Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-07-29
2001-04-03
Chan, Christina Y. (Department: 1644)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C536S024100, C536S024200, C435S320100
Reexamination Certificate
active
06211352
ABSTRACT:
FIELD OF INVENTION
The present invention relates to the identification, cloning and sequencing of nucleic acid molecules encoding an isoform of the enzyme glutamic acid decarboxylase and further relates to the use of these molecules and/or peptides and polypeptides encoded thereby in diagnostic tests for Insulin Dependent Diabetes Mellitus and other diseases in which glutamic acid decarboxylase is an autoantigen and in the treatment of patients suffering from these diseases.
BACKGROUND TO THE INVENTION
The enzyme glutamic acid decarboxylase (hereinafter referred to as “GAD”) catalyses the conversion of L-glutamic acid to the inhibitory neurotransmitter &ggr;-amino butyric acid (hereinafter referred to as “GABA”). GAD is expressed both in the GABA secretory neurons of the central nervous system (1-3), in the &bgr;-cells of the pancreas (4,5), and in spermatoza (6). Analysis of immunoaffinity-purified, enzymatically active brain GAD has identified several isomeric forms of GAD with M
r
54-67,000 (7,8). Using antisera raised to purified brain GAD to screen brain cDNA expression libraries, cDNAs encoding full length rat (9) and feline (10) GAD sequences have been isolated and sequenced. Comparisons of the deduced amino acid sequences of rat and feline GAD show that both proteins are 95% identical and, therefore, highly conserved during evolution.
Autoantibodies reactive with GAD in GABA-ergic neurons are present in the majority of sera from patients with the rare neurological disease Stiff Man Syndrome (hereinafter referred to “SMS”; 11,12). Patients positive for GAD autoantibodies have an increased frequency of polyendocrine autoimmunity especially Insulin Dependent Diabetes Mellitus (hereinafter referred to as “IDDM”). During the pre-clinical stage of IDDM and in patients with recent onset clinical IDDM, autoantibodies are frequently detected against an islet cell M
r
64,000 protein designated “64K” (13). In a recent report, the 64K autoantigen was presumptively identified as GAD (14). However, Genovese (15) has suggested that GAD is co-precipitated with a separate 64K protein, the latter distinguished by tryptic products of M
r
37,000/40,000 that are distinct from a M
r
50,000 product of GAD. GAD comprises at least two isoforms encoded by separate genes (16, 17, 18). The predicted molecular weights of the known isoforms are approximately 67,000 and 65,000 (referred to as the “67K” and “65K” isoforms, respectively). The distribution of GAD isoforms in different tissues in still not well defined, but it is likely that the 65K isoform accounts for the GAD component of the 64K autoantigen (17).
In work leading up to the present invention, the inventors sought to clone the 67K isoform of GAD from human and other species for potential diagnostic and/or therapeutic use. In accordance with the present invention, human brain (HB), human pancreatic islet (HI) and mouse brain (MB) GAD (hereinafter referred to as “HBGAD”, “HIGAD” and “MEGAD”, respectively) have been cloned and sequenced. In further accordance with the present invention, recombinant GAD proteins corresponding to the 67K isoform and their fragments and derivatives were used as an antigen to detect antibodies and T-cells reactive with GAD thereby forming a basis for a new range of diagnostics and therapeutics for diseases of the type including preclinical and clinical IDDM and SMS and other diseases in which GAD is an autoantigen.
SUMMARY OF THE INVENTION
Accordingly, a first aspect of the present invention provides a nucleic acid molecule comprising a nucleotide sequence which encodes or is complementary to a sequence which encodes human or mouse glutamic acid decarboxylase (GAD) corresponding to a 67K isoform of the enzyme or antigenically active fragments or derivatives thereof.
Another aspect of the present invention provides a synthetic peptide or polypeptide displaying the antigenicity of all or a portion of the 67K isoform of GAD or a fragment thereof and reactive with autoantibodies and/or T-cells.
