The invention relates to epitopes of the tau protein which are specifically occurring in a phosphorylated state in tau protein from Alzheimer paired helical filaments, to protein kinases which are responsible for the phosphorylation of the amino acids of the tau protein giving rise to said epitopes, and to antibodies specific for said epitopes. The invention further relates to pharmaceutical compositions for the treatment or prevention of Alzheimer's disease, to diagnostic compositions and methods for the detection of Alzheimer's disease and to the use of said epitopes for the generation of antibodies specifically detecting Alzheimer tau protein. Additionally, the invention relates to methods for testing drugs effective in dissolving Alzheimer paired helical filaments or preventing the formation thereof.

Description of the Invention

The invention relates to epitopes of the tau protein which are specifically occurring in a phosphorylated state in tau protein from Alzheimer paired helical filaments, to protein kinases which are responsible for the phosphorylation of the amino acids of the tau protein giving rise to said epitopes, and to antibodies specific for said epitopes. The invention further relates to pharmaceutical compositions for the treatment or prevention of Alzheimer's disease, to diagnostic compositions and methods for the detection of Alzheimer's disease and to the use of said epitopes for the generation of antibodies specifically detecting Alzheimer tau protein. Additionally, the invention relates to methods for testing drugs effective in dissolving Alzheimer paired helical filaments or preventing the formation thereof.

The brains of Alzheimer patients contain two characteristic types of protein deposits, the plaques and the tangles. These structures have been of peak importance in Alzheimer research during the last few years (for a recent review see Goedert et al., Current Opinion in Neurobiology 1 (1991), 441 to 447). A prominent component of the tangles are the paired helical filaments (PHFs). It seems now clear that the PHFs are largely made up of the microtubule-associated protein tau which is normally attached to the neuronal microtubule network and, furthermore, particularly enriched in the axons.

There are six isoforms of tau in human brain that arise from alternative splicing of a single gene. All these isoforms also occur in PHFs (Goedert et al., Neuron 3 (1989), 519-526). The main biochemical differences between normal and Alzheimer PHF tau protein known so far may be summarized as follows: (1) PHF tau protein is, in contrast to normal tau protein, highly insoluble which makes a biochemical analysis difficult; (2) PHF tau protein reacts with certain antibodies in a phosphorylation dependent manner, suggesting a special phosphorylation status (Grundke-Iqbal et al., Proc. Natl. Acad. Sci. USA 83 (1986), 4913-4917, Nukina et al., Proc. Natl. Acad. Sci. USA 84 (1987), 3415-3419); (3) PHF tau protein has a lower electrophoretic mobility in SDS gels, suggesting a higher M.sub.r value which may be related to its phosphorylation pattern (Steiner et al., EMBO J. 9 (1990), 3539-3544); (4) PHF tau protein forms paired helical filaments with a characteristic 78 nm crossover repeat (Crowther and Wischik, EMBO J. 4 (1985), 3661-3665).

Tau protein purified from brain has very little secondary structure (as judged by CD spectroscopy), and a sedimentation constant of 2.6S, pointing to a highly asymmetric shape (Cleveland et al., J. Mol. Biol. 1161 (1977), 227-247, in agreement with electron microscopic data (Hirokawa et al., J. Cell. Biol. 107 (1988), 1449-1459. The C-terminal half contains 3 or 4 internal repeats which are involved in microtubule binding and promoting their assembly (hence "assembly domain"). This domain can be phosphorylated by several protein kinases (Steiner et al., EMBO J. 9 (1990), 3539-3544), a point that may be significant in view of the abnormal phosphorylation of Alzheimer tau (see, e.g. Grundke-Iqbal et al., ibid.). Moreover, the repeat region also lies in the core of Alzheimer paired helical filaments (see, e.g. Goedert et al., ibid.; Jakes et al. EMBO J. 10 (1991), 2725-2729).

It has been hypothesized that PHF tau protein has a lower affinity for microtubules compared to normal tau proteins since a similar effect has been found when normal tau is phosphorylated in vitro by some kinases (Lindwall and Cole, J. Biol. Chem. 259 (1984), 5301-5305). Lack or reduced binding to microtubules might therefore be a result of abnormal phosphorylation of the tau protein. This abnormal state might lead to microtubule disassembly and interfere with vital neuronal processes, such as rapid axonal transport. The abnormally phosphorylated tau proteins might then aggregate into PHFs. As a consequence thereof the neurons would eventually die thus setting the stage for the generation of the Alzheimer's disease.

