoreactivity with the anti-GAD antibodies described below.

[0058] We have also, human islet cell protein has been detergent extracted from the cytoplasm Imunopureshipite by serum from people with IDD - distribution (immunoprecipitation) is often also found to result in the identification of M _r 67,000 protein. We are students of US schools and
Antibodies to this 67,000 M _r antigen were analyzed in a relative population of probands with IDD. Autoantibodies to the 67,000 M _r protein were detected in 60% (12/2) of newly diagnosed IDD patients.
Was observed in 0) and only in 1 of 18 controls (5% P <0.001). 67,000 in this IDD population
Autoantibodies has been observed in humans is a positive for often (73% 11/15) 64KA for the M _r, people of 67,000M _r autoantibody-positive is only found only one person in patients lacking 64KA There wasn't. 64 KA for autoantibodies to 67,000 M _r protein,
Only 1 in 34 non-diabetic first-degree relatives lacking both islet cell cytoplasmic and insulin autoantibodies
It only existed in people (3%). Coomassie SDS-PAGE
Analysis of the gel, the presence of autoantibodies against 67,000M _r protein was often associated with the protein of the strong staining of 70,000M _r. Gas phase amino acid sequencing of this 70,000M _r protein
This reveals that this is the immunoglobulin V-III region chain and is thereby directed to the IgM class of antibodies. Therefore, immunity to the 67,000 M _r antigen may be increased by IgM class antibodies but is not limited thereto. We also showed that almost all 67,000 M _r autoantibody-positive patients also contained 64KA, but 67,0
00M _r Autoantibody positive but 64KA negative is rare. We therefore suggest that there may be antigenic cross-reactivity between the 64,000 and 67,000 M _r islet cell antigens, and that autoantibodies against these proteins can identify these immunological cross-reactive epitopes. Believe in.

As used in this application, "immunologically equivalent to the 64K antigen" refers to peptides that react with 64KA. The term 64KA or 64K autoantibody refers to the compound or compounds that react with the 64K antigen.

Since the 64,000 M _r protein is amphipathic, we sought to observe whether the 64K protein was essentially restricted to the plasma membrane of the cell, and if so, normal islands. Surface expression of this protein in cells (surface e
Experiments were done to see if there was evidence for xpression).

Our biochemical characterization studies showed no evidence of glycosylation of the 64K protein, a common feature for surface expressed proteins. A series of broad enzyme spectra was used to detect common forms of protein carbohydrate glycosylation. This assay includes the enzymes N-GLYCANASE ^™ (for N-linked carbohydrates), treatments for O-linked carbohydrates, neuraminidase (for sialic acid groups) and β-endogalactosidase (for galactose groups). Stuff) was used. If one of the carbohydrate groups is present on the 64K protein, the enzyme will cleave that carbohydrate, the mobility of the 64K protein will increase during subsequent SDS-PAGE analysis, and the reduced molecular weight will Will indicate the protein of No difference in the mobility of the 64K protein in SDS-PAGE gels was detected after treatment of the protein with this particular set of enzymes. We also used a series of 19 broad and narrow spectrum carbohydrate binding lectins to thoroughly analyze the 64K protein for the presence of glycosylation. However, none of these lectins bound to the 64K protein. Thus, there appears to be no glycosylation in the 64K protein.

Finally, using standard techniques of radiolabeled cell surface proteins at ¹²⁵ I, any of the 64K proteins was immunoprecipitated from surface labeled islet cells with serum from diabetic patients.
It has been previously shown that failure to immunoprecipitate (Kolmanda [1987] Diabetes 36, 1432-1440). The study suggested that there is no evidence for surface representations. However, the general technique is labeling tyrosine (a relatively rare amino acid that may not be present in the extracellular portion of the protein), and the conditions were harsh in nature. Thus, it may be severe enough to destroy the antigen binding site of the 64K protein, and thus the antibody may no longer bind to the protein. However,
We used the newly described technique (Thompson J.
[1987] Biochemistry 26,743-750). It is much less harsh in chemistry and radiolabels either lysine (a much more common amino acid than tyrosine) or the NH ₂ -terminal amino acid, more than the method used in Kolman et al. It has been shown to label cell surface proteins with much higher specific activity. Cell surface amino groups ¹²⁵ I sulfo succinate stains diyl (hydroxyphenyl) Purobione - were derivatized with bets by ¹²⁵ I (hydroxyphenyl) propionyl groups. However, even with these improved assays, the 64K protein was not immunoprecipitated from intact cell surface labeled islet cells.

To confirm that the 64K protein is actually present in the plasma membrane, we made a crude membrane preparation of islet cells and used the reagent Triton X-114 from a patient with IDD. The 64K protein was immunoprecipitated using serum. Next we developed an improved method for the detection of autoantibodies to the 64K protein.
This method did not use detergents and produced results that were easier to interpret and faster to perform. The method involved making a crude membrane preparation of ³⁵ S methionine labeled islet cells followed by trypsinization for 60 minutes at 4 ° C (2 mg / ml 50 mM Tris buffer pH 7.
Four). This material is then centrifuged, followed by our standard immunoprecipitation technique. Following gel electrophoresis and autoradiography, the 40K band is observed in immunoprecipitates from sera of diabetic patients but not in controls. Isolated 64K treated with tyrosine
Since we have already shown that the protein also shows a 40K protein, this 40K immunoprecipitated directly from trypsinized islets
It is highly probable that the bands of the same represent the same protein. The assay for the 40K protein is not only an improvement for the easier detection of 64KA, but also immobilon
gas phase sequence analysis using a bilon) membrane yields a much more compatible product. The reason for this improvement is that the 40K fragment lacks the major hydrophobic region that has caused difficulty in our determination of the N-terminal amino acid sequence of the 64K protein bound to INOBILON ^™ using the gas phase sequencer of Applied Biosystems. This is because the possibility is very strong. Proteins with strongly hydrophobic regions are gas phase sequencers
It has been established that it is solubilized in the solvent used in, and is thus the technically dominant hardware for direct sequencing of this protein. The 64,000 M _r than the range, the 40,000 _r range is lower background staining than 64K protein in silver staining of the SDS page gels, much easier to be able to see the 40K protein Is.

