Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 25 or more amino acid residues in defined sequence
Reexamination Certificate
1999-07-07
2001-05-15
Duffy, Patricia A. (Department: 1645)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
25 or more amino acid residues in defined sequence
C530S327000, C530S329000, C530S402000
Reexamination Certificate
active
06232437
ABSTRACT:
The invention relates to new monoclonal antibodies directed against the human microtubule-associated protein tau, to the hybridomas secreting these monoclonal antibodies, and to the antigen recognition by these monoclonal antibodies and their applications. The invention also relates to a process for diagnosing brain diseases involving the particular epitope (of the tau protein) which is recognized by said monoclonal antibodies.
Alzheimer's disease (AD) is the most common form of adult-onset dementia. At present, no biochemical test is available for antemortem diagnosis of AD. The disease is therefore clinically diagnosed primarily by exclusion of other forms of dementia. The illness is characterized neuropathologically by the presence of neuritic (senile) plaques and neurofibrillary tangles (NFT).
Neurofibrillary tangles consist of paired helical filaments (PHF), of which the main protein component is a modified form of the microtubule-associated protein tau (Brion et al., 1985; Greenberg and Davies, 1990; Lee et al.,1991), which under normal circumstances promotes microtubule assembly and stability (Weingarten et al., 1975; Bré and Karsenti, 1990), which is synthesized in the neurons of several species, including humans (Kosik et al., 1989) and which is abundantly present in the axonal compartment of these neurons (Binder et al., 1985).
The protein exists as a family of different isoforms of which 4 to 6 isoforms are found in normal adult brain but only 1 isoform is detected in fetal brain (Goedert et al., 1989). The diversity of the isoforms is generated from a single gene by alternative mRNA splicing (Himmler, 1989). The most striking feature of tau protein, as predicted from molecular cloning, is a stretch of 31 or 32 amino acids occurring in the carboxy-terminal part of the molecule that is repeated 3 or 4 times. Additional diversity is generated through 29 or 58 amino acid-long insertions in the NH
2
-terminal part of the molecules (Goedert et al., 1989).
Tau variants of 64 and 69 kDa, which are abnormally phosphorylated, as revealed by the apparent increase in their molecular mass observed after alkaline phosphatase treatment, have been detected exclusively in brain areas showing neurofibrillary tangles and senile plaques (Flament et al., 1989, 1990). The sites of phosphorylation by 4 different kinases have been mapped in the C-terminal microtubule-binding half of tau, and it could be shown that the action of a calcium calmodulin-dependent kinase on bacterially expressed tau resulted in the phosphorylation of Ser(405) which induced a lower electrophoretical mobility (Steiner et al., 1990). Tau present in paired helical filaments, called PHT-tau is abnormally phosphorylated (Lee et al., 1991). This abnormal phosphorylation causes a conformational change in tau, resulting probably in self-association and the formation of PHFs. PHF-tau in AD is phosphorylated at several sites, one of which is the phosphoserine 199 and/or 202. This site is specifically recognized by a mAb called AT8 (Biernat et al., 1992). Therefore, AT8 is a discriminative marker for PHF-tau (Goedert et al., 1992).
Several antibodies have been reported that show reactivity to human tau either because they are directed to non-specific phosphorylated epitopes present on neurofilament and subsequently shown to cross-react with normal and abnormally phosphorylated tau (Nukina et al., 1987; Ksiezak-Reding et al., 1987) or because they recognized specific epitopes on normal and abnormally phosphorylated tau (Kosik et al., 1988). In addition to the tau antibodies directed towards non-specific epitopes, antibodies directed specifically to phosphorylated tau epitopes have been described (Mercken et al., 1992b).
