Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
1999-11-02
2002-12-31
Duffy, Patricia A. (Department: 1645)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C436S536000, C436S063000, C435S007100, C435S007920, C435S007930, C435S007940, C435S007950
Reexamination Certificate
active
06500674
ABSTRACT:
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. 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
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 (Iqbal et al., 1989; Lee et al., 1991; Hasegawa et al., 1992). In four of these sites, the phosphorylated residu is followed by a proline residu, indicating that a proline-directed kinase is involved in some of the abnormal phosphorylations of tau. In addition to these sites ten others are present in htau40, two of which are also abnormally phosphorylated, as indicated by antibody reactivity (Mab tau2: Watanabe et al., 1992; Mab AT8: Biernat et al., 1992, Goedert et al., 1993).
The abnormal phosphorylation of tau in Alzheimer's disease is due to a shift in the phosphatase/kinase equilibrium. In vitro several kinases can phosphorylate tau: cdc2-kinases (Vulliet et al, 1992; Ledesma et al, 1992), MAP kinases (Drewes et al, 1992, Roder and Ingram, 1991), glycogen synthase kinases (Mandelkow et al, 1992) and TPKI and TPKII (Ishiguro et al, 1992). The phosphatases are less well studied in Alzheimer's disease and so far only one phosphatase was able in vitro to dephosphorylate the abnormally phosphorylated sites, namely protein phosphatase 2A
1
(Goedert et al, 1992).
So far, the detection of PHF-tau in brain extracts, either via antibodies (Mab Alz50: Ghanbari et al., 1990; Mab Ab423: Harrington et al., 1991), or via the change in molecular weight (Flament et al., 1990, Delacourte et al., 1993), or else by functional assay (Bramblett et al. 1992) has been very useful to discriminate dementia with altered cytoskeletal properties from normal aged subjects or from patients with other types of dementia. Nevertheless the detection of PHF-tau in CSF remained impossible, even using antibodies directed at one of the abnormally phosphorylated sites such as serine 202 (Goedert et al., 1993). This can be ascribed to one or more of the following reasons: 1) the low concentration of PHF-tau in CSF, 2) non-even use of phosphorylation sites among all the potential phosphorylation sites, 3) differences in phosphatase sensitivity of these sites, and, 4) too low an affinity constant of the antibodies used.
The aim of the present invention is therefore to provide monoclonal antibodies which allow the reliable and sensitive detection of abnormally phosphorylated tau present in cerebrospinal fluid.
The invention also aims at providing the hybridomas which secrete the above-said monoclonal antibodies.
The invention furthermore aims at providing the epitopes of the abnormally phosphorylated tau protein present in brain homogenates or in body fluids such as cerebrospinal fluid, which are recognized by said monoclonal antibodies.
Finally, the invention aims at providing a process for the detection or diagnosis in vitro of brain diseases involving abnormally phosphorylated tau proteins.
The present invention relates more particularly to a monoclonal antibody which forms an immunological complex with a phosphorylated epitope of an antigen belonging to abnormally phosphorylated tau (PHF-tau) residing in the region spanning positions 143-254 with the following amino acid sequence:
(SEQ ID NO 1)
143 150
NH
2
- Lys Gly Ala Asp Gly Lys Thr Lys Ile Ala
Thr
160
Pro Arg Gly Ala Ala Pro Pro Gly Gln Lys Gly Gln
170
Ala Asn Ala Thr Arg Ile Pro Ala Lys
Thr
Pro Pro
180
Ala Pro Lys
Thr
Pro Pro Ser Ser Gly Glu Pro Pro
190 200
Lys Ser Gly Asp Arg Ser Gly Tyr Ser
Ser
Pro Gly
&em
Van De Voorde Andre
Vandermeeren Marc
Vanmechelen Eugeen
Duffy Patricia A.
Greer Burns & Crain Ltd.
Innogenetics N.V.
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