# Patent

## Title
Therapeutic applications of laminin and laminin-derived protein fragments

## Abstract

Laminin and specific laminin-derived protein fragments are disclosed as potent inhibitors of Alzheimer's disease type amyloidoses. A specific region is identified within laminin which interacts with the Alzheimer's disease beta-amyloid protein and contributes to the observed inhibitory and therapeutic effects.
A prominent  ̃130 kilodalton band in laminin was found in human serum and cerebrospinal fluid which primarily interacted with Aβ as determined by ligand blotting methodology. This  ̃130 kilodalton laminin fragment is known as the E8 fragment and is also believed to consist of the globular domains of the laminin A chain. The interaction of specific laminin fragments such as the newly discovered  ̃130 kDa protein is believed to bind Aβ in biological fluids and keep it in a soluble state.

## Background
This is a continuation of U.S. Application Ser. No. 08/947,057 filed Oct. 8, 1997 now abandoned, which claims priority to U.S. Provisional Application No. 60/027,981 filed Oct. 8, 1996.


TECHNICAL FIELD 
The invention relates to the discovery, identification and use of laminin, laminin-derived protein fragments, and laminin-derived polypeptides, as well as related peptides and antibodies, for the therapeutic intervention and diagnosis of Alzheimer's disease and other amyloidoses. In addition, the discovery and identification of an Alzheimer's beta-amyloid protein (Aβ) specific binding region within the globular domain repeats of the laminin A chain, has led to new diagnostic and therapeutic applications for Alzheimer's disease and other amyloidoses which are disclosed.
BACKGROUND OF THE INVENTION
Alzheimer's disease is characterized by the accumulation of a 39-43 amino acid peptide termed the beta-amyloid protein or Aβ, in a fibrillar form, existing as extracellular amyloid plaques and as amyloid within the walls of cerebral blood vessels. Fibrillar Aβ amyloid deposition in Alzheimer's disease is believed to be detrimental to the patient and eventually leads to toxicity and neuronal cell death, characteristic hallmarks of Alzheimer's disease. Accumulating evidence now implicates amyloid as a major causative factor of Alzheimer's disease pathogenesis. Discovery and identification of new compounds, agents, proteins, polypeptides or protein-derivatives as potential therapeutic agents to arrest Alzheimer's disease Aβ amyloid formation, deposition, accumulation and/or persistence is desperately sought.
It is known that Aβ is normally present in human blood and cerebrospinal fluid. However, it is not known why this potential fibrillar protein remains soluble in circulating biological fluids. Can the agent(s) responsible for this extraordinary solubility of fibrillar Aβ be applied to diagnostic and therapeutic regimens against the fibrillar Aβ amyloid present in Alzheimer's brain?
SUMMARY OF THE INVENTION
The present invention provides answers to these questions and relates to the novel and surprising discovery that laminin and specific laminin-derived protein fragments are indeed potent inhibitors of Alzheimer's disease amyloidosis, and therefore have potential use for the therapeutic intervention and diagnosis of the amyloidoses. In addition, we have identified a specific region within laminin which interacts with the Alzheimer's disease beta-amyloid protein and contributes to the observed inhibitory and therapeutic effects. In addition, specific laminin-derived protein fragments which also interact with the Aβ of Alzheimer's disease have been discovered to be present in human serum and cerebrospinal fluid, and implicate diagnostic applications which are described.
Laminin is a specific basement membrane component that is involved in several fundamental biological processes, and may play important roles in the pathogenesis of a number of different human diseases. Using a solid phase binding immunoassay, the present invention determined that laminin binds the Aβ of Alzheimer's disease with a single binding constant of Kd=2.7×10−9 M. In addition, using a Thioflavin T fluorometry assay (which quantitatively determines the amount of fibrillar amyloid formed), the present invention has determined that laminin is surprisingly an extremely potent inhibitor of Aβ fibril formation. In this latter study, 25 μM of Aβ (residues 1-40) was incubated at 37° C. for 1 week in the presence or absence of 100 nM laminin. Laminin was found to significantly (p<0.001) inhibit Aβ (1-40) amyloid fibril formation by 2.9-fold at 1 hour, 4.6-fold at 1 day, 30.6-fold at 3 days and 27.1-fold at 1 week. Other basement membrane components including perlecan, fibronectin and type IV collagen were not effective inhibitors of Aβ (1-40) fibrillogenesis in comparison to laminin, demonstrating the specificity of the inhibitory effect exhibited by laminin. The inhibitory effects of laminin on Aβ fibrillogenesis was also found to occur in a dose-dependent manner. In addition, laminin was found to cause dissolution of pre-formed Alzheimer's disease amyloid fibrils in a dose-dependent manner following 4 days of incubation. Laminin was digested with V8, trypsin or elastase to determine small protease-resistent fragments of laminin which still interacted with Aβ. A  ̃55 kilodalton (kDa) laminin fragment derived from V8 or elastase digested laminin was found to interact with biotinylated Aβ (1-40). Amino acid sequencing of the  ̃55 kDa fragment identified an Aβ-binding domain within laminin situated within the globular repeats of the laminin A chain.
Intact laminin was found to be present in human serum but not human cerebrospinal fluid, whereas laminin protein fragments ranging from  ̃120 kDa to  ̃200 kDa were found to be present in both human serum and cerebrospinal fluid. Of all the laminin protein fragments present in human biological fluids described above, a prominent  ̃130 kilodalton band was found in human serum and cerebrospinal fluid which primarily interacted with Aβ as determined by ligand blotting methodology. This  ̃130 kilodalton laminin fragment is known as the E8 fragment (i.e. generated following elastase digestion of laminin)(Yurchenco and Cheng, J. Biol. Chem. 268:17286-17299, 1993) and is also believed to consist of the globular domains of the laminin A chain. The interaction of specific laminin fragments such as the newly discovered  ̃130 kDa protein is believed to bind Aβ in biological fluids and keep it in a soluble state. The present invention describes the use of laminin, laminin-derived protein fragments, and laminin-derived polypeptides for the therapeutic intervention and diagnosis of Alzheimer's disease and other amyloidoses. In addition, the discovery and identification of a specific Alzheimer's Aβ-binding region within the globular domain repeats of the laminin A chain, and the discovery of the presence of laminin fragments containing this region in human serum and cerebrospinal fluid, has led to new diagnostic and therapeutic applications for Alzheimer's disease and other amyloidoses.
FEATURES OF THE INVENTION
A primary object of the present invention is to establish new therapeutic methods and diagnostic applications for the amyloid diseases. The amyloid diseases include, but are not limited to, the amyloid associated with Alzheimer's disease and Down's syndrome (wherein the specific amyloid is referred to as beta-amyloid protein or Aβ), the amyloid associated with chronic inflammation, various forms of malignancy and Familial Mediterranean Fever (wherein the specific amyloid is referred to as AA amyloid or inflammation-associated amyloidosis), the amyloid associated with multiple myeloma and other B-cell dyscrasias (wherein the specific amyloid is referred to as AL amyloid), the amyloid associated with type II diabetes (wherein the specific amyloid is referred to as amylin or islet amyloid), the amyloid associated with the prion diseases including Creutzfeldt-Jakob disease, Gerstmann-Straussler syndrome, kuru and animal scrapie (wherein the specific amyloid is referred to as PrP amyloid), the amyloid associated with long-term hemodialysis and carpal tunnel syndrome (wherein the specific amyloid is referred to as beta2-microglobulin amyloid), the amyloid associated with senile cardiac amyloid and Familial Amyloidotic Polyneuropathy (wherein the specific amyloid is referred to as transthyretin or prealbumin), and the amyloid associated with endocrine tumors such as medullary carcinoma of the thyroid (wherein the specific amyloid is referred to as variants of procalcitonin).
A primary object of the present invention is to use laminin, laminin-derived protein fragments and/or laminin-derived polypeptides as potent inhibitors of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease and other amyloidoses. “Laminin fragments, laminin-derived fragments, laminin-derived protein fragments and/or laminin-derived polypeptides”, may include, but are not limited to, laminin A (or A1) chain, laminin B1 chain, laminin B2 chain, laminin A2 chain (merosin), laminin G1 chain, the globular domain repeats within the laminin A1 chain, SEQ ID NO: 1 (11 amino acid sequence within the mouse laminin A chain), SEQ ID NO: 2 (fourth globular repeat with the mouse laminin A chain), SEQ ID NO: 3 (fourth globular repeat within the human laminin A chain), SEQ ID NO: 4 (mouse laminin A chain), SEQ ID NO: 5 (human laminin A chain), SEQ ID NO: 6 (human laminin B1 chain), SEQ ID NO: 7 (mouse laminin B1 chain), SEQ ID NO: 8 (rat laminin B2 chain), SEQ ID NO: 9 (human laminin B2 chain), SEQ ID NO: 10 (mouse laminin G1 chain), SEQ ID NO: 11 (human laminin G1 chain), and all fragments or combinations thereof.
Yet another object of the present invention is to use conformational dependent proteins, polypeptides, or fragments thereof for the treatment of Alzheimer's disease and other amyloidoses. Such conformational dependent proteins include, but are not limited to, laminin, laminin-derived fragments including laminin A1 chain (SEQ ID NO 4; SEQ ID NO: 5), the globular repeat domains within the laminin A1 chain (SEQ ID NO: 2, SEQ ID NO:3), an 11- amino acid peptide sequence within the globular domain of the laminin A chain (SEQ ID NO:1), laminin B1 chain (SEQ ID NO:6, SEQ ID NO: 7), laminin B2 chain (SEQ ID NO: 8, SEQ ID NO:9), laminin G1 chain (SEQ ID NO: 10, SEQ ID NO: 11) and/or portions thereof.
Yet another aspect of the present invention is to use peptidomimetic compounds modelled from laminin, laminin-derived protein fragments and/or laminin-derived polypeptides, including but not limited to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, and fragments thereof, as potent inhibitors of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease and other amyloidoses.
Yet another object of the present invention is to mimic the 3-dimensional Aβ-binding site(s) on laminin, laminin-derived protein fragments and/or laminin-derived polypeptides and use these mimics as potent inhibitors of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease and other amyloidoses.
Yet a further aspect of the present invention is to use anti-idiotypic antibodies to laminin, laminin-derived protein fragments and/or laminin-derived polypeptides as potent inhibitors of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease and other amyloidoses.
Another aspect of the invention is to provide new and novel polyclonal and/or monoclonal peptide antibodies which can be utilized in a number of in vitro assays to specifically detect Aβ-binding laminin derived protein fragments and/or Aβ-binding laminin derived polypeptides in human tissues and/or biological fluids. Polyclonal or monoclonal antibodies that are made specifically against a peptide portion or fragment of laminin which interacts with Aβ can be utilized to detect and quantify amyloid disease specific laminin fragments in human tissues and/or biological fluids. These antibodies can be made by administering the peptides in antigenic form to a suitable host. Polyclonal or monoclonal antibodies may be prepared by standard techniques known to those skilled in the art.
Another object of the present invention is to use laminin, the Aβ-binding laminin fragments and/or laminin-derived polypeptides referred to above, for the detection and specific localization of laminin peptides important in the amyloid diseases in human tissues, cells, and/or cell culture using standard immunohistochemical techniques.
Yet another aspect of the present invention is to use antibodies recognizing laminin, any of the Aβ-binding laminin fragments, and/or laminin-derived polypeptides including, but not limited to, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, and fragments thereof, for in vivo labelling; for example, with a radionucleotide, for radioimaging to be utilized for in vivo diagnosis, and/or for in vitro diagnosis.
Yet another aspect of the present invention is to make use of laminin, Aβ-binding laminin protein fragments and/or Aβ-binding laminin-derived polypeptides including, but not limited to, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, and fragments thereof, as potential therapeutics to inhibit the deposition, formation, and accumulation of fibrillar amyloid in Alzheimer's disease and other amyloidoses (described above), and to enhance the clearance and/or removal of preformed amyloid deposits in brain (for Alzheimer's disease and Down's syndrome amyloidosis) and in systemic organs (for systemic amyloidoses).
Another object of the present invention is to use Aβ-binding laminin-derived polypeptides or fragments thereof, in conjunction with polyclonal and/or monoclonal antibodies generated against these peptide fragments, using in vitro assays to detect amyloid disease specific autoantibodies in human biological fluids. Specific assay systems can be utilized to not only detect the presence of autoantibodies against Aβ-binding laminin-derived protein fragments or polypeptides thereof in biological fluids, but also to monitor the progression of disease by following elevation or diminution of laminin protein fragments and/or laminin-derived polypeptide autoantibody levels.
Another aspect of the invention is to utilize laminin, laminin-derived protein fragments and/or laminin-derived polypeptide antibodies and/or molecular biology probes for the detection of these laminin derivatives in human tissues in the amyloid diseases.
Yet another object of the present invention is to use the laminin-derived protein fragments of the present invention in each of the various therapeutic and diagnostic applications described above. The laminin-derived protein fragments include, but are not limited to, the laminin A1 chain, the globular repeats within the laminin A1 chain, the laminin B1 chain, the laminin B2 chain, the laminin G1 chain, the laminin A2 chain (also known as merosin), and all constituents or variations thereof, including but not limited to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, and fragments thereof, including peptides which have at least 70% homology to the sequences disclosed herein. Specific laminin-derived protein fragments or peptides as described above may be derived from any species including, but are not limited to, human, murine, bovine, porcine, and/or equine species.
Another object of the invention is to provide polyclonal and/or monoclonal peptide antibodies which can be utilized in a number of in vitro assays to specifically detect laminin protein fragments in human tissues and/or biological fluids. Polyclonal or monoclonal antibodies made specifically against a peptide portion or fragment of any of the laminin fragments described herein can be utilized to detect and quantify laminin-derived protein fragments in human tissues and/or biological fluids. A preferred embodiment is a polyclonal antibody made to the  ̃130 kilodalton Aβ-binding laminin fragment present in human serum and cerebrospinal fluid. These antibodies can be made by isolating and administering the laminin-derived fragments and/or polypeptides in antigenic form to a suitable host. Polyclonal or monoclonal antibodies may be prepared by standard techniques by one skilled in the art.
Yet another object of the present invention is to use laminin-derived fragment antibodies as described herein as a specific indicator for the presence and extent of laminin breakdown in brain by monitoring biological fluids including, but not limited to, cerebrospinal fluid, blood, serum, urine, saliva, sputum, and stool.
Yet another object of the present invention is to use laminin-derived fragment antibodies as described herein as a specific indicator for the presence, extent and/or progression of Alzheimer's disease and/or other brain amyloidoses by monitoring biological fluids including, but not limited to, cerebrospinal fluid, blood, serum, urine, saliva, sputum, and stool.
Yet another object of the present invention is to use laminin-derived fragment antibodies as described herein as a specific indicator for the presence and extent of laminin breakdown in systemic organs by monitoring biological fluids including, but not limited to, cerebrospinal fluid, blood, serum, urine, saliva, sputum, and stool.
Yet another object of the present invention is to use laminin-derived fragment antibodies as described herein as a specific indicator for the presence and extent of amyloidosis in type II diabetes by monitoring biological fluids including, but not limited to, cerebrospinal fluid, blood, serum, urine, saliva, sputum, and stool.
