WO1994028412A1 - Composition and method for in vivo imaging of amyloid deposits - Google Patents

Abstract

An amyloid binding composition for in vivo imaging of amyloid deposits comprising a labeled amyloid protein or variant thereof which binds to amyloid deposits in vivo; and a pharmaceutically acceptable carrier, is described. Methods of detecting amyloid deposits and for diagnosing Alzheimer's Disease and Down's Syndrome are also described.

Description

COMPOSITION AND METHOD FOR IN VIVO IMAGING OF AMYLOID DEPOSITS

Background of the Invention The present invention relates to the identification of compositions which are suitable for use in in vivo imaging of amyloid deposits and methods related thereto. More specifically, the present invention relates to a method of diagnosing Alzheimer's Disease.

Alzheimer's Disease ("AD") is the most common cause of dementia in the United States, and the presence of the disease is difficult to determine without invasive biopsies. The condition is characterized by impairments in memory, cognition, language and mobility, and these impairments progress over time.

Post-mortem slices of brain tissue from AD victims show that amyloid-containing senile plaques are a prominent feature of selective areas of the AD and the Down Syndrome brain. Divry, P., J. Neurol . Psych . , 27: 643-657 (1927); isniewski, et al . , "Reexamination of the pathogenesis of the senile plaque," In Zimmerman, H.M. (ed.) : Progress in Neuropathology, N.Y. (1973) , Grune and Stratton, pp. 1-26. These plaques range in size from approximately 9 μm to 50 μm in diameter, when viewed by immunocytochemical methods designed to detect amyloid, and they vary in morphology and density. Majocha et al . , Proc . Natl . Acad. Sci . USA, 85: 6182-6186 (1988). Classical staining methods can detect senile plaques as large as 200 μm. Tomlinson, et al., "Ageing and the dementias," In : Adams, J.H., et al . , (ed.). Green-field's Neuropathology, Edition 4, J. Wiley and Sons, N.Y., pp. 951-1006. These plaques are most often found in the cerebral cortex, but they also occur in deeper grey matter, including the amygdaloid nucleus, the corpus striatum, and the diencephalon. Plaques have also been described in the cerebellum. Pro, et al . , Neurology, 30: 820-825 (1980) . Senile plaques are composed of extracellular amyloid, reactive cells, and degenerating neurites that contain Paired Helical Filaments, lysosomes, abnormal mitochondria and astrocytic processes. Wisniewski, et al . - supra (1973). The mechanisms responsible for the excessive accumulation of amyloid, the major proteinaceous component of senile plaques, have been recently addressed at the protein chemistry, molecular biology and genetic level.

Specifically, amyloid is composed of fibrils of 4-8 nm in diameter that form the core of senile plaques. Mertz et al . , Acta Neuropathol . , 60: 113-124 (1983). The amyloid is readily demonstrated by application of thioflavin S or Congo red to brain sections. In the latter case, polarized light causes amyloid to appear with a characteristic yellow-green color. The staining property reflects the presence of twisted beta-pleated sheet fibrils, as noted above. A detailed discussion of the biochemistry and histochemistry of amyloid can be found in Glenner, N. Engl . J. Med. , 302: 1333-1343 (1980).

Vascular amyloidosis, referred to as congophilic angiopathy, has been recognized since the early part of this century as a significant aspect of the microscopic pathology of Alzheimer's Disease. Vinters, et al . Stroke , 18: 311-324 (1987). Over 90% of Alzheimer cases have congophilic angiopathy. Glenner, et al . , Ann . Pathol . , 1: 120-129 (1981). Similar to parenchymal amyloid deposits, vascular amyloid is demonstrated by characteristic thioflavin S and Congo red staining reactions. The parieto-occipital cortex is usually more affected than that in the frontal and temporal lobes. Tomlinson et al . , supra (1984).

In vascular amyloidosis, the amyloid appears to infiltrate the micro-vasculature, and affected vessels often pass from the leptomeninges into the cortex. Small cerebral vessels with arterioles that appear as thickened tubes are observed. The changes include the small pial and intracortical arterioles, the leptomeningeal vessels and the intracortical capillaries. Tomlinson et al . , supra (1984) . Immunocytochemical and electron microscopic studies have indicated that the amyloid component of senile plaques are often observed in close proximity to affected microvessels. Allsop, et al . Neurosci . Lett . , 68: 252-256 (1986). However, the angiopathy may occur without senile plaques. Montjoy, et al . , J . Neurol . Sci . , 57: 89-103 (1982).

The principle component of both cerebral (senile plaques) and vascular amyloid is the 4.2 kilodalton peptide, jS-amyloid, which is also referred to as /3/A4 and A4. Glenner et al . , Biochem . Biophys . Res . Commun . , 120: 885 (1984) . 3/A4 is derived from a parent molecule, the amyloid precursor protein (APP). Kang et al . , Nature , 325: 733-736 (1987) . At least three major variants of APP are known, having 695, 751 and 770 amino acids, respectively. In all three variants, the site of the |3/A4 peptide is in the same relative 3'-end location, as follows:

Transmembrane Site Intra- cytoplasmic Extracytoplasmic Domain Domain

0/4

Kang et al . , supra (1987) showed through cloning APP- 695 that APP has a large extracellular domain, a transmembrane domain (which gives rise to the 0/A4 peptide) and an intracytoplasmic domain (See Figure 9) . The signal sequence, for transport through the endoplasmic reticulum membrane, is followed by a region rich in cysteine, which suggests that disulfide bridges may stabilize this portion of the structure. Within the next 100 residues are a stretch of 7 uninterrupted threonine residues and a region containing 28 glutamic acid residues and 17 aspartic residues. Marotta, et al . J. Mol . Neurosci . , 3: 111-125 (1992) suggest that this domain could bind cations extensively and may have physiological significance. Sodium dodecyl sulfate ("SDS") may be bound to a lesser extent than usual due to this domain. The region from residue 290 to 597, at which point the 0/A4 site begins, contains two.potential N-glycosylation sites at positions 467-469 and 496-498. The 0/A4 peptide (residues 596-638 or 639) is either 42 or 43 amino acids in length and partly includes the putative transmembrane domain (amino acids 625-648) . The C-terminal region of the APP is relatively small, consisting of 57 residues. Following the transmembrane region, lysine residues are present (residues 649-651) which, according to Kang et al . supra (1987), could interact with phospholipid head groups in the membrane. This feature has been described for the junction between membrane and cytoplasmic domains of cell-surface receptors. One site (amino acids 684-686) is a potential glycosylation sequence.

Gandy et al. report that during in vitro studies of synthetic peptides corresponding to the cytoplasmic domain, it was observed that protein kinase C rapidly catalyzed the phosphorylation of a peptide corresponding to amino acid residues 645-661 on ser-655. Gandy et al . , Proc . Natl . Acad . Sci . USA, 85(16): 5218-5221 (1988), suggesting that this site may be an important control region for amyloid metabolism and its interaction with other intracellular regulatory elements.

Recent research has also focused on the biological activity of j8/A4. Specifically, it has been noted that this peptide and its fragments are trophic, toxic and or amnestic at various concentrations. Also, 0/A4 forms insoluble aggregates (self-aggregates) under various conditions, and the neurotoxicity of /S/A4 is related to the aggregation process. Kirshner, et al . , Proc . Natl . Acad. Sci . USA, 84: 6953-6957 (1987) and Maggio, Annu . Rev. Neurosci . , 11: 13-28 (1988). Maggio et al . , also studied the aggregation properties of radioiodinated synthetic 0/A4 peptides in vitro. Proc . Natl . Acad. Sci . USA, 89: 5462-5466 (June 1992).

Pike et al . , J. Neurosci . , 13(4): 1676-1687 (1993) tested the aggregation properties of an overlapping series of synthetic β-amyloid peptides and compared them with their neurotoxic properties in vitro. They discovered that few peptides assembled into aggregates immediately after solubilization but that over time peptides containing the highly hydrophobic B29-35 region formed stable aggregations. In short-term cultures, neurotoxicity was associated with those peptides demonstrating significant aggregations.

Thus far, diagnosis of AD has been achieved mostly through clinical criteria evaluation, brain biopsies and post mortem tissue studies. However, recent work has focused on i munoassay methods for detecting markers of AD in body fluids such as spinal fluid and also in in situ hybridization studies using nucleic acid probes. World Patent No. 92/17152 by Potter; Warner, M. , Anal . Chem. , 59: 1203A (1987); U.S. Patent No. 4,666,829 by Glenner et al . In U.S. application no. 105,751, the contents of which is hereby incorporated by reference, Marotta et al . describe anti-j8/A4 antibodies for purposes of in vitro and in vivo diagnostic methods. Glenner et al . , supra , teach the use of the β/A4 peptide, or fragments thereof, for the production of antibodies which recognize the antigenic determinants of the polypeptide or homologues thereof. Glenner et al . further teach the use of the disclosed polypeptide for the production of nucleic acid probes which hybridize with the gene encoding the polypeptide. One such polypeptide has the following amino acid sequence (SEQ ID N0:1) : H₂N-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Gln- Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly- Ser-Asn-Lys-COOH. The diagnostic methods taught in this patent are characterized as non-invasive.