Although overall tau mRNA levels are only slightly modulated in Alzheimer-affected brain regions (Goedert et al., 1988; Barton et al., 1990) it has been shown that total tau protein levels may differ at least 6-fold (Khatoon et al., 1992). This has been demonstrated by polyclonal antibodies against tau (Flament and Delacourte, 1990) and by monoclonal antibodies directed to well-defined epitopes. The Alz 50 monoclonal antibody recognizing a phosphate-independent epitope in the N-terminus of the tau molecule (Goedert et al., 1991) has been used in a sandwich immunoassay on brain homogenates and it has been shown that tau levels are higher in Alzheimer's patient brains (Ghanbari et al., 1990; patent application EP 444 856).
An antibody named “423”, raised against pronase-treated PHFs and specifically reactive with a 9.5 kDa and a 12 kDa fragment was also used to measure tau protein in Alzheimer's disease (patent application WO 89/03993). Similarly, it was found that increased mAb 423 immunoreactivity was observed in Alzheimer brain homogenates as compared with control brain homogenates.
Mercken et al. (1992b) describe a range of monoclonal antibodies which are either specific for a phosphatase-sensitive epitope (AT8) or which react with PHF-tau as well as with normal tau (AT1, AT4, AT6, AT9, ATI 1, AT12 and AT14) in Western blotting.
Moreover, the antibody tau 1 (Wischik et al., 1988; Harrington et al., 1990) was also used to measure tau in brain homogenates. In one case when tau levels were specifically measured in Alzheimer-affected brain sections, tau levels were eight-fold higher as compared with levels in normal brain homogenates (Khatoon et al., 1992).
In a first attempt to diagnose Alzheimer disease in cerebrospinal fluid, the PHF-tau-specific monoclonal antibody AT8 (Mercken et al., 1992b), was used. However, no PHF tau antigen could be demonstrated.
Thus far, none of the monoclonals that have been described have been successful in detecting tau in non-concentrated cerebrospinal fluid (CSF), although the presence of tau was observed in 100-fold concentrated CSF (Wolozin and Davies, 1987) or in CSF samples using polyclonal antibodies (Delacourte and Vermersch, 1991).
The aim of the present invention is therefore to provide monoclonal antibodies which allow reliable and sensitive detection of normal and abnormally phosphorylated tau present in brain extracts and in unconcentrated cerebrospinal fluid. The invention also aims at providing the hybridoma which secretes the above-said monoclonal antibodies.
The invention furthermore aims at providing the epitope of the tau protein present in brain homogenates or in body fluids such as cerebrospinal fluid, which is recognized by said monoclonal antibodies.
The invention aims at providing a process for the detection or diagnosis in vitro of brain diseases involving tau protein.
The monoclonal antibodies of the invention are characterized by the fact that they react with an epitope which is present in both normal and abnormally phosphorylated human tau protein. The monoclonal antibodies are furthermore characterized by the fact that they form an immunological complex with an epitope or an antigen belonging to normal and abnormally phosphorylated human tau protein. The monoclonal antibodies of the invention are also characterized by the fact that they do not form an immunological complex with other phosphorylated proteins such as MAP-1, MAP-2 and neurofilaments which share part of their sequence with tau protein (Nukina et al., 1987; Lewis et al., 1988) as determined by means of an ELISA. The monoclonal antibodies of the invention are also characterized by the fact that they are liable to detect human tau protein in CSF, with said tau protein being at a concentration as low as 1.0 pg/ml and with said tau protein being detected at 100% recovery upon the addition of a fixed amount of tau protein in tau protein-negative CSF (100% spiking recovery).
The monoclonal antibodies of the invention also enable the diagnosis of Alzheimer's disease (AD) on the basis of CSF, i.e., to detect tau and modified forms of tau in CSF. The problem associated herewith is that this antigen is present in a very low amount in CSF, therefore the detection assay must be very sensitive. This problem can be resolved by using the combination of the monoclonal antibody of th
Mercken Marc
Van De Voorde Andre
Vandermeeren Marc
Vanmechelen Eugeen
Bierman, Muserlian and Lucas
Duffy Patricia A.
N.V. Innogenetics S.A.
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