Yet another object of the present invention is to use laminin-derived fragment antibodies as described herein as a specific indicator for the presence and extent of amyloidosis in other systemic amyloidoses by monitoring biological fluids including, but not limited to, cerebrospinal fluid, blood, serum, urine, saliva, sputum, and stool.
Yet another object of the present invention is to make use of peptides or fragments of laminin as described herein, including but not limited to, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, and fragments thereof, as potential blocking therapeutics for the interaction of laminin and laminin-derived fragments in a number of biological processes and diseases (such as in Alzheimer's disease and other amyloid diseases described herein).
Yet another object of the invention is to utilize specific laminin-derived fragment antibodies, as described herein, for the detection of these laminin fragments in human tissues in the amyloid diseases.
Another object of the present invention is to use laminin, laminin-derived protein fragments, and laminin-derived polypeptides, as described herein, for the treatment of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease and other amyloidoses.
Another object of the present invention is to use pills, tablets, caplets, soft and hard gelatin capsules, lozenges, sachets, cachets, vegicaps, liquid drops, elixers, suspensions, emulsions, solutions, syrups, tea bags, aerosols (as a solid or in a liquid medium), suppositories, sterile injectable solutions,and sterile packaged powders, which contain laminin, laminin-derived protein fragments, and laminin-derived polypeptides, including, but not limited to, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, and fragments thereof, to treat patients with Alzheimer's disease and other amyloidoses.
Yet another object of the present invention is to use laminin, laminin-derived protein fragments, and laminin-derived polypeptides, including, but not limited to, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, and fragments thereof, as potent agents which inhibit amyloid formation, amyloid deposition, amyloid accumulation, amyloid persistence, and/or cause a dissolution of pre-formed or pre-deposited amyloid fibrils in Alzheimer's disease, and other amyloidoses.
Yet another object of the present invention is to provide the use of laminin, laminin-derived protein fragments, and laminin-derived polypeptides, as described herein, for inhibition of amyloid formation, deposition, accumulation, and/or persistence, regardless of its clinical setting.
Yet another object of the present invention is to provide compositions and methods involving administering to a subject a therapeutic dose of laminin, laminin-derived protein fragments, and laminin-derived polypeptides, which inhibit amyloid deposition, including but not limited to, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, and fragments thereof. Accordingly, the compositions and methods of the invention are useful for inhibiting amyloidosis in disorders in which amyloid deposition occurs. The proteins or polypeptides of the invention can be used therapeutically to treat amyloidosis or can be used prophylactically in a subject susceptible to amyloidosis. The methods of the invention are based, at least in part, in directly inhibiting amyloid fibril formation, and/or causing dissolution of preformed amyloid fibrils.
Yet another object of the present invention is to provide pharmaceutical compositions for treating amyloidosis. The pharmaceutical compositions include a therapeutic compound of the invention in an amount effective to inhibit amyloid deposition and a pharmaceutically acceptable vehicle.
These and other features and advantages of the present invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying figures.



BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention.
FIG. 1 is a binding curve demonstrating the binding interaction of EHS laminin to substrate bound Aβ (1-40). A single binding site with a Kd=2.7×10−9 M is determined.
FIG. 2 demonstrates the potent inhibition of Aβ amyloid fibril formation by laminin as determined by a Thioflavin T fluorometry assay over a 1 week experimental period.
FIG. 3 compares the potent inhibition of Aβ amyloid fibril formation by laminin to other basement membrane components including fibronectin, type IV collagen and perlecan. Only laminin is found to have a potent inhibitory effect on Aβ fibrillogenesis as early as 1 hour after incubation.
FIG. 4 is a graph of a 1 week Thioflavin T fluorometry assay utilized to determine the potential dose-dependent effects of laminin on inhibition of Aβ amyloid fibril formation. Significant dose-dependent inhibition of Aβ (1-40) amyloid fibril formation is observed at 1 day, 3 days and 1 week of treatment with increasing concentrations of laminin.
FIG. 5 is a graph of a Thioflavin T fluorometry assay utilized to determine the potential dose-dependent effects of laminin on dissolution of pre-formed Aβ (1-40) amyloid fibrils within a 4 day incubation period. Laminin causes dissolution of pre-formed Aβ amyloid fibrils in a dose-dependent manner.
FIG. 6 is a graph of a 1 week Thioflavin T fluorometry assay utilized to determine the effects of laminin on islet amyloid polypeptide (amylin) fibrillogenesis, and determine whether laminin causes a dose-dependent inhibition of amylin fibril formation. Laminin does not significantly inhibit amylin fibrillogenesis suggesting its specificity for Alzheimer's disease amyloidosis.
FIG. 7 is a black and white photograph of laminin digested with V8 protease, separated by SDS-PAGE and following interaction with biotinylated Aβ (1-40). The smallest fragment of V8-resistent laminin that interacts with Aβ is a  ̃55 kilodalton fragment.
FIG. 8 is a black and white photograph of laminin digested with trypsin, separated by SDS-PAGE and following interaction with biotinylated Aβ (1-40). The smallest fragment of trypsin-resistent laminin that interacts with Aβ is a  ̃30 kilodalton fragment.
FIGS. 9A & 9B are black and white photographs of laminin digested with elastase, separated by SDS-PAGE and following interaction with biotinylated Aβ(1-40). A  ̃55 kilodalton laminin fragment (arrow) that binds biotinylated Aβ was identified and sequenced. Note also the presence of a  ̃130 kDa fragment (arrowheads) that binds Aβ following 1.5 hours of elastase digestion (lane 2). FIG. 9A is a ligand blot using biotinylated Aβ as a probe, whereas FIG. 9B is Coomassie blue staining of the same blot in FIG. 9A to locate the specific band(s) for sequencing.
FIG. 10 (SEQ. ID NO: 12) shows the complete amino acid sequence of the mouse laminin A chain. Sequencing of the  ̃55 kilodalton Aβ-binding band shown in FIGS. 9A & 9B leads to the identification of an 11 amino acid segment (underline and arrowhead) within the laminin A chain. This Aβ binding region of laminin is situated within the globular domain repeats of the laminin A chain.
FIG. 11 shows schematic diagrams of laminin and the newly discovered “Aβ-binding region” of laminin (shown in left panel; between the two arrowheads) which is situated within the last three globular domains of the laminin A chain.
FIG. 12 is a black and white photograph of a Western blot demonstrating the presence of laminin (arrowheads) and/or laminin-derived protein fragments (bands between the two arrows) in human serum (lanes 1-7; left side) and human cerebrospinal fluid (lanes 1-7; right side) obtained from Alzheimer's disease, type II diabetes and normal aged patients. A  ̃110-130 kilodalton range of laminin positive protein fragments (between the two arrows) is present in both human serum and cerebrospinal fluid, whereas intact laminin (arrowheads) is only present in serum but not in cerebrospinal fluid.
FIG. 13 is a black and white photograph demonstrating that intact laminin (arrow) and a prominent  ̃130 kilodalton band (arrowhead) present in human Alzheimer's disease, type II diabetes and normal aged patient serum, bind Aβ. The Aβ-binding laminin and specific Aβ-binding laminin fragments in human serum were identified following separation by SDS-PAGE and interaction with nanomolar concentrations of biotinylated Aβ (1-40).
FIG. 14 is a black and white photograph demonstrating the presence of a prominent  ̃130 kilodalton band (arrow) in human Alzheimer's disease and normal aged patient cerebrospinal fluid, identified following separation by SDS-PAGE and following interaction with nanomolar concentrations of biotinylated Aβ (1-40). This same  ̃130 kilodalton Aβ-binding protein is also present in human serum (FIG. 13).



DETAILED DESCRIPTION OF THE INVENTION
The following sections are provided by way of background to better appreciate the invention.
Alzheimer's Disease
Alzheimer's disease is the most common cause of dementia in middle and late life, and is manifested by progressive impairment of memory, language, visuospatial perceptions and behavior (A Guide to the Understanding of Alzheimer's Disease and Related Disorders, edited by Jorm, New York University Press, New York 1987). A diagnosis of probable Alzheimer's disease can be made on clinical criteria (usually by the exclusion of other diseases, memory tests etc), but a definite diagnosis requires the histological examination of specific abnormalities in the brain tissue usually obtained at autopsy.
In Alzheimer's disease, the parts of the brain essential for cognitive processes such as memory, attention, language, and reasoning degenerate, robbing victims of much that makes us human, including independence. In some inherited forms of Alzheimer's disease, onset is in middle age, but more commonly, symptoms appear from the mid-60's onward. Alzheimer's disease is characterized by the deposition and accumulation of a 39-43 amino acid peptide termed the beta-amyloid protein, Aβ or β/A4 (Glenner and Wong, Biochem. Biophys. Res. Comm. 120:885-890, 1984; Masters et al, Proc. Natl. Acad. Sci. USA 82:4245-4249, 1985; Husby et al, Bull. WHO 71:105-108, 1993). Aβ is derived from larger precursor proteins termed beta-amyloid precursor proteins (or βPPs) of which there are several alternatively spliced variants. The most abundant forms of the βPPs include proteins consisting of 695, 751 and 770 amino acids (Tanzi et al, Nature 331:528-530, 1988; Kitaguchi et al, Nature 331:530-532, 1988; Ponte, et al, Nature 331:525-528, 1988). The small Aβ peptide is a major component which makes up the amyloid deposits of neuritic “plaques” and in the walls of blood vessels (known as cerebrovascular amyloid deposits) in the brains of patients with Alzheimer's disease. In addition, Alzheimer's disease is characterized by the presence of numerous neurofibrillary “tangles”, consisting of paired helical filaments which abnormally accumulate in the neuronal cytoplasm (Grundke-Iqbal et al, Proc. Natl. Acad. Sci. USA 83:4913-4917, 1986; Kosik et al, Proc. Natl. Acad. Sci. USA 83:4044-4048, 1986; Lee et al, Science 251:675-678, 1991). The pathological hallmarks of Alzheimer's disease is therefore the presence of “plaques” and “tangles”, with amyloid being deposited in the central core of plaques and within the blood vessel walls. It is important to note that a so-called “normal aged brain” has some amyloid plaques and neurofibrillary tangles present. However, in comparison, an Alzheimer's disease brain shows an over abundance of plaques and tangles. Therefore, differentiation of an Alzheimer's disease brain from a normal brain from a diagnostic point of view is primarily based on quantitative assessment of “plaques” and “tangles”.
In an Alzheimer's disease brain, there are usually thousands of neuritic plaques. The neuritic plaques are made up of extracellular deposits consisting of an amyloid core usually surrounded by enlarged axons and synaptic terminals, known as neurites, and abnormal dendritic processes, as well as variable numbers of infiltrating microglia and surrounding astrocytes. The neurofibrillary tangles present in the Alzheimer's disease brain mainly consist of tau protein, which is a microtubule-associated protein (Grundke-Iqbal et al, Proc. Natl. Acad. Sci. USA 83:4913-4917, 1986; Kosik et al, Proc. Natl. Acad. Sci. USA 83:4044-4048, 1986; Lee et al, Science 251:675-678, 1991). At the ultrastructural level, the tangle consists of paired helical filaments twisting like a ribbon, with a specific crossing over periodicity of 80 nanometers. In many instances within a neurofibrillary tangle, there are both paired helical filaments and straight filaments. In addition, the nerve cells will many times die, leaving the filaments behind. These tangles are known as “ghost tangles” since they are the filamentous remnants of the dead neuron.
The other major type of lesion found in the brain of an Alzheimer's disease patient is the accumulation of amyloid in the walls of blood vessels, both within the brain parenchyma and in the walls of the larger meningeal vessels which lie outside the brain. The amyloid deposits localized to the walls of blood vessels are referred to as cerebrovascular amyloid or congophilic angiopathy (Mandybur, J. Neuropath. Exp. Neurol. 45:79-90, 1986; Pardridge et al, J. Neurochem. 49:1394-1401, 1987).
In addition, Alzheimer's disease patients demonstrate neuronal loss and synaptic loss. Furthermore, these patients also exhibit loss of neurotransmitters such as acetylcholine. Tacrine, the first FDA approved drug for Alzheimer's disease is a cholinesterase inhibitor (Cutler and Sramek, New Engl. J. Med. 328:808-810, 1993). However, this drug has showed limited success, if any, in the cognitive improvement in Alzheimer's disease patients and initially had major side effects such as liver toxicity.
For many years there has been an ongoing scientific debate as to the importance of “amyloid” in Alzheimer's disease and whether the “plaques” and “tangles” characteristic of this disease, were a cause or merely the consequences of the disease. Recent studies during the last few years have now implicated that amyloid is indeed a causative factor for Alzheimer's disease and not merely an innocent bystander. The Alzheimer's disease Aβ protein in cell culture has been shown to cause degeneration of nerve cells within short periods of time (Pike et al, Br. Res. 563:311-314, 1991; J. Neurochem. 64:253-265, 1994). Studies suggest that it is the fibrillar structure, a characteristic of all amyloids, that is responsible for the neurotoxic effects. The Aβ has also been found to be neurotoxic in slice cultures of hippocampus (the major memory region affected in Alzheimer's)(Harrigan et al, Neurobiol. Aging 16:779-789, 1995) and induces nerve cell death in transgenic mice (Games et al, Nature 373:523-527, 1995; Hsiao et al, Neuron 15:1203-1218, 1995). In addition, injection of the Alzheimer's Aβ into rat brain causes memory impairment and neuronal dysfunction (Flood et al, Proc. Natl. Acad. Sci. U.S.A. 88:3363-3366, 1991; Br. Res. 663:271-276, 1994), two additional hallmarks of Alzheimer's disease. Probably, the most convincing evidence that amyloid (ie. beta-amyloid protein) is directly involved in the pathogenesis of Alzheimer's disease comes from genetic studies. It has been discovered that the production of Aβ can result from mutations in the gene encoding, its precursor, known as the beta-amyloid precursor protein (Van Broeckhoven et al, Science 248:1120-1122, 1990; Europ. Neurol. 35:8-19, 1995; Murrell et al, Science 254:97-99, 1991; Haass et al, Nature Med. 1:1291-1296, 1995). This precursor protein when normally processed usually only produces very little of the toxic Aβ. The identification of mutations in the amyloid precursor protein gene which causes familial, early onset Alzheimer's disease is the strongest argument that amyloid is central to the pathogenetic process underlying this disease. Four reported disease-causing mutations have now been discovered which demonstrate the importance of the beta-amyloid protein in causing familial Alzheimer's disease (reviewed in Hardy, Nature Genet. 1:233-234, 1992). All of these studies suggest that providing a drug to reduce, eliminate or prevent fibrillar Aβ formation, deposition, accumulation and/or persistence in the brains of human patients should be considered an effective therapeutic.