U.S. Patent Nos. 5,039,511 and 4,933,156 by Quay et al . describe the in vitro and in vivo use of iodinated imaging compounds derived from bisdiazobenzidine compounds to detect the presence and location of amyloid deposits in an organ or area of a patient.

Although β/A4 has been considered for use in in vitro diagnostic methods, this polypeptide has never been described in connection with in vivo diagnostic imaging methods. Therefore, a need exists for a diagnostic in vivo imaging method that exploits the self-aggregation properties of amyloid proteins such as β/A4.

Summary of the Invention

One object of the present invention is to provide an amyloid binding composition for in vivo imaging of amyloid deposits comprising a labeled amyloid protein which binds to amyloid deposits in vivo and a pharmaceutically acceptable carrier.

Another object of the present invention is to provide an in vivo method for detecting amyloid deposits in a subject comprising the steps of administering to a subject a detectable quantity of an amyloid binding composition comprising a labeled amyloid binding protein and a pharmaceutically acceptable carrier and detecting the binding of the labeled protein to the amyloid deposit. Another object of the present invention is to provide a method of diagnosing an amyloidosis-associated disease, such as Alzheimer's Disease and Down Syndrome, by applying the above method to the detection of amyloid deposits in subjects suspected of having an amyloidosis- associated disease.

The amyloid binding protein of the present invention includes all variants of the amyloid protein which bind to amyloid deposits in vivo.

Brief Description of the Drawings Figure l shows a chart setting forth chemically documented amyloidosis with protein types.

Figure 2 depicts a PAGE-SDS gel of the A4-0 synthetic amyloid peptide. The amyloid polypeptide of 28 residues, corresponding to the previously reported sequence of Masters et al . , Proc . Natl . Acad . Sci . USA 82: 4245-4249 (1985) was synthesized on a Biosearch SAM2 synthesizer using the general procedure of Merrifield, J. Am . Chem . Soc , 85: 2149-2154 (1963). Purification was achieved with a 3 X 65 cm column of Sephadex G50 (10-40 μ) . The peptide (10 μg) was suspended in sample buffer containing 2% SDS (Brown, et al., J. Neurochem . , 40: 299-308 (1983)) and 9.5 M urea. Electrophoresis was carried out on a uniform 10% gel containing 0.1% SDS. (A) Molecular weight markers: phosphorylase B (94 kd) , bovine serum albumin (68 kd) , ovalbumin (43 kd) , carbonic anhydrase (29 kd) , trypsin inhibitor (22 kd) , lysozyme (14.5 kd) . (B) The synthetic amyloid peptide ran as a sharp band at the front of the gel and as an aggregated form of higher molecular weight.

Figure 3 depicts gel electrophoresis of the synthetic peptides A4-0 and P2 (APP amino acids 413-429) . (Panel a) : Twenty ug of A4-0 (lane 1) and P2 (APP amino acids 413-429) (lane 2) were analyzed by 18% SDS-PAGE (acrylamide : bisacrylamide = 30 : 0.8). (Panel b) : Analysis of A4-0 and P2 (APP amino acids 413-429) on 11% SDS/urea-PAGE (acrylamide : bisacrylamide = 20 : 1) . Lanes 1-3 containing A4-0 (10 ug) were incubated with 2% SDS and 5% 2-ME at 95°C for 5 minutes (Lane 1) , 30 min (Lane 2) and 60 minutes (Lane 3) . Lane 4 contained P2 (lOug) . Gels were stained with Coomassie Brilliant blue R-250. Molecular weights are shown on the right (Kd) .

Figure 4 shows slot blots of immunostained A4-0 after addition of itself or a second A4 homologue. This assay depicts the increase staining intensity after A4 homologues are added to one another. This reflects the ability of homologues to self-aggregate and thus increase the staining intensity. In each case, the slot contained 1 ug of A4 peptide. To each, 2.5, or 10.0 ug of exogenous peptide was added, as indicated. The blots were then immunostained (see descriptions of Figures 5, 6 and 7) .

Figure 5 shows immunoblots with and without exogenous A4-0 peptide. Density values of the immunoreaction products of A4-0 with and without exogenous peptides after reaction with 10H3. The values of the bars correspond to the density of blots shown in Figure 4. The description of Figure 4 indicates the condition of each blot with regard to the exogenous peptide that was added to the blotted peptide prior to addition of 10H3. The height of the bars above the black bar (no peptide addition) is a measure of the extent to which the exogenous peptide bound to the attached peptide on the filter paper and increased the density of immunostaining of the complex.

At the three indicated concentrations (2.5, 5.0 and 10.0 ug/ml) A4-0 was added to the A4-0 that was already present at a concentration of 1 ug. The blot was then immunostained to develop the colored reaction product. The data were derived from scanning Figure 4.

Figure 6 shows immunoblots with and without exogenous A4-H peptide. At the three indicated concentrations (2.5, 5.0 and 10.0 ug/ml) A4-H as added to the A4-0 that was already present at a concentration of 1 ug. The blot was then immunostained to develop the colored reaction product. The data were derived from scanning Figure 4. See description of Figure 5 for more details. Figure 7 shows immunoblots with and without exogenous Opl peptide. At the three indicated concentrations (2.5, 5.0 and 10.0 ug/ml) Opl as added to the A4-0 that was already present at a concentration of 1 ug. The blot was then immunostained to develop the colored reaction product. The data were derived from scanning Figure 4. See description of Figure 5 for more details.

Figure 8 shows the reactivity of 10H3 towards A4-0 (upper panel) .

Figure 9 (SEQ ID NOS:11 and 12) is the nucleotide sequence and predicted amino acid sequence of cDNA encoding the precursor protein (APP) of the /3/A4 with the 0/A4 region boxed, as set forth in Kang et al . , supra , (1987) . Detailed Description of the Preferred Embodiments

Applicants have discovered that an amyloid binding composition comprising a labeled amyloid protein may be used in vivo for detecting the presence and location of amyloid deposits. The amyloid binding composition of the present invention comprises a labeled amyloid protein and a pharmaceutically acceptable carrier. This protein is any natural or synthetic protein which binds to amyloid deposits in vivo. In one embodiment, the protein is the β-amyloid polypeptide (β/A4 peptide) , which in its longest form has 42 to 43 amino acid residues, as shown in Figure 9. See Masters, et al. , Proc . Nat . Acad . Sci . , USA . , 82: 4245-4249 (1985) . As noted above, B/A4 is derived from a larger amyloid precursor protein having from 695 to 770 amino acids. See Kang et al . , Nature , 325: 733 (1987). The term "amyloid deposit" includes amorphous, eosinophilic materials that are associated with amyloidosis, a disease complex including over 20 different clinically defined syndromes, as discussed above. Chemically, amyloid deposits are proteinaceous, and their chemical compositions are unique for each of the clinical syndromes with which they are associated, as set forth in Figure 1. Preferably, the amyloid deposit of the present invention is that found in the brain of Alzheimer's Disease patients. As noted above, such amyloid deposits are found in senile plaques in selected areas of the AD brain and are composed of fibrils of 4-8 nm diameter. These plaques are detected by application of thioflavin S or Congo red to brain sections and in the latter case, appear yellow-green under polarized light. They have twisted beta-pleated sheet fibrils and are further characterized by Glenner, N . Eng. J. Med . , 302: 1333-1343 (1980). In another embodiment, the amyloid deposit of the present invention is that which is associated with vascular amyloidosis, as described in Vinters, Stroke , 18: 311-324 (1987). Vascular amyloid deposits infiltrate the cerebral micro- vasculature. Similar to amyloid deposits found in senile plaques in the parenchyma of the AD brain, vascular amyloid deposits have characteristic thioflavin S and Congo red staining reactions. Montjoy et al . , J. Neurol . Sci . , 57: 89-103 (1988).

The term "amyloidosis-associated disease" includes any disease characterized by local or systemic amyloid deposits. (See Figure 1) Preferably, the amyloidosis- associated disease of the present invention is Alzheimer's Disease or Down Syndrome.

