In general, the invention features antibodies specific for PrP^Sc and diagnostic, therapeutic, and decontamination uses thereof. The invention also features synthetic peptides useful as immunogens for generating antibodies specific for PrP^Sc and therapeutic for the treatment of prion diseases.

SUMMARY OF THE INVENTION

As is discussed herein, evidence is provided demonstrating that a YYX continuous epitope of PrP is useful for generating antibodies specific for PrP^Sc. In particular, we have demonstrated that immunization protocols utilizing a short continuous synthetic peptide from the PrP sequence resulted in the generation of high-affinity polyclonal and monoclonal antibodies specific to PrP^Sc. Moreover, such antibodies were also observed to lack detectable reactivity with PrP^C. In one example, a peptide including a YYR sequence was chosen based on molecular modeling analysis of the conformational change from PrP^C to PrP^Sc, that predicted a sequence which is solvent-accessible on the molecular surface of the PrP^Sc isoform of the protein.

Accordingly, the invention features epitope-specific anti-PrP antibodies or fragments thereof that bind with high binding affinity to a continuous YYX epitope of a mammalian PrP^Sc. Preferably, the antibody binds to a YYR epitope; a YYQ epitope; or a YYD epitope of a mammalian PrP^Sc. In preferred embodiments, the antibody is a monoclonal or a polyclonal antibody. Such antibodies include IgG, IgM, IgE, IgD, or IgA antibodies, as well as fragments such as Fab or Fv fragments. Such anti-PrP antibodies are advantageously directed against a particular PrP^Sc epitope. In addition, antibodies that bind to PrP^Sc can be used to quantitate PrP^Sc in any standard diagnostic assay.

In still another related aspect, the invention features the use of epitope-specific anti-PrP antibodies in an immunological detection procedure for the diagnosis of infective disease-specific prions. Anti-PrP antibodies that react specifically with PrP^Sc can be prepared using an appropriately adapted PrP peptide as has been illustrated herein. The invention particularly relates to diagnostic aids that contain the PrP peptide, and/or epitope-specific anti-PrP antibodies. In addition, the invention relates to antibodies that selectively bind to disease-specific prion protein and not normal prion protein.

In another aspect, the invention features a prion protein peptide with the sequence tyrosine-tyrosine-arginine (YYR). Preferably, the peptide is a tripeptide that is linked to a carrier, making the peptide more immunogenic, allowing for the preparation of high-affinity anti-PrP antibodies. The synthesis of such a tripeptide is described herein. According to the invention, such short peptides (e.g., the YYR, YYQ, or YYD tripeptides) represent determinants that are accessible in the PrP^Sc isoform of the prion protein, but not in the normal PrP^C isoform, and/or are clustered in PrP^Sc in a manner which allows antibody detection. For example, the YYR tripeptide is contained within two of the three prion protein epitopes; however, this tripeptide has not been previously identified as the specific basis of PrP^Sc immunoreactivity. Moreover, such sequences are highly conserved across a number of species including, but not limited to bovine, man, sheep, mouse, and hamster (FIG. 2).

In still another aspect, the invention further features a synthetic peptide having the formula:

A-Tyr-Tyr-B-(Tyr-Tyr-B)_n (SEQ ID NOS: 1-11)

wherein A is either any amino acid or is absent; B is either any amino acid or is absent; and n is from 0 to 10, inclusive. In preferred embodiments, at least one of A and B is not Tyr. In other preferred embodiments, A or B are chosen from Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp. In other preferred embodiments, the peptide is linked to an immunological carrier. Such peptides include, without limitation, A-Tyr-Tyr-Arg (SEQ ID NO: 12); A-Tyr-Tyr-Gln (SEQ ID NO: 13); A-Tyr-Tyr-Asp (SEQ ID NO: 14); or any pharmaceutically acceptable salt thereof.

In yet another aspect, the invention features a synthetic peptide having the formula:

A-Tyr-Tyr-B-C-Tyr-Tyr-D-Tyr-Tyr-(Tyr-Tyr-B)_n (SEQ ID NOS: 15-24)

wherein A is either any amino acid or is absent; B is either any amino acid or is absent; C is either any amino acid or is absent; D is either any amino acid or is absent; and n is 0 to 10, inclusive. In preferred embodiments, at least one of A, B, C, and D is not Tyr. In other preferred embodiments, A, B, C, or D are chosen from Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp. In still other preferred embodiments, A is chosen from Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp, and B, C, and D are chosen from Arg, Gln, Asp, Glu, Phe, or Trp. An exemplary peptide includes, without limitation, A-Tyr-Tyr-Arg-Arg-Tyr-Tyr-Arg-Tyr-Tyr (SEQ ID NO: 25); or a pharmaceutically acceptable salt thereof. In other embodiments, the peptide is linked to an immunological carrier.

