Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1997-03-24
2000-09-19
Duffy, Patricia A.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 792, 435331, 435975, 436547, 436548, 436811, 436503, 5303879, 5303881, G01N 3353, C07K 1600, C12P 2108
Patent
active
06121003&
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to the field of diagnosis of Alzheimer's disease.
The invention relates to new monoclonal antibodies specific for a particular type of phosphorylated epitopes present in a particular form or subclass of phosphorylated tau, to the hybridomas secreting these monoclonal antibodies, and to the antigen recognition pattern of these monoclonal antibodies and their applications. The invention also relates to a process for diagnosing brain diseases involving the monoclonal antibodies of the invention, more particularly in brain and body fluid such as cerebrospinal fluid (CSF). The invention also relates to a region of the tau molecule modifiable in vivo by the process of phosphorylation, which is found to be associated with the formation of "neurofibrillary tangles" (NFT) and "paired helical filaments" (PHF) as they occur in several types of dementia and which is specifically recognized by the monoclonal antibodies of the invention.
BACKGROUND OF THE INVENTION
Alzheimer's disease (AD) is the most common form of adult-onset dementia. At present, no reliable biochemical test is available for antemortem diagnosis of AD. The disease is usually diagnosed clinically on the basis of exclusion of other forms of dementia. The diagnosis can only be confirmed irrevocally by neuropathologic examination and the demonstration of large amounts of neuritic (senile) plaques and neurofibrillary tangles (NFT) in particular brain regions (McKhann et al, 1984), the latter appearing to correlate better with the severity and the duration of AD.
Neurofibrillary tangles consist of paired helical filaments (PHF). The microtubule-associated protein tau is a major protein component of PHF and NFT (Brion et al., 1985b; Delacourte and Defossez, 1986; Grundke-Iqbal et al., 1986; Kosik et al., 1986; Wood et al., 1986; Kondo et al., 1988).
Tau protein exists in different isoforms, of which 4 to 6 are found in adult brain but only 1 isoform is detected in fetal brain. The diversity of the isoforms is generated from a single gene on human chromosome 17 by alternative mRNA splicing (Himmler, 1989; Goedert et al., 1989; Andreadis et al., 1992). The most striking feature of tau protein, as deduced from molecular cloning, is a stretch of 31 or 32 amino acids, occurring in the carboxy-terminal part of the molecule, which can be repeated either 3 or 4 times. Additional diversity is generated through 29 or 58 amino acid-long insertions in the NH.sub.2 -terminal part of tau molecules (Goedert et al., 1989). For simplicity, all numbering in this patent application refers to the human tau variant htau40 containing all exons (441 amino acids long) according to Goedert et al (1989).
In vivo tau promotes microtubule assembly and stability in the axonal compartment of neurons by interactions involving its microtubule binding domain which is localized in the repeat region of tau (255-381) (Lewis et al, 1990).
In normal circumstances adult brain contains 2 a 3 mol phosphate per mole of tau (Selden and Pollard, 1983; Ksiezak-Reding et al, 1992). Phosphorylation of different sites in normal tau as studied in rat and humans is dependent on the developmental state (Lee et al, 1991; Bramblett et al, 1993; Goedert et al, 1993a). Tau variants of 60, 64 and 68 kDa arising as a consequence of phosphorylation have been detected in brain areas showing neurofibrillary tangles (Delacourte et al, 1990; Goedert et al., 1992; Flament et al., 1990b, Greenberg & Davies, 1990). In tau isolated from PHF (PHF-tau), phosphorylation can occur at several positions (Iqbal et al., 1989; Lee et al., 1991; Hasegawa et al., 1992).
Sofar, the detection of PHF-tau in brain extracts, either via antibodies (Mab Alz50: Ghanbari et al., 1990; Mab Ab423: Harrington et al., 1991; Mab AT120: Vandermeeren et al., 1993; Mab AT180; Mab AT270: International application No. PCT/EP 94/04146 filed on Dec. 14, 1994 and Mab AT8: International application published under WO 93/08302), or via the change in molecular weight (Flament et al., 1990, Delacourte
REFERENCES:
Harlow et al, "Antibodies A Laboratory Manual", Cold Spring Harbor Laboratory 1988, pp. 274-275.
Mercken et al., "Monoclonal Antibodies with Selective Specificity for Alzheimer Tau are Directed Against Phosphate-Sensitive Epitopes," pp. 265-272, Acta Neuropathologica, vol. 84, No. 6, Berlin, Germany (1992).
Drewes et al., "Mitogen Activated Protein (MAP) Kinase Transforms Tau Protein into Alzheimer-like State," pp. 2131-2138, The EMBO Journal, vol. 11, No. 6, Oxford, England (Jun. 1992).
Roder et. al., "Brain Protein Kinase PK40erk Converts Tau into a PHF-like Form as Found in Alzheimer's Disease," pp. 639-647, Biochemical and Biophysical Research Communications, vol. 193, No. 2, Duluth, Minnesota (Jun. 15, 1993).
Hanger et al., "Glycogen Synthase Kinase-3 Induces Alzheimer's Disease-like Phosphorylation of Tau: Generation of Paired Helical Filament Epitopes and Neuronical Localization of the Kinase," pp. 58-62, Neuroscience Letters, vol. 147, No. 1, Shannon, Ireland (Nov. 23, 1992).
Vandermeeren et al., "Detection of Tau Proteins in Normal and Alzheimer's Disease Cerebrospinal Fluid with a Sensitive Sandwich Enzyme-linked Immunosorbent Assay," pp. 1828-1834, Journal of Neurochemistry, vol.61, No. 8, New York, New York (Nov. 1993).
Lichtenberg-Kragg et al., "Phosphorylation-dependent Epitopes of Neurofilament Anitbodies on Tau Protein and Relationship with Alzheimer Tau," pp. 5384-5388, Proceedings of the National Academy of Sciences of the USA, vol. 89, No. 12, Washington, DC (Jun. 1992).
Van De Voorde Andre
Vanmechelen Eugeen
Duffy Patricia A.
Innogentics N.V.
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