Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Using tissue cell culture to make a protein or polypeptide
Reexamination Certificate
1996-03-15
2001-05-29
Duffy, Patricia A. (Department: 1645)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Using tissue cell culture to make a protein or polypeptide
C435S326000, C435S331000, C530S388100
Reexamination Certificate
active
06238892
ABSTRACT:
The invention relates to new monoclonal antibodies directed against a particular epitope present on the human microtubule-associated protein tau, to the hybridoma secreting these monoclonal antibodies, to the process for diagnosing brain diseases involving the particular epitope of the tau protein, and to the monoclonal antibodies recognizing said epitope.
Tau is a microtubule-associated protein which is synthesized in the neurons (Kosik, K. S. et al., 1989) of several species, including humans and which is abundantly present in the axonal compartment of these neurons (Binder, L. I. et al., 1985). Functionally the tau protein is involved in the polymerization of tubulin (Weingarten, M. D. et al., 1975) and presumably in reducing microtubule instability (Bre, M. H. and Karsenti, E. 1990).
Tau protein is also the major constituent of paired helical filaments (PHF), characteristic structures found as neurofibrillary tangles in tissue sections of the brain of Alzheimer patients (Greenberg, S. and Davies, P., 1990; Lee, V. M.-Y. et al., 1991). 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, M. et al., 1989). The diversity of the isoforms is generated from a single gene by alternative mRNA splicing (Himmler, A., 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, M. et al., 1989).
Tau variants of 64 and 69 kDa, which are abnormally phosphorylated as revealed by the decrease in their molecular mass observed after alkaline phosphatase treatment, have been detected exclusively in brain areas showing neurofibrillary tangles and senile plaques (Flament, S. et al., 1989 and 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 a phosphorylation of Ser(405) which induced a lower electrophoretical mobility (Steiner, B. et al., 1990).
Several antibodies are reported that show reactivity to human tau either because they are directed to nonspecific phosphorylated epitopes present on neurofilament and subsequently shown to cross-react with normal and abnormally phosphorylated tau (Nukina, N. et al., 1987; Ksiezak-Reding et al., 1987) or because they recognized specific epitopes on normal and abnormally phosphorylated tau.
The Alz50 monoclonal antibody (Wolozin, B. L. et al., 1986; Nukina et al., 1988) recognizing a phosphate-independent epitope present on tau variants of bovine origin and of normal and abnormally phosphorylated tau from human origin (Ksiezak-Reding, H. et al., 1988, Flament, S. and Delacourte, A. 1990) belongs to the latter class of antibodies. The epitope recognized by this monoclonal is specifically expressed in the somatodendritic domain of degenerating cortical neurons during Alzheimer disease (Delacourte, A. et al., 1990).
The Alz50 epitope has recently been mapped to the NH
2
-terminal part of the tau molecule (Ksiezak-Reding, H. et al., 1990; Goedert, M. et al., 1991). Due to its cross-reactivity with normal tau, this antibody is only able to discriminate normal from abnormally phosphorylated tau by the use of Western blotting detection of brain homogenates or by ammonium sulfate-concentrated CSF, or else by using a sandwich immunoassay on brain homogenates (Ghanbari et al., 1990; Wolozin, B. and Davies, P. 1987; European patent publication (“EP”) 444 856). A CSF-based assay using antibodies directed against PHF was first described by Mehta et al., 1985, but shows considerable overlap between Alzheimer CSF and CSF from controls. The epitope recognized by this antibody was identified as part of ubiquitin (Perry et al., 1989).
Other monoclonal antibodies have been developed to recognize tau protein. For instance, monoclonal antibody 5E2 was raised by immunization with human fetal heat-stable microtubule-associated proteins and recognizes an epitope spanning amino acids 156-175 which is present in normal and abnormally phosphorylated tau (Kosik, K. S. et al., 1988).
Other antibodies such as tau 1 and several others were raised by immunization with bovine tau, bovine heat-stable microtubule-associated protein, or rat brain extracts (Binder, L. I. et al., 1985; Kosik, K. S. et al., 1988), and most of the antibodies recognize the normal and the abnormally phosphorylated tau (Ksiezak-Reding, R. et al., 1990).
An antibody named “423”, raised against the core of PHF, reacted specifically with a 9.5 and 12-kDa fragment of the tau protein, localized in the repetitive elements of tau, but recognized neither normal human tau nor the abnormally phosphorylated tau in Alzheimer's brain (Wischik, C. H. et al., 1988).
This antibody has been used to discriminate Alzheimer PHF pathology from normal controls in brain homogenates (Harrington, G. R. et al., 1990; patent WO89/03993).
Thus far, none of all the antibodies described heretofore has had an absolute specificity for the abnormally phosphorylated tau either by immunohistology, Western blotting, or ELISA. Quantitative measurements of normal and abnormally phosphorylated tau have until now only been able to detect tau in brain homogenates, in brain extracts containing PHF, or in concentrated CSF samples after Western blotting (Ghanbari H. A. et al., 1990; Harrington C. R. et al., 1990, Wisniewski, H. M. et al., 1989; Wolozin, B. and Davies, P. 1987).
The aim of the present invention is therefore to provide monoclonal antibodies which are specifically able to detect only abnormally phosphorylated tau present in brain tissue sections, in brain extracts, or in body fluids such as cerebrospinal fluid.
The invention also provides the hybridoma secreting such monoclonal antibodies.
The invention further provides the epitope of tau protein which is expressed in abnormally phosphorylated tau in brain tissue sections or in brain homogenates or in body fluids, such as cerebrospinal fluid, and which is recognized by such monoclonal antibodies.
The invention still further provides the epitope of tau protein expressed in the brain of patients affected with neurological disorders such as Alzheimer's disease and Down syndrome.
The invention yet further provides 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 abnormally phosphorylated human tau. The monoclonal antibodies are furthermore characterized by the fact that they form an immunological complex with abnormally phosphorylated human tau, and more specifically with a non-structural epitope present in abnormally phosphorylated human tau.
By “non-structural” epitopes is meant epitopes which depend for their antibody recognition both on their primary structure as well as on post-translational modifications and conformation in such a way that particular treatments (e.g. formalin fixation, detergent treatment, dephosphorylation) may alter or destroy the epitopes.
The expression “form an immunologically complex with” means that a monoclonal antibody of the invention binds to the aforementioned antigen under the conditions used in any one of the following techniques:
Light immunomicroscopy:
Brain tissue samples, obtained at surgery or autopsy, are fixed by immersion in 4% formalin or Bouin's fixative and embedded in paraffin. Four-mm-thick sections are prepared. The monoclonal antibodies of the invention are applied in conjunction with a technique to visualize the formed immune complexes such as the avidin-biotinylated peroxidase complex technique (Hsu, S. M., et al., 1981) using 3,3′-diaminobenzidine tetrahydrochloride for developmen
Mandelkow Eva-Maria
Mercken Marc
Van De Voorde Andre
Vandermeeren Marc
Vanmechelen Eugeen
Duffy Patricia A.
Merchant & Gould P.C.
N.V. Innogenetics S.A.
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