Gas phase amino acid sequencing of either the intact 64K protein or the 40K tyrosine cleaved fragment revealed NH ₂ -terminal blockade. This information, together with the solubility of the 40K fragment, suggests that the 40K protein is attached to the β-cell plasma membrane near its COOH terminus. Specifically, we would predict that a string of hydrophobic amino acids would be present in the 230 amino acids closest to the COOH terminus, followed by a series of 3-4 highly charged amino acids. .

Example 7-64K Phosphorylation of Proteins Many key regulatory proteins exist in cells in either phosphorylated or dephosphorylated form, and their steady-state level of phosphorylation interconverts. This is reflected in the relative activities of protein kinase and protein phosphatase that catalyze the process. Phosphorylation triggers small conformational changes in these proteins that alter biochemical and biological properties. Hormones and other extracellular signals convey information via this mechanism.

Although the full role of phosphorylation within human islets has not yet been identified, serum and islet cells are a method of identifying all phosphorylated proteins that have a major role in IDD-induced autoimmune destruction. An in vitro (in vitro) kinase assay using protein was applied.

Two groups of individuals were selected for the in vitro phosphorylation assay test. Diagnosed according to established National Diabetes Data Group (NDDG) criteria (National Diabetes Data Group Diabetes. 1979; 28: 1039-5
7), there were 7 newly initiated IDD patients referred to at the University of Florida Diabetes Clinics. We also tested eight non-diabetic controls without any known family history of autoimmune disease. Human pancreatic islets isolated from cadaver pancreas were 91.2 ± 2.3% pure and had an insulin content of 0.4 ± 0.1 mU per islet. Following 48 hours of in vitro culture, including the use of normal human serum alone, islet cells were immunoprecipitated as follows. Immune complexes were prepared with sera from either patients with IDD or controls but the sera were incubated with islet cell detergent extract,
Subsequent incubation with gamma- ³² P-ATP and in vitro phosphorylated products were analyzed on SDS-PAGE gels. 50 mM Tris buffer (2% Triton X-114, aprotinin, PMSF, NaF) containing islet cells (1,000 / test serum)
Dissolved in (3 hours, 4 ° C.). Detergent lysates were incubated with normal human serum (100 μl / 1,000 islands) and subsequently adsorbed to excess protein A Sepharose CL-4B (Pharmacia). Aliquots containing unbound (precleared) lysate were incubated with either IDD or control serum (25 μl, 18 hours, 4 ° C.). Following incubation of immunoglobulins with protein A Sepharose CL-4B (2 hours, 4 ° C.), the complex was then supplemented with 0.1% SDS, 1.
50 mM with 0% Triton X-114 and 2 mM EDTA
Tris-HCl (pH7.4) 3 times, and 50 mM Tris-HCl
It was washed twice with (pH 7.4). Kinase reaction is 10 mM MnC
50 mM H containing l ₂ , 10 mM MgCl ₂ , 1% Triton X-114 and 20 μCi of adenosine (gamma- ³² P) 5'triphosphate (Amersham,> 5,000 Ci / mmol)
It was started by adding 10 μl of EPES buffer (pH 7.4). The precipitate was suspended and incubated at 30 ° for 10 minutes. The reaction was stopped by the addition of electrophoresis sample buffer and heated at 95 ° C. for 4 minutes. Discontinuous SDS of ³² P-labeled product
Separation was carried out on a 15% polyacrylamide separation gel, followed by autoradiography for 6-72 hours with an intensifying screen (Kodak, X-omat AR5). Sera from IDD patients have a specific M _{r of} 64,000
The protein was immunoprecipitated, which was not observed in control sera.

Finally, to analyze whether the identity of the M _r 64,000 protein detected by the in vitro kinase reaction was the same as that observed in the immunoprecipitation of metabolically labeled islet cells, A tryptic peptide mapping experiment was performed. IDD serum treatment ³⁵ S methionine labeled islet cell M _r 64,000 protein immunoprecipitated showed original similar tryptic peptide mapping of the in vitro phosphate warp Le of M _r 64,000 protein.

Specifically, 64,000M _r and 125,000M _r
Cleave the protein from the SDS-PAGE gel and 1 mg of protein
/ Ml trypsin (3-30 min, 4 ° C). The resulting protein was run again on an SDS-PAGE gel. 125,000M _r
Protein was unaffected by this treatment. But 64,0
The 00M _r protein increased the mobility in the 40,000M _r range. Therefore the 64,000 ³² P-labeled islet cell protein shows the M _r 64,000 ³⁵ S-methionine labeled island cellular protein the same M _r as we have observed (40,000) when treated with trypsin.

Example 8 Alternate biochemistry of 64K autoantigen
Identification of 64K self-antigen is glutamate decarboxylase (GA
We have established that it shows homology to D) and is immunoreactive with anti-GAD antibodies. GAD enzymes are localized in the brain and in the β cells of the pancreas. Biochemical characterization of GAD in the literature has been limited to its role in the formation of gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter (neurotransmitter) in the mammalian brain. This GAD sequence has been published by Kobayashi et al. (Kobayashi Y., DL Kaufman and AJ Tobin [1987] "Glutamic acid decarboxylase cDNA.
A: Nucleotide sequence encoding enzymatically active fusion protein "J. Neuroscience 7 (9): 2768-2772) is reproduced herein in Table 6.