Up to now, it was not known which protein kinases are responsible for the abnormal phosphorylation. Ishiguro et al. (Neuroscience Letters 128, (1991), 195-198) have isolated a kinase fraction from bovine brain extracts which contain a protein kinase recognizing the serine/threonine proline motif. This kinase phosphorylated residues Ser 144, Thr 147, Ser 177 and Ser 315 of the tau protein. These residues differed from the ones reported by others (Lee et al., Science 251 (1991), 675-678). Therefore, it remains unclear which protein kinase and which target amino acid residue(s) are involved in the generation of Alzheimer's disease, if at all.

It is, moreover, of utmost importance for the diagnosis of Alzheimer's disease, in particular at an early stage of the disease process, to develop antibodies which are specifically directed to epitopes on the protein which are characteristic of the Alzheimer state. A monoclonal antibody, TAU1, has been isolated which is capable of distinguishing between phosphorylated and non-phosphorylated forms of the tau protein (see, e.g., Lee et al., ibid.). However, this antibody specifically recognizes dephosphorylated tau protein which is seemingly not associated with the Alzheimer state. Another antibody, Alz 50 (Ksiezak-Reding et al., J. Biol. Chem. 263 (1988), 7943-7947) reacts with PHFs as well as with tau protein. Sternberger et al., Proc. Natl. Acad. Sci. USA 82 (1985), 4774-4776, have isolated an antibody, SMI 34, which recognizes a phosphorylated epitope common to Alzheimer tau protein and neurofilament protein. Finally, Lee et al. (ibid.) made antibodies directed to a phosphorylated peptide comprising the KSPV motif in the C-terminal region of the tau protein. All these antibodies known in the art have the disadvantage that for none of them it is known whether they recognize an epitope which is uniquely characteristic for the Alzheimer's disease state.

Furthermore, no reliable data on the fine structure of Alzheimer paired helical filaments, nor on the mode or regulation of their formation from tau proteins is available so far. For the prevention of the formation of PHFs it would be highly advantageous if the mode of assembly of PHFs from tau protein and the regulatory mechanisms underlying said assembly were known.

Thus, the technical problem underlying the present invention was to provide a phosphorylated epitope characteristic for the Alzheimer tau protein, a kinase activity which specifically catalyzes this phosphorylation, pharmaceutical compositions comprising inhibitors to said kinases, antibodies for recognizing said epitopes, diagnostic compositions containing said epitopes, methods involving kinases and/or antibodies for the in vitro diagnosis of Alzheimer's disease, methods for the in vitro conversion of normal tau protein into Alzheimer tau protein and methods for testing drugs effective in dissolving Alzheimer PHFs or preventing the formation thereof.

The solution to the above technical problem is achieved by providing the embodiments characterized in the claims.) Accordingly, the present invention relates to an epitope of the tau protein which is specifically occurring in a phosphorylated state in tau protein from Alzheimer paired helical filaments.

The term "phosphorylated state in tau proteins from Alzheimer paired helical filaments" refers to a state of the tau protein where tau shows an upward M.sub.r shift, has a reduced binding to microtubules and is phosphorylated at ser or thr followed by pro, or certain serines in the repeat region (see below).

Note: Amino acids are denoted by the one-letter or three-letter code; see e.g. Lehninger, Biochemistry, 2nd edition, Worth Publishers, New York, 1975, page 72.

There may be one or more epitopes of the tau protein which specifically occur in a phosphorylated state in Alzheimer paired helical filaments. These epitopes may, moreover, be phosphorylated by a single or different enzymes displaying phosphorylating activity.

In a preferred embodiment of the present invention, said epitopes are specifically phosphorylated by a protein kinase from mammalian brain having the following biochemical properties: (a) it phosphorylates ser-pro and thr-pro motifs in tau protein; (b) it has an M.sub.r of 42 kD; (c) it is activated by ATP and has a K.sub.m of 1.5 mM; (d) it is activated by tyrosine phosphorylation; (e) it is recognized by an anti-MAP kinase antibody; and (f) it is deactivated by phosphatase PP2a.

The term "ser-pro and thr-pro motifs" as used herein refers to a phosphorylatable ser or thr residue followed by a pro residue. These types of sites are phosphorylated by the isoforms of MAP kinase, GSK-3, and cdk2 (see below).