In addition to amyloid protein purified from natural sources such as cerebrovascular tissue, as described hereinafter, amyloid protein of the present invention includes recombinant and synthetic amyloid protein and variants of the naturally occurring, recombinant and synthetic protein. In a preferred embodiment, the amyloid protein of the invention comprises the 0-amyloid polypeptide and variants thereof. The category of "variants" includes, for example, a fragment of the β- amyloid polypeptide or any homologous amino acid sequence or amino acid addition, wherein the resulting polypeptide has the same or similar function as the natural occurring polypeptide in that it binds to amyloid deposits in vivo . In one embodiment, the amyloid protein of the present invention is comprised of the 0-amyloid polypeptide or variant thereof and amino acids from the APP protein which are from regions of the APP protein which are either adjacent or non-adjacent to the 0-amyloid polypeptide. For example, in one embodiment, the amyloid protein of the present invention comprises:

(A) The 0/A4 peptide alone (SEQ ID N0:2): Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His- Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys- Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val-Ile-Ala- Thr ; or

(B) The 0/A4 peptide plus the amino acids of the transmembrane domain of the APP (SEQ ID NO:3): Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His- Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys- Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val-Ile-Ala- Thr-Val-Ile-Val-Ile-Thr-Leu-Val-Met-Leu; or

(C) The 0/A4 peptide plus the remaining c-terminal amino acids of the entire APP (SEQ ID NO:4) :

Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His- Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys- Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val-Ile-Ala- Thr-Val-Ile-Val-Ile-Thr-Leu-Val-Met-Leu-Lys-Lys-Lys-Gln- Tyr-Thr-Ser-Ile-His-His-Gly-Val-Val-Glu-Val-Asp-Ala-Ala- Val-Thr-Pro-Glu-Glu-Arg-His-Leu-Ser-Lys-Met-Gln-Gln-Asn- Gly-Tyr-Glu-Asn-Pro-Thr-Tyr-Lys-Phe-Phe-Glu-Gln-Met-Gln- Asn; or

(D) The 0/A4 peptide with the preceeding 10 amino acids of the APP (SEQ ID NO:5):

Lys-Thr-Glu-Glu-Ile-Ser-Glu-Val-Lys-Met-Asp-Ala-Glu-Phe- Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val- Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile- Gly-Leu-Met-Val-Gly-Gly-Val-Val-Ile-Ala-Thr; or (E) The 0/A4 peptide with any other APP amino acids attached to it that are not normally ajacent

(SEQ ID NO:6) :

X-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-

His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn- Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val-Ile-

Ala-Thr-Y, wherein X and Y are one or more APP amino acids which are not ajacent to 0/A4 in the nature; and

(F) any fragment of (A)-(E), wherein said fragment is large enough to bind amyloid deposit in vivo. The term "fragment" includes a linear amino acid subsequence of the β-amyloid polypeptide, wherein such fragment binds amyloid deposits in vivo . A variant which contains an amino acid sequence variation or substitution is a homologous sequence. "Homology" between two sequences connotes a likeness short of identity indicative of a derivation of the first sequence from the second. For example, a polypeptide is "homologous" to β- amyloid polypeptide if it contains an amino acid sequence similar enough to the natural sequence so as to confer the same or similar amyloid binding property as the natural β-amyloid polypeptide. Such a sequence may be only a few amino acids long and may be a single linear sequence or one or more linear sequences which confer binding activity to the polypeptide when amino acids from separated portions of a linear sequence are spatially juxtaposed after protein folding. The variants encompassed by this invention can be ascertained, for example, by the in vitro quantitative assays describe below in Examples 3-7. That is, applicants have conducted a series of studies involving the addition of increasing concentrations of β-amyloid polypeptide variants to a solid support containing a specific peptide called A4-0. The increase in density of immunostain using an anti-A4-0 monoclonal antibody, 10H3, described in U.S. Patent application no. 105,751 by Marotta, et al . , incorporated by reference above, was measured. Based upon this work, it was possible to determine which peptides were suitable for use in the in vivo methods, according to the invention. Other poly- and/or monoclonal antibodies suitable for this assay can be produced by methods well known in the art. See Kennett et al . , Monoclonal Antibodies- Hybridomas: A New Dimension in Biological Analysis , Plenum Press (1980) Protein which qualifies as "amyloid protein" according to the above criteria can be produced by methods known and emerging in the art, including conventional reverse genetic techniques, i.e., by designing a genetic sequence based upon an amino acid sequence or by conventional genetic splicing techniques. For example, β-amyloid polypeptide variants can be produced by techniques which involve site-directed mutagenesis or oligonucleotide-directed mutagenesis. See, for example, "Mutagenesis of Cloned DNA," in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 8.0.3 et seq. (Ausubel, et al . eds. 1989) ("Ausubel").

Other amyloid protein variants within the present invention are molecules that correspond to a portion of the β-amyloid polypeptide, but are not coincident with the natural molecule, and display the binding activity of the natural molecule when presented alone or, alternatively, when linked to a carrier or biologically active signal sequence that permits proteins to pass through membranes. See von Heijne, G., J. Mol . Biol . , 184: 99-105 (1985). An amyloid protein variant of this type could represent an actual fragment, as discussed above, or could be a polypeptide synthesized de novo or recombinantly.

To be used in recombinant expression of amyloid protein or amyloid protein variant, a polynucleotide molecule encoding such a molecule would preferably comprise a nucleotide sequence, corresponding to the desired amino acid sequence, that is optimized for the host of choice in terms of codon usage, initiation of translation, and expression of commercially useful amounts of, for instance, β-amyloid polypeptide or fl¬ amyloid polypeptide variant. Also, the vector selected for transforming the chosen host organism with such a polynucleotide molecule should allow for efficient maintenance and transcription of the sequence encoding the polypeptide. The encoding polynucleotide molecule may code for a chimeric protein; that is, it can have a nucleotide sequence encoding a biologically active part of the β-amyloid molecule operably linked to a coding sequence for a non-β-amyloid moiety, such as a signal peptide for the host cell. For instance, in order to isolate a DNA segment which encodes a β-amyloid molecule, total DNA from cerebrovascular tissue can be prepared according to published methods. See, for example, Maniatis, et al . , MOLECULAR CLONING: A LABORATORY MANUAL (Cold Spring Harbor Laboratories, NY 1982); Baess, Acta Pathol . Microbiol . Scand. (Sect. B) , 82: 78084 (1974). The DNA thus obtained can be partially digested with a restriction enzyme to provide an assortment of genomic fragments. An enzyme with a tetranucleotide recognition site, such as Sau3A (MJbol) , is suitable for this purpose. The fragments from such a partial digestion then can be size-fractionated, for example, by sucrose gradient centrifugation (see Maniatis, supra) or by pulsed field gel electrophoresis (See Anad, Trends in Genetics, November 1986, at pages 278-83), to provide fragments of a length commensurate with that of DNA encoding the β- amyloid molecule. Molecular cloning of amyloid cDNA derived drom mRNA of the Alzheimer brain and the expression thereof is described in detail in Zain et al . , Proc. Natl . Acad . Sci . USA. , 85: 929-933 (1988) and Marotta et al . , Proc. Natl . Acad . Sci . USA. , 86: 337-341 (1989) , respectively, both of which are herein incorporated by reference.

According to well-known methods described, for example, in Ausubel at 5.0.1 et seq. , the selected fragments can be cloned into a suitable cloning vector. A DNA sequence thus obtained could be inserted, for example, at the BamHl site of the pUClδ cloning vector which is transfected into appropriate host cells such as E. coli or a mammalian cell. A variety of screening mechanisms known in the art of the invention can then be used to identify clones containing the β-amyloid gene.

In another embodiment of the invention, amyloid protein of the present invention is purified from tissue samples. For example, brains from Alzheimer's Disease post-mortum patients are histologically sectioned and stained with Congo Red dye. Upon visualization with a polarizing microscope, amyloid deposits can be identified by their green color. Brains exhibiting extensive cerebrovascular amyloidosis are used as source for purified amyloid protein. After removal of contaminants from the amyloid containing vessels of the meninges, the meningeal tissues are homogenized and centrifuged to yield a brownish layer rich in amyloid fibrils. This layer is then digested with collagenase, solubilized in 6M guanidine HCl, pH 8.0 and centrifuged. The supernatant containing the solubilized protein is desalted by dialysis and gel exclusion column chromatography and high performance liquid chromatography is used to purify the polypeptide. The amino acids for the purified protein (e.g., β-amyloid polypeptide) are then sequentially cleaved in an automated amino acid sequencer, such as a Beckman 890 C spinning cup sequencer, and analyzed by high performance liquid chromatography in order to determine the amino acid sequence of the amyloid protein. See Glenner & Wong, Biochem . Biophys . Chem . Res . Commun . , 120: 885 (1984).

In another embodiment, amyloid protein and variants thereof can be produced in accordance with published methods. For instance, Kirschner et al . , Proc. Natl . Acad. Sci . USA, 84: 6953-57 (1987) used an ABI

Synthesizer model 380 B (Applied Biosystems, Foster City, CA) to synthesize synthetic β-amyloid peptides consisting of residues 1-28 and homologues thereof. General methods for peptide synthesis can be found in Clark Lewis et al . , Science , 231: 134 (1986). See also, Hilbich et al . , J. Mol . Biol . , 218: 149-163 (1991); Majocha et al . , Proc. Natl . Acad. Sci . USA, 85: 6182-6186 (1988); and U.S. Patent application No. 105,751 by Marotta et al .