Antigenic cross-reactivity between GAD and 64K protein was confirmed. A series of immunoprecipitations using autoantibodies to the 64K protein and monoclonal antibodies directed against GAD confirmed competition for binding to the 64K protein. Monoclonal antibodies that react with the GAD protein are readily available to those of skill in the art, for example, American Type Culture Collection (ATCC) 12852 Rockville Parkland Drive, Midland, USA 12301.
Monoclonal antiserum GAD-1 (ATCC HB184) is specific for binding to GAD.

For these studies human islet cells were metabolically labeled with ³⁵ S methionine and the detergent phase separated as described above. The islet cell extract was then split into two samples of equal volume. Each sample was precleared twice with either control (no 64K autoantibody) or IDD (64K autoantibody containing) serum. Pre-clear with this IDD serum
The step removes or significantly reduces the amount of 64K protein from the islet cell protein preparation, while the preclear in control sera does not significantly affect the amount of 64K protein.
Both precleared samples (control and IDD) were individually immunoprecipitated with a monoclonal antibody against GAD. Results: Preclearing with control (64K autoantibody negative) serum did not affect the ability of monoclonal anti-GAD antibodies to immunoprecipitate 64K proteins in islet cells. However, IDD sera containing 64K autoantibodies as pre-clearing agents significantly reduced the ability of monoclonal anti-GAD antibodies to immunoprecipitate 64K proteins in islet cells. This means that 64K autoantigen and GAD
Have demonstrated antigenic cross-reactivity between.

Sera from 15 freshly-initiated IDD patients and individuals with a sample obtained 2-3 years after the start of IDD, 5
Sera from 65 individuals, including 10 individuals with IDD of -10 year duration, 20 control individuals and 5 individuals at high risk of IDD, were tested for anti-GAD activity. Immunization in the form of autoantibodies to GAD was clearly associated with IDD.

As described in Example 6 above, immunoprecipitation of islet cells with IDD serum not only resulted in the identification of the 64K protein, but some immunoprecipitated 67,000 M _r of protein. It was This observation was particularly noted when the detergent phase preparation did not produce a sharp separation between aqueous (primarily cytosolic) and detergent phase (membrane associated) proteins. Thus, the 64K protein can be found in islet cells in two forms. 67,000 M _r soluble (cytosolic) type and 6
4,000M _r Hydrophobic (membrane) type. The major autoantigen in IDD is of type 64,000M _r , but there is also autoimmunity to type 67,000.

Gas phase amino acid sequencing of 64K autoantigens isolated from ³⁵ S-labeled islet cells is shown in Table 6.
It shows homology between the K protein and GAD sequences. This homology was discovered as follows.

The 64K autoantigen was isolated from ³⁵ S-labeled human islet cells as described above. After SDS-PAGE, the proteins from the gel were transferred to an IMOBILON-P ^™ membrane for visualization by autoradiography. The 64K band was localized, excised, and treated with cyanogen bromide, which cleaves the peptide chain at methionine residues. The resulting peptide fragment was eluted from the PBDF membrane and rerun on SDS-PAGE before transferring to IMOBILON-P ^™ . Oh the band - autoradiography - and it so that can be seen in, and cut out the fragments in the M _r 33,000, and subjected to gas phase peptide sequencing. For example, the following sequence profile was obtained. MET-ILE-PRO-GLU-VAL-LYS

We have found a high degree of similarity between the 64K amino acid sequence and GAD at two levels. As previously mentioned in the first, when our 64K protein treated with cyanogen bromide, it produces peptide fragments of 33,000M _r. One would expect this fragment to consist of approximately 300 codons (900 nucleotides). Examination of the GAD sequences shown in Table 6 reveals an open reading frame of 625 codons and 1875 nucleotides in the total protein. Nucleotide positions 916-933 of the sequence are coded for the next amino acid. PHE-PHE-PRO-GLU-VAL-LYS

The 64K peptide sequence thus shows a high degree of similarity with the GAD sequence in amino acid sequence. Furthermore, this GAD peptide sequence can be cleaved with cyanogen bromide if a PHE or MET amino acid substitution is made in that part,
It is located in the region of the protein that is expected to observe the 33,000M _r peptide. Second, nucleotides have differences between the resolved amino acid sequences (MET-PHE-PRO-GLU-VAL-LYS), and nucleotide sequences 916-933 are accompanied by only minor nucleotide substitutions. In fact, methionine rather than phenylalanine appears to be in the first position of the 64K sequence of this protein, since the peptide is cleaved with cyanogen bromide, which is specific for methionine. Therefore we have our DNA
The above sequence information was used to construct a probe to identify the gene coding for the human pancreatic 64K protein from the library.

A mixed oligonucleotide of the following sequence was synthesized. This synthetic oligonucleotide was radiolabeled using T4 polynucleotide kinase and gamma- ³² P-ATP and used as a probe for a human island cDNA library. Approximately 120,000 clones were screened and 3 positive plaques were detected. In this way, the DNA coding for the 64K protein was identified and isolated.

The DNA coded for the 64K protein can be introduced into an appropriate prokaryotic expression vector for the production of large quantities of recombinant protein. This product may be used in assays (eg ELISA, RIA) to determine the level of anti-64K antibodies in prediabetic patients or to diagnose prediabetic individuals.
Etc.) can be used for development. Appropriate eukaryotic expression vectors can also be used for production in native 64K eukaryotic cells. This overcomes the possibility that bacteria will not be able to properly process the 64K precursor and will be recognized by autoantibodies.