The term "anti-MAP kinase antibody" refers to an antibody which specifically recognizes a mitogen activated protein kinase (MAP kinase). This kinase probably belongs to a family of closely related enzymes which have been referred to in the art by different names, e.g. MAP2 (microtubule-associated protein 2, see e.g. de Miguel et al., DNA and Cell Biology 10 (1991), 505-514) kinase, MBP (myelin basic protein) kinase or ERK1 (for a review, see Hunter, Meth. Enzym. 200 (1991), 1-37). MAP kinase is similar with respect to its biochemical properties to functionally similar enzymes from a variety of sources (Hunter, ibid.).

In another preferred embodiment of the present invention said epitope includes the phosphorylatable serine residues 46, 199, 202, 235, 396, 404 and/or 422 and/or the phosphorylatable threonine residues 50, 69, 111, 153, 175, 181, 205, 212, 217 and/or 231; see FIG. 1a (see Original Patent).

The numbering of the amino acids was done in line with the largest human tau isoform, htau 40, see Goedert et al. (1989 ibid.).

In a particularly preferred embodiment said epitope includes the phosphorylatable serine residue of amino acid position 262. This is phosphorylated by the brain extract and the 35KD and 70KD kinases prepared from it; see below. In accordance with the present invention it has been shown that phosphorylation of said residue significantly interferes with binding of tau protein to microtubuli. This epitope may be used for diagnostic in vitro methods to test for the onset of Alzheimer disease.

In another particularly preferred embodiment said epitope includes the phosphorylatable serine residues 262, 293, 324 and 409.

Accordingly, another object of the invention is to provide a method for testing the onset of Alzheimer disease by assaying the phosphorylation status of serine in position 262 and the other Ser-Pro or Thr-Pro motifs named above. This may e.g. be done by incubating a sample of cerebrospinal fluid of a patient or a sample of nerve tissue after biopsy with a monoclonal or polyclonal antibody capable of distinguishing between a phosphorylated and a non-phosphorylated serine 262 comprising epitope.

The epitopes of the invention may comprise one or more of the residues enumerated above. Moreover, the epitopes of the present invention may comprise only one or more phosphorylated serine residues, one or more phosphorylated threonine residues or a combination thereof. The actual composition of the epitope may be determined by methods which are known in the art. It is also clear to the person skilled in the art that other amino acids of the protein may contribute to the epitope which is recognized by an antibody directed against the sites of tau protein which are phosphorylated by MAP kinase.

In a further preferred embodiment of the present invention, said epitope comprises the amino acid sequences

KESPLQ (SEQ ID NO: 2), YSSPGSP (SEQ ID NO: 3), PGSPGT (SEQ ID NO: 4), YSSPGSPGTPGS (SEQ ID NO: 5), PKSPSS (SEQ ID NO: 6), YKSPVVS (SEQ ID NO: 7), GDTSPRH (SEQ ID NO: 8), MVDSPQL (SEQ ID NO: 9), PLQTPTE (SEQ ID NO: 10), LKESPLQTPTED (SEQ ID NO: 11), AKSTPTA (SEQ ID NO: 12), IGDTPSL (SEQ ID NO: 13), KIATPRGA (SEQ ID NO: 14), PAKTPPA (SEQ ID NO: 15), APKTPPS (SEQ ID NO: 16), PAKTPPAPKTPPS (SEQ ID NO: 17), SPGTPGS (SEQ ID NO: 18), RSRTPSL (SEQ ID NO: 19), SLPTPPT (SEQ ID NO: 20), RSRTPSLPTPPT (SEQ ID NO: 21), VVRTPPK (SEQ ID NO: 22), VVRTPPKSPSSA (SEQ ID NO: 23), KIGSTENLK (SEQ ID NO: 24), KCGSKDNIK (SEQ ID NO: 25), KCGSLGNIH (SEQ ID NO: 26), KIGSLDNITH (SEQ ID NO: 27).

Again, it is to be understood that not all of the amino acids of the peptide necessarily contribute to the specific site actually recognized by the antibody.

Another object of the present invention is to provide a protein kinase which is capable of specifically converting tau protein to Alzheimer tau protein by phosphorylation of the amino acid motif ser-pro or thr-pro.