Other Amyloid Diseases
The “amyloid diseases” consist of a group of clinically and generally unrelated human diseases which all demonstrate a marked accumulation in tissues of an insoluble extracellular substance known as “amyloid”, and usually in an amount sufficient to impair normal organ function. Rokitansky in 1842 (Rokitansky, “Handbuch der pathologischen Anatomie”, Vol. 3, Braumuller and Seidel, Vienna) was the first to observe waxy and amorphous looking tissue deposits in a number of tissues from different patients. However, it wasn't until 1854 when Virchow (Virchow, Arch. Path. Anat. 8:416, 1854) termed these deposits as “amyloid” meaning “starch-like” since they gave a positive staining with the sulfuric acid-iodine reaction, which was used in the 1850's for demonstrating cellulose. Although cellulose is not a constituent of amyloid, nonetheless, the staining that Virchow observed was probably due to the present of proteoglycans (PGs) which appear to be associated with all types of amyloid deposits. The name amyloid has remained despite the fact that Friederich and Kekule in 1859 discovered the protein nature of amyloid (Friedrich and Kekule, Arch. Path. Anat. Physiol. 16:50, 1859). For many years, based on the fact that all amyloids have the same staining and structural properties, lead to the postulate that a single pathogenetic mechanism was involved in amyloid deposition, and that amyloid deposits were thought to be composed of a single set of constituents. Current research has clearly shown that amyloid is not a uniform deposit and that amyloids may consist of different proteins which are totally unrelated (Glenner, N. England J. Med. 302:1283-1292, 1980).
Although the nature of the amyloid itself has been found to consist of completely different and unrelated proteins, all amyloids appear similar when viewed under the microscope due to amyloid's underlying protein able to adapt into a fibrillar structure. All amyloids regardless of the nature of the underlying protein 1) stain characteristically with the Congo red dye and display a classic red/green birefringence when viewed under polarized light (Puchtler et al, J. Histochem. Cytochem. 10:355-364, 1962), 2) ultrastructurally consists of fibrils with a diameter of 7-10 nanometers and of indefinite length, 3) adopt a predominant beta-pleated sheet secondary structure. Thus, amyloid fibrils viewed under an electron microscope (30,000 times magnification) from the post-mortem brain of an Alzheimer's disease patient would look nearly identical to the appearance of amyloid present in a biopsied kidney from a rheumatoid arthritic patient. Both these amyloids would demonstrate a similar fibril diameter of 7-10 nanometers.
In the mid to late 1970's amyloid was clinically classified into 4 groups, primary amyloid, secondary amyloid, familial amyloid and isolated amyloid. Primary amyloid, is amyloid appearing de novo, without any preceding disorder. In 25-40% of these cases, primary amyloid was the antecedent of plasma cell dysfunction such as the development of multiple myeloma or other B-cell type malignancies. Here the amyloid appears before rather than after the overt malignancy. Secondary amyloid, appeared as a complication of a previously existing disorder. 10-15% of patients with multiple myeloma eventually develop amyloid (Hanada et al, J. Histochem. Cytochem. 19:1-15, 1971). Patients with rheumatoid arthritis, osteoarthritis, ankylosing spondylitis can develop secondary amyloidosis as with patients with tuberculosis, lung abscesses and osteomyelitis (Benson and Cohen, Arth. Rheum. 22:36-42, 1979; Kamei et al, Acta Path. Jpn. 32:123-133, 1982; McAdam et al, Lancet 2:572-575, 1975). Intravenous drug users who self-administer and who then develop chronic skin abscesses can also develop secondary amyloid (Novick, Mt. Sin. J. Med. 46:163-167, 1979). Secondary amyloid is also seen in patients with specific malignancies such as Hodgkin's disease and renal cell carcinoma (Husby et al, Cancer Res. 42:1600-1603, 1982). Although these were all initially classified as secondary amyloid, once the amyloid proteins were isolated and sequenced many of these turned out to contain different amyloid proteins.
The familial forms of amyloid also showed no uniformity in terms of the peptide responsible for the amyloid fibril deposited. Several geographic populations have now been identified with genetically inherited forms of amyloid. One group is found in Israel and this disorder is called Familial Mediterranean Fever and is characterized by amyloid deposition, along with recurrent inflammation and high fever (Mataxas, Kidney 20:676-685, 1981). Another form of inherited amyloid is Familial Amyloidotic Polyneuropathy, and has been found in Swedish (Skinner and Cohen, Biochem. Biophys. Res. Comm. 99:1326-1332, 1981), Portuguese (Saraiva et al, J. Lab. Clin. Med. 102:590-603, 1983; J. Clin. Invest. 74:104-119, 1984) and Japanese (Tawara et al, J. Lab. Clin. Med. 98:811-822, 1981) nationalities. Amyloid deposition in this disease occurs predominantly in the peripheral and autonomic nerves. Hereditary amyloid angiopathy of Icelandic origin is an autosomal dominant form of amyloid deposition primarily affecting the vessels in the brain, and has been identified in a group of families found in Western Iceland (Jennson et al, Clin. Genet. 36:368-377, 1989). These patients clinically have massive cerebral hemorrhages in early life which usually causes death before the age of 40.
The primary, secondary and familial forms of amyloid described above tend to involve many organs of the body including heart, kidney, liver, spleen, gastrointestinal tract, skin, pancreas, and adrenal glands. These amyloid diseases are also referred to as “systemic amyloids” since so many organs within the body demonstrate amyloid accumulation. For most of these amyloidoses, there is no apparent cure or effective treatment and the consequences of amyloid deposition can be detrimental to the patient. For example, amyloid deposition in kidney may lead to renal failure, whereas amyloid deposition in heart may lead to heart failure. For these patients, amyloid accumulation in systemic organs leads to eventual death generally within 3 to 5 years.
Isolated forms of amyloid, on the other hand, tend to involve a single organ system. Isolated amyloid deposits have been found in the lung, and heart (Wright et al, Lab. Invest. 30:767-773, 1974; Pitkanen et al, Am. J. Path. 117:391-399, 1984). Up to 90% of type II diabetic patients (non-insulin dependent form of diabetes) have isolated amyloid deposits in the pancreas restricted to the beta cells in the islets of Langerhans (Johnson et al, New Engl. J. Med. 321:513-518, 1989; Lab. Invest. 66:522-535, 1992). Isolated forms of amyloid have also been found in endocrine tumors which secrete polypeptide hormones such as in medullary carcinoma of the thyroid (Butler and Khan, Arch. Path. Lab. Med. 110:647-649, 1986; Berger et al, Virch. Arch. A Path. Anat. Hist. 412:543-551, 1988). A serious complication of long term hemodialysis is amyloid deposited in the medial nerve and clinically associated with carpal tunnel syndrome (Gejyo et al, Biochem. Biophys. Res. Comm. 129:701-706, 1985; Kidney Int. 30:385-390, 1986). By far, the most common type and clinically relevant type of organ-specific amyloid, and amyloid in general, is that found in the brains of patients with Alzheimer's disease (see U.S. Pat. No. 4,666,829 and Glenner and Wong, Biochem. Biophys. Res. Comm. 120:885-890, 1984; Masters et al, Proc. Natl. Acad. Sci., USA 82:4245-4249, 1985). In this disorder, amyloid is predominantly restricted to the central nervous system. Similar deposition of amyloid in the brain occurs in Down's syndrome patients once they reach the age of 35 years (Rumble et al, New England J. Med. 320:1446-1452, 1989; Mann et al, Neurobiol. Aging 10:397-399, 1989). Other types of central nervous system amyloid deposition include rare but highly infectious disorders known as the prion diseases which include Creutzfeldt-Jakob disease, Gerstmann-Straussler syndrome, and kuru (Gajdusek et al, Science 197:943-960, 1977; Prusiner et al, Cell 38:127-134, 1984; Prusiner, Scientific American 251:50-59, 1984; Prusiner et al, Micr. Sc. 2:33-39, 1985; Tateishi et al, Ann. Neurol. 24:35-40, 1988).
It was misleading to group the various amyloidotic disorders strictly on the basis of their clinical features, since when the major proteins involved were isolated and sequenced, they turned out to be different. For example, amyloid seen in rheumatoid arthritis and osteoarthritis, now known as AA amyloid, was the same amyloid protein identified in patients with the familial form of amyloid known as Familial Mediterranean Fever. Not to confuse the issue, it was decided that the best classification of amyloid should be according to the major protein found, once it was isolated, sequenced and identified.
Thus, amyloid today is classified according to the specific amyloid protein deposited. The amyloid diseases include, but are not limited to, the amyloid associated with Alzheimer's disease, Down's syndrome and hereditary cerebral hemorrhage with amyloidosis of the Dutch type (wherein the specific amyloid is now known as the beta-amyloid protein or Aβ), the amyloid associated with chronic inflammation, various forms of malignancy and Familial Mediterranean Fever (AA amyloid or inflammation-associated amyloidosis), the amyloid associated with multiple myeloma and other B-cell abnormalities (AL amyloid), the amyloid associated with type II diabetes (amylin or islet amyloid), the amyloid associated with the prion diseases including Creutzfeldt-Jakob disease, Gerstmann-Straussler syndrome, kuru and animal scrapie (PrP amyloid), the amyloid associated with long-term hemodialysis and carpal tunnel syndrome (beta2-microglobulin amyloid), the amyloid associated with senile cardiac amyloid and Familial Amyloidotic Polyneuropathy (prealbumin or transthyretin amyloid), and the amyloid associated with endocrine tumors such as medullary carcinoma of the thyroid (variants of procalcitonin).
Laminin and Its Structural Domains
Laminin is a large and complex 850 kDa glycoprotein which normally resides on the basement membrane and is produced by a variety of cells including embryonic, epithelial and tumor cells (Foidart et al, Lab. Invest. 42:336-342, 1980; Timpl et al, Methods Enzymol. 82:831-838, 1982). Laminin-1 (is derived from the Engelbreth-Holm-Swarm tumor) and is composed of three distinct polypeptide chains, A, B1 and B2 (also referred to as alpha1, β1 and gamma-1, respectively), joined in a multidomain structure possessing three shorts arms and one long arm (Burgeson et al, Matrix Biol. 14:209-211, 1994). Each of these arms is subdivided into globular and rodlike domains. Studies involving in vitro self-assembly and the analysis of cell-formed basement membranes have shown that laminin exists as a polymer, forming part of a basement membrane network (Yurchenco et al, J. Biol. Chem. 260:7636-7644, 1985; Yurchenco et at, J. Cell Biol. 117:1119-1133, 1992; Yurchenco and Cheng, J. Biol. Chem. 268: 17286-17299, 1993). Laminin is believed to play important roles in a number of fundamental biological processes including promotion of neural crest migration (Newgreen and Thiery, Cell Tissue Res. 211:269-291, 1980; Rovasio et al, J. Cell Biol. 96:462-473, 1983), promotion of neurite outgrowth (Lander et al, Proc. Natl. Acad. Sci. 82:2183-2187, 1985; Bronner-Fraser and Lallier, Cell Biol. 106:1321-1329, 1988), the formation of basement membranes (Kleinman et al, Biochem. 22:4969-4974, 1983), the adhesion of cells (Engvall et al, J. Cell Biol. 103: 2457-2465, 1986) and is inducible in adult brain astrocytes by injury (Liesi et al, EMBO J. 3:683-686, 1984). Laminin interacts with other components including type IV collagen (Terranova et al, Cell 22:719-726, 1980; Rao et at, Biochem. Biophys. Res. Comm. 128:45-52, 1985; Charonis et at, J. Cell Biol. 100: 1848-1853, 1985; Laurie et at, J. Mol. Biol. 189:205-216, 1986), heparan sulfate proteoglycans (Riopelle and Dow, Brain Res. 525:92-100, 1990; Battaglia et al, Eur. J. Biochem. 208:359-366, 1992) and heparin (Sakashita et al, FEBS Lett. 116:243-246, 1980; Del Rosso et al, Biochem. J. 199:699-704, 1981; Skubitz et al, J. Biol. Chem. 263:4861-4868, 1988).
Several of the functions of laminin have been found to be associated with the short arms. First, the short arms have been found to participate in laminin polymerization (Yurchenco et al, J. Cell Biol. 117:1119-1133, 1992; Yurchenco and Cheng, J. Biol. Chem. 268: 17286-17299, 1993). A recently proposed three-arm interaction hypothesis of laminin polymerization (Yurchenco and Cheng, J. Biol. Chem. 268: 17286-17299, 1993) further holds that self-assembly is mediated through the end regions of each of the three short arms. A prediction of this model is that each short arm can independently and competitively inhibit laminin polymerization. However, it has not been possible to formally test this prediction using conventional biochemical techniques because of an inability to separate the alpha and gamma chains. Second, several heparin binding sites have been thought to reside in the short arms (Yurchenco et al, J. Biol. Chem. 265:3981-3991, 1990; Skubitz et al, J. Cell Biol. 115:1137-1148, 1991), although the location of these sites have remained obscure. Third, the alpha1β1 integrin has been found to selectively interact with large short arm fragments containing all or most of the short arm domains (Hall et al, J. Cell Biol. 110:2175-2184, 1990; Goodman et al, J. Cell Biol. 113:931-941, 1991).
Most functional activities of laminin appear to be dependent upon the conformational state of the glycoprotein. Specifically, self-assembly and its calcium dependence, nidogen (entactin) binding to laminin, alpha6β1 integrin recognition of the long arm, heparin binding to the proximal G domain (cryptic) and RGD-dependent recognition of the short A chain of laminin (cryptic) have all been found to be conformationally dependent (Yurchenco et al, J. Biol. Chem. 260:7636-7644, 1985; Fox et al, EMBO J. 10:3137-3146, 1991; Sung et al, J. Cell Biol. 123:1255-1268, 1993). Two consequences of improperly folded laminin, loss of normal functional activity and the activation of previously cryptic activities, suggest that it is important to map and characterize biological activities using correctly folded laminin or conformational homologues to any particular laminin or laminin fragment.
Laminin may also be involved in the pathogenesis of a number of important diseases. For example, in diabetes significant decrease in the levels of laminin on the glomerular basement membranes indicates that a molecular imbalance occurs (Shimomura and Spiro, Diabetes 36:374-381, 1987). In experimental AA amyloidosis (ie. inflammation-associated amyloidosis), increased levels of laminin are observed at the sites of AA amyloid deposition (Lyon et al, Lab. Invest. 64:785-790, 1991). However, the role(s) of laminin in systemic amyloidosis is not known. In Alzheimer's disease and Down's syndrome, laminin is believed to be present in the vicinity of Aβ-containing amyloid plaques (Perlmutter and Chui, Brain Res. Bull. 24:677-686, 1990; Murtomaki et al, J. Neurosc. Res. 32:261-273, 1992; Perlmutter et al, Micro. Res. Tech. 28:204-215, 1994).
Previous studies have indicated that the various isoforms of the beta-amyloid precursor proteins of Alzheimer's disease, bind both the basement membrane proteins perlecan (Narindrasorasak et al, J. Biol. Chem. 266:12878-12883, 1991) and laminin (Narindrasorasak et al, Lab. Invest. 67:643-652, 1992). With regards to laminin, it was not previously known whether laminin interacts with Aβ, whether a particular domain of laminin (if any) participates in Aβ interactions, and whether laminin had any significant role(s) in Aβ amyloid fibrillogenesis.
