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Purified antigen for alzheimer's disease and methods of obtaining and using same

Purified antigen for alzheimer's disease and methods of obtaining and using same description/claims

The invention relates to a preparation comprising Alzheimer's disease antigen (A68), to methods of obtaining this purified antigen, and to methods of using this purified antigen preparation, for instance, in diagnosing Alzheimer's disease. The antigen preparation according to the invention is purified in that it is substantially free of immunoglobulin G. The invention further relates to methods of making Alzheimer disease antigens that can be used instead of or along with the A68 antigen preparation (e.g., for diagnosing AD), such as recombinant human tau, tau isolated from various species including human, and phosphorylated recombinant human tau or isolated tau, as well as A68 anti-idiotypic antibodies.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder affecting 7% of the population over 65 years of age and characterized clinically by progressive loss of intellectual function and pathologically by a continuing loss of neurons from the cerebral cortex. This pathological impairment usually is correlated with increased numbers of neuritic plaques in the neocortex and with the loss of presynaptic markers of cholinergic neurons. Neuritic plaques are composed of degenerating axons and nerve terminals, as well as possible astrocytic elements, and these plaques often exhibit a central amyloid core.

Another characteristic pathological feature of Alzheimer's disease is development of neurofibrillary tangles. A neurofibrillary tangle is an intraneuronal mass composed of normal intermediate filaments and paired helical filaments having unusual properties, which twist and form tangles. Neurofibrillary tangles are comprised of several different proteins.

Neurochemical studies confirm that neurotransmitter systems are deleteriously affected by Alzheimer's disease. The most consistently and severely affected system is that of the cholinergic neurons located in the Nucleus Basalis of Meynert. In addition, a reduction in somatostatin, substance P, and corticotropin releasing factor are observed.

None of the above-mentioned pathologic states such as neurochemical alterations, neuritic plaques or neurofibrillary tangles are unique to Alzheimer's disease. These impairments also occur in the brains of normal aged individuals and are associated with other diseases such as Guam Parkinson's Disease, Dementia Pugilistica and Progressive Supra-nuclear Palsy. For example, paired helical filaments, the twisted filaments that form the tangles and fill the neurites of plaques, also occur in certain other diseases. In fact, immunologic studies have shown that AD epitopes of paired helical filaments exist in Pick bodies, the spherical structures found in affected neurons in the temporal cortex of brains affected by Pick's Disease. In addition, the densities of neurofibrillary tangles and neuritic plaques within the cerebral cortex of an Alzheimer's disease patient correlates only weakly with the stages of the illness.

Accordingly, the diagnosis of Alzheimer's disease has been extremely difficult. Ante-mortem diagnosis of the disease is performed primarily by exclusion of other diseases. An article entitled, “The Neurochemistry of Alzheimer's Disease and Senile Dementia”, by Peter Davies in Medicinal Research Reviews, Vol. 3, No. 3, pp. 221-236 (1983), discusses Alzheimer's disease and at page 223 states: The problem in the diagnosis of Alzheimer's disease is that there is no positive test: the clinician has to rule out other causes of dementia such as strokes, microvascular disease, brain tumors, thyroid dysfunction, drug reactions, severe depression and a host of other conditions that can cause intellectual deficits in elderly people. Only when all of these problems have been eliminated as a cause of the symptoms should a diagnosis of Alzheimer's disease be accepted.

Post-mortem diagnosis of Alzheimer's disease has been based on determination of the number of neuritic plaques and tangles in brain tissue using specialized staining techniques. However, such diagnostic methods, based on neurohistopathological studies, require extensive staining and microscopic examination of several brain sections. Moreover, the plaques and tangles are not confined to individuals having Alzheimer's disease, but also may occur in the brains of normal, elderly individuals or individuals with other diseases. Thus, a more definitive and reliable method for making the diagnosis is needed.

U.S. Pat. No. 4,666,829 issued to Glenner et al. discloses attempts to identify an antigen specific for Alzheimer's disease. However, the antigen described by Glenner et al. also is present in adults of advanced age who do not have Alzheimer's disease (see Ghanbari et al., Journal of the American Medical Association, 263, pp. 2907-2910 (1990)). Therefore, a need still exists for a method of diagnosing Alzheimer's disease as distinct from other diseases or age-related indicia.

Similarly, U.S. Pat. No. 5,492,812 issued to Voorheis et al. describes a diagnostic method for Alzheimer's disease that is carried out by screening for tau peptides in the blood of a patient. This method calls for the use of an antibody or Fab fragment that specifically binds tau peptide derived from either the amino terminal 200 amino acids or carboxy terminal 50 amino acids of a tau protein. This method requires that “the whole of the 200 amino acid N-terminal residues of the various tau proteins as well as some portion of their 50 amino acid most C-terminal residues will be released when cleaved from the filaments by ubiquitin-recognizing proteases or other proteases during degeneration and rupture of the affected neurons” (col. 5, lines 12-19). The method further requires that the cleaved segments find their way into body fluids outside the brain (col. 5, lines 19-22). Accordingly, the method is dependent upon, and is ineffective in the absence of, proteolytic fragmentation of the tau complex of proteins. The method further is dependent upon, and is ineffective in the absence of, the subsequent release of the proteolytic fragments into body fluids. It thus is desirable that a direct means of assay for Alzheimer disease-associated antigens be identified, particularly a means that does not require proteolytic fragmentation and subsequent release into the bloodstream of fragments.

Along these lines, PCT International Application WO 96/20218 of Ghanbari et al. describes the isolation of an antigen associated with Alzheimer's disease and a monoclonal antibody directed against this antigen. This antigen is specific for Alzheimer's disease, being present in high quantities in Alzheimer's Disease patients, and being nearly non-detectable in non-Alzheimer's Disease patients. However, the antigen is described as being only “partially purified” in the preparation of Ghanbari et al., consisting of an aggregate of proteins, with the predominant protein having a molecular weight of about 68,000 daltons, and including tau and hyperphosphorylated tau (see, e.g, Example 2). Accordingly, for some applications, a more purified preparation of this Alzheimer's Disease antigen may be desirable and/or required.

It therefore is an object of this invention to provide, among other things, a purified preparation of an Alzheimer's disease antigen. It is another object of this invention to provide, among other things, a method of diagnosing Alzheimer's disease using the purified preparation. It is a further object of this invention to provide, methods of obtaining the purified preparation of the Alzheimer's disease antigen. These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.

The invention provides, among other things, a preparation comprising Alzheimer's disease antigen (A68), as well as methods of obtaining this purified antigen (Ag), and methods using the purified Ag, for instance, for diagnosing Alzheimer's Disease (AD). This Ag is purified in that it is substantially free of immunoglobulin G (IgG). The invention additionally provides methods of making AD Ags that can be used instead of or along with A68 (e.g., for binding AD autoantibodies), such as recombinant human tau, tau isolated from various species including human, and phosphorylated recombinant human tau or isolated tau, as well as A68 anti-idiotype antibodies (Abs). The invention further describes treatments of these Ags that enhance their reactivity with autoantibodies directed against A68. These treatments include treatment with hypericin, free fatty acids, and/or hydroxynonenal or other advanced glycation end products.

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