Preferably, said protein kinase belongs to the class of MAP kinases. These kinases can be used for various purposes, e.g. for the in vitro conversion of tau protein into Alzheimer tau protein. The Alzheimer tau protein thus obtainable may be used to study e.g. substances which are capable of inhibiting its formation or the formation of PHFs. Moreover, they may be used for the development of drugs capable of dissolving said PHFs or for converting Alzheimer tau protein into normal tau protein. It is also conceivable that a system based on the ability of the protein kinase of the invention to convert normal into Alzheimer tau protein will provide a well defined in vitro system for Alzheimer's disease.

In a preferred embodiment of the invention, said protein kinase has the following biochemical properties: (a) it phosphorylates ser-pro and thr-pro motifs in tau protein; (b) it has an M.sub.r of 42 kD; (c) it is activated by ATP and has a K.sub.m of 1.5 mM; (d) it is activated by tyrosine phosphorylation; (e) it is recognized by an anti-MAP kinase antibody; and (f) it is deactivated by phosphatase PP2a. The term "M.sub.r" is defined as the relative molecular weight determined by SDS gel.electrophoresis.

In still another preferred embodiment of the invention, said protein kinase is obtainable by carrying out the following steps: (a) homogenizing porcine brain in 10 mM Tris-HCl, pH 7,2, 5 mM EGTA, 2 mM DTT and a cocktail of protease inhibitors (leupeptin, aprotinin, pepstatin A, .alpha.2-macroglobulin, PMSF (phenyl methyl sulphonyl fluoride)); (b) centrifugating the homogenate at 100,000.times.g for 30 minutes at 4.degree. C.; (c) removing the supernatant after centrifugation; (d) precipitating the crude protein by ammonium sulfate precipitation; (e) desalting the crude preparation by gel filtration; (f) activating the crude enzyme by incubation in activation buffer; (g) further purifying the crude preparation by ion exchange chromatography; and (h) identifying the enzyme by Western blotting.

The term "activation buffer" is defined as a buffer comprising 25 mM Tris, 2 mM EGTA, 2 mM DDT, 40 mM p-nitro-phenylphosphate, 10 .mu.M okadaic acid, 2 mM MgATP, and pro-tease inhibitors.

Another preferred embodiment of the present invention relates to a protein kinase which is capable of specifically converting tau protein to Alzheimer tau protein by phosphorylating IGS and/or CGS motifs in the repeat region of tau protein.

In a further preferred embodiment of the kinase of the invention, said kinase is obtainable by carrying out the following steps: (A) Subjecting mammalian brain extract to ion exchange chromatography on Mono Q (Pharmacia); (B) testing the fractions eluted for binding to microtubules and phosphorylation of the protein; (C) further purifying the fractions binding to microtubules and capable of phosphorylating tau protein by gel chromatography; (D) subjecting the fraction eluting at about 35 kDal to ion exchange chromatography on Mono Q; (E) collecting the major peak eluting between 200 and 250 mM NaCl; and has the following characteristics: (a) it binds to Mono Q but not to Mono S; (b) it has an acidic pI; (c) it shows a major band (>95%) at 35 kDal and a minor band (<5%) at 41 kDal on silver-stained gels; (d) it incorporates a phosphate amount of 3.2 Pi into htau34, 3.4 Pi into htau40, 3.3 Pi into htau23 and 2.8 Pi into mutant htau23 (Ser262.fwdarw.Ala); and (e) it phosphorylates serine residues 262, 293, 324 and 409 of tau protein.

Said brain extract may e.g. be human or bovine brain extract.

In still another preferred embodiment, the kinase of the present invention is obtainable by the following steps: (A) preparation of high spin supernatant of extract from mammalian brain; (B) subjecting the brain extract to chromatography on ion exchange Q-Sepharose (Pharmacia); (C) testing the fractions and flowthrough for phosphorylation of tau protein and influence on binding to microtubules; (D) chromatography of flowthrough on S-Sepharose, wherein the kinase activity elutes at 250 mM NaCl; (E) chromatography on heparin agarose, wherein the kinase activity elutes at 250 mM NaCl; (F) gel filtration, wherein the kinase activity elutes at 70 kDal; (G) chromatography on Mono Q, wherein the kinase activity elutes at 150 mM NaCl; and has the following characteristics: (a) it does not bind to Q-Sepharose but to S-Sepharose; (b) it has an alkaline pI; (c) it shows a major band around 70 kDal on SDS gels; (d) it incorporates 3-4 phosphates into htau34, htau40, htau23, and the construct K19 (i.e., the four-repeat microtubule binding region); (e) it does not phosphorylate a mutant of K19 where Ser 262, 293, 324, and 409 are mutated into Ala; and (f) it phosphorylates Ser 262, 293, 324, and 409 or tau protein.