In another aspect, the invention relates to short synthetic prion peptides (e.g., three to ten amino acids or four to twelve amino acids, inclusive) having antigencity as a PrP^Sc, including one or more of the following: a threonine tetrarepeat found at T189-193 of mouse PrP or the corresponding amino acid residues of human, sheep, goat, or bovine PrP; the M128, M133, or M153 amino acid residues of mouse PrP or the corresponding amino acid residues of human, sheep, goat, or bovine PrP; the H186 amino acid residue of mouse PrP or the corresponding amino acid residue of human, sheep, goat, or bovine PrP; the Q159, Q167, Q185, or Q216 amino acid residues of mouse PrP or the corresponding amino acid residues of human, sheep, goat, or bovine PrP; the N158 amino acid residue of mouse PrP or the corresponding amino acid residue of human, sheep, goat, or bovine PrP; the M128, M133, or M153 amino acid residues of mouse PrP or the corresponding amino acid residues of human, sheep, goat, or bovine PrP; the L124 or L129 amino acid residues of mouse PrP or the corresponding amino acid residues of human, sheep, goat, or bovine PrP; or the 1181 or 1183 amino acid residues of mouse PrP or the corresponding amino acid residues of human, sheep, goat, or bovine PrP.

Peptides according to the invention can be prepared by chemical synthesis according to methods known in the art.

In addition, a PrP peptide, according to the invention, can be used for preparing epitope-specific anti-PrP^Sc antibodies. In particular, the peptide of the invention provides the advantages of a highly pure substance, and is suitable for preparing anti-PrP antibodies which can be used to detect PrP^Sc in a sample, for example, in standard immunological assays such as immunoprecipitations, ELISA, and flow cytometry. The PrP peptide according to the invention (e.g., YYR) can be used to prepare both polyclonal epitope-specific anti-PrP^Sc antibodies (antisera) and monoclonal epitope-specific anti-PrP antibodies. These antibodies are prepared according to standard methods known in the art, and are preferably bound to a carrier material for the generation of antibodies.

Moreover, compounds which exploit the PrP^Sc-specific exposure of YYX can be rationally designed or obtained from combinatorial libraries which mimic the interaction of YYX with anti-YYX antibodies. These compounds are useful in prion diagnostics or as therapies for prion diseases.

In another aspect, the invention features a pharmaceutical preparation for the therapy and prevention of prion diseases comprising a PrP peptide of the invention or structurally related compounds, or a polyclonal or monoclonal antibody in a pharmaceutical carrier. Such pharmaceuticals contain a PrP peptide or epitope-specific anti-PrP antibodies according to the invention.

If desired, the peptides and antibodies of the invention can be provided in the form of pharmaceutically acceptable salts. Examples of preferred salts are those with therapeutically acceptable organic acids, e.g., acetic, lactic, maleic, citric, malic, ascorbic, succinic, benzoic, salicylic, methanesulfonic, toluenesulfonic, or pamoic acid, as well as polymeric acids such as tannic acid or carboxymethyl cellulose, and salts with inorganic acids such as the hydrohalic acids, e.g., hydrochloric acid, sulfuric acid, or phosphoric acid. In addition, any of the peptides or antibodies of the invention may be administered to a mammal, particularly a human, in one of the traditional modes (e.g., orally, parenterally, transdermally, or transmucosally), in a sustained release formulation using a biodegradable biocompatible polymer, or by using micelles, gels, and liposomes.

In yet another aspect, the invention features a method of treating or preventing a prion disease in an animal (for example, a human, a bovine, sheep, pig, goat, dog, or cat). In one preferred embodiment, the method involves administering to the animal a therapeutically effective amount of epitope-specific anti-PrP antibody or PrP peptide that blocks the conversion of PrP^C to PrP^Sc, inhibits PrP^Sc:PrP^Sc aggregate formation, or blocks the recruitment of PrP^C to PrP^Sc. The PrP peptide may also be used to immunize the host against prion disease by stimulating the production of host antibodies specific for PrP^Sc.

In related aspects, the invention features methods and kits for detecting PrP^Sc in a biological sample.

In still another aspect, the invention features methods and kits for decontaminating PrP^Sc from a biological sample. In a preferred embodiment, the method involves the steps of: (a) treating the biological sample with the polyclonal or monoclonal antibody (or a fragment or analog thereof), the treatment permitting antibody:PrP^Sc complex formation; and (b) recovering the antibody:PrP^Sc complex from the biological sample. Such a decontamination procedure may also involve the use of perfusing a biological sample with antibody (or a fragment or analog thereof) for the removal or inactivation of PrP^Sc.