The present invention has discovered that laminin binds Aβ with relatively high affinity and surprisingly laminin is a potent inhibitor of Aβ amyloid formation, and causes dissolution of pre-formed Alzheimer's disease amyloid fibrils. In addition, a 55-kilodalton elastase resistent fragment of laminin which also binds Aβ has been localized to the globular domain repeats within the A chain of laminin. This region is believed to be responsible for many of the inhibitory effects that laminin has on Alzheimer's disease amyloidosis. These findings indicate that laminin, laminin-derived protein fragments and/or laminin-derived polypeptides, particularly those containing the disclosed Aβ-binding site within the globular domain repeats within the laminin A chain, may serve as novel inhibitors of Aβ amyloidosis in Alzheimer's disease and other amyloidoses. In addition, the discovery and identification of an Alzheimer's Aβ-binding region within the globular domain repeats of the laminin A chain, and the discovery of its presence in human serum and cerebrospinal fluid, as a  ̃130 kDa laminin-derived fragment, leads to novel diagnostic and therapeutic applications for Alzheimer's disease and other amyloidoses.
EXAMPLES
The following examples are provided to disclose in detail preferred embodiments of the binding interaction of laminin with Aβ, and the potent inhibitory effects of laminin and disclosed fragments on Aβ fibril formation. However, it should not be construed that the invention is limited to these specific examples.
Example 1
Binding of Laminin to the Beta-Amyloid Protein (Aβ) of Alzheimer's Disease
2 μg of Aβ (1-40)(Bachem Inc., Torrance, Calif. USA; Lot #WM365) in 40 μl of Tris-buffered saline (TBS)(pH 7.0) was allowed to bind overnight at 4° C. to microtiter wells (Nunc plates, Maxisorb). The next day all of the microtiter wells were blocked by incubating with 300 μl of Tris-buffered saline containing 100 mM Tris-HCl, 50 MM NaCl, 0.05% Tween-20, and 3 mM NaN3 (pH 7.4)(TTBS) plus 2% bovine serum albumin (BSA). Various dilutions (ie. 1:10, 1:30, 1:90, 1:270, 1:810, 1:2430 and 1:7290) of Engelbreth-Holm-Swarm (EHS) mouse tumor laminin (1 mg/ml)(Sigma Chemical Co., St. Louis, Mo., USA) in 250 μl of TBS (pH 7.4) were placed in wells (in triplicate) either containing substrate bound Aβ (1-40) or blank, and allowed to bind overnight at 4° C. overnight. The next day, the wells were rinsed 3 times with TTBS, and then probed for 2 hours with 100 μl of rabbit anti-laminin antibody (Sigma Chemical Company, St. Louis, Mo.) diluted 1:10,000 in TTBS. After 3 rinses with TTBS, the wells were then incubated for 2 hours on a rotary shaker with 100 μl of secondary probe consisting of biotinylated goat anti-rabbit (1:1000) and strepavidin-peroxidase (1:500 dilution of a 2 μg/ml solution) in TTBS containing 0.1% BSA. The wells were then rinsed 3 times with TTBS and 100 μl of a substrate solution (OPD-Sigma Fast from Sigma Chemical Co., St. Louis, Mo.) was added to each well and allowed to develop for 10 minutes or until significant color differences were observed. The reaction was stopped with 50 μl of 4N H2SO4 and read on a Model 450 microplate reader (Biorad, Hercules, Calif. USA) at 490 nm. Data points representing a mean of triplicate determinations were plotted and the affinity constants (ie. Kd) were determined using Ultrafit (version 2.1, Biosoft, Cambridge, U.K.) as described below.
The binding data were analyzed assuming a thermodynamic equilibrium for the formation of the complex BL, from the laminin ligand in solution, L, and the uncomplexed Aβ adsorbed to the microtiter well, B, according to the equation: Kd=[B]×[L]/[BL]. We elected to determine Kd's by using an enzyme-linked immunoassay that gives a color signal that is proportional to the amount of unmodified laminin bound to Aβ (Engel, J. and Schalch, W., Mol. Immunol. 17:675-680, 1980; Mann, K. et al, Eur. J. Biochem. 178:71-80, 1988; Fox, J. W. et al, EMBO J. 10:3137-3146, 1991; Battaglia, C. et al, Eur. J. Biochem. 208:359-366, 1992).
To account for potential non-specific binding, control wells without Aβ (in triplicate) were included for each concentration of laminin used in each binding experiment. Optical densities of the control wells never exceeded 0.050 at all laminin concentrations employed for these experiments. The optical densities of the control wells were subtracted from the optical densities of the Aβ-containing wells that received similar laminin concentrations. Non-specific absorbance obtained from Aβ containing wells that did not receive laminin were also subtracted from all data points. Thus, the equation in the form of: ODexp=ODo+(S×[laminin])+(ODmax×[laminin]/([laminin]+Kd) where (S×[laminin]) represents non-specific binding (control wells) and ODo is the non-specific absorbance, becomes ODexp=ODmax×[laminin]/([laminin]+Kd). Therefore, at 50% saturation ODexp=0.50 ODmax and Kd=[laminin]. Determination of [laminin] at 50% saturation was performed by non-linear least square program (Ultrafit from Biosoft, UK) using a one-site model.
As demonstrated in FIG. 1, EHS laminin bound immobilized Aβ (1-40) with a single binding constant with an apparent dissociation constant of Kd=2.7×10−9 M. Several repeated experiments utilizing this solid phase binding immunoassay indicated that laminin bound Aβ (1-40) repetitively with one apparent binding constant.
Example 2
Inhibition of Alzheimer's Disease Aβ Fibril Formation by Laminin
The effects of laminin on Aβ fibrillogenesis was also determined using the previously described method of Thioflavin T fluorometry (Naiki et al, Lab. Invest. 65:104-110, 1991; Levine III, Protein Sci. 2:404-410, 1993; Levine III, Int. J. Exp. Clin. Invest. 2:1-6, 1995; Naiki and Nakakuki, Lab. Invest. 74:374-383, 1996). In this assay, Thioflavin T binds specifically to fibrillar amyloid and this binding produces a fluorescence enhancement at 480 nm that is directly proportional to the amount of amyloid fibrils formed (Naiki et al, Lab. Invest. 65:104-110, 1991; Levine III, Protein Sci. 2:404-410, 1993; Levine III, Int. J. Exp. Clin. Invest. 2:1-6, 1995; Naiki and Nakakuki, Lab. Invest. 74:374-383, 1996). In a first study, the effects of EHS laminin on Aβ (1-40) fibrillogenesis was assessed. For this study, 25 μM of freshly solubilized Aβ (1-40)(Bachem Inc., Torrance, Calif., USA; Lot #WM365) was incubated in microcentrifuge tubes at 37° C. for 1 week (in triplicate), either alone, or in the presence of 100 nM EHS laminin (Sigma Chemical Company, St. Louis, Mo., USA) in 100 mM Tris, 50 mM NaCl, pH 7.0 (TBS). 100 nM of laminin utilized for these studies represented a Aβ:laminin molar ratio of 250:1. 50 μl aliquots were then taken from each tube for analysis at 1 hr, 1 day, 3 days, and 1 week. In a second set of studies, the effects of laminin on Aβ (1 -40) fibril formation was directly compared to other basement membrane components including fibronectin, type IV collagen and perlecan. For these studies, 25 μM of freshly solubilized Aβ (1-40) was incubated in microcentrifuge tubes for 1 week (in triplicate) either alone, or in the presence of 100 nM of EHS perlecan (isolated as previously described)(Castillo et al, J. Biochem. 120:433-444, 1996), fibronectin (Sigma Chemical Company, St. Louis, Mo., USA) or type IV collagen (Sigma Chemical Company, St. Louis, Mo., USA). 50 ul aliquots were then taken for analysis at 1 hour, 1 day, 3 days and 1 week. In a third set of studies, 25 μM of freshly solubilized Aβ (1-40) was incubated in microcentrifuge tubes for 1 week (in triplicate) either alone, or in the presence of increasing concentrations of laminin (i.e. 5 nM, 15 nM, 40 nM and 100 nM). 50 μl aliquots were taken for analysis at 1 hour, 1 day, 3 days and 1 week.
For each determination described above, following each incubation period, Aβ peptides +/− laminin, perlecan, fibronectin or type IV collagen, were added to 1.2 ml of 100 μM Thioflavin T (Sigma Chemical Co., St. Louis, Mo.) in 50 mM phosphate buffer (pH 6.0). Fluorescence emission at 480 nm was measured on a Turner instrument-model 450 fluorometer at an excitation wavelength of 450 nm. For each determination, the fluorometer was calibrated by zeroing in the presence of the Thioflavin T reagent alone, and by seting the 50 ng/ml riboflavin (Sigma Chemical Co., St. Louis, Mo.) in the Thioflavin T reagent to 1800 fluorescence units. All fluorescence determinations were based on these references and any background fluorescence given off by laminin, perlecan, type IV collagen, or fibronectin alone in the presence of the Thioflavin T reagent was always subtracted from all pertinent readings.
As shown in FIG. 2, freshly suspended Aβ (1-40) alone, following a 1 hour incubation at 37° C., demonstrated an initial fluorescence of 41 fluorescence units. During the 1 week incubation period there was a gradual increase in the fluorescence of 25 μM Aβ (1-40) alone, increasing 6.7-fold from 1 hour to 1 week, with a peak fluorescence of 379 fluorescence units observed at 1 week. This increase was significantly inhibited when Aβ (1-40) was co-incubated with laminin, in comparison to Aβ alone. Aβ (1-40) co-incubated with laminin displayed fluorescence values that were 2.9-fold lower (p<0.001) at 1 hour, 4.6-fold lower (p<0.0001) at 1 day, 30.6-fold lower (p<0.0001) at 3 days and 27.1-fold lower (p<0.0001) at 1 week. This study indicated that laminin was a potent inhibitor of Aβ amyloid fibril formation, nearly completely inhibiting amyloid fibril formation even after 1 week of incubation.
To determine whether the inhibitory effects of laminin was specific to this basement membrane component, an direct comparison was made to other known basement membrane components including perlecan, fibronectin, and type IV collagen. In these studies 25 μM of Aβ (1-40) was incubated in the absence or presence of either 100 nM of laminin, 100 nM of fibronectin, 100 nM of type IV collagen and 100 nM of perlecan (FIG. 3). Freshly solubilized Aβ (1-40) when incubated at 37° C. gradually increased in fluorescence levels from 1 hour to 1 week (by 10.8-fold)(FIG. 3), as previously demonstrated (FIG. 2). Perlecan was found to significantly accelerate Aβ (1-40) amyloid formation at 1 day and 3 days, whereas fibronectin and type IV collagen only showed significant inhibition of Aβ (1-40) fibrillogenesis at 1 week. Laminin, on the other hand, was again found to be a very potent inhibitor of Aβ fibrillogenesis causing a 9-fold decrease at 1 and 3 days, and a 21-fold decrease at 1 week. This study reconfirmed the potent inhibitory effects of laminin on Aβ fibrillogenesis, and demonstrated the specificity of this inhibition, since none of the other basement membrane components (including fibronectin, type IV collagen and perlecan) were very effective inhibitors.
To determine whether the inhibitory effects of laminin on Aβ fibrillogenesis occurred in a dose-dependent manner, different concentrations of laminin (i.e. 5 nM, 15 nM, 40 nM and 100 nM) were tested. As shown in FIG. 4, freshly solubilized Aβ (1-40) when incubated at 37° C. gradually increased from 1 hour to 1 week, as previously demonstrated (FIGS. 2 and 3). 100 nM of laminin significantly inhibited Aβ fibril formation at all time points studied, including 1 hour, 1 day, 3 days and 7 days. Laminin was also found to inhibit Aβ fibril formation in a dose-dependent manner which was significant (p<0.05) by 3 days of incubation. At 3 days and 7 days, both 100 nM and 40 nM of laminin significantly inhibited Aβ fibril formation. This study reconfirmed that laminin was a potent inhibitor of Aβ fibril formation and that this inhibition occurred in a dose-dependent manner.
Example 3
Laminin Causes Dose-Dependent Dissolution of Pre-Formed Alzheimer's Disease Amyloid Fibrils
The next study was implemented to determine whether laminin was capable of causing a dose-dependent dissolution of pre-formed Alzheimer's disease Aβ (1-40) amyloid fibrils. This type of activity would be important for any potential anti-Alzheimer's amyloid drug which can be used in patients who already have substantial amyloid deposition in brain. For example, Alzheimer's disease patients in mid-to late stage disease have abundant amyloid deposits in their brains as part of both neuritic plaques and cerebrovascular amyloid deposits. A therapeutic agent capable of causing dissolution of pre-existing amyloid would be advantageous for use in these patients who are at latter stages of the disease process.
For this study, 1 mg of Aβ (1-40)(Bachem Inc., Torrance, Calif., USA; Lot #WM365) was dissolved in 1.0 ml of double distilled water (1 mg/ml solution) and then incubated at 37° C. for 1 week. 25 μM of fibrillized Aβ was then incubated at 37° C. in the presence or absence of laminin (from EHS tumor; Sigma Chemical Company, St. Louis, Mo., USA) at concentrations of 125 nM, 63 nM, 31 nM and 16 nM containing 150 mM Tris HCl, 10 mM NaCl, pH 7.0. Following a 4 day incubation, 50 μl aliquots were added to 1.2 ml of 100 μM Thioflavin T (Sigma Chemical Co., St. Louis, Mo.) in 50 mM NaPO4 (pH 6.0) for fluorometry readings as described in example 2.
As shown in FIG. 5, dissolution of pre-formed Alzheimer's disease Aβ amyloid fibrils by laminin occurred in a dose-dependent manner. A significant (p<0.001) 41% dissolution of pre-formed Aβ amyloid fibrils was observed with 125 nM of laminin, whereas 63 nM of laminin caused a significant (p<0.001) 39% dissolution. Furthermore, 31 nM and 16 nM of laminin still caused a significant (p<0.01) 28% and 25% dissolution of pre-formed Aβ amyloid fibrils. These data demonstrated that laminin causes dissolution of pre-formed Alzheimer's disease amyloid fibrils in a dose-dependent manner following a 4-day incubation.
Example 4
Laminin Does Not Significantly Inhibit Islet Amyloid Polypeptide (Amylin) Fibril Formation
In the next study, the specificity of the laminin inhibitory effects on Alzheimer's disease amyloid was determined by testing laminin's potential effects on another type of amyloid. Amyloid accumulation occurs in the islets of Langerhans in  ̃90% of patients with type II diabetes (Westermark et al, Am. J. Path. 127:414-417, 1987). The major protein in islet amyloid is a 37 amino acid peptide, termed islet amyloid polypeptide or amylin which is known to be a normal secretory product of the beta-cells of the pancreas (Cooper et al, Proc. Natl. Acad. Sci., 84:8628-8632, 1987). The dose-dependent effects of laminin on amylin fibrillogenesis was determined using the Thioflavin T fluorometry assay. 25 μM of Aβ (1-40) (Bachem Inc., Torrance , Calif., USA; Lot #WM365) was incubated in microcentrifuge tubes at 37° C. for 1 week (in triplicate), either alone, or in the presence of 5 nM, 15 nM, 40 nM and 100 nM of laminin in 150 mM Tris HCl, 10 mM NaCl, pH 7.0 (TBS). 50 μl aliquots were taken from each tube for analysis at 1 hr, 1 day, 3 days, and 1 week using Thioflavin T fluorometry as described in example 2.