Recently, considerable interest has been focused on proteins recognized by effectors of the immune system (Benjamin D., J. Benzofsky, I. East, F. Garr.
De, C. Hannam, S. Leech [1984] "Protein antigen structure recognized by T cells: antigen structure of protein; re-evaluation"
Ann. Rev. Immunol. 2: 67-101). Based on algorithms that predict T and β cell epitopes, a great deal of knowledge is available on the binding epitopes of T cells and antibodies (Delissi, S. and JA Verzovsky [1985] "T.
Cellular antigenic sites tend to be amphipathic in structure. '' Proc.
Natl. Acad. Sci. 82: 7048-7052; Hopp, TP and K.
R. Woods [1981] "Prediction of Protein Antigenic Determinants from Amino Acid Sequences" Proc. Natl. Acad. Sci. 78: 3824
-3828). Furthermore, experimental studies on model protein antigens such as cytochrome C have allowed the precise localization of T and B cell loci and the characterization of critical amino acid residues important for recognition ( Milichi, DR [1989] "Synthetic T and B cell recognition sites; implications for vaccine development" Dixon, FJ, Advances in Immunology. 45 Academic Press, London 1989; 195-282). But,
In autoimmune diseases specific to human organs, the lack of knowledge of the nature and availability of self-antigens has limited the study of epitopes (McKay, IR and ME Gerschwin [1989] “Nuclear Molecular basis for mitochondrial autoreactivity during crotch of the sex-bearing bladder "Immunol. Today 10:
315-318). In organ-specific autoimmune diseases, the determination of the nature of the autoantigen region recognized by T cells and autoantibodies allows the design of antagonists and immunomodulatory compounds to drive tissue injury disease.

One aspect of the invention is the identification of immunoreactive peptides consisting of the epitope of the 64K protein. For these studies, the GAD cloned cDNA template can be used in conjunction with DNA amplification technology to express selected segments of the 64K antigen as recombinant proteins in E. coli. On average 80 amino acid residues,
Larger fragments can be produced, containing multiple overlapping different recombinant fragments, and the entire extracellular region of the molecule, and the autoantibody binding site labeled with a radioisotope. Analyzed by immunoprecipitation of the expressed bacterial product growing in the presence of the precursor. Using these procedures allows epitopic mapping of the correct antigen amino acid sequence of the GAD molecule. 64K
Synthetic peptides consisting of antigenic epitopes of proteins can be used for both diagnostic and therapeutic purposes as described herein.

One such peptide of the invention consists of the PRO-GLU-VAL-LYS sequence described above. Thus, one peptide that may be used as described herein for diagnostic or therapeutic purposes may contain from about 8 to about 30 amino acids containing the PRO-GLU-VAL-LYS sequence. Most advantageously, such a peptide may be from about 10 to about 20 amino acids. The peptide may have one sequence of the GAD protein or the 64K islet cell protein itself. It is well known that there are several forms of GAD that may differ in the exact amino acid sequence. The isolation of GAD from rats, pigs, cattle, cats and humans has all been reported. For example, Crum, DS, LD Burnett, JL Joseph, LC Harrison (1991) "Human glutamate decarboxylase cD from brain and pancreatic islets.
NA cloning and partial nucleotide sequences "Bioche
m. Biophys. Res. Comm. 176 (3): 1239-1244; Spink
DC, TG Porter, S.U., DL Martin (1987)
"Multiple types of glutamate decarboxylase with different kinetics in rat brain Brain Res. 421: 235-244; Denna LA, SC way, HS phosphorus, CT phosphorus, JYU (1987)" Brain L-glutamate decarboxylase, purification and subunit structure "Proc. Natl. Acad,
Sci. USA 84: 668-672; Chan YC, DI Gottley
B. (1988) "Characterization of proteins purified with monoclonal antibodies against glutamate decarboxylase" J.
Neurosci. 88: 2123-2130; Spink DC, TG Porter, SJ W, DL Martin (1985) "Three kinetic distinctions of glutamate decarboxylase from pig brain. Characterization of morphology "Biochem. J. 231: 695-
703; Blinda-Man JM, M. Maitre, L. Osora, P. Mandel (1978) "L-glutames from the human brain.
Purification and some properties of todecarboxylase, '' Eur. J.
Biochem. 86: 143-152; and Fan WM, L. Leed Hokwet, E. U, JY W (1990) Proc. Natl. Acad.
Sci. USA 87: 8491-8495. Each of these GAD sequences has 6
It may include an epitope that is immunoreactive with a 4K autoantibody.
Thus, peptides according to the invention from these various forms of GAD can be used for therapeutic or diagnostic purposes.
Also, these various GAD sequences can be used to provide information about the types of amino acid substitutions that can be made in the 64K sequence while maintaining critical immunoreactivity. The synthetic peptide of the present invention can be represented as follows. A-Pro-Glu-Val-Lys-B

In the formula, both A and B both have a length of 0 to
It consists of a sequence of about 20 amino acids, where A and B are such that the synthetic peptides are totally immunoreactive with 64K autoantibodies or are recognized by T cells or β cells,
It has sufficient homology with 64K. This homology can be, for example, about 90% with a 64K or GAD protein. The synthetic peptides described herein are typically less than about 50 amino acids and, in any event, less than the full length of the GAD or 64K protein.

The 64K proteins we describe here exist in at least two forms. That is, a form or forms having a size of about 63-65K, and a form having a size of about 67K. These forms are clearly distinguished. The lower molecular weight form was observed as a doublet as described above. The presence of multiple forms of 64K, as described above, may prevent people at risk of developing diabetes from some form.
64K has been favored for diagnostic purposes in determining whether one has antibodies to the 64K antigen. Although the various forms of 64K share homology, they also have heterologous regions that can give rise to different immunoreactivity. Therefore, most adequate diagnoses or treatment plans are 64
Multiple peptides (or hybrid peptides) consisting of sequences that are immunoreactive with each form of autoantibody to K
Would include the use of. In making peptides according to the present invention for diagnostic or therapeutic applications, one of skill in the art can use sequences from various forms of GAD or 64K, or also sequences modified with various amino acid substitutions. Can be used. To be within the scope of the invention, these peptides are
It is immunoreactive with antibodies to the 64K protein.

