Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Using tissue cell culture to make a protein or polypeptide
Patent
1996-06-20
1999-12-28
Duffy, Patricia A.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Using tissue cell culture to make a protein or polypeptide
435331, 436548, 5303879, 5303881, C12P 2104, C12P 2108, C12N 500, C07K 1600
Patent
active
060080248
DESCRIPTION:
BRIEF SUMMARY
The invention relates to new monoclonal antibodies specific for PHF-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 monoclonal antibodies of the invention, more particularly in cerebrospinal fluid (CSF) samples. 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 Alzheimer's disease or with other types of dementia and which is specifically recognized by the monoclonal antibodies 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 therefore diagnosed clinically on the basis of exclusion of other forms of dementia. The diagnosis can be confirmed neuropathologically by the demonstration of large amounts of neuritic (senile) plaques and neurofibrillary tangles (NFT) in particular brain regions (McKhann et al, 1984).
Neurofibrillary tangles consist of paired helical filaments (PHFs). Immunocytochemical evidence suggests that 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). Definite proof that the tubulin-binding domain of tau is tightly associated with the core of PHFs was obtained via amino acid sequencing (Kondo et al., 1988). Nevertheless it has been suggested that tau peptides may represent only a small portion of the major component of PHF (Wischik 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 (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 tau variant htau40 containing all exons (441 amino acids long) according to Goedert et al (1989).
Under normal circumstances tau promotes microtubule assembly and stability in the axonal compartment of neurons. The microtubule-binding domain in tau is localized in the repeat region of tau (255-381) (Lewis et al, 1990) and is modulated by adjacent regions: the carboxyterminal tail (382-414) and the proline-rich region (143-254) (Drubin & Kirschner, 1991). Stability and bundling of the microtubules is mediated by a short hydrophobic zipper in the carboxyterminal tail of tau (Lewis et al, 1989). Both assembly and stability are regulated by alternative mRNA splicing and phosphorylation.
In normal circumstances adult brain contains 2 to 3 mol phosphate per mole of tau (Selden and Pollard, 1983; Ksiezak-Reding et al, 1992) present amongst others at serine 404 (Poulter et al, 1993), while other results demonstrate that phosphorylation of different sites in normal tau follows different developmental profiles (Lee et al, 1991; Bramblett et al, 1993; Goedert et al, 1993a). Abnormal tau variants of 60, 64 and 68 kDa have been detected exclusively in brain areas showing neurofibrillary changes and senile plaques (Delacourte et al, 1990). The abnormal electrophoretical behavior of tau is due to phosphorylation since alkaline phosphatase treatment of these tau molecules changes their molecular mass to that of normal tau (Goedert et al., 1992; Flament et al., 1990b, Greenberg & Davies, 1990). Currently abnormal phosphorylation sites have been detected in PHF-tau at positions 46, 231, 235, 263 and 396
REFERENCES:
Goedert et al., "The abnormal phosphorylation of tau protein at Ser-202 in Alzheimer disease recapitulates phosphorylation during development", Proceedings of the National Academy of Sciences of the USA, vol. 90, No. 11, Jun. 1, 1993, Washington, D.C., pp. 5066-5070.
Ghanbari et al., "Detection and Measurement of Alzheimer's Disease-Associated Protein (ADAP) in Cerebrospinal Fluid (CSF): An Antemortem Marker for Alzheimer Disease", Society for Neuroscience, Symposia, vol. 17, 1991, Bethesda, MD, p. 1259.
Goedert et al., "Cloning and sequencing of the cDNA encoding an isoform of microtubule-associated protein tau containing four tandem repeats: differential expression of tau protein mRNAs in human brain", The Embo Journal, vol. 8, No. 2, Feb. 1989, Oxford, GB, pp. 393-399.
Vandermeeren et al., "Detection of t Proteins in Normal and Alzheimer's Disease Cerebrospinal Fluid with a Sensitive Sandwich Enzyme-Linked Immunosorbent Assay", Journal of Neurochemistry, vol. 61, No. 5, Nov. 1993, New York, NY pp. 1828-1834.
Vandermeeren et al, (Journal of Neurology, 61: 1828-1834, 1993.
Mercken et al, Acta Neuropathol, 84: 265-272, 1992.
Hasegawa et al, Journal of Neurochemistry, 60: 2068-2077, 1993.
Harlow et al "Antibodies, A Laboratory Manual", Cold Spring Harbor Laboratory Press, 1988 pp. 271-276.
Van De Voorde Andre
Vandermeeren Marc
Vanmechelen Eugeen
Duffy Patricia A.
Innogenetics N.V.
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