As shown in FIG. 6, freshly suspended amylin alone following a 1-hour incubation at 37° C. reached a maximum fluorescence of 1800 fluorescence units, which did not significantly change during the 1 week experimental period. The initial high fluorescence of amylin was attributed to amylin's ability to spontaneously form amyloid fibrils within a very short incubation period. Laminin at 100 nM did not significantly inhibit amylin fibril formation at all time points within the 1 week experimental period (FIG. 6). In addition, no significant inhibition of amylin fibrillogenesis by laminin at decreasing concentrations (i.e. 40 nM, 15 nM and 5 nM) was observed, even though a decrease (but not significant) in amylin fibril formation was observed with 40 nM of laminin at 1 day, 3 days and 1 week (FIG. 6). This study demonstrated that the inhibitory effects of laminin did not occur with amylin fibril formation, and demonstrated the specificity of the observed laminin inhibitory effects on Alzheimer's disease amyloid.
Example 5 
Identification of V8 and Trypsin-Resistent Laminin Fragments which Interact with the Beta-Amyloid Protein of Alzheimer's Disease
In the next set of studies, we determined whether small fragment(s) of laminin generated by V8 or trypsin digestion would bind to Aβ. This would enable one to determine the domain(s) of laminin which bind Aβ and likely play a role in inhibition of Aβ fibril formation and causing dissolution of preformed Alzheimer's amyloid fibrils (as demonstrated in the invention).
For these experiments, Aβ (1-40) was biotinylated according to the manufacturer's protocol (Pierce, Rockford, Ill.). For the ligand studies, intact EHS laminin was left undigested, or digested with V8 or trypsin (Sigma Chemical Company, St. Louis, Mo., USA). More specifically, 2 μg of trypsin or V8 protease in 2 μl of 50 MM Tris-HCl buffer (pH 8.0) were added to 50 μl of laminin (50 μg)(in the same buffer) and incubated overnight at 37° C. The next day, 10 μl of protease-digested laminin (or undigested laminin) was mixed with 10 μl of 2× sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, and heated for 5 minutes in a boiling water bath. SDS-PAGE was performed according to the method of Laemmli (Laemmli, U.K. Nature 227:680-685, 1970), or according to the method of Schägger and Jagow (Schägger and Jagow, Anal. Biochem. 166:368-379, 1987) using a Mini-Protean II electrophoresis system (Biorad) with precast 4-15% Tris-Glycine or 10-20% tricine polyacrylamide gels, respectively, and under non-reducing conditions. Electrophoresis occurred at 200V for 45 minutes along with pre-stained molecular weight standards.
After SDS-PAGE (10-20% tricine or 4-15% Tris-Glycine gels) was performed as described above, the separated laminin and its fragments (total protein of 10 μg/lane) were transferred to polyvinylidine difluoride membrane (PVDF) using a Mini transblot electrophoresis transfer cell (Biorad, Hercules, Calif., U.S.A.). Electrotransfer was performed at 100V for 2 hours. Following transfer, membranes were rinsed with methanol and dried. The fragment(s) of laminin involved in binding to Aβ were then detected by using biotinylated-Aβ (1-40), as described above. Blots were probed for 2 hours with 2 μM biotinylated Aβ (1-40) in TTBS. The membranes were then rinsed three times (10 seconds each) with TTBS, probed for 30 minutes with strepavidin alkaline phosphatase conjugate (Vectastain), rinsed again (as described above), and followed by the addition of an alkaline phosphatase substrate solution (Vectastain). Following color development, the reaction was stopped by flushing the membranes with double distilled water.
As shown in FIG. 7, V8-digested laminin produced multiple protein fragments which interacted with biotinylated Aβ (1-40). Using a 4-15% Tris-Glycine gel system (FIG. 7, lane 1), V8-resistent laminin fragments which interacted with Aβ included fragments of  ̃400 kDa (which probably represented intact laminin which was left undigested),  ̃100-130 kDa,  ̃85 kDa, and a prominent fragment at  ̃55 kDa. Using a 10-20% tricine gel system (FIG. 7, lane 2), V8-resistent laminin fragments which interacted with Aβ included fragments of  ̃130 kDa,  ̃85 kDa, and a prominent fragment at  ̃55 kDa (FIG. 7, lane 2, arrow). It is important to note that molecular size expressed in kilodaltons (kDa) are generally approximate. This study demonstrated that the smallest V8-resistent protein fragment of laminin which interacted with Aβ (1-40) was  ̃55 kDa.
As shown in FIG. 8, trypsin-digested laminin produced multiple protein fragments which interacted with biotinylated Aβ (1-40). Using a 4-15% Tris-Glycine gel system (FIG. 8, lane 1), trypsin-resistent laminin fragments which interacted with Aβ included fragments of  ̃400 kDa (which probably represented intact laminin which was left undigested),  ̃150-200 kDa,  ̃97 kDa,  ̃65 kDa and a prominent fragment at  ̃30 kDa. Using a 10-20% tricine gel system (FIG. 8, lane 2), trypsin-resistent laminin fragments which interacted with Aβ included fragments of  ̃97 kDa,  ̃90 kDa,  ̃65 kDa and a prominent fragment at  ̃30 kDa (FIG. 8, lane 2, arrow). This study demonstrated that the smallest trypsin-resistent fragment of laminin which interacted with Aβ (1-40) was  ̃30 kDa.
Example 6
Identification of Elastase-Resistent Laminin Fragments Which Interact with the Beta-Amyloid Protein of Alzheimer's Disease
In the next set of studies, we determined whether small fragment(s) of laminin generated by elastase digestion would bind to Aβ. In addition, we sequenced and identified the region within elastase-resistent laminin which interacted with Aβ. For these experiments, Aβ (1-40) was biotinylated according to the manufacturer's protocol (Pierce, Rockford, Ill.). For the ligand studies, intact EHS laminin was left undigested, or digested with elastase (Sigma Chemical Company, St. Louis, Mo., USA). For elastase digestion, 2 μg of elastase in 8 μl of 50 mM Tris-HCl buffer (pH 8.0) was added to 50 μl of laminin (50 μg)(in the same buffer) and incubated for 1.5 hours or 2.5 hours at 37° C. In addition, as a control, 2 μg of elastase in 50 μl of 50 mM Tris-HCl buffer (pH 8.0) was incubated for 2.5 hours at 37° C. Following the appropriate incubation times as described above, 10 μl of each of the above incubations were mixed with 10 μl of 2× SDS-PAGE electrophoresis sample buffer, and heated for 5 minutes in a boiling water bath. SDS-PAGE was performed according to the method of Laemmli (Laemmli, Nature 227:680-685, 1970) using a Mini-Protean II electrophoresis system with precast 4-15% Tris-Glycine polyacrylamide gels, and under non-reducing conditions. Electrophoresis occurred at 200V for 45 minutes along with pre-stained molecular weight standards (Biorad).
After SDS-PAGE was performed as described above, the separated laminin fragments were transferred to PVDF using a Mini transblot electrophoresis transfer cell (Millipore, Bedford, Mass., U.S.A.). Electrotransfer was performed at 100V for 2 hours. Following transfer, membranes were rinsed with methanol, dried and cut into two equal parts which were used for Aβ ligand blotting, or Coomassie blue staining and subsequent amino acid sequencing. The fragment(s) of laminin involved in binding to Aβ were then detected by using biotinylated-Aβ (1-40), as described above. Blots were probed for 2 hours with 2 μM biotinylated Aμ (1-40) in TTBS. The membranes were then rinsed three times (10 seconds each) with TTBS, probed for 30 minutes with strepavidin alkaline phosphatase conjugate (Vectastain), rinsed again (as described above), and followed by the addition of an alkaline phosphatase substrate solution (Vectastain). Following color development, the reaction was stopped by flushing the membranes with double distilled water.
For Coomassie blue staining, PVDF membranes were immersed with 0.2% Coomassie Brilliant blue (w/v) in 50% methanol, 10% acetic acid, and 40% distilled water for 2 minutes, and then rinsed with 50% methanol, 10% acetic acid, and 40% distilled water until visible bands were observed, and no background staining was present. The 55 kDa Aβ-binding laminin fragment, described below, was sent to the Biotechnology Service Center (Peptide Sequence Analysis Facility at the University of Toronto, Toronto, Ontario, Canada) and subjected to amino acid sequencing using a Porton 2090 Gas-Phase Microsequencer (Porton Instruments, Tarzana, Calif.) with on-line analysis of phenylthiohydantoin derivatives.
In FIG. 9A represents an Aβ ligand blot whereas FIG. 9B represents the equivalent Coomassie blue stained blot. As shown in FIG. 9A (lanes 2 and 3), elastase-digested laminin produced multiple protein fragments which bound biotinylated Aβ(1-40). FIG. 9A, lane 1 represents undigested mouse EHS laminin, whereas lanes 2 and 3 represents laminin which had been digested with elastase for 1.5 hours or 2.5 hours, respectively. FIG. 9A, lane 4 represents elastase digestion for 2.5 hours in the absence of laminin. Undigested laminin (FIG. 9A, lane 1) which interacted with Aβ included multiple bands from >400 kDa to > ̃86 kDa, with the most prominent Aβ-interaction occurring with intact laminin (i.e. 400 kDa). Elastase-resistant laminin protein fragments which interacted with Aβ (FIG. 9A, lanes 2 and 3) included fragments of > ̃400 kDa,  ̃130 kDa (arrowhead),  ̃80-90 kDa,  ̃65 kDa and a prominent band at  ̃55 kDa (arrow). The interaction of these elastase-resistant laminin protein fragments with Aβ were only observed under non-reducing conditions suggesting that the Aβ interaction was also conformation dependent. The 130 kDa elastase resistant laminin fragment which interacts with Aβ, is also believed to be part of the E8 fragment (see FIG. 11), and is the same protein fragment of laminin that appears to be present in human serum and cerebrospinal fluid (see Examples 10 and 11). FIG. 9A, lane 4 demonstrates that the band observed at  ̃29 kDa represents non-specific Aβ binding due to the presence of the elastase enzyme alone.
FIG. 9B demonstrates all of the multiple protein bands which were stained by Coomassie blue. Note, for example, in FIG. 9B, lanes 2 and 3, that elastase digestion of laminin produced multiple protein fragments between  ̃55 kDa and  ̃90 kDa which did not bind Aβ, and were not observed in the Aβ ligand blot (FIG. 9A, lanes 2 and 3).
Example 7
An Aβ-Binding Domain Within Laminin is Identified Within the Globular Repeats of the Laminin A Chain
The 55 kDa laminin fragment (ie. produced following 1.5 hours of elastase digestion) that demonstrated positive Aβ binding interaction by ligand blotting was then prepared (FIG. 9, Panel B, lane 2, arrow) in large amounts for amino acid sequencing (as described in example 6). Sequence data determined the exact location within laminin that was involved in binding to Aβ. An 11-amino acid sequence was determined from sequencing of the 55 kDa band. The sequence identified was:
Leu-His-Arg-Glu-His-Gly-Glu-Leu-Pro-Pro-Glu (SEQ ID NO:1).
The specific Aβ-binding domain within laminin was then identified by comparison to known mouse laminin sequence (Sasaki and Yamada, J. Biol. Chem. 262:17111-17117, 1987; Sasaki et al, Proc. Natl. Acad. Sci. 84:935-939, 1987; Durkin, et al, Biochem. 27:5198-5204, 1988; Sasaki et al, J. Biol. Chem. 263:16536-16544, 1988), since mouse EHS laminin was utilized in the studies of the present invention. In addition, the complete amino acid sequence within laminin was retrieved from the National Center for Biotechnology Information, Bethesda, Md., U.S.A.
FIG. 10 shows the complete amino acid sequence of mouse laminin A chain (Genebank accession number P19137; SEQ ID NO: 4). The 11 amino acid protein fragment sequenced from the  ̃55 kDa protein within laminin which binds Aβ is identified (FIG. 10; bold underline and arrowhead; SEQ ID NO: 1) and matches exactly to the region within the third globular domain repeat of laminin A chain (FIG. 11). The fourth globular domain repeat of mouse laminin A chain is shown as SEQ ID NO: 2 (Genebank Accession Number P19137; amino acids #2746-2922), whereas the fourth globular domain repeat of human laminin A chain is shown as SEQ ID NO: 3 (Genebank Accession Number P25391; amino acids #2737-2913).
FIG. 11 shows two schematic representations of laminin (Colognato-Pyke et al, J. Biol. Chem. 270:9398-9406, 1995) and the newly discovered Aβ-binding region of laminin (shown in left panel; between the two arrowheads) which is situated within the last three globular domains of the laminin A chain. The left panel of FIG. 11 illustrates laminin and fragments generated following protease digestions. Elastase fragments E1′, E1X (dark line border), E-alpha-35 and E4 all correspond to regions of the short arms of laminin. Long arm fragments are E8, E3 and cathepsin G fragment C8-9. The E8 fragment produced by elastase digestion of laminin contains the long arm fragments containing the distal part of the long arm and the G subdomains 1-3, and consists of a 130-150 kda (Yurchenco and Cheng, J. Biol. Chem. 268:17286-17299, 1993). The E3 fragments also produced by elastase digestion of laminin contains the distal long arm globule with G subdomains 4 and 5. The E3 fragment shown in FIG. 11, Panel A, has previously shown to be a doublet at  ̃60 kDa and  ̃55 kDa (Yurchenco and Cheng, J. Biol. Chem. 268:17286-17299, 1993). This also confirms our discovery whereby the  ̃55 kDa fragment which we found to bind Aβ is localized within the E3 region of laminin (FIG. 11, Left Panel).
The right panel of FIG. 11 depicts the function map with the alpha (A chain), β (B1 chain) and gamma (B2 chain) chains of laminin shown in shades of decreasing darkness. EGF repeats are indicated by bars in the rod domains of the short arm. Domains, based on sequence analysis, are indicated in small Roman numerals and letters. The locations of heparin-binding, polymer-forming, and the active alpha1β1 integrin-binding sites are shown in bold-face for the alpha-chain short arm. The long arm functions of heparin binding (heparin), alpha6β1 integrin-recognition site (alpha6β1), and dystroglycan (DG), mapped in other studies, are indicated in gray-shaded labels. It is interesting to note that the Aβ-binding region of laminin is also a region involved in binding to heparin.
It should also be emphasized that the globular domain repeats of the laminin A chain likely interacts with Aβ in a conformation dependent manner, since the interaction of the  ̃55-kilodalton elastase-resistent protein fragments with Aβ was only observed under non-reducing conditions.