The term "in vivo imaging" refers to any method that permits the detection of a labeled amyloid protein which binds to amyloid deposits located in a subject's body. A "subject" is a mammal, preferably a human. Often, particularly when the composition and method of the invention is directed to the diagnosis of Alzheimer's Disease or Down Syndrome, the subject will manifest clinical symptoms of the suspected amyloidosis. These clinical symptoms are well-known to the practitioner of this invention and include loss of memory, and other impairments described above. The amyloid binding composition of the present invention must be of a "detectable quantity." A detectable quantity is that which is sufficient to enable detection of the site of amyloid deposit location when compared to a background signal. The dosage of the amyloid binding composition will vary depending upon such considerations as age, condition, sex, extent of disease in the patient, counterindications, and other variables, to be adjusted by the individual physician. Dosage can vary from 0.01 mg/kg to 2,0000 mg/kg, preferably 0.1 mg/kg to 1,000 mg/kg.

In accordance with this invention, the amyloid protein may be labeled by any of several techniques known to the art. See, e . g. , Wagner et al . , J. Nucl . Med . , 20: 428 (1979); Sundberg et al., J. Med. Chem . , 17: 1340 (1974) and Saha et al . , J. Nucl . Med. , 6: 542 (1976).

The label is chosen based upon the type of detection instrument employed. For instance, a chosen radionucleotide must have a type of decay which is detectable for a given type of instrument. Another consideration relates to the half-life of the isotope. The half-life should be long enough so that it is still detectable at the time of maximum uptake by the target, but short enough so that the host does not sustain deleterious radiation. Preferably, the chosen label will lack a particulate emission, but will produce a large number of photons in a 140-200 keV range, which may be readily detected by, for instance, conventional gamma camera. Suitable radioisotopes for purposes of this invention include, gamma-emitters, position-emitters, x- ray emitters and fluorescence-emitters. These radioisotopes include Iodine-131, Iodine-123, Iodine-126, Iodine-133, Bromine- 77, Indium-Ill, Indium-113m, Gallium-67, Gallium-68, Ruthenium-95, Rutheium-97, Ruthenium-103, Ruthenium-105, Mercury-107, Mercury-203, Rhenium-99m, Rhenium-105, Rhenium 101, Tellurium-121m, Tellurium-122m, Tellurium-125m, Thulium-165, Thulium-167, Thulium-168, Technetium-99m and Fluorine-18. The preferred radiolabel is Technetium-99m. Suitable paramagnetic isotopes for use in Magnetic Resonance Imaging (MRI) , according to this invention, include ¹⁵⁷Gd, ^5SMn, ^,62Dy, ⁵²Cr, and ∞Fe.

Administration to the subject may be accomplished intraventricularly, intravenously, intraarterially, via the spinal fluid or the like. Administration may also be intradermal or intracavitary, depending upon the body site under examination. After a sufficient time has lapsed for the labeled amyloid protein to bind with amyloid deposits, for example 30 minutes to 48 hours, the area of the subject under diagnosis is examined by routine imaging techniques such as MRI, SPECT and planar scintillation imaging. The exact protocol will necessarily vary depending upon factors specific to the patient, as noted above, and depending upon the body site under examination, method of administration and type of label used; the determination of specific procedures would be routine to the skilled artisan. The distribution of the bound radioactive isotope and its decrease with time is then monitored and recorded. By comparing the results with data obtained from studies of clinically normal individuals, the presence and location of amyloid deposits can be determined.

Thus, in one embodiment, the methods of the present invention is used to diagnoses an amyloidosis-associated disease. Where the site of examination is the brain, the in vivo detection of amyloid deposits according to the methods of the present invention signifies a diagnosis of Alzheimer's Disease. The detection of amyloid deposits in the brain of patients manifesting clinical symptoms of Down Syndrome, signifies a diagnosis of Down Syndrome. In that regard, applicants note that the gene for APP, located on chromosome 21, is over-represented in Down Syndrome individuals (Serra et al . , Araer. J. Med. Gen . Supp. , 7: 11-19 (1990). Accumulations of amyloid occur in young Down Syndrome patients, with nearly 90% of Down Syndrome subjects aged less than 30 years showing amyloid accumulation (Hyman, Prog. Clin . Biol . Res . 379: 123-142 (1992)). The Down Syndrome patient displays amyloid accumulations early in life, often by late teenage years. As adults, nearly 100% will develop Alzheimer Disease (Cork, →Amer. J. Med. Gen . Supp. , 7: 282-539 (1990)). The neuropathology of Down Syndrome is essentially identical to that of Alzheimer Disease and includes 0/A4 amyloid deposits in senile plaques. The Alzheimer - like lesions represent a major neuropathologic trait of the brain of the Down Syndrome patient (Serra et al . , Supra (1990)).

The amyloid-binding compositions of the present invention are advantageously administered in the form of injectable compositions. A typical composition for such purpose comprises a pharmaceutically acceptable carrier. For instance, the composition may contain about 10 mg of human serum albumin and from about 20 to 200 micrograms of the labeled amyloid protein per milliliter of phosphate buffer containing NaCl. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like, as described in REMINGTON'S PHARMACEUTICAL SCIENCES, 15th Ed. Easton: Mack Publishing Co. pp 1405- 1412 and 1461-1487 (1975) and THE NATIONAL FORMULARY XIV., 14th Ed. Washington: American Pharmaceutical Association (1975) , the contents of which are hereby incorporated by reference. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride. Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobials, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components of the binding composition are adjusted according to routine skills in the art. See GOODMAN AND GILMAN'S THE PHARMACOLOGICAL BASIS FOR THERAPEUTICS (7th ed.). The skilled artisan would also readily appreciate that a suitable excipient or carrier would need to prevent aggregation of the binding composition prior to contacting the target amyloid deposit in vivo.

Particularly preferred amyloid binding compositions of the present invention are those that, in addition to binding to amyloid deposits in in vivo, are also non- toxic at appropriate dosage levels, have a satisfactory duration of effect, and display an adequate ability to cross the blood-brain barrier. In this regard. United States Patent No. 4,540,564 discloses an approach for enhancing blood-brain barrier-penetrating ability by attaching a centrally acting drug species to a reduced. biooxidizable, lipoidal form of dihydropyridine pyridinium salt redox carrier. Thus, in one embodiment, the composition of the present invention includes such a blood-brain barrier crossing enhancer carrier. In vivo animal testing provides yet a further basis for determining dosage ranges, efficacy of transfer through the blood barrier and binding ability. Particularly preferred for this purpose is the "senile animal" model for cerebral amyloidosis — animals such as aged dogs or monkeys, which are known to develop variable numbers of Alzheimer-type cerebral senile plaques, see Wisniewski, et al . , J. Neuropathol . & Exp. Neurol . , 32: 566 (1973), Selkoe, et al . , Science , 235: 873 (1987) are tested for binding and detection efficacy. This in vivo assay requires control-biopsy monitoring to confirm and quantify the presence of amyloid deposits.

Also, cellular models of amyloidosis have been prepared that overproduce 0-amyloid polypeptide in animals for purposes of testing the efficacy of the amyloid binding compositions and methods of the present invention. See Marotta, et al . Proc. Natl . Acad. Sci . USA, 86: 337-341 (1989). Such cell models have been adapted to a behavior paradigm. See Tate-Ostroff, Proc . Natl . Acad. Sci . USA 89: 7090-7094, (1992). That is, because AD patients suffer circadian rhythm dysfunction, this behavioral deficit was modeled in rats by a cell grafting techniques. PC12 cells transfected with the 0- amyloid polypeptide C-terminal region of the APP were implanted into the suprachiasmatic nuclei ("SCN") of rats; the SCN is a primary circadian oscillator in mammals. Animals receiving a yloidotic cell grafts, but not animals receiving control cell grafts, exhibited disrupted activity rhythms, although temperature rhythms were unaffected. The specificity of the disruption was similar to circadian dysfunction seen in AD patients. The data supported an association between a defined behavioral disruption and amyloid overexpression either directly or through the release of cellular factors as a consequence of amyloid overproduction.

Other suitable animal models for use in testing the compositions and methods of the present invention are produced transgenically. For instance, Quon et al . , Nature , 352: 239-241 (1991) used rat neural-specific enolase promoter inhibitor domain to prepare transgenic mice. See also, Wirak et al . , Science , 253: 323-325 (1991) . Still other models have been produced by Intracranial administration of the 0/A4 peptide directly to animals (Tate et al . , Bull . Clin . Neurosci . , 56: 131- 139 (1991).

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following examples are merely illustrative and not limitative of the remainder of the disclosure in any way whatsoever.