In another preferred embodiment of the invention, the 70 kDal kinase which phosphorylates the two IGS motifs and the two CGS motifs of tau protein (Serines 262, 293, 324, 409) may be obtained as follows: (A) Preparation of high spin supernatant of brain extract; (B) chromatography on Q-Sepharose; (C) chromatography of flowthrough on S-Sepharose, wherein the kinase activity elutes at 250 mM NaCl; (D) chromatography on heparin agarose, wherein the kinase activity elutes at 250 mM NaCl; (E) gel filtration, wherein the kinase activity elutes at 70 kDal; (F) chromatography on Mono Q, wherein the kinase activity elutes at 150 mM NaCl. (See FIG. 45 (see Original Patent))

The brain extract in step A may be e.g. human or another mammalian brain extract.

The purification steps noted above are conventional ones known in the art as described throughout this specification.

Thus, preparation of the brain extract was carried out as described in Example 11, whereas binding studies between tau and taxol-stabilized microtubules may be done as described in Example (6).

Furthermore, assays of tau-phosphorylation such as in-gel assays may be carried out as described in detail in Example 11.

Chromatography on Mono Q may be carried out as described in Example 11.

With respect to the actual conditions used for obtaining said kinase, a person skilled in the art will be able to deviate from the protocol outlined above and still obtain the kinase of the invention. Such a deviation may, e.g., concern the composition of the protease inhibitor cocktail of step (a): It is conceivable to use different inhibitors under the proviso that the kinase activity is not diminished or destroyed.

In a most preferred embodiment the present invention relates to a protein kinase which specifically phosphorylates serine residues 46, 199, 202, 235, 262, 396, 404, 422 and threonine residues 50, 69, 111, 153, 175, 181, 205, 212, 217, 231 of the tau protein.

In another most preferred embodiment, said kinase phosphorylates serine residue 262.

A further preferred embodiment relates to a protein kinase which is glycogen synthase kinase-3, that is, isoform .alpha., 51 kD or .beta. (45 kD) and/or cdk2-cyclin A (33 kD).

In another preferred embodiment of the present invention, said kinase is a protein kinase from human brain, porcine brain, or another source.

Another object of the invention is to provide pharmaceutical compositions containing a specific inhibitor for the protein kinase of the invention, optionally in combination with a pharmaceutically acceptable carrier and/or diluent.

The term "specific inhibitor for the protein kinase" refers to substances which specifically inhibit the enzymatic action of the protein kinase of the present invention. Inhibitors to enzymes such as protein kinases and their mode of action are well known in the art. For example, such an inhibitor may bind to the catalytic domain of the enzyme thus rendering it incapable of converting its substrate. Examples of such inhibitors are peptide inhibitors and deactivating phosphatases such as PP2a.

Another example is the deactivation of kinases by their phosphatases, e.g., PP-2a in the case of MAP kinase.

Said pharmaceutical composition may be administered to a patient in need thereof by a route and in a dosage which is deemed appropriate by the physician familiar with the case. Pharmaceutically acceptable carriers and/or diluents are well known in the art and may be formulated according to the route of administration or the special disease status of the patient.

In a preferred embodiment the present invention relates to a pharmaceutical composition for use in the treatment of Alzheimer's disease.

Again, said pharmaceutical composition may be administered to a patient in need thereof by route and in a dosage which is deemed appropriate by the physician handling the case.

In another preferred embodiment of the present invention, said pharmaceutical composition contains as the specific inhibitor at least one oligo- or polypeptide comprising an epitope of the invention.

The term "oligo- or polypeptide comprising an epitope of the invention" refers to peptides which in their two- or three-dimensional structure reconstitute the epitope of the invention which is specifically recognized by an antibody directed thereto. Moreover, said oligo- or polypeptides may solely consist of the amino acids representing said epitope(s) or they may comprise additional amino acids. The construction of such oligo- or polypeptides is well known in the art.

Another object of the invention is an antibody which specifically recognizes an epitope of the invention.