In another aspect, the invention features a method for identifying a compound for the treatment of a prion disease. The method includes the steps of (a) measuring the binding of an anti-YYX antibody to PrP^Sc in the presence of a test compound; and (b) measuring the binding of the anti-YYX antibody to PrP^Sc in the absence of the test compound; wherein a level of binding of the anti-YYX antibody to PrP^Sc in the presence of the test compound that is less than the level of binding of the anti-YYX antibody to PrP^Sc in the absence of the test compound is an indication that the test compound is a potential therapeutic compound for the treatment of a prion disease. Preferably, the anti-YYX antibody is an anti-YYR antibody, anti-YYD antibody, or anti-YYQ antibody.

In another aspect, the invention features a method for identifying a compound for diagnosing a prion disease. The method includes the steps of: (a) measuring the binding of an anti-YYX antibody to PrP^Sc in the presence of a test compound; and (b) measuring the binding of the anti-YYX antibody to PrP^Sc in the absence of the test compound; wherein a level of binding of the anti-YYX antibody to PrP^Sc in the presence of the test compound that is less than the level of binding of the anti-YYX antibody to PrP^Sc in the absence of the test compound is an indication that the test compound is a potential compound useful for diagnosing a prion disease.

DETAILED DESCRIPTION OF THE INVENTION

We have determined that the orientation of selected aromatic side chains of tyrosines of a YYX epitope (e.g., YYR) at one or more sites in PrP defines a continuous immunologic epitope specific for the molecular surface of PrP^Sc, whereas the same tyrosine side chains are known to be inaccessible in the PrP^C conformation, according to published PrP^C NMR structure solutions (Riek et al., Nature 382:180, 1996; Donne et al., Proc. Natl. Acad. Sci. USA, 94:13452-7, 1997; Zahn et al, Proc. Natl. Acad. Sci. USA, 97:145-50, 2000). This discovery facilitates the generation of PrP^Sc-specific antibodies which may be used for diagnostic and therapeutic purposes, as well as the development of screens for novel compounds useful to detect or combat prions and their related diseases and disorders.

Generation of Epitope-Specific Antibodies to PrP^Sc

Antibodies specifically recognize proteins via unique amino acid determinants or epitopes. These determinants or epitopes may be of a linear amino acid sequence or distinct conformations formed by amino acids in three-dimensional space. Considering conversion of PrP^C to PrP^Sc involves a major change in protein conformation, it is likely that unique epitopes will be formed or revealed upon conversion. Therefore, as is discussed herein, we have developed a so-called side chain hypothesis pertaining to prion protein conversion. According to this scheme, side chains normally sequestered in the solvent-inaccessible interior of PrP^C may be solvent accessible in PrP^Sc. The preponderance of newly exposed side chains are therefore expected to be hydrophobic, as evidenced by increased solvent exposure of hydrophobic residues in a stable PrP^Sc-like intermediate (Swietnicki et al, J. Biol. Chem. 272:27517-20, 1997). The extrusion of these hydrophobic side chains, alone or in combination with side chains that are normally present on the molecular surface of PrP^C, form the basis of unique epitopes for antibody recognition of PrP^Sc. Moreover, these surface-accessible hydrophobic side chains are expected to change the solubility and aggregation characteristics of PrP^Sc, commensurate with the known properties of this structural isoform. Newly accessible side chains may also participate in the process of recruitment of PrP^C to PrP^Sc.

Testing this hypothesis began in vitro by examining the orientation of tryptophan and tyrosine rings, two hydrophobic amino acid side chains for which information was obtained by fluorescence spectroscopic studies. First, a beta sheet transition of mouse recombinant PrP^C was induced by low pH in order to model the structural changes that characterize the conversion of PrP^C to PrP^Sc (Hornemann and Glockshuber, Proc. Natl. Acad. Sci. 95:6010, 1998). FIG. 1A demonstrates a shift in molecular ellipticity by circular dichroism from a pH of 7.0 to 3.0 consistent with a change of PrP^C from predominantly alpha helix to predominantly beta sheet (spectra shifting from double to single minima at appropriate respective frequencies).

Second, the solvent accessibility of tyrosine and tryptophan side chains in recombinant PrP^C with pH titration was examined using standard fluorescence spectroscopy (FIG. 1B). This study is based on the principle that aromatic side chains which are neighboring other amino acid side chains (i.e., in the interior of the protein) will possess a different specific fluorescence than aromatic side chains exposed to water (for example, Chin et al, Biochemistry 31:1945-51,1992). When recombinant mouse PrP^C was subjected to low pH, tryptophan and tyrosine aromatic groups displayed opposing behaviors consistent with differential solvent exposure.

