Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1995-06-07
2002-09-24
Saunders, David (Department: 1644)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007400, C435S975000, C436S506000, C530S326000
Reexamination Certificate
active
06455267
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to glutamic acid decarboxylase
65
(GAD
65
) polypeptides and methods of using GAD
65
polypeptides diagnostically and therapeutically in autoimmune disease.
2. Description of the Background Art
Insulin-dependent diabetes mellitus (IDDM; type I diabetes) is one of the most common metabolic disorders. In the United States, IDDM affects approximately one in 300 to 400 people, and epidemiological studies suggest that its incidence is increasing. The disease results from the autoimmune destruction of the insulin-producing &bgr;-cells of the pancreas. More specifically, the preonset stage is characterized by “insulitis”, in which lymphocytes infiltrate the pancreatic islets and selectively destroy the &bgr;-cells. Insulitis may be present for many years before the onset of clinical symptoms. The typical IDDM presentation of hyperglycemia appears only fter at least 80% of the insulin-producing &bgr;-cells are lost. The remaining &bgr;-cells are destroyed during the next few years.
Although insulin therapy allows most IDDM patients to lead normal lives, this replacement is imperfect and does not completely restore metabolic homeostasis. Thus, severe complications which result in dysfunctions of the eye, kidney, heart, and other organs are common in IDDM patients undergoing insulin therapy. Because of this, it is highly desirable to extend the latency period and prevent progression (e.g., through administration of immunosuppressant drugs to interfere with the autoimmune process and insulin to achieve better control of the effects of sustained hypoglycemia) between the start of &bgr;-cell destruction and the actual requirement of insulin replacement (i.e., when 80% of the &bgr;-cells are destroyed). Therefore, a diagnostic test which determines the beginning of &bgr;-cell destruction would allow the clinician to administer immunosuppressant drugs (Silverstein, et al.,
New England Journal of Medicine
, 319:599-604, 1988) or prophylactic insulin therapy (Keller, et al.,
Lancet
, 341:927, 1993) to extend this latency period and thus significantly delay the onset of insulin replacement side effects.
Many IDDM patients have sera which contain antibodies to a 64 kD molecule (Baekkeskov, et al.,
J.Clin.Invest
., 79:926-934, 1987; Atkinson, et al.,
Lancet
, 335:1357-1360, 1990), to islet cell cytoplasmic (ICA) molecules or islet cell surface (ICSA) molecules (Bottazzo, et al,
Lancet
, 1:668-672, 1980), or to insulin (Palmer, et al.,
Science
, 222:1137-1139, 1983; Atkinson, et al.,
Diabetes
, 35:894-898, 1986). Atkinson and coworkers (Atkinson, et al.,
Lancet
, 335:1357-1360, 1990) have demonstrated that the presence of antibodies to the 64 kD molecule in human sera appears to be the earliest and most reliable indicator that onset of IDDM symptoms will eventually occur.
Recently, Baekkeskov and coworkers established that the 64 kD molecule and glutamic acid decarboxylase (GAD) have several antigenic epitopes in common and thus they may be identical or very similar molecules. Although this identiffication is an important finding, the use of this information as a diagnostic tool for predicting IDDM is quite. cumbersome and limited unless knowledge of the molecular biology of GAD is known. Studies by Kaufman, et al., (
J. Clin. Invest
., 89:283, 1992) established that the 64 kD molecule was intact GAD
65
. Consequently, the cloning and subsequent production of large quantities of GAD
65
or a GAD molecule which is antigenically substantially identical to the GAD
65
molecule or fragments of the GAD
65
molecule, both of which can be easily purified, will allow the development of a diagnostic kit designed to predict IDDM as well as effective therapeutic modalities. The present invention provides a means for accomplishing these results.
SUMMARY OF THE INVENTION
The present invention arose out of the discovery that recombinant DNA technology could be used to produce eukaryotic GAD
65
polypeptide and that GAD
65
polypeptide could be used in the diagnosis and therapy of patients with autoimmune disease. Particularly relevant is the use of eukaryotic GAD
65
polypeptide in the diagnosis and therapy of patients having, or at risk of having, GAD-associated autoimmune disorders such as insulin-dependent diabetes mellitus (IDDM) or stiff man disease.
A major advantage of the present invention is that it provides the art with a ready source of eukaryotic GAD
65
polypeptide corresponding to that purified from natural sources, while avoiding the problems associated with the isolation of naturally occurring eukaryotic GAD
65
polypeptide when separating it from other eukaryotic non-GAD
65
polypeptides. This absence of other eukaryotic non-GAD
65
polypeptides is significant in that it allows the development of test systems which will only detect antibodies specifically reactive with GAD
65
polypeptides.
Another advantage of providing eukaryotic GAD
65
polypeptide in host cells is that by so doing, it is possible to obtain much larger quantities of the polypeptide than are currently practicably available from natural sources. As a consequence, not only is it possible to use the polypeptide of the invention to more accurately classify and treat patients with such autoimmune diseases as IDDM, but it is also now possible to provide commercially useful quantities of GAD polypeptide for use in diagnostic systems and pharmaceutical compositions.
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Legay et al., “Evidence for Two Distinct Forms of Native Glutamic Acid Decarboxylase in Rat Brain Soluble Extract: An Immunoblotting Study”, J. of Neurochemistry (1987), 48:1022-1026.
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Erlander Mark G.
Kaufman Daniel L.
Tobin Allan J.
Burns Doane Swecker & Mathis L.L.P.
Diamyd Medical AB
Saunders David
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