Said antibody may be a serum derived or a monoclonal anti-body. The production of both monoclonal and polyclonal antibodies to a desired epitope is well known in the art (see, e.g. Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988). Furthermore, said antibody may be a natural or an antibody derived by genetic engineering, such as a chimeric antibody derived by techniques which are well understood in the art. Moreover, said antibody also refers to a fragment of an antibody which has retained its capacity to bind the specific epitope, such as a Fab fragment.

In a preferred embodiment, the antibody of the present invention recognizes the protein kinase of the present invention.

The term "recognizes the protein kinase of the present invention" as used herein means that the antibody does not or insignificantly cross-reacts with other substances such as different protein kinases present in the same biological environment. Moreover, it means that the antibody does not or insignificantly cross-reacts with different protein kinases when tested in in vitro systems.

In another preferred embodiment, the antibody of the present invention is a monoclonal antibody.

Another object of the invention is to provide diagnostic compositions for the detection and/or monitoring of Alzheimer's disease comprising an epitope of the invention; a kinase of the invention; and/or an antibody of the invention.

The diagnostic composition of the invention may comprise for example an antibody of the invention which specifically recognizes one of the kinases of the invention or an enhanced level of said kinases in a sample to be tested. In another embodiment, said diagnostic composition may comprise an antibody of the invention directed to one of the epitopes of the invention. Thus, an Alzheimer correlated disease state of a sample may be detected by treating said sample with an antibody recognizing the epitope of the invention. The antibody-epitope (hapten) complex may be visualized using a second antibody directed to the antibody of the invention and being labelled according to methods known in the art (see, e.g., Harlow and Lane, ibid.).

In still another embodiment of the present invention, said diagnostic composition may consist of an epitope of the invention and an antibody of the invention. Treatment of a sample with said antibody may give rise to conclusions with regard to the disease state of the corresponding patent, if the binding of said antibody to said sample is brought in relation to binding of said antibody to said epitope of the invention used as a reference sample.

In still another embodiment, the diagnostic composition may comprise an epitope of the invention, a kinase of the invention and an antibody of the invention. Kinase activity may be monitored with respect to phosphorylation of the sample as compared to the phosphorylation of the epitope of the invention. From the quantitated kinase activity the phosphorylation state of the tau protein contained in said sample and therefore the disease state of the patient may be deduced. The kinase activity may e.g. be deduced by including a substrate analog in the same reaction, which is visually detectable upon enzymatic conversion. Such substrate analogs are widely used in the art. Alternatively, the amount of phosphorylated tau protein in the sample may be detected after treatment with the kinase of the invention by employing an antibody of the invention directed to the phosphorylated epitope and using the amount of antibody-epitope complex provided by the diagnostic composition as an internal standard, or by determining the amount of phosphate incorporated into tau protein by the kinase, e.g. by radio-active tracer methods which are well known in the art.

The person skilled in the art is in the position to design other test systems which combine any of the above objects of the invention. It is to be understood that all conceivable combinations fall within the scope of protection of the pre-sent invention.

Another object of the invention is to provide a method for the in vitro diagnosis and/or monitoring of Alzheimer's disease comprising assaying a cerebrospinal fluid isolate of a patient or carrying out a biopsy of nerve tissue for the presence of a phosphorylated Alzheimer tau protein containing an epitope of the invention; for the presence of a protein kinase of the invention; or for the presence of phosphatases PP2a, PP1 and/or calcineurin.

The "cerebrospinal fluid isolate of a patient" is obtained by standard medical procedures.

An example for a nerve tissue suitable for said biopsy is the olfactory epithelium. The person skilled in the art may carry out said method employing e.g. the diagnostic tools illustrated in connection with the diagnostic compositions, supra.

In a preferred method of the present invention, the Alzheimer tau protein and the phosphorylation of serine residue 262 of tau protein, respectively, is detected by using an antibody of the invention.

Said antibody preferably is an antibody directed to an epitope of the invention.

In another preferred embodiment of the invention, the protein kinase is detected by using an oligo- or polypeptide comprising an epitope of the invention and/or by using an antibody of the invention.

Still another object of the invention is to provide a method for the in vitro conversion of normal tau protein into Alzheimer tau protein wherein normal tau protein is treated with a protein kinase of the present invention under conditions which allow the phosphorylation of said normal tau protein.

The term "Alzheimer tau protein" refers to tau protein that is abnormally phosphorylated (e.g. at ser-pro or thr-pro motifs) and recognized by Alzheimer-specific antibodies.