Yet another aspect of the present invention contemplates a method for the detection of antibodies to GAD in a sample which method comprises contacting a peptide or polypeptide corresponding to all or an antigenic portion of the 67K isoform of GAD with said sample for a time and under conditions sufficient for a complex to form between the peptide or polypeptide and an antibody reactive to GAD and then detecting the complex
Still yet another aspect of the present invention provides a method for detecting diseases of the type including IDDM and SMS, or for screening asymptomatic individuals, by the detection and/or determination of the titre of autoantibodies in a biological sample from said individual, said method comprising contacting said sample with a peptide or polypeptide corresponding to all or an antigenic portion of the 67K isoform of GAD for a time and under conditions sufficient to form a complex between the peptide or polypeptide and an antibody reactive to GAD and then detecting the complex and/or the amount of peptide or polypeptide which has been bound in a complex.
Even yet another method of reducing autoantibodies and/or autoreactive T-cells to GAD in a patient in need thereof and/or to desensitise or induce tolerance to eliminate or diminish reactivity of autoreactive T-cells or autoantibodies to the autoantigen, said method comprising administering to said patient an effective amount of an antigenic peptide or polypeptide corresponding to all or part of the 67K isoform of GAD.
The present invention also provides a method of reducing autoantibodies and/or autoreactive T-cells to GAD in a patient in need thereof and/or to desensitise or induce tolerance to eliminate or diminish reactivity of autoreactive T-cells or autoantibodies to the autoantigen, said method comprising administering to said patient GAD reactive T-cell lines or clones or cell membranes and/or receptors for the antigen from said GAD reactive T-cell lines or clones for a time and under conditions sufficient to act as immunogens to induce inhibition and/or reduction of T-cells responses to GAD autoantigen.
REFERENCES:
patent: 5705626 (1998-01-01), Tobin et al.
patent: WO 92/04632 (1991-09-01), None
patent: WO 92/03733 (1992-03-01), None
patent: WO 92/05446 (1992-04-01), None
Sambrook et al., Molecular Cloning, A Laboratory Manual, Second Edition, 1989, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 16.3-16.4.*
Baekkeskov, et al. (Mar. 1987) “Antibodies to a 64,000 MrHuman Islet Cell Antigen Precede the Clinical Onset of Insulin-Dependent Diabetes”,J. Clin. Invest. 79:926-934.
Baekkeskov, et al. (Sep. 13, 1990) “Identification of the 64K Autoantigen in Insulin-Dependent Diabetes as the GABA-Synthesizing Enzyme Glutamic Acid Decarboxylase”,Nature 347:151-156.
Bond, et al. (May 1988) “Pattern of Expression of Glutamic Acid Decarboxylase mRNA in the Developing Rat Brain”,Proc. Natl. Acad. Sci. USA 85:3231-3234.
Chang, et al. (Jun. 1988) “Characterization of the Proteins Purified with Monoclonal Antibodies in Glutamic Acid Decarboxylase”,The Journal of Neuroscience 8(6):2123-2130.
Erlander, et al. (Jul. 1991) “Two Genes Encode Distinct Glutamate Decarboxylases”,Neuron 7:91-100.
Jackson, et al. (1990) “Drosophila GABAergic Systems: Sequence and Expression of Glutamic Acid Decarboxylase”,Journal of Neurochemistry 54(3) : 1068-1078.
Julien, et al. (1987) “Molecular Cloning, Expression and in situ Hybridization of Rat Brain Glutamic Acid Decarboxylase Messenger RNA”,Neuroscience Letters 73:173-180.
Julien, et al. (1990) “Rat Brain Glutamic Acid Decarboxylase Sequence Deduced from a Cloned cDNA”,Journal of Neurochemistry 54(2) : 703-705.
Katarova, et al. (1990) “Molecular Identification of the 62 kd Form of Glutamic Acid Decarboxylase from the Mouse”,European Journal of Neuroscience 2(3) : 190-202.
Kaufman, et al. (May 1986) “Brain Glutamate Decarboxylase Cloned in &lgr;-11: Fusion Protein Produces &ggr;-Aminobutyric Acid”,Science 232:1138-1140.
Kaufman, et al. (Jan. 1992) “Autoimmunity to Two forms of Glutamate Decarboxylase in Insul
Cram David
De Aizpurua Henry
Harrison Leonard
Honeyman Margot
Amrad Corporation Limited
Chan Christina Y.
Clemens Karen
Scully Scott Murphy & Presser
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