The term analog as used in this application refers to
Refers to a compound that is substantially the same as another compound but has been modified, for example, by the addition of additional amino acids or side chains. As used herein, a mutant (mut
The terms ants and variants refer to amino acid sequences that are substantially the same as another sequence, but which have amino acid substitutions at certain positions in the amino acid sequence. A fragment refers to a portion of a longer amino acid sequence.

The present invention includes the use of specific GAD and 64K amino acid sequences. The invention further encompasses analogs and mutants of these sequences, as well as fragments and analogs and mutants of the sequences. These analogs, mutants, and fragments are included within the scope of the invention as long as the analogs, fragments or mutants retain substantially the same immunoreactivity as antibodies to 64K. For example, making conservative amino acid substitutions is within the skill of one in the art. For example, amino acids can be divided into the following classes: basic, hydrophobic, acidic, polar,
And amide. Substitutions in which one class of amino acid is replaced by another amino acid of the same type are included within the scope of the invention, as long as the substitution does not substantially alter the biological activity of the compound. The following table lists examples of amino acids belonging to each class.

Table 5 Amino Acid Classes Examples of Amino Acids Nonpolar Ala, Val, Leu, Ile, Pro, Met, Phe, Trp Polar Gly, Ser, Thr, Cys, Tyr, Asn, Gln Negatively charged Asp , Glu plus charged Lys, Arg, His

In some cases, non-conservative permutations may also be performed. Construction of various mutants can occur by cassette mutagenesis of synthetic genes. This step is known to any person skilled in the art. To the extent that these substitutions do not substantially alter the immunological reactivity with the antibody to 64K, the resulting compounds are included within the scope of the invention. Conservative amino acid substitutions are merely an example of one type of modification that is within the scope of the present subject matter.

These synthetic peptides will be used in the development of diagnostic reagents to be used in similar assays developed with the recombinant proteins described above. Synthetic peptides can also be used to make vaccines for the prevention of IDD, similar to their purpose for vaccine development in general (Mirich [1989], supra).

Example 9 Insulin-dependent diabetes mellitus
Peripheral blood against glutamate decarboxylase
Identification of T Lymphocyte Responses Insulin-dependent diabetes mellitus (IDD) appears to result from autoimmune destruction of insulin-producing pancreatic β-cells. The high frequency of early appearance of antibodies immunoreactive with glutamate decarboxylase (GAD) indicates that
It suggests that this protein may be a target antigen in diabetes. However, since the disease is believed to be dependent on T lymphocytes, we have peripheral blood for children with newly diagnosed IDD and recombinant GAD from people at high and low risk of developing IDD. The mononuclear cell response was analyzed.

Mononuclear infiltration of pancreatic islet cells at the clinical onset of IDD (Insularity) is a pathological proof of disease. This lesion is proposed primarily for T-lymphocytes, and it is these cells that are thought to play a pivotal role in the pathogenesis of IDD.
Evidence supporting this idea includes the observation that ICA titers do not rise, but that the disease recurs in long-term diabetic patients who have received pancreatic transplants. Immunosuppressants have also been shown to be effective in improving the metabolic potential of individuals with IDD. In a spontaneous animal model of IDD (NOD mice), diabetes can be introduced into non-diabetic mice through conversion of CD4 and CD8 T lymphocytes. Disease can also be prevented in NOD mice through the use of anti-CD4 therapy. However, the nature of the target antigen playing a role in this autoimmune phenomenon remains unclear.

We are here at the beginning of IDD
We also tested whether T-lymphocyte reactivity was characteristic within the population at increased risk of illness.

Recombinant GAD Antigen was Recognized by PET on Rat Brain GAD65
Manufactured from expression. Peripheral blood mononuclear cells were
Ficoll-Hypaque "isolated by density centrifugation
(St. Louis, Sigma, Missouri). 1 × 10 ⁶ mononuclear cells were cultured in a 24-well tissue culture tray in the presence of 10 μg / ml of antigen for 7 days. After 6 days in culture, 1.0 μCi ³ H thymidine (NEN, Wilmington, Del.) Was added to each well. The culture medium was harvested semi-automatically after 18 hours and thymidine incorporation was assessed by liquid scintillation counting. Cell proliferation was expressed as the stimulation index (SI) minus the cpm incorporated in the presence of antigen divided by the cpm incorporated in the absence of antigen (medium only). An SI greater than or equal to 3 (≧ 3) was determined as a statistically positive response.

Our study showed that T-lymphocyte reactivity to GAD occurs at the onset of IDD and in persons with an increased risk of IDD. This T cell responsiveness to GAD is not present in healthy people.
These findings support the hypothesis that GAD contains an antigenic epitope of T-lymphocytes and may therefore be involved in the pathogenesis of IDD disease. T- in newly initiated IDD patients
There is no correlation between the degree of lymphocyte response and ICA titer, age at onset of disease, sex or HLA type. Thus, the reactivity of T-lymphocytes to this GAD contributes to provide additional information and provides another way to diagnose disease.

Examples 10-64K Organisms for detection of autoantibodies
Body fluid collection Greater than 500 microliter volumes of whole blood were collected from humans for testing for 64KA autoantibodies.
Blood was drawn directly into glass evacuated vessels, or drawn for injection, and subsequently transferred to glass evacuated vessels. The common vacuum vessel tubes used to obtain serum (blood without coagulation factors) are called red top tubes (no sodium heparin present) or serum separator (STS) tubes. When the common red top tube was used, this tube was allowed to clot (for more than 10 minutes) and clots were removed.
During this period, either sample tube can be centrifuged for 5 minutes at 1000 rpm at room temperature. The serum in the sample was removed, placed in a plastic storage bottle, and sealed tightly.
Samples can be frozen at -20 ° C until 64K autoantibody analysis.