The term "conditions which allow the phosphorylation of said normal tau protein" refers to conditions allowing the activity, preferably the optimal activity, of protein kinase. This activity results in phosphorylation of the substrate at the ser-pro and/or thr-pro motifs. The phosphorylated substrate may then be recognized by Alzheimer-specific antibodies.

Normal tau protein may be derived from natural or recombinant sources. It is, for the purpose of carrying out the method of the present invention, however, expedient to use recombinant material.

The method of the present invention provides sufficient amounts of Alzheimer tau protein for a variety of purposes: With the method of the present invention an in vitro model for the study of the generation of the Alzheimer state of proteins may be established (see above). Moreover, inhibitors may be tested which prevent the conversion of normal to Alzheimer tau protein. These "inhibitors" may be specific for the epitope to be phosphorylated by e.g. blocking the epitope or may be directed to various domains on the protein kinase, as long as they prevent or disturb its biological activity. Another type of inhibition is the antagonistic action of phosphatases on tau or its kinases. Furthermore, the Alzheimer tau protein generated by the method of the present invention may be employed in binding studies to microtubule structures thus contributing to the elucidation of the molecular basis underlying Alzheimer's disease.

The person skilled in the art knows how to employ the method of the present invention for a variety of different-purposes which all fall under the scope of protection of the present invention.

The present invention relates, moreover, to the use of an epitope of the invention for the generation of Alzheimer tau protein specific antibodies or antibodies to a tau protein specific for the onset of Alzheimer disease.

The methods for obtaining said antibodies are well known in the art; thus, the generation of polyclonal or monoclonal antibodies may be conducted using standard methods (see, e.g., Harlow and Lane, ibid.). If an oligo- or polypeptide is used for the generation of antibodies it is desirable to couple the peptide comprising the epitope to a suitable carrier molecule capable of inducing or enhancing the immune response to said epitope, such as bovine serum albumin or keyhole limpet hemocyanin. The methods of coupling hapten (comprising or being identical to the epitope) and carrier are also well known in the art (Harlow and Lane, ibid.). It is also to be understood any animal suitable to generate the desired antibodies may be used therefor.

In another aspect, the present invention relates to a pharmaceutical composition for use in the treatment or prevention of Alzheimer's disease comprising an inhibitor of the formation of Alzheimer paired helical filaments from tau protein dimers.

In accordance with the present invention, it was found that tau proteins form antiparallel dimers via assembly of their repeat units located in the C-terminal domain of the protein. Whereas dimerization of tau proteins appears to be a physiological process, the formation of higher order structures such as PHFs seems to be due to deregulation in the assembly process. Consequently, PHFs are formed from a number of tau dimers wherein the cross-linking of dimers may occur via intermolecular disulfide bridging.

Deregulation of the assembly process with subsequent formation of PHFs from tau dimers appears to be due to abnormal phosphorylation of tau proteins because, as has been found in accordance with the present invention, truncated tau proteins consisting merely of the repeat units are able to form PHFs, whereas tau proteins or tau-like proteins comprising the N-terminus and C-terminus as well are unable to do so.

An inhibitor useful in the composition of the present invention is therefore any inhibitor capable of inhibiting the formation of PHFs from tau dimers regardless of the molecular mechanism it interferes with. Such an inhibitor may be, for example, an inhibitor to a protein kinase responsible for abnormal phosphorylation of tau proteins as a compound interfering with the formation of intermolecular cross-links or association of tau dimers.

A further object of the present invention is to provide a method for testing drugs effective in dissolving Alzheimer paired helical filaments comprising the following steps: (a) allowing the formation of Alzheimer paired helical filaments from polypeptides comprising tau-derived sequences under appropriate conditions; (b) incubating the Alzheimer paired helical filaments with the drug to be tested; and (c) examining the result of the incubation of step (b) with respect to the dissolution of the Alzheimer-like paired helical filaments.

The term "effective in dissolving Alzheimer paired helical filaments" as used herein is intended to also include partially dissolved PHFs. For the object of the present invention it is sufficient that the drug to be tested is effective in the reduction of the size or the break-up of PHFs, thus fulfilling a supplementary function in therapy, although a total dissolution by the drug is preferred.

The term "polypeptides comprising tau derived sequences" refers to any polypeptide which comprises sequences from tau protein capable of forming PHFs regardless of the length of said sequences or of mutations, deletions, insertions or heterologous sequences as long as the function of said polypeptides to form PHFs remains intact.

