The present invention provides a therapeutic method for removing amyloid fibrils from a patient. The present invention also provides a transgenic animal that develops systemic AA amyloidosis within three weeks for use as a tool to investigate AA amyloidosis and to evaluate agents that may be potentially useful in preventing and treating amyloid-related disorders. Further, the present invention provides diagnostic assays for monitoring immunoglobulin light chain fibrillogenesis in real-time and for identification of the chemical nature of the protein in amyloid deposits which enables the determination of the type of amyloidosis for therapeutic and prognostic purposes.

Description of the Invention

SUMMARY OF THE INVENTION

At the present, no therapeutic agent has been demonstrated to effectively treat amyloidosis in vivo. Thus, there still exists a need for developing a method of treating amyloidoses and a method of identifying therapeutic agents that are effective in treating amyloidosis in vivo.

The present invention provides a therapeutic method for removing in vivo amyloid fibrils from a patient. The present invention also provides a transgenic animal that develops systemic AA amyloidosis within three weeks for use as a tool to investigate AA amyloidosis and to evaluate agents that may be potentially useful in preventing and treating amyloid-related disorders.

The present invention provides a transgenic non-human animal, preferably a transgenic mouse, that develops extensive AA amyloid deposits three weeks after administration of amyloid enhancing factor (AEF).

The present invention discloses a method of increasing the rate of development of amyloid deposits in a transgenic animal carrying an IL-6 gene under the control of a promoter or enhancer, comprising administering to the animal an effective amount of amyloid enhancing factor (AEF), wherein the increase in rate of development of amyloid deposits is relative to a transgenic animal not administered with AEF.

The present invention also provides a method of identifying an agent effective in preventing amyloidosis comprising administering a test agent to a transgenic animal carrying an IL-6 gene under the control of a promoter or enhancer, determining the life span of the transgenic animal, and comparing its life span to that of a control transgenic animal, wherein a longer life span of the transgenic animal administered with the test agent indicates that the test agent is effective in preventing AA amyloidosis.

The present invention teaches a method of identifying an agent effective in preventing amyloidosis comprising administering a test agent and AEF to a transgenic animal carrying an IL-6 gene under the control of a promoter or enhancer, determining the life span of the transgenic animal, and comparing its life span to that of a control transgenic animal, wherein a longer life span of the transgenic animal administered with the test agent indicates that the test agent is effective in preventing amyloidosis.

The present invention also discloses a method of identifying an agent effective in treating amyloidosis comprising administering a test agent to a transgenic animal carrying an IL-6 gene under the control of a promoter or an enhancer and having amyloid deposits in its body, determining the life span of the transgenic animal, and comparing its life span to that of a control transgenic animal, wherein a longer life span of the transgenic animal administered with the test agent indicates that the test agent is effective in treating amyloidosis.

Additionally, the present invention provides a method of identifying an agent effective in treating amyloidosis comprising administering AEF to a transgenic animal carrying an IL-6 gene under the control of a promoter or enhancer, administering a test agent, preferably three weeks later, determining the life span of the transgenic animal, and comparing its life span to that of a control newborn transgenic animal, wherein a longer life span of the transgenic animal administered with the test agent indicates that the test agent is effective in treating amyloidosis.

As an alternative embodiment, the agent effective to prevent or treat amyloidosis is identified by detecting the development of amyloid deposits by radiographic imaging, such as MRI, CT or SPECT scan, of the transgenic animal. In one aspect of the invention, a decrease or a constant level of amyloid deposits in the transgenic animal as compared to a control animal indicates that the test agent is effective in treating amyloidosis.

In a preferred embodiment, the transgenic animal is a transgenic mouse; the IL-6 gene is a human IL-6 gene; the promoter is a mouse metallothionein-I (MT-1) promoter; the enhancer is a human E.mu. enhancer.

Also, the methods of the present invention can be used to identify agents effective to prevent or treat AA amyloidosis.

Moreover, the present invention provides a method of removing amyloid deposits from a patient comprising administering to the patient amyloid fibrils in an effective amount to generate an immune response that will promote the removal of in vivo amyloid fibrils from the patient. In a preferred embodiment, the amyloid fibril comprises an amyloid light chain polypeptide or whole light chain. The present invention also provides a vaccine or pharmaceutical composition comprising an amyloid fibril and a carrier. The preferred amyloid fibrils of the present invention are preformed amyloid fibrils. They may be obtained by recombinant or synthetic means.