Example 11-Used to Test Biological Fluids
Preparation of 64K Antigens for Human islet cell preparations must be isolated and metabolically labeled to provide a source of 64K antigens before samples can be tested for 64K autoantibodies. Human islet cell preparations were prepared by the method of Ricody et al. (Ricordi, C., R.
Lacy, PE, Finke, EH, Olac
k), BJ, and Scharp, DW [1988] "An automated method for the isolation of human pancreatic islets" Diabetes 37: 413-420), as outlined in detail in It can be isolated from a pancreas donation. 6,000 islets in 50 ml CMRL medium
Can be stored in T-150 plastic tissue culture based flasks at a concentration of islets / T-150 flask.

Remove and discard CMRL medium before testing. 50 ml of RPMI1640 medium (16 mM glucose, 100 μU / m in each flask)
l Penicillin, 100 μG / ml streptomycin and 2% v / v
(Supplemented with normal human serum) was added and incubated overnight at 37 ° C. in culture medium in an atmosphere of 95% air / 5% CO ₂ . The islets were collected in centrifuge tubes at a concentration of 50 ml medium per test tube.

Cap the test tube and stir at room temperature at 1000 rpm for 3
Centrifuge for minutes. The supernatant was removed and discarded. Small amount (approximately
4 ml of RPMI labeling medium (16 mM glucose, 20 mM HEPES, 2
% Human serum supplemented, amino acid methionine is not present) was added to each tube for approximately 30 seconds per tube with gentle mixing. 50 ml of all islet cells
Collect in polypropylene centrifuge tubes, and spin at 1000 rpm for 5
Centrifuge for minutes. The supernatant was removed and discarded. Then the island cells
Resuspended in RPMI labeling medium (1000 islands / ml medium) and transferred to 100 mm plastic tissue culture medium Petri dishes. Island 15 minutes 37
Incubate at 95 ° C (95% air / 5% CO ₂ ) and then add ³⁵ S methionine.
The medium was added to the medium at a concentration of 0.5 mCi / 1 × 10 ⁴ cells. Island cells again
Incubated for 5 hours at 37 ° C (95% air / 5% CO ₂ ). The medium in the Petri dish was aspirated without agitating (or collecting) the islet cells and the medium was discarded.

The cells were then added with an equal volume of RPMI1640 medium at a concentration of 1000 islets / ml and 37 ° C. (95% air / 5% CO ₂ ).
Incubated for 30 minutes. The islet cells and their medium were collected in 15 ml conical polypropylene centrifuge tubes. Test tube up to 15 ml marking 20 mM Tris (pH 7.4), 150 mM NaCl, 1000K
Fill with IE / ml aprotin and 2 mM phenylmethylsulfonylfluoride, cap and centrifuge (1000 rpm, 5 min),
And the supernatant was discarded. The test tube containing the pellet was then frozen (-80 ° C).

Example 12-64K Antigen Detergent Extraction and 64K Self
Testing Biological Fluids Against Antibodies Islet cells were slowly thawed in vitro on ice to perform immunoprecipitation of the 64K antigen using test serum. Once detergent extraction buffer Once extracted (20 mM Tris (pH7.4), 150mM NaCl, 1000KIE / ml Apurochin and 2mM phenylmethylsulfonyl fluoride and 2% Triton ^TM X-114) was added to a test tube, an island on ice Dissolved for 3 hours. For a total of 60 seconds (4 times with a 1-minute break on ice for 15 seconds between each sonication),
Using an ultrasonic probe inserted in the detergent extract,
The islet cells were sonicated in vitro. At 15 minute intervals, the island was also destroyed mechanically.

An equal volume of extract was pipetted into a centrifuge tube, the balanced test tube was placed in a Beckman type 50 rotor and the tube was ultracentrifuged (100,000 xg, 30 min, 4 min).
Insoluble material was removed from the detergent-extracted islands by The supernatant was removed and placed in a new polypropylene test tube. The pellet can be discarded. The islet cell lysate (supernatant) can then be separated into an aqueous phase, a sucrose phase and a detergent phase before the immunoprecipitation test.

A method for extraction of the detergent phase is described in Bordeal (Bordier, C. [1981] Triton ^TM X-11.
4 Phase separation of integral membrane proteins in solution ”J. Biol. Chem. 256: 1
The method of 604-07) was directly followed.