The term "appropriate conditions" in connection with the formation of Alzheimer PHFs refers to any condition which allows said formation. Said conditions may include the availability of a MAP kinase if natural tau protein is used.

In a preferred embodiment, the conditions applied in step (a) of said method comprise an environment of 0.3 to 0.5 M Tris-HCl and pH 5.0 to 5.5 without additional salts.

Still another object of the invention is to provide a method for testing drugs effective in the prevention or reduction of the formation of Alzheimer paired helical filaments comprising the following steps: (a) incubating the drug to be tested with polypeptides comprising tau-derived sequences under conditions which allow the formation of Alzheimer paired helical filaments in the absence of said drug; and (b) examining the result of the incubation of step (a) with respect to the presence or absence of Alzheimer paired helical filaments in the incubation mixture.

The term "conditions which allow the formation of Alzheimer paired helical filaments in the absence of said drug" refers to any condition which allows the formation of PHFs provided said drug is not included in the incubation mixture. A preferred example of such a condition is an environment of 0.3 to 0.5 M Tris-HCl and pH 5.0 to 5.5 without additional salts.

The term "presence or absence of Alzheimer paired helical filaments" as used herein is intended to include results wherein only a limited amount of PHFs has been formed as compared to control experiments where no such drug has been used.

In a preferred embodiment in the above methods, said polypeptides comprise essentially the repeats from the C-terminal part of the tau protein only.

In accordance with the present invention, it was found that the repeats comprised in the C-terminal domain of the tau protein are responsible for dimerization of the protein under physiological conditions and subsequent oligomerization leading to Alzheimer-like paired helical filaments. The term "Alzheimer-like paired helical filaments" is used here as opposed to "Alzheimer paired helical filament" solely to indicate that non-repeat unit parts of the tau protein normally present in PHFs are absent from PHFs generated by said polypeptides.

Accordingly, the polypeptides comprising essentially the repeat units only provide an ideal in vitro system to study PHF formation and studies on the fine structure of PHFs.

In a particularly preferred embodiment, said polypeptides are comprising mainly the repeat regions of tau, such as K11 and/or K12.

K11 and K12 are ideally suited for the above testing purposes because they are essentially comprised of repeat units from the tau protein only.

For the method of the invention, K11 and K12 may be used alone or in combination.

In a further aspect, the present invention relates to a method for testing drugs effective in dissolving Alzheimer paired helical filaments comprising the following steps: (a) introducing a functional gene encoding a MAP kinase under the control of suitable regulatory regions into a cell expressing or overexpressing tau protein; (b) allowing the formation of phosphorylated tau protein and of Alzheimer paired helical filaments; (c) isolating said Alzheimer paired helical filaments; (d) applying the drug to be tested to said paired helical filaments under appropriate conditions; and (e) examining the effect of said drug on said paired helical filaments.

The term "cell expressing tau protein" as used in step (a), supra, refers to cells which endogenously express tau or which have the capacity to express tau and into which a functional tau gene has been introduced. In the latter case the person skilled in the art is aware of the fact that the sequence of the introduction of the genes encoding the MAP-kinase and tau is irrelevant for the purpose of the method of the invention.

The term "under appropriate conditions" in step (c), supra, refers to conditions which allow the drug to be effective in dissolving PHFs and are particularly optimal conditions.

Said method is particularly advantageous, since the system involved which is based on the use of continuously growing cell lines providing a close image of the in vitro situation provide an ample supply of phosphorylated tau protein.

In a preferred embodiment said cell expressing tau protein is a neuroblastoma or chromocytoma cell or a primary culture of nerve cells.

Such cells or cell lines are well known in the art. Preferred examples are the neuroblastoma cell lines N21 and PC12.

These cell lines are particularly preferred because they express tau endogenously.

A further object of the invention is a pharmaceutical composition for the treatment of Alzheimer disease comprising a PP2a and/or PP-1 and/or calcineurin phosphatase as the active or one of the active ingredients.
 

Claim 1 of 4 Claims

1. An immunogenic composition characterized by the ability to generate an antibody which distinguishes between phosphorylated and dephosphorylated tau comprising: (a) a tau peptide consisting of the tau amino acid sequence Lys-Ile-Gly-Ser-Thr-Glu-Asn-Leu-Lys (residues 259-267 in SEQ ID NO: 1) conjugated to (b) a carrier molecule, wherein the carrier molecule induces or enhances an immune response to the peptide of (a).

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