Example 8
Identification of Laminin and Laminin Protein Fragments in Human Serum and Cerebrospinal Fluid Derived from Alzheimer's disease, Type II Diabetes, and/or Normal Aged Patients
In the next study, western blotting techniques using a polyclonal antibody against laminin was used to determine whether intact laminin and/or laminin fragments were present in human serum and cerebrospinal fluid obtained from Alzheimer's disease, type II diabetes and/or normal aged patients. In this study, human serum was obtained from the Alzheimer's disease Research Center at the University of Washington from either living aged patients who may have had corresponding mini-mental state examinations (where a score of 30 is normal, a score of 15 suggests moderate dementia and a score <10 suggests severe dementia), or from living aged patients who had subsequently died and were diagnosed at autopsy with Alzheimer's disease (following examination of their brains obtained postmortem). In addition, human serum was obtained from the Diabetes Endocrinology Research Center at the University of Washington. The following human serums were obtained and analyzed as part of this study: 1) patient #9; a normal 67 yr old female with a mini-mental score of 30; 2) patient #5226 —a 70 year old female with confirmed moderate Alzheimer's disease who also had a mini-mental score of 12; 3) patient #5211—a 66 year old male with confirmed Alzheimer's disease who also had a mini-mental score of 25; 4) patient B—a 63 year old male who had confirmed type II diabetes; 5) patient #5223—a 68 year old female with confirmed Alzheimer's disease who also had a mini-mental score of 22; 6) patient #22—an 83 yr old normal aged female who also had a mini-mental score of 30; 7) patient #C—a 68 year old male with confirmed type II diabetes. Each of these serums were utilized in this study and represent lanes 1-7 (left side) of FIG. 12 (in the same order as above).
In addition, cerebrospinal fluid was obtained from the Alzheimer's disease Research Center at the University of Washington from either living aged patients who may have had corresponding mini-mental state examinations, or from living aged patients who had subsequently died and were diagnosed at autopsy with Alzheimer's disease (following examination of their brains obtained postmortem). The following human cerebrospinal fluids were obtained as part of this study: 1) patient #6—a normal 64 year old female who had a mini-mental score of 30; 2) patient #7—a normal 67 year old male who had a mini-mental score of 30; 3) patient #8—a normal 80 year old female who had a mini-mental score of 30; 4) patient #9—a normal 67 year old female who had a mini-mental score of 30; 5) patient #1111P—a normal 78 year old female who had a mini-mental score of 30; 6) patient #50—a 66 year old male patient with probable moderate Alzheimer's disease as indicated by a mini-mental score of 15; 7) patient #54—a 73 year old male with probable severe Alzheimer's disease as indicated by a mini-mental score of 8. Each of these cerebrospinal fluid samples were utilized in this study and represent lanes 1-7 (right side) of FIG. 12 (in the same order as above).
For the study described above, 10 μl of human serum diluted at 1:10, or 10 μl of undiluted human cerebrospinal fluid was added to 10 μl of SDS-PAGE buffer and ligand blots were prepared as in Example 6. Blots were probed for 2 hours with a polyclonal antibody (used at a dilution of 1:10,000 in TTBS) against EHS laminin (Sigma Chemical Company, St. Louis, Mo.). The membranes were then rinsed 3 times (10 seconds each) with TTBS and incubated for 1 hour with a biotinylated goat anti-rabbit IgG secondary antibody diluted 1:1,000 with TTBS. The membranes were then rinsed three times (10 seconds each) with TTBS, probed for 30 minutes with strepavidin alkaline phosphatase conjugate (Vectastain), rinsed again (as described above), followed by the addition of an alkaline phosphatase substrate solution (Vectastain). Following color development, the reaction was stopped by flushing the membranes with double distilled water.
As shown in FIG. 12, intact laminin (arrowheads) was present in human serum (lanes 1-7; left side) but not in human cerebrospinal fluid (lanes 1-7; right side). Qualitative observations suggest that intact laminin (as described above) may have been decreased in serum of Alzheimer's disease patients in comparison to controls (i.e. compare intact laminin in FIG. 12, lane 1, left side-normal individual; to FIG. 12, lane 2, left side-Alzheimer's disease patient). In addition to intact laminin, human serum derived from Alzheimer's disease, type II diabetes and normal aged patients also contained laminin immunoreactivity in a series of band from  ̃120 kDa to  ̃200 kDa (FIG. 12, bands observed between the two arrows). On the other hand, cerebrospinal fluid samples did not contain intact laminin (FIG. 12; lanes 1-7; right side) but only contained a series of laminin immunoreactive protein fragments from  ̃120 kDa to  ̃200 kDa (i.e. FIG. 12, bands observed between the two arrows). This study determined that a series of laminin protein fragments are present in both human serum and cerebrospinal fluid of Alzheimer's disease, type II diabetes and normal aged patients, whereas intact laminin is only present in human serum. The novel discovery of the laminin fragments in human cerebrospinal fluid suggests that it may be used as a marker to determine the extent of laminin breakdown in the brain during Alzheimer's disease and other brain disorders.
Example 9
Identification of a  ̃130 Kilodalton Laminin Protein Fragment in Human Serum of Alzheimer's disease, Type II Diabetes and Normal Aged Patients which Binds Aβ
In the next study, Aβ ligand blotting techniques were utilized to identify whether laminin or laminin protein fragments present in human serum bind Aβ. In this study, human serum was obtained from the Alzheimer's disease Research Center at the University of Washington from either living patients who may have had corresponding mini-mental state examinations (where a score of 30 is normal, a score of 15 suggests moderate dementia and a score <10 suggests severe dementia), or from living patients who had subsequently died and were diagnosed at autopsy with Alzheimer's disease (following examination of their brains obtained postmortem). In addition. human serum was obtained from the Diabetes Endocrinology Research Center at the University of Washington. The first six human serum samples (i.e. FIG. 13, lanes 1-6) were the same serum samples as indicated in Example 8. In addition, FIG. 13 lanes 7-10 consisted of human serum obtained from lane 7) patient #E—a 54 year old male with confirmed type II diabetes, lane 8) patient #5230—a 72 year old female with confirmed moderate Alzheimer's disease who had a mini-mental score of 19, lane 9) patient #E—a 54 year old male with confirmed type II diabetes, and lane 10) patient #F—a 69 year old male with confirmed type II diabetes.
For this study, Aβ (1-40) was biotinylated according to the manufacturer's protocol (Pierce, Rockford, Ill.). For the ligand studies, following SDS-PAGE as described above in Example 8, separated laminin and its fragments present in human serum were transferred to polyvinylidine difluoride membrane (PVDF) using a Mini transblot electrophoresis transfer cell. Electrotransfer was performed at 100V for 2 hours. Following transfer, membranes were rinsed with methanol and dried. The fragment(s) of laminin in human serum involved in binding to Aβ were then detected by using biotinylated-Aβ (1-40). Blots were probed for 2 hours with 1 μM biotinylated Aβ (1-40) in TTBS. The membranes were then rinsed three times (10 seconds each) with TTBS, probed for 30 minutes with strepavidin alkaline phosphatase conjugate (Vectastain), rinsed again (as described above), and followed by the addition of an alkaline phosphatase substrate solution (Vectastain). Following color development, the reaction was stopped by flushing the membranes with double distilled water.
As shown in FIG. 13, Aβ interacted with intact human laminin (arrow) in most samples of human serum. However, it was surprising to note that intact laminin was virtually absent in 2 of the 4 Alzheimer's disease patients serum (FIG. 13, lanes 5 and 8), suggesting that laminin-derived fragments may be important in Alzheimer's disease as a diagnostic marker. The most interesting discovery was that of all the laminin immunoreactive protein fragments found in human serum (i.e  ̃120 kDa to  ̃200 kDa, bands observed between the arrows, FIG. 12, lanes 1-7, right side), only a prominent  ̃130 kDa band was found to interact with Aβ (FIG. 13, arrowhead). This same prominent band is approximately the same molecular weight of the E8 band generated from mouse laminin following elastase digestion (see FIG. 9), and which also contains the globular domain repeats of the laminin A chain. This study therefore determined that besides intact laminin, human serum contains a  ̃130 kDa laminin fragment which binds to Aβ, and may be important for keeping Aβ soluble in biological fluids such as blood. This study also suggests that qualitative and quantitative assessment of laminin fragments in human serum may prove diagnostic for the extent and progression of Alzheimer's disease, type II diabetes and other amyloidoses.
Example 10
Identification of a  ̃130 Kilodalton Laminin Protein Fragment in Human Cerebrospinal Fluid of Alzheimer's disease and Normal Aged Patients which Binds Aβ
In the next study, Aβ ligand blotting techniques were utilized to identify whether laminin protein fragments (<200 kDa) present in human cerebrospinal fluid bind Aβ. In this study, human cerebrospinal fluid was obtained from the Alzheimer's disease Research Center at the University of Washington from either living aged patients who may have had corresponding mini-mental state examinations (where a score of 30 is normal, a score of 15 suggests moderate Alzheimer's disease and a score <10 suggests moderate Alzheimer's disease), or from living aged patients who had subsequently died and were diagnosed at autopsy with Alzheimer's disease (following examination of their brains obtained postmortem). The following human cerebrospinal fluids were obtained and analyzed as part of this study (depicted in FIG. 14, lanes 1-10): 1) patient #65—a 71 yr old male with probable severe Alzheimer's disease as indicated by a mini-mental score of 0; 2) patient #54—a 73 yr old male with probable severe Alzheimer's disease as indicated by a mini-mental score of 8.; 3) patient #6—a normal 64 yr old female who had a mini-mental score of 30; 4) patient #7—a normal 67 yr old male who had a mini-mental score of 30; 5) patient #8—a normal 80 yr old female who had a mini-mental score of 30; 6) patient #9—a normal 67 yr old female who had a mini-mental score of 30; 7) patient #1111P—a normal 78 yr old female who had a mini-mental score of 30; 8) patient #50—a 66 yr old male patient with probable moderate Alzheimer's disease as indicated by a mini-mental score of 15; 9) patient #52—a 69 yr old male with probable moderate Alzheimer's disease as indicated by a mini-mental score of 16; 10) patient #64—a 64 yr old male with probable severe Alzheimer's disease as indicated by a mini-mental score of 0. Each of these cerebrospinal fluid samples were utilized in this study and represent lanes 1-10 of FIG. 14 (in the same order as above).
For this study, Aβ ligand blotting was employed as described in Example 9. The fragment(s) of laminin in human cerebrospinal fluid involved in binding to Aβ were detected by using biotinylated-Aβ (1-40). Blots were probed for 2 hours with 50 nM of biotinylated Aβ (1-40) in TTBS. The rest of the Aβ ligand blotting procedure is as described above in Example 9.
As shown in FIG. 14, Aβ interacted with laminin fragment bands between  ̃120 kDa and  ̃200 kDa in most samples of human cerebrospinal fluid. As observed in human serum, most samples of human cerebrospinal fluid also contained a prominent  ̃130 kDa laminin fragment (FIG. 14, arrow) which interacted with Aβ. No intact Aβ-binding laminin was found in human cerebrospinal fluid (not shown), as previously demonstrated (FIG. 12, Example 8). Again, this same prominent  ̃130 kDa Aβ-binding laminin fragment present in human cerebrospinal fluid is approximately the same molecular weight of the E8 band generated from laminin, and which also contains the globular domain repeats of the laminin A chain. This study therefore determined that human cerebrospinal fluid also contains a  ̃130 kDa laminin fragment which binds to Aβ, and may be important for keeping Aβ soluble in biological fluids such as cerebrospinal fluid.
Further Aspects and Utilizations of the Invention
Laminin-Derived Protein Fragments and Polypeptides
One therapeutic application of the present invention is to use laminin, laminin protein fragments which bind Aβ or other amyloid proteins, and/or laminin polypeptides derived from amino acid sequencing of the laminin fragments which bind Aβ (such as the  ̃130 kilodalton protein described herein) or other amyloid proteins, as potent inhibitors of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease and other amyloidoses. The amyloid diseases include, but are not limited to, the amyloid associated with Alzheimer's disease and Down's syndrome (wherein the specific amyloid is referred to as beta-amyloid protein or Aβ), the amyloid associated with chronic inflammation, various forms of malignancy and Familial Mediterranean Fever (wherein the specific amyloid is referred to as AA amyloid or inflammation-associated amyloidosis), the amyloid associated with multiple myeloma and other B-cell dyscrasias (wherein the specific amyloid is referred to as AL amyloid), the amyloid associated with type II diabetes (wherein the specific amyloid is referred to as amylin or islet amyloid), the amyloid associated with the prion diseases including Creutzfeldt-Jakob disease, Gerstmann-Straussler syndrome, kuru and animal scrapie (wherein the specific amyloid is referred to as PrP amyloid), the amyloid associated with long-term hemodialysis and carpal tunnel syndrome (wherein the specific amyloid is referred to as beta2-microglobulin amyloid), the amyloid associated with senile cardiac amyloid and Familial Amyloidotic Polyneuropathy (wherein the specific amyloid is referred to as transthyretin or prealbumin), and the amyloid associated with endocrine tumors such as medullary carcinoma of the thyroid (wherein the specific amyloid is referred to as variants of procalcitonin).
The polypeptides referred to above may be a natural polypeptide, a synthetic polypeptide or a recombinant polypeptide. The fragments, derivatives or analogs of the polypeptides to any laminin fragment referred to herein may be a) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue and such substituted amino acid residue may or may not be encoded by the genetic code, or b) one in which one or more of the amino acid residues includes a substituent group, or c) one in which the mature polypeptide is fused with another compound, such as a compound used to increase the half-life of the polypeptide (for example, polylysine), or d) one in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of the invention.
The tertiary structure of proteins refers to the overall 3-dimensional architecture of a polypeptide chain. The complexity of 3-dimensional structure arises from the intrinsic ability of single covalent bonds to be rotated. Rotation about several such bonds in a linear molecule will produce different nonsuperimpossable 3-dimensional arrangements of the atoms that are generally described as conformations.
Protein conformation is an essential component of protein-protein, protein-substrate, protein-agonist, protein-antagonist interactions. Changes in the component amino acids of protein sequences can result in changes that have little or no effect on the resultant protein conformation. Conversely, changes in the peptide sequences can have effects on the protein conformation resulting in reduced or increased protein-protein, etc. interactions. Such changes and their effects are generally disclosed in Proteins: Structures and Molecular Properties by Thomas Creightonm W. H. Freeman and Company, New York, 1984 which is hereby incorporated by reference.
“Conformation” and “conformation similarity” when used in this specification and claims refers to a polypeptide's ability (or any other organic or inorganic molecule) to assume a given shape, through folding and the like, so that the shape, or conformation, of the molecule becomes an essential part of it's functionality, sometimes to the exclusion of its chemical makeup. It is generally known that in biological processes two conformational similar molecules may be interchangeable in the process, even the chemically different. “Conformational similarity” refers to the latter interchangeability or substitutability. For example, laminin and laminin-derived protein fragments are among the subjects of the invention because they have been shown to bind the Aβ protein and render it inactive in fibril formation; it is contemplated that other molecules that are conformationally similar to laminin, or any claimed laminin fragment or polypeptide, may be substituted in the claimed method to similarly render the Aβ inactive in fibrillogenesis and other amyloid processes. In general it is contemplated that levels of conformational similarity at or above 70% are sufficient to assume homologous functionality in the claimed processes, though reduced levels of conformational similarity may be made to serve as well. Conformational similar levels at or above 90% should provide some level of additional homologue functionality.