EXAMPLES As noted above, A4 is intended to be the same as 0/A4, throughout the examples. The peptides used in the following Examples have the following structures: A4-0 (peptides 1-28), SEQ ID NO:7:

N-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His- His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Ser- Ala-COOH

The A4-0(l-28) polypeptide that was reported in Masters, et al . Proc. Nat ' l . Acad. Sci . U.S.A. , 82 : 4245- 4249 (1985) is the first 28 amino acids of the 4.2 Kd peptide derived from senile plaque cores of an AD brain. Masters, et al . have also shown that the naturally occurring peptide aggregates even in denaturing gels. The A4-0(l-28) sequence of this invention was synthesized by Biosearch in San Rafael, CA. The underlined amino acids differ from A4-P(l-28) , as shown below. A4-H (peptides 1-28) :

The A4-H peptide is the same as A4-0(l-28) except that it was synthesized by the Harvard Microchemistry Laboratory.

A4-P (peptides 1-28), SEQ ID NO:8:

N-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Gln-Val-His- His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn- Lvs-COOH

This sequence was reported by Glenner and Wong, supra , (1984) and derived from vascular amyloid of the AD brain and from a Down Syndrome brain. Three of 28 amino acids are different from the A4-0/A4-H peptides(underlined) .

A4-B (peptides 1-28), SEQ ID NO:9:

N-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His- His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn- Lvs-COOH This sequence was obtained from Bachem and is the 28 amino acid structure that is commonly determined from molecular cloning studies (Kang, et al . , Nature (London) 325 : 733-736 (1987)). Unlike the Glenner and Wong, supra , sequence (A4-P(l-28) ) , it has Glu, not Gin, at position 11. And, unlike A4-0/A4-H, it has Asn-Lys and not Ser-Ala at the C-terminus.

Opl (peptides 1-10), SEQ ID NO:10: N-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-COOH

A4(l-10) consists of the first 10 amino acids of the amyloid peptide derived from any source and is described in U.S. Patent No. 4,666,829 by Glenner et al . Thus far, this sequence appears conserved in all reports on amyloid that is derived from non-Familial AD cases. The A4-(l- 10) antigen used in the present studies was synthesized by the Harvard Microchemistry Laboratory. Summary of sequence variations: dashed line indicates sequence conservation among the peptides shown.

A4-0 (1-28) N Glu Ser-Ala- (Masters) A4-H (1-28) Glu Ser-Ala- (Masters)

A4-P (1-28) N Gin Asn-Lys- (Glenner) A4-B (1-28) N Glu Asn-Lys- (Kang) Opl N 1

Exa ple 1. Self-aσσreσation of the A4-0 amyloid peptide in SDS/urea acrylamide gels

The synthetic β-amyloid 28-mer polypeptide, A4-0 (Masters, et al . , supra . ) was analyzed by polyacrylamide gel electrophoresis (PAGE) procedures (Brown, et al . , J. Neurochem . , 40 : 299-308 (1983)) and was noted to have unusual aggregation properties. The peptide was dissolved in a PAGE sample buffer containing SDS and urea and was electrophoresed on a 10% gel containing SDS (See description of Figure 2) . After staining with Coomassie blue, the peptide appeared as a broad band at approximately 23-25 kd and a narrow band that migrated at the get front during electrophoresis (See Figure 2) . The higher molecular weight species appeared to be an aggregate since it was eliminated by adding urea to the separating get and, subsequently, a 3-4 kd band was obtained (not shown) . Polyclonal antiserum to the 28-mer was prepared and applied to nitrocellulose blots of an overloaded gel. The latter contained a series of aggregated peptides of various apparent molecular weights, all of which reacted with the antiserum. Thus, the synthetic 28-mer had aggregational properties not unlike the naturally occurring A4-θ amyloid protein of 4 kd (Masters, et al . , supra) . Applicants' studies demonstrating the aggregation properties of the A4-0 peptide were previously reported (Salim, et al . , "Molecular Cloning of Amyloid cDNA from Alzheimer Brain Messenger RNA" in Familial Alzheimer' s Disease, J.P. Blass et al . eds.. Marcel Dekker, NY pp 153-165 (1988).

Based upon the results shown in Figure 2, applicants concluded that even in the presence of strong denaturing agents and after electrophoresis, A4-0 strongly bound to itself. Example 2: Self-aggregation of A4-0 peptide on highly cross-linked SDS/urea acrylamide gels

Applicants obtained confirmatory data using the highly cross-linked acrylamide gel system described by Honda and Marotta, Neurochem . Res . , 17: 367-374 (1992). When analyzed by this gel system containing SDS and urea, the synthetic peptide A4-0 migrated as a broad series of bands below an apparent molecular weight of 15kDa (data not shown) . However, when 6M urea was added to the PAGE system the peptide appeared as a sharp single band of 15kDa (Figure 3) and smaller size bands were not observed even after silver staining (data not shown) . By contrast, peptide P2(413-429), used as a control and corresponding to an extracytoplasmic region of the B/A4 precursor protein, migrated with the bromphenol blue dye front on both SDS-PAGE and SDS/urea-PAGE systems (Figure 3, lane 4). Since the theoretical molecular weight of the 28 amino acid peptide A4-0 is 3,178 Da the results indicate that the band of 15kDa is an aggregate. Migration of A4-0 peptide bands on both gel systems was not affected by 2-ME nor by pre-treatment with 80% formic acid (data not shown) .

The 15kDa was visible after peptide A4-0 were treated for 5 minutes at 95°C prior to electrophoresis (Figure 3, lane 1). When boiling time was increased to 30 minutes or 60 minutes the aggregate partly dissociated to a smaller size (Figure 3, lanes 2 and 3). This dissociation was not dependent on the presence of SDS and 2-ME in the sample buffer but rather on the time of heat denaturation. These data were previously reported (Honda and Marotta, supra) .

Applicants concluded that Figure 3 confirms that even in the presence of strong denaturing agents and heat treatment after electrophoresis, A4-0 strongly bound to itself. Example 3: Self-aggregation of A4 peptides on immunoblots

Due to the desirability of obtaining a quantitative assay for the selection of β-amyloid polypeptides for the composition and methods of the present invention, applicants elected to use quantitative slot blots to test aggregation of peptides rather than tissue slices. The general immunoblotting procedure utilizing A4 peptides attached to a solid support and detectable by applied anti-amyloid antibodies was reported earlier (Majocha, et al . , supra , (1988). In all cases, the monoclonal antibody used to detect A4 aggregates was 10H3 (2 ug/ml) .

One microgram of each of the indicated peptides were added overnight at room temperature to Millipore P filter paper to which A4-0 was attached. The peptides were dissolved in ICC buffer: 2% BSA, 0.3M NaCl, 20mM Tris, 0.01% Triton. The blots were immunoprocessed (Majocha, et al. supra . ) and then optically scanned for density; the areas under the curves were integrated by means of an LKB Laser Densito eter.

Peptides A4-0, A4-H and Opl were applied to filters to which was bound peptide A4-0, the antigen used to prepare mab 10H3. The experiment was designed to test the competence of each of the applied peptides to bind to the bound peptide. While A4-0 and A4-H have the same primary structure, it has been noted that peptides with identical sequences that are obtained from different sources may have non-identical properties. (See Figure

4). The density of staining (the optical density of the immunoreaction product) is quantitated in Figures 5, 6 and 7. The OD is a measure of the extent of the aggregation since it will be related to the antibody concentration and thus the color reaction. The density values shown in Figure 5 were obtained by densitometric scanning of the reaction product on blots from which the control value (no primary antibody) was subtracted. A further control was one in which the mab 10H3 was added to blots containing Opl in the absence of added exogenous peptide. This control value represents the antibody-antigen reaction without interference from added peptides.

Based upon the results presented in Figure 5, applicants concluded that A4-0 bound to itself with at an optimal concentration of 5.0 ug/ml. Example 4: Self-aggregation of A4-H peptides on immunoblots

The experiment of Example 3 was repeated except that the exogenous peptide was A4-H. The data are shown in Figure 6 and based upon these results, applicants concluded that A4-H bound to A4-0 at an optimal concentration of 2.5 ug/ml.

Example 5: Self-aggregation of Opl peptides on immunoblots

The experiment of Example 3 was repeated except that the exogenous peptide was Opl. The data are shown in Figure 7 and based upon these results, applicants concluded that Opl bound to A4-0 at an optimal concentration of 2.5 ug/ml.

Based upon the results presented in Figures 5, 6 and 7, applicants concluded that three peptides bound the filter-bound A4-0 peptide and increased the extent to which 10H3 reacted. The reaction is concentration- dependent. The three peptides, A4-0, A4-H and Opl, aggregated to the attached A4-0. The Opl 10-mer reacted nearly as well or better, at 2.5 ug, as the larger 28- ers.