After extraction, the detergent phase of the islet cell lysate was then placed in a capped 1.5 ml Epindorf tube containing normal human serum (10 μl test serum per 100 μl detergent phase supernatant, 1 hour, 4 ° C.
[Ice bath]) and subsequently adsorbed to pre-expanded protein A Sepharose CL-4B (which can be purchased from Pharmacia, Sweden). 100 μl swollen Protein A Sepharose CL-4B, 15 ml covered
In a conical centrifuge tube of 400 μl each was added to each test serum / detergent phase mixture and kept on a shaking platform at 4 ° C. (300 rpm for 2 hours). At that time the test tube is 1000r
It can be centrifuged for 1 minute at pm and the supernatant removed. The Protein A Sepharose pellet can be discarded. An aliquot of unbound (pre-cleared) lysate (supernatant) (100μ containing 5-10x10 ⁶ cpm)
l) into a 1.5 ml Epindorf centrifuge tube and 64K
Incubated with 25 μl of serum to be tested for the presence of autoantibodies. Then, the test tube was capped and incubated at 4 ° C for 18 hours (in an ice bath). 100 μl of pre-swollen Protein A Sepharose CL-4B was added to each assay tube, and the reaction mixture was incubated at 4 ° C. for 2 hours on a shaking platform at 300 rpm. To the tube was added 900 μl of 0.5% Triton ^™ X-114 buffer to wash the Protein A Sepharose CL-4B. Swirl the test tubes for 10 seconds on each, followed by 1000 seconds for 10 seconds.
Centrifuge at rpm. The supernatant was aspirated and the pellet was left in the test tube. Another 900 μl of 0.5% Triton
^TM wash buffer was added to the test tube and the wash procedure was repeated 5 more times. One final wash step was performed with ice-cold water washes instead of 0.5% Triton ^™ buffer. At this point the tube should only contain protein A sepharose CL-4B and any immune complexes formed between the antibody and / or islet protein. Bound immune complexes can be removed, 80 mM Tris (pH 6.8), 3.0% (w / v) sodium dodecyl sulfate (SDS), 15% sucrose, 0.001 bromophenyl blue and
(5.0% v / v) β-mercaptoethanol with the same stopper 1.5
It can be denatured by boiling for 3 minutes in 100 μl of sample buffer contained in a ml Epindorf tube. The beads of sepharose are then removed by centrifuging the tubes (1000 rpm, 1 minute) and the supernatant carefully removed. A 50 μl sample is then electrophoresed through a discontinuous SDS 10% polyacrylamide separation gel, followed by the method of Laemli [Laemmli, UK 1970. Cleavage of structural proteins during assembly of the bacteriophage T4 head Nature.
227: 680-85], followed by Coomassie-Brilliant Bull-staining followed by fluorography using the method described in detail in the commercially available product Enhance (New England New York). Clear-).

Following electrophoresis, place the gel on filter paper and
Dry for 60 hours at 60 ° C in an electrophoresis gel dryer. Once dry, remove the gel, place on X-ray film (Kodak XR OMAT) in a film cassette with intensifying screen, and place at -80 ° C for 3 weeks. The film (called the fluororadiogram) is processed using standard X-ray film photographic processing. Test samples are scored as positive or negative after comparison to their respective positive and negative controls.

Examples 13-Methods for Detecting 64KA In addition to using the immunoprecipitation technique outlined in Example 11, the present invention can be practiced using any procedure that facilitates the detection of the presence of 64KA. For example, another immunological method that can be used is the enzyme-linked immunosorbent assay (ELIS
A) and radioimmunoassay (RIA). The principles and experimental methods of these procedures are well known to those skilled in the art. This assay can be performed quickly and efficiently by using natural or recombinant proteins that bind to 64KA.
Both whole cell and cell lysate procedures are well known to those conducting research in this field and can be readily used to detect 64KA of the present invention.

For example, as described in Example 8, the amino acid sequences shown in Table 6 can be analyzed to confirm immunologically reactive epitopes. These episodes are 64
It is an amino acid sequence that will immunologically react with KA.
Synthetic peptides that can be used according to these procedures are described in the Examples.
See 8 for details. These sequences can then be produced by recombination. For recombinant production, the DNA encoding the epitope is inserted into a vector, which vector is then used to transform a suitable host cell, which then hosts the desired amino acid sequence. I can be expressed. Bacterial, insect, yeast and mammalian cells can all serve as suitable hosts, although eukaryotic cells are the preferred host if protein folding is an important factor in epitopic reactivity.

Purified protein or lysates of cells producing the protein can be used in the assay.

Another method of using the 64K antigen for detecting 64KA is to use an antibody raised against 64KA, which is otherwise known as an anti-64KA antibody. This antibody is immunoprecipitated with 64KA, and detection can be performed as described above.

Example 14-64K Autoantigen in Immune Complex-Self
Assays for detecting autoantibodies Patients with IDD or in the process of developing this disease can be expected to have immune complexes containing the 64K antigen in their serum. These immune complexes typically consist of a 64K autoantigen surrounded by several immunoglobulins. Since individuals who do not have this disease or who are not going to develop this disease will not have these immune complexes, detecting these complexes is the basis of assays for detecting this disease. I will provide a.

Blood is drawn from patients or controls who are initially tested for IDD. Serum is obtained by spinning out (centrifuging) red blood cells from a blood sample. ID
For patients with D or those who are about to develop the disease, this serum can be expected to contain immune complexes containing the 64K antigen. The serum sample can then be incubated with protein A sepharose. The volume of sample incubated with serum is small, eg 25 μl. Following incubation of immunoglobulins with protein A Sepharose CL-4B (2 hours, 4 ° C.), the complex was then incubated with 50 mM Tris-HCl (0.1% SDS, 1.0% Triton X-114, and 2 mM EDTA). pH 7.
It can be washed 3 times with 4) and twice with 50 mM Tris-HCl (pH 7.4). By washing the cepharose as described above, the external serum proteins are washed away, and only the immunoglobulin bound to the protein A sepharose remains. Radioactive phosphonylation of the 64K autoantigen can then be achieved using the kinase reaction. The kinase reaction was 10 mM MnCl ₂ , 10 mM MgCl ₂ ,
1% Triton X-114, and 20 μCi adenosine (γ- ³² P) 5 '
50m containing triphosphoric acid (Amersham> 5000Ci / mmol)
It can be started by adding 10 μl of M HEPES buffer (pH 7.4). The precipitate is suspended and can be incubated at 30 ° C. for 10 minutes. The reaction can then be stopped by the addition of electrophoresis sample buffer and heated at 95 ° C for 4 minutes. ^{The 32} P-labeled products can be separated on a discontinuous SDS 15% polyacrylamide separation gel, followed by autoradiography for 6-72 hours in an enhanced screen (Kodak, X-omat AR5). Sera from IDD patients specifically immunoprecipitate the M _r 64,000 protein, which is not observed in control sera.