Thus, one skilled in the art would envisage that changes can be made to the laminin sequence, or fragments or polypeptides thereof, that would increase, decrease or have no effect on the binding of laminin or fragments thereof, to Aβ amyloid. In addition, one skilled in the art would envisage various post-translational modifications such as phosphorylation, glycosylation and the like would alter the binding of laminin, laminin fragments or laminin polypeptides to Aβ amyloid.
The polypeptides of the present invention include the polypeptides or fragments of laminin described herein, including but not limited to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, and fragments thereof, as well as polypeptides which have at least 70% similarity (preferably 70% identity) and more preferably a 90% similarity (more preferably a 90% identity) to the polypeptides described above.
Fragments or portions of the polypeptides or fragments of laminin of the present invention may be employed for producing the corresponding full-length polypeptides by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full length polypeptides.
The polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. Polypeptides of the invention may also include an initial methionine amino acid residue.
Chemical polypeptide synthesis is a rapidly evolving area in the art, and methods of solid phase polypeptide synthesis are well-described in the following references, hereby entirely incorporated by reference (Merrifield, J. Amer. Chem. Soc. 85:2149-2154, 1963; Merrifield, Science 232:341-347, 1986; Fields, Int. J. Polypeptide Prot. Res. 35, 161, 1990).
Recombinant production of laminin polypeptides can be accomplished according to known method steps. Standard reference works seting forth the general principles of recombinant DNA technology include Watson, Molecular Biology of the Gene, Volumes I and II, The Benjamin/Cummings Publishing Company Inc., publisher, Menlo Park, Calif. 1987; Ausubel et al, eds., Current Protocols in Molecular Biology, Wiley Interscience, publisher, New York, N.Y. 1987; 1992; and Sambrook et al, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory, publisher, Cold Spring Harbor, N.Y. 1989, the entire contents of which references are herein incorporated by reference.
The polypeptides of the present invention may also be utilized as research reagents and materials for discovery of treatments and diagnostics for human diseases.
Antibodies
Antibodies generated against the polypeptides corresponding to specific sequences recognizing the laminin fragments of the present invention which bind Aβ or other amyloid proteins can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodies can then be used to isolate the polypeptides from tissue expressing that polypeptide. Preferred embodiments include, but are not limited to, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, and fragments thereof, as well as polypeptides which have at least 70% similarity (preferably 70% identity) and more preferably a 90% similarity (more preferably a 90% identity) to the polypeptides described above.
The term “antibody” is meant to include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, anti-idiotypic antibodies to antibodies specific for laminin-derived protein fragments or polypeptides of the present invention.
Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen.
A monoclonal antibody contains a substantially homogeneous population of antibodies specific to antigens, which population contains substantially similar epitope binding sites. For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, Nature 256:495-497, 1975), the trioma technique, the human B-cell hybridoma technique (Kozbor et al, Immunology Today 4:72, 1983), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, GILD and any subclass thereof.
Chimeric antibodies are molecules different portions of which are derived from different animal species, such as those having variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region, which are primarily used to reduce immunogenicity in application and to increase yields in production. Chimeric antibodies and methods for their production are known in the art (ex. Cabilly et al, Proc. Natl. Acad. Sci. U.S.A 81:3273-3277, 1984; Harlow and Lane: Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory 1988).
An anti-idiotypic antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding site of an antibody. An anti-iodiotypic antibody can be prepared by immunizing an animal of the same species and genetic type (e.g., mouse strain) as the source of the monoclonal antibody with the monoclonal antibody to which an anti-iodiotypic antibody is being prepared. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody by producing an antibody to these idiotypic determinants (the anti-idiotypic antibody). See, for example, U.S. Pat. No. 4,699,880, which is herein incorporated by reference.
The term “antibody” is also meant to include both intact molecules as well as fragments thereof, such as, for example, Fab and F(ab′)2, which are capable of binding antigen. Fab and F(ab′)2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al, J. Nucl. Med. 24:316-325, 1983).
The antibodies or fragments of antibodies, useful in the present invention may be used to quantitatively or qualitatively detect laminin or laminin-derived fragments in a sample or to detect presence of cells which express a laminin polypeptide of the present invention. This can be accomplished by immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric or fluorometric detection.
One of the ways in which a laminin fragment antibody can be detectably labeled is by linking the same to an enzyme and use in an enzyme immunoassay (EIA). This enzyme, in turn, when later exposed to an appropriate substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric, or by visual means. Enzymes which can be used detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. The detection can be accomplished by colometric methods which employ a chromogenic substrate for the enzyme. Detection can be accomplished by colometric methods which employ a chromogenic substrate for the enzyme. Detection can also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate with similarly prepared standards (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory 1988; Ausubel et al, eds., Current Protocols in Molecular Biology, Wiley Interscience, N.Y. 1987, 1992).
Detection may be accomplished using any of a variety of other immunoassays. For example, by radiolabeling of the antibodies or antibody fragments, it is possible to detect R-PTPase through the use of a radioimmunoassay (RIA). A good description of RIA may be found in Laboratory Techniques and Biochemistry in Molecular Biology, by Work et al, North Holland Publishing Company, NY (1978) with particular reference to the chapter entitled “An Introduction to Radioimmune Assay and Related Techniques” by Chard, incorporated entirely by reference herein. The radioactive isotope can be detected by such means as the use of a gamma-counter, a scintillation counter or by autoradiography.
It is also possible to label a laminin fragment polypeptide antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labelling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine, commercially available, e.g., from Molecular Probes, Inc. (Eugene, Oreg., U.S.A.).
The antibody can also be detectably labeled using fluorescence emitting metals such as 152EU, or other of the lanthanide series. These metals can be attached to the antibody using such metal groups as diethylenetriamine pentaacetic acid (EDTA).
The antibody can also be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction, Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, and oxalate ester.
Likewise, a bioluminescent compound may be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
The antibodies (or fragments thereof) useful in the present invention may be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of a laminin fragment of the present invention. In situ detection may be accomplished by removing a histological specimen from a patient, and providing the labeled antibody of the present invention to such a specimen. The antibody (or fragment) is preferably provided by applying or by overlaying the labeled antibody (or fragment) to a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of a laminin fragment polypeptide but also its distribution on the examined tissue. Using the present invention, those of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.
In accordance with yet a further aspect of the present invention there are provided antibodies against laminin, laminin fragments and/or laminin-derived polypeptides which interact with Aβ or other amyloid proteins, or derivatives thereof. These antibodies can be used for a number of important diagnostic and/or therapeutic applications as described herein. In one aspect of the invention, polyclonal and/or monoclonal antibodies made against laminin, laminin fragments and/or laminin-derived polypeptides which bind Aβ or other amyloid proteins, may be utilized for Western blot analysis (using standard Western blotting techniques knowledgeable to those skilled in the art) to detect the presence of amyloid protein-binding laminin fragments or amyloid protein-binding laminin polypeptides in human tissues and in tissues of other species. Western blot analysis can also be used to determine the apparent size of each amyloid protein-binding laminin fragment. In addition, Western blotting following by scanning densitometry (known to those skilled in the art) can be used to quantitate and compare levels of each of the laminin fragments in tissue samples, biological fluids or biopsies obtained from individuals with specific diseases (such as the amyloid diseases) in comparison to tissue samples, biological fluids or biopsies obtained from normal individuals or controls. Biological fluids, include, but are not limited to, blood, plasma, serum, cerebrospinal fluid, sputum, saliva, urine and stool.
In yet another aspect of the invention, polyclonal and/or monoclonal antibodies made against laminin, laminin fragments and/or laminin-derived peptides which bind Aβ or other amyloid proteins, can be utilized for immunoprecipitation studies (using standard immunoprecipitation techniques known to one skilled in the art) to detect laminin, laminin fragments and/or laminin-derived peptides which bind Aβ or other amyloid proteins, in tissues, cells and/or biological fluids. Use of the laminin, laminin fragment and/or laminin-derived peptide antibodies for immunoprecipitation studies can also be quantitated to determine relative levels of laminin, laminin fragments and/or laminin-derived peptides which interact with Aβ or other amyloid proteins, in tissues, cells and/or biological fluids. Quantitative immunoprecipitation can be used to compare levels of laminin, laminin fragments and/or laminin amyloid protein-binding peptides in tissue samples, biological fluids or biopsies obtained from individuals with specific diseases (such as the amyloid diseases) in comparison to tissue samples, biological fluids or biopsies obtained from normal individuals or controls.
Therapeutic Applications
Yet another aspect of the present invention is to make use of laminin, laminin fragments and/or laminin-derived polypeptides as amyloid inhibitory therapeutic agents. The laminin-derived peptide sequences or fragments can be synthesized utilizing standard techniques (ie. using an automated synthesizer). Laminin, laminin fragments and/or laminin-derived polypeptides which bind Aβ or other amyloid proteins, can be used as potential blocking therapeutics for the interaction of laminin in a number of biological processes and diseases (such as in the amyloid diseases described above). In a preferred embodiment, specific peptides made against the amino acid sequence of laminin contained within the  ̃55 kDa laminin fragment (i.e. globular repeats within the laminin A chain; SEQ ID NO 3) described in the present invention, may be used to aid in the inhibition of amyloid formation, deposition, accumulation, and/or persistence in a given patient. Likewise, in another preferred embodiment anti-idiotypic antibodies made against laminin, laminin fragments and/or laminin-derived polypeptides (as described above) may be given to a human patient as potential blocking antibodies to disrupt continued amyloid formation, deposition, accumulation and/or persistence in the given patient.
Preparations of laminin-derived polypeptides for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions, which may contain axillary agents or excipients which are known in the art. Pharmaceutical compositions such as tablets, pills, tablets, caplets, soft and hard gelatin capsules, lozenges, sachets, cachets, vegicaps, liquid drops, elixers, suspensions, emulsions, solutions, syrups, tea bags, aerosols (as a solid or in a liquid medium), suppositories, sterile injectable solutions, sterile packaged powders, can be prepared according to routine methods and are known in the art.
In yet another aspect of the invention, laminin, laminin fragments and/or laminin-derived polypeptides may be used as an effective therapy to block amyloid formation, deposition, accumulation and/or persistence as observed in the amyloid diseases. For example, the invention includes a pharmaceutical composition for use in the treatment of amyloidoses comprising a pharmaceutically effective amount of a laminin, laminin fragment and/or laminin-derived polypeptide anti-idiotypic antibody and a pharmaceutically acceptable carrier. The compositions may contain the laminin, laminin fragments and/or laminin-derived polypeptide anti-idiotypic antibody, either unmodified, conjugated to a potentially therapeutic compound, conjugated to a second protein or protein portion or in a recombinant form (ie. chimeric or bispecific laminin, laminin fragment and/or laminin polypeptide antibody). The compositions may additionally include other antibodies or conjugates. The antibody compositions of the invention can be administered using conventional modes of administration including, but not limited to, topical, intravenous, intra-arterial, intraperitoneal, oral, intralymphatic, intramuscular or intralumbar. Intravenous administration is preferred. The compositions of the invention can be a variety of dosage forms, with the preferred form depending upon the mode of administration and the therapeutic application. Optimal dosage and modes of administration for an individual patient can readily be determined by conventional protocols.
Laminin, laminin-derived protein fragments, and laminin-derived polypeptides, or antibodies of the present invention may be administered by any means that achieve their intended purpose, for example, to treat laminin involved pathologies, such as Alzheimer's disease and other amyloid diseases, or other related pathologies, using a laminin-derived polypeptide described herein, in the form of a pharmaceutical composition.
For example, administration of such a composition may be by various parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, transdermal or buccal routes. Alternatively, or concurrently, administration may be by the oral route. Parenteral administration can be by bolus injection or by gradual perfusion over time.
A preferred mode of using a laminin-derived polypeptide, or antibody pharmaceutical composition of the present invention is by oral administration or intravenous application.
A typical regimen for preventing, suppressing or treating laminin-involved pathologies, such as Alzheimer's disease amyloidosis, comprises administration of an effective amount of laminin-derived polypeptides, administered over a period of one or several days, up to and including between one week and about 24 months.
It is understood that the dosage of the laminin-derived polypeptides of the present invention administered in vivo or in vitro will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. The most preferred dosage will be tailored to the individual subject, as is understood and determinable by one of skill in the art, without undue experimentation.
The total dose required for each treatment may be administered by multiple doses or in a single dose. A laminin-derived polypeptide may be administered alone or in conjunction with other therapeutics directed to laminin-involved pathologies, such as Alzheimer's disease or amyloid diseases, as described herein.
Effective amounts of a laminin-derived polypeptide or composition, which may also include a laminin-fragment derived antibody, are about 0.01 μg to about 100 mg/kg body weight, and preferably from about 10 μg to about 50 mg/kg body weight, such as 0.05, 0.07, 0.09, 0.1, 0.5, 0.7, 0.9., 1, 2, 5, 10, 20, 25, 30, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 mg/kg.
Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions, which may contain axillary agents or excipients which are known in the art. Pharmaceutical compositions comprising at least one laminin-derived polypeptide, such as 1-10 or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 laminin-derived polypeptides, of the present invention may include all compositions wherein the laminin-derived polypeptide is contained in an amount effective to achieve its intended purpose. In addition to at least one laminin-derived polypeptide, a pharmaceutical composition may contain suitable pharmaceutically acceptable carriers, such as excipients, carriers and/or axillaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
Pharmaceutical compositions comprising at least one laminin-derived polypeptide or antibody may also include suitable solutions for administration intravenously, subcutaneously, dermally, orally, mucosally, rectally or may by injection or orally, and contain from about 0.01 to 99 percent, preferably about 20 to 75 percent of active component (i.e. polypeptide or antibody) together with the excipient. Pharmaceutical compositions for oral administration include pills, tablets, caplets, soft and hard gelatin capsules, lozenges, sachets, cachets, vegicaps, liquid drops, elixers, suspensions, emulsions, solutions, and syrups.
The laminin, laminin-derived protein fragments, and laminin-derived polypeptides for Alzheimer's disease and other central nervous system amyloidoses may be optimized to cross the blood-brain barrier. Methods of introductions include but are not limited to systemic administration, parenteral administration i.e., via an intraperitoneal, intravenous, perioral, subcutaneous, intramuscular, intraarterial, intradermal, intramuscular, intranasal, epidural and oral routes. In a preferred embodiment, laminin, laminin-derived protein fragments, and laminin-derived polypeptides may be directly administered to the cerebrospinal fluid by intraventricular injection. In a specific embodiment, it may be desirable to administer laminin, laminin-derived protein fragments, and laminin-derived polypeptides locally to the area or tissue in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, by injection, by infusion using a cannulae with osmotic pump, by means of a catheter, by means of a suppository, or by means of an implant.
In yet another embodiment laminin, laminin-derived protein fragments, and laminin-derived polypeptides may be delivered in a controlled release system, such as an osmotic pump. In yet another embodiment, a controlled release system can be placed in proximity to the therapeutic target, ie. the brain, thus requiring only a fraction of the systemic dose.
In yet another aspect of the present invention, peptidomimetic compounds modelled from laminin, laminin fragments and/or laminin-derived polypeptides identified as binding Aβ or other amyloid proteins, may serve as potent inhibitors of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease and other amyloidoses. Peptidomimetic modelling is implemented by standard procedures known to those skilled in the art.