Example 6: Specificity of 10H3 for both A4-0 and Qpl

The results shown in Figure 7 indicate that a small peptide, a ten-mer, was able to bind at least as well as 1-28-mers to an A4 substrate. Thus, this assay, which measures the optical density of the reaction product between the added 10H3 mab and the Opl peptide on the solid surface, reflected the presence of the exogenous peptide, as applicants previously demonstrated for the reaction between 10H3 and A4-0.

With respect to Opl, additional studies were carried out to confirm the reactivity of 10H3. On separate solid supports (Millipore P paper) either the A4-0 antigen (2ug/slot) or the Opl antigen (2 ug/slot) were absorbed using a slot blot apparatus. The results are shown in Figure 8, as follows:

Reactivity of 10H3 towards A4-0 (upper panels : Blot no:

1. Immunostain lacking the primary antibody (10H3) showed no reactivity with the blot, as expected. 2. 10H3 was very strongly reactive with its own antigen, A4-0.

3. Soluble A4-0 antigen added to the mix caused inhibition of 10H3 towards A4-0.

5 4. Soluble Opl added to the mix caused inhibition of 10H3 towards A4-0.

5. 10H3 was reactive towards the Opl antigen.

6. Soluble Opl added to the mix showed inhibition of 10H3 towards Opl.

10 The slot blots were quantified by densitometry and numerical values were obtained that indicated the extent of the reaction between 10H3 and antigens. These values are given below in Table I in which each numbered item refers to the blot number in Figure 8 and the description

15 given above:

Table I: Optical Density of Reaction Between 10H3 and Either A4-0 or Opl Antigens in Blots of Figure 7

Slot Number: 1 2 3 4 5 6

OD Units: 0.03 0.70 0.31 0.18 0.16 0.07

Based upon the results presented in Figure 8 and Table I, applicants concluded that 10H3 is reactive with its own A4-0 antigen as well as with the Opl peptide.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: THE MIRIAM HOSPITAL

(ii) TITLE OF INVENTION: Composition and Method for in Vivo Imaging of Amyloid Deposits

(iii) NUMBER OF SEQUENCES: 13

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Foley & Lardner

(B) STREET: 3000 K Street, N.W. , Suite 500

(C) CITY: Washington, D.C.

(E) COUNTRY: USA

(F) ZIP: 20007-5109

(V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: 27 May 1994

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: SAXΞ, Bernhard D.

(B) REGISTRATION NUMBER: 28,665

(C) REFERENCE/DOCKET NUMBER: 57548/103/MIHO

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (202)672-5300

(B) TELEFAX: (202)672-5399

(C) TELEX: 904136

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Gin Val His His Gin Lys 1 5 10 15

Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys 20 25

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 43 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gin Lys 1 5 10 15

Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala He He 20 25 30

Gly Leu Met Val Gly Gly Val Val He Ala Thr 35 40

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 52 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gin Lys 1 5 10 15

Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala He He 20 25 30

Gly Leu Met Val Gly Gly Val Val He Ala Thr Val He Val He Thr 35 40 45

Leu Val Met Leu 50

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 99 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gin Lys 1 5 10 15

Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala He He 20 25 30

Gly Leu Met Val Gly Gly Val Val He Ala Thr Val He Val He Thr 35 40 45

Leu Val Met Leu Lys Lys Lys Gin Tyr Thr Ser He His His Gly Val 50 55 60

Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys 65 70 75 80

Met Gin Gin Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gin 85 90 95

Met Gin Asn

(2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 53 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Lys Thr Glu Glu He Ser Glu Val Lys Met Asp Ala Glu Phe Arg His 1 5 10 15

Asp Ser Gly Tyr Glu Val His His Gin Lys Leu Val Phe Phe Ala Glu 20 25 30

Asp Val Gly Ser Asn Lys Gly Ala He He Gly Leu Met Val Gly Gly 35 40 45

Val Val He Ala Thr 50

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: /note= "Xaa at position 1 corresponds to 1 or more APP amino acids which are not adjacent to B/A4 in nature."

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 45

(D) OTHER INFORMATION: /note= "Xaa at position 45 corresponds to 1 or more APP amino acids which are not adjacent to B/A4 in nature."

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Xaa Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gin 1 5 10 15

Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala He 20 25 30

He Gly Leu Met Val Gly Gly Val Val He Ala Thr Xaa 35 40 45

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(vii) IMMEDIATE SOURCE: (B) CLONE: A4-0 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gin Lys 1 5 10 15

Leu Val Phe Phe Ala Glu Asp Val Gly Ser Ser Ala 20 25

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(vii) IMMEDIATE SOURCE: (B) CLONE: A4-P

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Gin Val His His Gin Lys 1 5 10 15

Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys 20 25

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(vii) IMMEDIATE SOURCE: (B) CLONE: A4-B

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gin Lys 1 5 10 15

Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys 20 25

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(vii) IMMEDIATE SOURCE: (B) CLONE: Opl

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

Asp Ala Glu Phe Arg His Asp Ser Gly Tyr 1 5 10

(2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3353 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 147..2234

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

AGTTTCCTCG GCAGCGGTAG GCGAGAGCAC GCGGAGGAGC GTGCGCGGGG CCCCGGGAGA 60

CGGCGGCGGT GGCGGCGCGG GCAGAGCAAG GACGCGGCGG ATCCCACTCG CACAGCAGCG 120

CACTCGGTGC CCCGCGCAGG GTCGCG ATG CTG CCC GGT TTG GCA CTG CTC CTG 173

Met Leu Pro Gly Leu Ala Leu Leu Leu 1 5

CTG GCC GCC TGG ACG GCT CGG GCG CTG GAG GTA CCC ACT GAT GGT AAT 221 Leu Ala Ala Trp Thr Ala Arg Ala Leu Glu Val Pro Thr Asp Gly Asn 10 15 20 25

GCT GGC CTG CTG GCT GAA CCC CAG ATT GCC ATG TTC TGT GGC AGA CTG 269 Ala Gly Leu Leu Ala Glu Pro Gin He Ala Met Phe Cys Gly Arg Leu 30 35 40

AAC ATG CAC ATG AAT GTC CAG AAT GGG AAG TGG GAT TCA GAT CCA TCA 317 Asn Met His Met Asn Val Gin Asn Gly Lys Trp Asp Ser Asp Pro Ser 45 50 55

GGG ACC AAA ACC TGC ATT GAT ACC AAG GAA GGC ATC CTG CAG TAT TGC 365 Gly Thr Lys Thr Cys He Asp Thr Lys Glu Gly He Leu Gin Tyr Cys 60 65 70

CAA GAA GTC TAC CCT GAA CTG CAG ATC ACC AAT GTG GTA GAA GCC AAC 413 Gin Glu Val Tyr Pro Glu Leu Gin He Thr Asn Val Val Glu Ala Asn 75 80 85

CAA CCA GTG ACC ATC CAG AAC TGG TGC AAG CGG GGC CGC AAG CAG TGC 461 Gin Pro Val Thr He Gin Asn Trp Cys Lys Arg Gly Arg Lys Gin Cys S»0 95 100 105

AAG ACC CAT CCC CAC TTT GTG ATT CCC TAC CGC TGC TTA GTT GGT GAG 509 Lys Thr His Pro His Phe Val He Pro Tyr Arg Cys Leu Val Gly Glu 110 115 120

TTT GTA AGT GAT GCC CTT CTC GTT CCT GAC AAG TGC AAA TTC TTA CAC 557 Phe Val Ser Asp Ala Leu Leu Val Pro Asp Lys Cys Lys Phe Leu His 125 130 135

CAG GAG AGG ATG GAT GTT TGC GAA ACT CAT CTT CAC TGG CAC ACC GTC 605 Gin Glu Arg Met Asp Val Cys Glu Thr His Leu His Trp His Thr Val 140 145 150

GCC AAA GAG ACA TGC AGT GAG AAG AGT ACC AAC TTG CAT GAC TAC GGC 653 Ala Lys Glu Thr Cys Ser Glu Lys Ser Thr Asn Leu His Asp Tyr Gly 155 160 165

ATG TTG CTG CCC TGC GGA ATT GAC AAG TTC CGA GGG GTA GAG TTT GTG 701 Met Leu Leu Pro Cys Gly He Asp Lys Phe Arg Gly Val Glu Phe Val 170 175 180 185

TGT TGC CCA CTG GCT GAA GAA AGT GAC AAT GTG GAT TCT GCT GAT GCG 749 Cys Cys Pro Leu Ala Glu Glu Ser Asp Asn Val Asp Ser Ala Asp Ala 190 195 200

GAG GAG GAT GAC TCG GAT GTC TGG TGG GGC GGA GCA GAC ACA GAC TAT 797 Glu Glu Asp Asp Ser Asp Val Trp Trp Gly Gly Ala Asp Thr Asp Tyr 205 210 215

GCA GAT GGG AGT GAA GAC AAA GTA GTA GAA GTA GCA GAG GAG GAA GAA 845 Ala Asp Gly Ser Glu Asp Lys Val Val Glu Val Ala Glu Glu Glu Glu 220 225 230