Example 15-Hybrid Protein Vaccine
Treatment of IDD used The specific event or agent that triggers the onset of diabetes is unknown. Viruses having antigens similar to the 64K antigen can produce both a normal immune response to the virus and an abnormal autoimmune response to the 64K antigen through molecular mimicry with the virus. Thus, genetic susceptibility is expressed by exaggerated or prolonged immune responses to environmental factors that initiate the disease process.
It is also possible that the 64K protein may have a delayed expression in the development of islet cells during ontogeny, and since resistance to it has not developed in early stages of life, it may be possible to use it as an antigen. .

Regardless of the mechanism of disease initiation, β-cell destruction can proceed in at least two ways. 64KA binds to foreign antigens initiated and stimulates other compounds of the immune system to destroy antibody-bound β cells. Or T-lymphocytes
It recognizes the 64K antigen and kills cells directly. 64K in either case
Uses proteins to interfere with the destruction process, thereby causing IDD
Can be delayed or prevented.

The novel method of treatment of the present invention involves the injection into the bloodstream of toxin bound to a purified form of the 64K antigen. The antigen-toxin complex rapidly reaches the lymph nodes, where the complex is normally taken up by immune cells, which produce 64KA. Also, the antigen-toxin complex will be bound by T-lymphocytes that recognize the 64K antigen on β cells. Therefore, β
Certain immune cells involved in cell destruction are poisoned and inactivated, leaving non-destructive immune cells innocuous.

The hybrid protein may consist, for example, of diphtheria toxin together with the 64K antigen. Construction of such hybrid toxins is described, for example, in US Pat.
5,382 (Murphy) hybrid protein.

Another method involves creating an altered virus (from the disease-initiating class of viruses) that lacks expression of a 64K-like protein, but retains infectivity. This attenuated virus can be used to vaccinate people at high genetic risk for IDD, ie, HLA homologous siblings or those in the early stages of IDD. Candidates for viruses that mimic the 64K protein, and thus candidates for initiating the course of the disease, include Kosakisaki virus, rubella virus and EMC virus. The vaccine composition can be made up of an attenuated virus such that the non-attenuated virus elicits an immune response to the epitope in common with the 64K protein. In an attenuated form, the vaccine virus produces an immune response against the virus but not against the 64K protein.

Example 16-Use of 64KA in association with pancreas transplantation A preferred method of treatment of patients with IDD is to transplant normal islets as a replacement for compromised or destroyed β-cells. Partial and whole pancreas transplants have been successfully performed in many patients with diabetes. However, permanent immunosuppressive therapy is always needed to maintain the transplant and prevent rejection. Partial or total pancreatic transplantation under continuous immunosuppressive therapy has produced normal levels of blood glucose in some patients with diabetes. Pancreas transplants are performed late in the course of diabetes, probably not reversing complications such as kidney disease, and in fact exacerbating retinopathy.

Importantly, successful transplantation of the pancreas between monozygotic twins has been maintained without immunosuppression, but autoimmune island cell destruction resulted in recurrent diabetes. Therefore, even when transplantation is not rejected, immunotherapy is essential to prevent disease recurrence. Destruction (rejection) of the transplanted islets is due at least in part to the re-emergence of self-antigens responsible for autoimmune destruction. No specific immunotherapy currently exists to prevent autoimmune destruction (rejection of transplanted islets / pancreas). Specific immunotherapy can be used to prevent islet cell destruction, using a hybrid toxin as described in detail in Example 11 to prevent autoimmune destruction of the transplanted islet cells or pancreas. The combined use of immunotherapy makes islet cell / pancreas transplantation a therapeutic tool for the treatment of IDD.

Examples 17-64 Kits for Assaying KA The novel procedures used herein can be used to provide reagent kits that facilitate the convenient analysis of serum samples. Kits can be made to use intact, recombinantly or synthetically produced 64K proteins or immunoreactive peptides that serve as antigens for 64KA detection. Alternatively, antibodies specifically developed to detect 64KA are also useful. The principles and methods of ELISA and RIA techniques for detecting antibodies are well established.

By way of example, for an ELISA assay, such a kit would consist of the following components: 1.64K protein, peptide or anti-64KA antibody 2. Enzyme (for example peroxidase) 3. Conjugate (complexed) animal anti-human immunoglobulin, and 4. Positive and negative controls 96-well plastic plate
, Colorimetric reagent, ELISA reader, blocking reagent and wash buffer.

Also, for example, for RIA, such a kit may consist of the following components: 1. Radiolabeled 64K protein, peptide or anti-64KA antibody 2. Wash buffer 3. Polyethylene glycol 4. Anti-human precipitation (second) antibody of goat or sheep, and 5. Positive and negative controls Any of the above kits Can be modified to include any of the appropriate laboratory equipment.

The examples and embodiments described herein are for illustration purposes only, and various modifications or changes will be suggested to those skilled in the art in light of the above and fall within the spirit and scope of the present application. It should be and fall within the scope of the appended claims.

Table 6. Cat glutamate decarboxylase
mRNA nucleotides and derived sequences

[Table 1]

[Table 1]

[Table 1]

[Table 1]

Continuation of front page    (72) Inventor Noel Kay. McLaren             United States 32618 Florida             Archer Box 10 Route 2 (72) Inventor William Castarn             United States 32608 Gue, Florida             Insville 47 S Road S. Dubli             U. 8123

Claims (1)

[Claims] 1. A drug containing a GAD protein or peptide for use in the treatment or prevention of IDD or for the manufacture of a medicament thereof. 2. The agent according to claim 1, wherein the agent comprises an immune system activating fragment, and the immune system activating fragment comprises the amino acid sequence Pro-Glu-Val-Lys.