Inspection of the amino acid sequence of human, bovine, and murine PrP^C revealed thirteen tyrosine residues (FIG. 2). Eleven tyrosines in human and bovine PrP and 10 in murine PrP are contained in the C-terminal ⅔ of the protein, which comprises the protease-resistant structured domain necessary and sufficient for prion infectivity. Remarkably, six tyrosines in this domain are present in the unusual "YY" paired motif (FIG. 2). Two of the three pairs are in conjunction with a C-terminal arginine (R), whereas the third YY motif is in conjunction with a C-terminal aspartate (D) in mice and hamsters or a glutamine (Q) in cattle, sheep, and humans. R contains a terminal guanido group, whereas Q and D contain carboxamide and carbonyl bonds, which are planar. Such a terminal planar amino acid in the YY motif may interact with the exposed tyrosine rings to stabilize or shepherd them for immune recognition.

In addition, inspection of the NMR-resolved structures of murine, hamster, and human PrP^C revealed that none of the identified tyrosine pairs are oriented with both of their rings in an orthogonally tandem configuration accessible on the molecular surface (Riek et al., Nature, 382:180, 1996; Donne et al., Proc. Natl. Acad. Sci., 94:13452, 1997; Zahn et al, Proc. Natl. Acad. Sci. USA, 97:145-50, 2000) (FIG. 3). It is reasonable to surmise that the increased exposure of tyrosine on the surface of acid-treated PrP^C or PrP^Sc is associated with some of the tyrosine pairs. One stable conformation of tyrosine rings is referred to as pi-stacking, in which the two rings are stacked in slightly displaced parallel manner (schematically illustrated in FIG. 4). Although stable, a preliminary search of the structural databases for pi-stacked surface accessible tyrosine rings identified only 4 other proteins with similar orientation, none of which are in the ectodomain of membrane proteins. Therefore, a novel PrP^Sc-specific epitope is thought to be tyrosine pairs in a pi-stacking orientation, with or without the contribution of side chains of arginine, glutamine, and aspartate (which, being planar, may also participate in a pi-stacking interaction with its preceding tyrosine).

In addition to changes in orientation of tyrosine side chains in PrP^Sc, it is also possible that the three YYR motifs become more immunologically accessible in PrP^Sc because of shifts in their proximity to each other. A typical IgG antibody is comprised of two identical antigen-binding regions that are connected to one constant region by a flexible hinge region. During the conformational change of PrP^C to PrP^Sc, YYR motifs probably move relative to each other (Korth et al., Nature, 390:74-77, 1997), moving from relatively separated to relatively close. In addition, IgG recognition of YYR motifs in PrP^C may be unfavorable because two critical motifs are on different sides of the molecule, rendering the recognition to be of a low-avidity univalent nature, rather than the high-avidity interaction in which both IgG antigen binding regions participate in recognition.

Our data with bovine PrP^C and PrP^Sc showed that anti-YYR antibodies specifically recognize PrP^Sc by immunoprecipitation and ELISA testing (see below), consistent with changes in the accessibility of tyrosine side chains in PrP^Sc and/or proximity of the YYR epitopes. The amino acid residue, arginine, is also thought to be important in the generation and recognition specificity of the YYR antibody. It is believed that electrostatic interaction between polar tyrosine side chains and the highly basic side chain of arginine contribute to the nature of the YYR epitope in both immunization by the YYR tripeptide, and the recognition of PrP^Sc by the derived antibody. It is notable that the third YY dimer motif in the terminal PrP loop is associated with a glutamine in some species (including humans and cattle), which is a partially conservative substitution with arginine, and aspartate in some other species (including mice and hamsters), which is not a conservative substitution. Exemplary amino acids having planar side chains include arginine, aspartate, and glutamine.

The phenomenon of amino acid side chain exposure incident on conformational conversion of PrP^C to PrP^Sc may not uniquely apply to tyrosine pairs. It is possible that other amino acids with bulky side chains may find these side chains to be poorly tolerated in the core of PrP^Sc, and that these side chains, alone or in combination with other local moieties, form the basis of unique immunoreactivity of PrP^Sc. Immunological epitopes differing between PrP^C and PrP^Sc form the basis of a diagnostic test for PrP^Sc, and are also useful in the treatment and immunization of humans and animals against prion disease. Hydrophobic side chain exposure is thought to be responsible for the increased hydrophobicity and enhanced aggregation of PrP^Sc compared to PrP^C.

Examples of bulky side chains not fully accessible to solvent in PrP^C include the following (amino acid residues are numbered according to the mouse PrP sequence):

• 1. Tyrosines not contained in the YYX motif, including Y127, Y156, Y217.
• 2. A threonine tetrarepeat, T189-193, partially not exposed to solvent.
• 3. Histidine H186.
• 4. Glutamine Q159, Q167, Q185, Q216.
• 5. Asparagine N158.
• 6. Methionine M128, M133, M153.
• 7. Leucine L124, L129.
• 8. Isoleucine I181, I183.

Claim 1 of 26 Claims

1. An antibody or fragment thereof that binds with high binding affinity to a YYX epitope of a mammalian PrP^Sc.

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