GTG GCT GAG GTG GAA GAA GAA GAA GCC GAT GAT GAC GAG GAC GAT GAG 893 Val Ala Glu Val Glu Glu Glu Glu Ala Asp Asp Asp Glu Asp Asp Glu 235 240 245

GAT GGT GAT GAG GTA GAG GAA GAG GCT GAG GAA CCC TAC GAA GAA GCC 941 Asp Gly Asp Glu Val Glu Glu Glu Ala Glu Glu Pro Tyr Glu Glu Ala 250 255 260 265

ACA GAG AGA ACC ACC AGC ATT GCC ACC ACC ACC ACC ACC ACC ACA GAG 989 Thr Glu Arg Thr Thr Ser He Ala Thr Thr Thr Thr Thr Thr Thr Glu 270 275 280

TCT GTG GAA GAG GTG GTT CGA GTT CCT ACA ACA GCA GCC AGT ACC CCT 1037 Ser Val Glu Glu Val Val Arg Val Pro Thr Thr Ala Ala Ser Thr Pro 285 290 295

GAT GCC GTT GAC AAG TAT CTC GAG ACA CCT GGG GAT GAG AAT GAA CAT 1085 Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp Glu Asn Glu His 300 305 310

GCC CAT TTC CAG AAA GCC AAA GAG AGG CTT GAG GCC AAG CAC CGA GAG 1133 Ala His Phe Gin Lys Ala Lys Glu Arg Leu Glu Ala Lys His Arg Glu 315 320 325

AGA ATG TCC CAG GTC ATG AGA GAA TGG GAA GAG GCA GAA CGT CAA GCA 1181 Arg Met Ser Gin Val Met Arg Glu Trp Glu Glu Ala Glu Arg Gin Ala 330 335 340 345

AAG AAC TTG CCT AAA GCT GAT AAG AAG GCA GTT ATC CAG CAT TTC CAG 1229 Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala Val He Gin His Phe Gin 350 355 360

GAG AAA GTG GAA TCT TTG GAA CAG GAA GCA GCC AAC GAG AGA CAG CAG 1277 Glu Lys Val Glu Ser Leu Glu Gin Glu Ala Ala Asn Glu Arg Gin Gin 365 370 375

CTG GTG GAG ACA CAC ATG GCC AGA GTG GAA GCC ATG CTC AAT GAC CGC 1325 Leu Val Glu Thr His Met Ala Arg Val Glu Ala Met Leu Asn Asp Arg 380 385 390

CGC CGC CTG GCC CTG GAG AAC TAC ATC ACC GCT CTG CAG GCT GTT CCT 1373 Arg Arg Leu Ala Leu Glu Asn Tyr He Thr Ala Leu Gin Ala Val Pro 395 400 405

CCT CGG CCT CGT CAC GTG TTC AAT ATG CTA AAG AAG TAT GTC CGC GCA 1421 Pro Arg Pro Arg His Val Phe Asn Met Leu Lys Lys Tyr Val Arg Ala 410 415 420 425

GAA CAG AAG GAC AGA CAG CAC ACC CTA AAG CAT TTC GAG CAT GTG CGC 1469 Glu Gin Lys Asp Arg Gin His Thr Leu Lys His Phe Glu His Val Arg 430 435 440

ATG GTG GAT CCC AAG AAA GCC GCT CAG ATC CGG TCC CAG GTT ATG ACA 1517 Met Val Asp Pro Lys Lys Ala Ala Gin He Arg Ser Gin Val Met Thr 445 450 455 CAC CTC CGT GTG ATT TAT GAG CGC ATG AAT CAG TCT CTC TCC CTG CTC 1565 His Leu Arg Val He Tyr Glu Arg Met Asn Gin Ser Leu Ser Leu Leu 460 465 470

TAC AAC GTG CCT GCA GTG GCC GAG GAG ATT CAG GAT GAA GTT GAT GAG 1613 Tyr Asn Val Pro Ala Val Ala Glu Glu He Gin Asp Glu Val Asp Glu 475 480 485

CTG CTT CAG AAA GAG CAA AAC TAT TCA GAT GAC GTC TTG GCC AAC ATG 1661 Leu Leu Gin Lys Glu Gin Asn Tyr Ser Asp Asp Val Leu Ala Asn Met 490 495 500 505

ATT AGT GAA CCA AGG ATC AGT TAC GGA AAC GAT GCT CTC ATG CCA TCT 1709 He Ser Glu Pro Arg He Ser Tyr Gly Asn Asp Ala Leu Met Pro Ser 510 515 520

TTG ACC GAA ACG AAA ACC ACC GTG GAG CTC CTT CCC GTG AAT GGA GAG 1757 Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro Val Asn Gly Glu 525 530 535

TTC AGC CTG GAC GAT CTC CAG CCG TGG CAT TCT TTT GGG GCT GAC TCT 1805 Phe Ser Leu Asp Asp Leu Gin Pro Trp His Ser Phe Gly Ala Asp Ser 540 545 550

GTG CCA GCC AAC ACA GAA AAC GAA GTT GAG CCT GTT GAT GCC CGC CCT 1853 Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val Asp Ala Arg Pro 555 560 565

GCT GCC GAC CGA GGA CTG ACC ACT CGA CCA GGT TCT GGG TTG ACA AAT 1901 Ala Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser Gly Leu Thr Asn 570 575 580 585

ATC AAG ACG GAG GAG ATC TCT GAA GTG AAG ATG GAT GCA GAA TTC CGA 1949 He Lys Thr Glu Glu He Ser Glu Val Lys Met Asp Ala Glu Phe Arg 590 595 600

CAT GAC TCA GGA TAT GAA GTT CAT CAT CAA AAA TTG GTG TTC TTT GCA 1997 His Asp Ser Gly Tyr Glu Val His His Gin Lys Leu Val Phe Phe Ala 605 610 615

GAA GAT GTG GGT TCA AAC AAA GGT GCA ATC ATT GGA CTC ATG GTG GGC 2045 Glu Asp Val Gly Ser Asn Lys Gly Ala He He Gly Leu Met Val Gly 620 625 630

GGT GTT GTC ATA GCG ACA GTG ATC GTC ATC ACC TTG GTG ATG CTG AAG 2093 Gly Val Val He Ala Thr Val He Val He Thr Leu Val Met Leu Lys 635 640 645

AAG AAA CAG TAC ACA TCC ATT CAT CAT GGT GTG GTG GAG GTT GAC GCC 2141 Lys Lys Gin Tyr Thr Ser He His His Gly Val Val Glu Val Asp Ala 650 655 660 665

GCT GTC ACC CCA GAG GAG CGC CAC CTG TCC AAG ATG CAG CAG AAC GGC 2189 Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met Gin Gin Asn Gly 670 675 680

TAC GAA AAT CCA ACC TAC AAG TTC TTT GAG CAG ATG CAG AAC TAGACCCCCG 2241 Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gin Met Gin Asn 685 690 695

CCACAGCAGC CTCTGAAGTT GGACAGCAAA ACCATTGCTT CACTACCCAT CGGTGTCCAT 2301

TTATAGAATA ATGTGGGAAG AAACAAACCC GTTTTATGAT TTACTCATTA TCGCCTTTTG 2361

ACAGCTGTGC TGTAACACAA GTAGATGCCT GAACTTGAAT TAATCCACAC ATCAGTAATG 2421

TATTCTATCT CTCTTTACAT TTTGGTCTCT ATACTACATT ATTAATGGGT TTTGTGTACT 2481 GTAAAGAATT TAGCTGTATC AAACTAGTGC ATGAATAGAT TCTCTCCTGA TTATTTATCA 2541

CATAGCCCCT TAGCCAGTTG TATATTATTC TTGTGGTTTG TGACCCAATT AAGTCCTACT 2601

TTACATATGC TTTAAGAATC GATGGGGGAT GCTTCATGTG AACGTGGGAG TTCAGCTGCT 2661

TCTCTTGCCT AAGTATTCCT TTCCTGATCA CTATGCATTT TAAAGTTAAA CATTTTTAAG 2721

TATTTCAGAT GCTTTAGAGA GATTTTTTTT CCATGACTGC ATTTTACTGT ACAGATTGCT 2781

GCTTCTGCTA TATTTGTGAT ATAGGAATTA AGAGGATACA CACGTTTGTT TCTTCGTGCC 2841

TGTTTTATGT GCACACATTA GGCATTGAGA CTTCAAGCTT TTCTTTTTTT GTCCACGTAT 2901

CTTTGGGTCT TTGATAAAGA AAAGAATCCC TGTTCATTGT AAGCACTTTT ACGGGGCGGG 2961

TGGGGAGGGG TGCTCTGCTG GTCTTCAATT ACCAAGAATT CTCCAAAACA ATTTTCTGCA 3021

GGATGATTGT ACAGAATCAT TGCTTATGAC ATGATCGCTT TCTACACTGT ATTACATAAA 3081

TAAATTAAAT AAAATAACCC CGGGCAAGAC TTTTCTTTGA AGGATGACTA CAGACATTAA 3141

ATAATCGAAG TAATTTTGGG TGGGGAGAAG AGGCAGATTC AATTTTCTTT AACCAGTCTG 3201

AAGTTTCATT TATGATACAA AAGAAGATGA AAATGGAAGT GGCAATATAA GGGGATGAGG 3261

AAGGCATGCC TGGACAAACC CTTCTTTTAA GATGTGTCTT CAATTTGTAT AAAATGGTGT 3321

TTTCATGTAA ATAAATACAT TCTTGGAGGA GC 3353

(2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 695 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg 1 5 10 15

Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro 20 25 30

Gin He Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val Gin 35 40 45

Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys He Asp 50 55 60

Thr Lys Glu Gly He Leu Gin Tyr Cys Gin Glu Val Tyr Pro Glu Leu 65 70 75 80

Gin He Thr Asn Val Val Glu Ala Asn Gin Pro Val Thr He Gin Asn 85 90 95

Trp Cys Lys Arg Gly Arg Lys Gin Cys Lys Thr His Pro His Phe Val 100 105 110

He Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu

115 120 125

Val Pro Asp Lys Cys Lys Phe Leu His Gin Glu Arg Met Asp Val Cys 130 135 140

Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu 145 150 155 160

Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly He 165 170 175

Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu 180 185 190

Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val 195 200 205

Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys 210 215 220

Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu 225 230 235 240

Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu 245 250 255

Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser He 260 265 270

Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg 275 280 285

Val Pro Thr Thr Ala Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu 290 295 300

Glu Thr Pro Gly Asp Glu Asn Glu His Ala His Phe Gin Lys Ala Lys 305 310 315 320

Glu Arg Leu Glu Ala Lys His Arg Glu Arg Met Ser Gin Val Met Arg 325 330 335

Glu Trp Glu Glu Ala Glu Arg Gin Ala Lys Asn Leu Pro Lys Ala Asp 340 345 350

Lys Lys Ala Val He Gin His Phe Gin Glu Lys Val Glu Ser Leu Glu 355 360 365

Gin Glu Ala Ala Asn Glu Arg Gin Gin Leu Val Glu Thr His Met Ala 370 375 380

Arg Val Glu Ala Met Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn 385 390 395 400

Tyr He Thr Ala Leu Gin Ala Val Pro Pro Arg Pro Arg His Val Phe 405 410 415

Asn Met Leu Lys Lys Tyr Val Arg Ala Glu Gin Lys Asp Arg Gin His 420 425 430

Thr Leu Lys His Phe Glu His Val Arg Met Val Asp Pro Lys Lys Ala 435 440 445

Ala Gin He Arg Ser Gin Val Met Thr His Leu Arg Val He Tyr Glu 450 455 460

Arg Met Asn Gin Ser Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala 465 470 475 480

Glu Glu He Gin Asp Glu Val Asp Glu Leu Leu Gin Lys Glu Gin Asn 485 490 495 Tyr Ser Asp Asp Val Leu Ala Asn Met He Ser Glu Pro Arg He Ser 500 505 510

Tyr Gly Asn Asp Ala Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr 515 520 525

Val Glu Leu Leu Pro Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gin 530 535 540

Pro Trp His Ser Phe Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn 545 550 555 560

Glu Val Glu Pro Val Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr 565 570 575

Thr Arg Pro Gly Ser Gly Leu Thr Asn He Lys Thr Glu Glu He Ser 580 585 590

Glu Val Lys Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val 595 600 605

His His Gin Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys 610 615 620

Gly Ala He He Gly Leu Met Val Gly Gly Val Val He Ala Thr Val 625 630 635 640

He Val He Thr Leu Val Met Leu Lys Lys Lys Gin Tyr Thr Ser He 645 650 655

His His Gly Val Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg 660 665 670

His Leu Ser Lys Met Gin Gin Asn Gly Tyr Glu Asn Pro Thr Tyr Lys 675 680 685

Phe Phe Glu Gin Met Gin Asn 690 695

(2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 11

(D) OTHER INFORMATION: /note= ""Xaa at Position 11 is either Glu or Gin.""

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 27

(D) OTHER INFORMATION: /note= ""Xaa at postion 27 is either Ser or Asn.""

(i ) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 28

(D) OTHER INFORMATION: /note= ""Xaa at position 28 is either Ala of Lys."" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Xaa Val His His Gin Lys 1 5 10 15

Leu Val Phe Phe Ala Glu Asp Val Gly Ser Xaa Xaa 20 25

Claims

hat Is Claimed Is:

1. An amyloid binding composition for in vivo imaging of amyloid deposits comprising:

(a) a labeled amyloid protein or variant thereof that binds to amyloid deposits in vivo; and

(b) a pharmaceutically acceptable carrier.

2. The composition of claim 1, wherein said amyloid protein is β-amyloid polypeptide or a variant thereof.

3. The composition of claim 2, wherein said β- amyloid polypeptide variant has the following amino acid sequence (SEQ ID NO:13):

N-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-X-Val-His-His- Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Yl-Y2- COOH; wherein X is either Glu or Gin; Yl is either Ser or Asn; and Y2 is either Ala or Lys.

4. The composition of claim 3, wherein said β- amyloid polypeptide variant is selected from the group consisting of (1) a variant wherein when X is Glu, Yl is Ser and Y2 is Ala, (2) a variant wherein when X is Glu, Yl is Asn and Y2 is Lys, and (3) a variant wherein when X is Gin, Yl is Asn and Y2 is Lys.

5. The composition of claim 2, wherein said β- amyloid polypeptide or variant thereof has an amino acid sequence selected from the following group of amino acid sequences:

(A) (SEQ ID NO:2) Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-

Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val- Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn- Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val- Gly-Gly-Val-Val-Ile-Ala-Thr;

(B) (SEQ ID NO:3) Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-

Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val- Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn- Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val- Gly-Gly-Val-Val-Ile-Ala-Thr-Val-Ile- Val-I le-Thr-Leu-Val-Met-Leu ;

(C) (SEQ ID NO : 4 ) Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-

Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val- Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn- Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val- Gly-Gly-Val-Val-Ile-Ala-Thr-Val-Ile- Val-Ile-Thr-Leu-Val-Met-Leu-Lys-Lys- Lys-Gln-Tyr-Thr-Ser-Ile-His-His-Gly- Val-Val-Glu-Val-Asp-Ala-Ala-Val-Thr- Pro-Glu-Glu-Arg-His-Leu-Ser-Lys-Met- Gln-Gln-Asn-Gly-Tyr-Glu-Asn-Pro-Thr- Tyr-Lys-Phe-Phe-Glu-Gln-Met-Gln-Asn;

(D) (SEQ ID NO: 5) Lys-Thr-Glu-Glu-Ile-Ser-Glu-Val-Lys-

Met-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser- Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu- Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser- Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met- Val-Gly-Gly-Val-Val-Ile-Ala-Thr;

(E) (SEQ ID NO: 6) X-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-

Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu- Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser- Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met- Val-Gly-Gly-Val-Val-Ile-Ala-Thr-Y, wherein X and Y are one or more APP amino acids which are not ajacent to 0/A4 in the nature; and

(F) any fragment of (A)-(E), wherein said fragment is large enough to bind amyloid deposit in vivo.

6. The composition of claim 1, wherein said labeled amyloid protein is radiolabeled amyloid protein.

7. The composition of claim 1, wherein said radiolabeled amyloid protein is Technetium 99m-labeled amyloid protein.

8. An in vivo method for detecting amyloid deposits in a subject comprising the steps of (a) administering to a subject a detectable quantity of an amyloid binding composition comprising a labeled amyloid protein or variant thereof and a pharmaceutically acceptable carrier; and

(b) detecting the binding of the labeled protein or variant thereof to the amyloid deposit.

9. The method of claim 8, wherein said amyloid protein is the β-amyloid polypeptide or variant thereof.

10. The method of claim 8, wherein said amyloid protein is radiolabeled.

11. The method of claim 10, wherein said detecting involves radioactive imaging.

12. The method of claim 8, wherein said administering is selected from the group consisting of intravenous injection, intraventricular injection and a combination of both intravenous and intraventricular injection.

13. The method of claim 8, wherein said amyloid deposits are located in the brain of a subject.

14. A method of diagnosing an amyloidosis-associated disease by detecting amyloid deposits in a subject suspected of having amyloid deposits, said method comprising the steps of:

(a) administering to a subject a detectable quantity of an amyloid binding composition comprising a labeled amyloid protein or variant thereof and a pharmaceutically acceptable carrier; and

(b) detecting the binding of said labeled protein to said amyloid deposit.

15. The method of claim 14, wherein said amyloidosis-associated disease is selected from the group consisting of Alzheimer's Disease and Down Syndrome.