In yet another aspect of the present invention, compounds that mimic the 3-dimensional Aβ binding site on laminin using computer modelling, may serve as potent inhibitors of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease and other amyloidoses. Design and production of such compounds using computer modelling technologies is implemented by standard procedures known to those skilled in the art.
Recombinant DNA technology, including human gene therapy, has direct applicability to the laminin proteins and their fragments, of this invention. One skilled in the art can take the peptide sequences disclosed herein and create corresponding nucleotide sequences that code for the corresponding peptide sequences. These sequences can be cloned into vectors such as retroviral vectors, and the like. These vectors can, in turn, be transfected into human cells such as hepatocytes or fibroblasts, and the like. Such transfected cells can be introduced into humans to treat amyloid diseases. Alternatively, the genes can be introduced into the patients directly. The basic techniques of recombinant DNA technology are known to those of ordinary skill in the art and are disclosed in Recombinant DNA Second Edition, Watson, et al., W. H. Freeman and Company, New York, 1992, which is hereby incorporated by reference.
Diagnostic Applications
Another aspect of the invention is to provide polyclonal and/or monoclonal antibodies against laminin, laminin fragments and/or laminin-derived polypeptides which bind Aβ or other amyloid proteins, which would be utilized to specifically detect laminin, laminin fragments and/or laminin-derived peptides in human tissues and/or biological fluids. In one preferred embodiment, polyclonal or monoclonal antibodies made against a peptide portion or fragment of laminin, can be used to detect and quantify laminin, laminin fragments and/or laminin-derived polypeptides in human tissues and/or biological fluids. Polyclonal and/or monoclonal peptide antibodies can also be utilized to specifically detect laminin fragments and/or laminin-derived polypeptides in human tissues and/or biological fluids. In a preferred embodiment, a polyclonal or monoclonal antibody made specifically against a peptide portion or fragment of  ̃55 kDa elastase-resistent protein which binds Aβ (as described herein), can be used to detect and quantify this laminin fragment in human tissues and/or biological fluids. In another preferred embodiment, a polyclonal or monoclonal antibody made specifically against a peptide portion or fragment of  ̃130 kDa laminin-derived protein which is present in human biological fluids and binds Aβ (as described herein), can be used to detect and quantify this laminin fragment in human tissues and/or biological fluids. Other preferred embodiments include, but are not limited to, making polyclonal or monoclonal antibodies made specifically against a peptide portion or fragment of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, as well as polypeptides which have at least 70% similarity (preferably 70% identity) and more preferably a 90% similarity (more preferably a 90% identity) to the polypeptides described above.
For detection of laminin fragments and/or laminin-derived polypeptides described above in human tissues, cells, and/or in cell culture, the polyclonal and/or monoclonal antibodies can be utilized using standard immunohistochemical and immunocytochemical techniques, known to one skilled in the art.
For detection and quantitation of laminin, laminin fragments and/or laminin-derived polypeptides in biological fluids, including cerebrospinal fluid, blood, plasma, serum, urine, sputum, and/or stool, various types of ELISA assays can be utilized, known to one skilled in the art. An antibody molecule of the present invention may be adapted for utilization in an immunometric assay, also known as a “two-site” or “sandwich” assay. In a typical immunometric assay, a quantity of unlabeled antibody (or fragment of antibody) is bound to a solid support or carrier, and a quantity of detectable labeled soluble antibody is added to permit detection and/or quantitation of the ternary complex formed between solid-phase antibody, antigen, and labeled antibody.
In a preferred embodiment, a “sandwich” type of ELISA can be used. Using this preferred method a pilot study is first implemented to determine the quantity of binding of each laminin-fragment monoclonal antibody to microtiter wells. Once this is determined, aliquots (usually in 40 μl of TBS; pH 7.4) of the specific laminin-fragment antibody are allowed to bind overnight to microtiter wells (Maxisorb C plate from Nunc) at 4° C. A series of blank wells not containing any laminin-fragment specific monoclonal antibody are also utilized as controls. The next day, non-bound monoclonal antibody is shaken off the microtiter wells. All of the microtiter wells (including the blank wells) are then blocked by incubating for 2 hours with 300 μl of Tris-buffered saline containing 0.05% Tween-20 (TTBS) plus 2% bovine serum albumin, followed by 5 rinses with TTBS. 200 μl of cerebrospinal fluid, blood, plasma, serum, urine, sputum, and/or stool and/or any other type of biological sample is then diluted (to be determined empirically) in TTBS containing 2% bovine serum albumin and placed in wells (in triplicate) containing bound laminin-fragment antibody (or blank) and incubated for 2 hours at room temperature. The wells are then washed 5 times with TTBS. A second biotinylated-monoclonal antibody against the same laminin-derived fragment (but which is against a different epitope) is then added to each well (usually in 40 μl of TBS; pH 7.4) and allowed to bind for 2 hours at room temperature to any laminin-fragment captured by the first antibody. Following incubation, the wells are washed 5 times with TTBS. Bound materials are then detected by incubating with 100 μl of peroxidase-avidin complex (1:250 dilution in TTBS with 0.1% BSA) for 1 hour on a rotary shaker. After 5 washes with TTBS, a substrate solution (100 μl, OPD-Sigma Fast from Sigma Chemical Co., St. Louis, Mo., USA) is added and allowed to develop significant color (usually 8-10 minutes). The reaction is stopped with 50 μl of 4N sulfuric acid and read on a standard spectrophotometer at 490 nm. This ELISA can be utilized to determine differences in specific laminin fragments (and/or Aβ-binding laminin fragments) in biological fluids which can serve as a diagnostic marker to follow the progression on a live patient during the progression of disease (ie. monitoring of amyloid disease as an example). In addition, quantitative changes in laminin fragments can also serve as a prognostic indicator monitoring how a live patient will respond to treatment which targets a given amyloid disease such as Alzheimer's disease. Such assays can be provided in a kit form.
A competition assay may also be employed wherein antibodies specific to laminin, laminin fragments and/or laminin-derived polypeptides are attached to a solid support and labelled laminin, laminin fragments and/or laminin-derived polypeptides and a sample derived from a host are passed over the solid support and the amount of label detected attached to the solid support can be correlated to the quantity of laminin, laminin fragments and/or laminin-derived polypeptides in the sample. This standard technique is known to one skilled in the art.
Another object of the present invention is to use laminin, laminin fragments and/or laminin-derived polypeptides, in conjunction with laminin, laminin fragment and/or laminin-derived peptide antibodies, in an ELISA assay to detect potential laminin, laminin fragment and/or laminin-derived peptide autoantibodies in human biological fluids. Such a diagnostic assay may be produced in a kit form. In a preferred embodiment, peptides containing the sequences of laminin, laminin-derived fragments and laminin-derived polypeptides as in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, as well as polypeptides which have at least 70% similarity (preferably 70% identity) and more preferably a 90% similarity (more preferably a 90% identity) to the polypeptides described above, will be used to initially bind to microtiter wells in an ELISA plate. A pilot study is first implemented to determine the quantity of binding of each laminin fragment polypeptide to microtiter wells. Once this is determined, aliquots (usually 1-2 μg in 40 μl of TBS; pH 7.4) of specific laminin fragment polypeptides (as described herein) are allowed to bind overnight to microtiter wells (Maxisorb C plate from Nunc) at 4° C. All the microtiter wells (including blank wells without the laminin fragment polypeptides) are blocked by incubating for 2 hours with 300 μl of Tris-buffered saline (pH 7.4) with 0.05% Tween-20 (TTBS), containing 2% albumin. This is followed by 5 rinses with TTBS. The patients' biological fluids (i.e., cerebrospinal fluid, blood, plasma, serum, sputum, urine, and/or stool) are then utilized and 200 μl are diluted (to be determined empirically) with TTBS containing 2% bovine serum albumin, and placed in microtiter wells (in triplicate) containing a specific laminin fragment polypeptide or blank wells (which do not contain peptide), and are incubated at 1.5 hours at room temperature. Any autoantibodies present in the biological fluids against the laminin fragment will bind to the substrate bound laminin fragment polypeptide (or fragments thereof). The wells are then rinsed by washing 5 times with TTBS. 100 μl of biotinylated polyclonal goat anti-human IgGs (Sigma Chemical company, St. Louis, Mo., USA), diluted 1:500 in TTBS with 0.1% bovine serum albumin, is then aliquoted into each well. Bound materials are detected by incubating with 100 μl of peroxidase-avidin complex (1:250 dilution in TTBS with 0.1% bovine serum albumin) for 1 hour on a rotary shaker. Following 5 washes with TTBS, substrate solution (100 μl, OPD-Sigma Fast from Sigma Chemical Company, St. Louis, Mo., USA) is added and allowed to develop significant color (usually 8-10 minutes). The reaction is stopped with 50 μl of 4N sulfuric acid added to each well and read on a standard spectrophotometer at 490 nm. This assay system can be utilized to not only detect the presence of autoantibodies against laminin fragments in biological fluids, but also to monitor the progression of disease by following elevation or diminution of laminin fragment autoantibody levels. It is believed that patients demonstrating excessive laminin fragment formation, deposition, accumulation and/or persistence as may be observed in the amyloid diseases, will also carry autoantibodies against the laminin fragments in their biological fluids. Various ELISA assay systems, knowledgeable to those skilled in the art, can be used to accurately monitor the degree of laminin fragments in biological fluids as a potential diagnostic indicator and prognostic marker for patients during the progression of disease (ie. monitoring of an amyloid disease for example). Such assays can be provided in a kit form. In addition, quantitative changes in laminin fragment autoantibody levels can also serve as a prognostic indicator monitoring how a live patient will respond to treatment which targets a given amyloid disease.
Other diagnostic methods utilizing the invention include diagnostic assays for measuring altered levels of laminin, laminin fragments and/or laminin-derived polypeptides in various tissues compared to normal control tissue samples. Assays used to detect levels of laminin, laminin fragments and/or laminin-derived polypeptides in a sample derived from a host are well-known to those skilled in the art and included radioimmunoassays, competitive-binding assays, Western blot analysis and preferably ELISA assays (as described above).
Yet another aspect of the present invention is to use the antibodies recognizing laminin, laminin fragments and/or laminin-derived polypeptides for labellings, for example, with a radionucleotide, for radioimaging or radioguided surgery, for in vivo diagnosis, and/or for in vitro diagnosis. In one preferred embodiment, radiolabelled peptides or antibodies made (by one skilled in the art) against laminin, laminin fragments and/or laminin-derived polypeptides may be used as minimally invasive techniques to locate laminin, laminin fragments and/or laminin-derived polypeptides, and concurrent amyloid deposits in a living patient. These same imaging techniques could then be used at regular intervals (ie. every 6 months) to monitor the progression of the amyloid disease by following the specific levels of laminin, laminin fragments and/or laminin-derived polypeptides.
Yet another aspect of the present invention is to provide a method which can evaluate a compound's ability to alter (diminish or eliminate) the affinity of a given amyloid protein (as described herein) or amyloid precursor protein, to laminin, laminin-derived fragments or laminin-derived polypeptides. By providing a method of identifying compounds which affect the binding of amyloid proteins, or amyloid precursor proteins to such laminin-derived fragments, the present invention is also useful in identifying compounds which can prevent or impair such binding interaction. Thus, compounds can be identified which specifically affect an event linked with the amyloid formation, amyloid deposition, and/or amyloid persistence condition associated with Alzheimer's disease and other amyloid diseases as described herein.
According to one aspect of the invention, to identify for compounds which allow the interaction of amyloid proteins or precursor proteins to laminin-derived fragments or laminin polypeptides, either amyloid or laminin fragments are immobilized, and the other of the two is maintained as a free entity. The free entity is contacted with the immobilized entity in the presence of a test compound for a period of time sufficient to allow binding of the free entity to the immobilized entity, after which the unbound free entity is removed. Using antibodies which recognize the free entity, or other means to detect the presence of bound components, the amount of free entity bound to immobilized entity can be measured. By performing this assay in the presence of a series of known concentrations of test compound and, as a control, the complete absence of test compound, the effectiveness of the test compound to allow binding of free entity to immobilized entity can be determined and a quantitative determination of the effect of the test compound on the affinity of free entity to immobilized entity can be made. By comparing the binding affinity of the amyloid-laminin fragment complex in the presence of a test compound to the binding affinity of the amyloid-laminin fragment complex in the absence of a test compound, the ability of the test compound to modulate the binding can be determined.
In the case in which the amyloid is immobilized, it is contacted with free laminin-derived fragments or polypeptides, in the presence of a series of concentrations of test compound. As a control, immobilized amyloid is contacted with free laminin-derived polypeptides, or fragments thereof in the absence of the test compound. Using a series of concentrations of laminin-derived polypeptides, the dissociation constant (Kd) or other indicators of binding affinity of amyloid-laminin fragment binding can be determined. In the assay, after the laminin-derived polypeptides or fragments thereof is placed in contact with the immobilized amyloid for a sufficient time to allow binding, the unbound laminin polypeptides are removed. Subsequently, the level of laminin fragment-amyloid binding can be observed. One method uses laminin-derived fragment antibodies, as described in the invention, to detect the amount of specific laminin fragments bound to the amyloid or the amount of free laminin fragments remaining in solution. This information is used to determine first qualitatively whether or not the test compound can allow continued binding between laminin-derived fragments and amyloid. Secondly, the data collected from assays performed using a series of test compounds at various concentrations, can be used to measure quantitatively the binding affinity of the laminin fragment-amyloid complex and thereby determine the effect of the test compound on the affinity between laminin fragments and amyloid. Using this information, compounds can be identified which do not modulate the binding of specific laminin fragments to amyloid and thereby allow the laminin-fragments to reduce the amyloid formation, deposition, accumulation and/or persistence, and the subsequent development and persistence of amyloidosis.
Therefore a kit for practicing a method for identifying compounds useful which do not alter laminin, laminin-derived fragments or laminin-derived polypeptides to an immobilized amyloid protein, said kit comprising a) a first container having amyloid protein immobilized upon the inner surface, b) a second container which contains laminin, laminin-derived fragments or laminin-derived polypeptides dissolved in solution, c) a third container which contains antibodies specific for said laminin, laminin-derived fragments or laminin-derived polypeptides, said antibodies dissolved in solution, and d) a fourth container which contains labelled antibodies specific for laminin, laminin-derived fragments or laminin-derived polypeptides, said antibodies dissolved in solution.

## Claims

1. A method for inhibiting or reducing beta-amyloid protein fibril formation, deposition or accumulation in vitro, the method comprising administering to vitro a cell culture containing beta-amyloid protein, an effective amount of a polypeptide consisting of SEQ ID NO: 3, the amount administered effective for the inhibition or reduction of the beta-amyloid protein fibril formation, deposition or accumulation, as compared to cell culture containing beta-amyloid protein in absence of polypeptide SEQ ID NO: 3.

2. The method of claim 1 wherein the polypeptide SEQ ID NO: 3 is synthetic.

3. The method of claim 1 wherein the polypeptide SEQ ID NO: 3 is taken from a globular domain repeat within a laminin A chain.

4. The method of claim 3, wherein the polypeptide of SEQ ID NO: 3 is taken from a 4th globular domain repeat within human laminin A chain.