Furthermore, the present invention provides diagnostic assays for monitoring immunoglobulin light chain fibrillogenesis in real-time. In one embodiment, the present invention discloses a method of identifying an agent that inhibits fibrillogenesis of a polypeptide comprising incubating a test agent with a polypeptide known to form fibrils and ThT, and measuring the fluorescence intensity as a function of time to determine whether the agent inhibits fibrillogenesis of the polypeptide. In another embodiment, a method of determining whether a compound is fibrillogenic comprising incubating the compound with ThT, and measuring fluorescence intensity as a function of time to determine whether the compound is fibrillogenic.

The present invention also discloses a method for identifying the chemical nature of the protein in amyloid deposits which enables the determination of the type of amyloidosis for therapeutic and prognostic purposes. The present invention includes a method of identifying the chemical nature of proteins in amyloid deposits comprising extracting the proteins from ultra-thin sections of formalin fixed, paraffin-embedded tissue biopsy specimens; isolating the proteins; and determining the amino acid sequence of each of the proteins.

DETAILED DESCRIPTION OF THE INVENTION

A. General Description

The present invention discloses that transgenic animals carrying the human IL-6 gene under the control of a promoter, such as the metallothionein-I promoter, have markedly increased concentrations of sAA and develop amyloid in the liver, spleen, and kidneys at an early stage in life. Furthermore, these transgenic animals develop extensive amyloid deposits in the heart and pancreas in addition to the liver, spleen, and kidneys only six weeks after treatment with AEF. The present invention provides a useful transgenic animal model for investigating AA amyloidosis. The pathologic deposits in AEF-treated transgenic animals are irreversible and contribute to the death of the animal. These transgenic animals are useful not only for investigating the disease process, but also as tools for evaluating the efficacy of drugs and other agents designed to prevent and treat this disease.

The present invention also includes methods of using the transgenic animals to identify potential therapeutic agents for treating or prevent AA amyloidosis and for inhibiting the aggregation of AA amyloid fibrils. The present invention also discloses the use of the transgenic animal to investigate the disease process.

Moreover, the present invention teaches compositions, vaccines, and methods of removing amyloid fibrils from a patient comprising administering to the patient an amyloidogenic protein in fibrillar form (a synthetic amyloid fibril) that will generate an immune response effective for removal of amyloid fibrils in vivo, often in only a few days.

Further, the present invention provides diagnostic assays for monitoring immunoglobulin light chain fibrillogenesis in real-time and for identification of the chemical nature of the protein in amyloid deposits which enables the determination of the type of amyloidosis for therapeutic and prognostic purposes.

B. Specific Embodiments

1. The Transgenic Rapid, Inducible Amyloid Deposition (TRIAD) Mouse

The present invention is based in part on the discovery that transgenic mice carrying the human IL-6 gene under the control of the metallothionein-I promoter have markedly increased concentrations of sAA and developed amyloid in the liver, spleen, and kidneys by about 3 months of age (Solomon et al. 1999). At the time of death at about 9 months of age, organs obtained from these animals have extensive amyloid deposits. The AA nature of the amyloid has been evidenced immunohistochemically and has been unequivocally established by sequence analysis of protein extracted from the fibrils. This disease process is apparent radiographically using small animal computer axial tomography (CT) and magnetic resonance imaging (MRI) equipment. The availability of this unique in vivo experimental model of AA amyloidosis provides the means to assess the therapeutic efficacy of agents designed to reduce or prevent the fibrillar deposits found in AA and other types of amyloid-associated disease.

The usefulness of the transgenic mouse AA model has been extended by the discovery that the pathologic process can be induced in young, i.e., 6-week old mice, through i.v. administration of AEF. Such mice develop extensive amyloid deposits in the heart and pancreas in addition to the liver, spleen, and kidneys; these pathologic deposits, in contrast to those induced by chemical stimuli, are irreversible and lead to death of the animal.

The present invention provides a method of obtaining transgenic animals that develop AA amyloidosis after administration of AEF. As used herein the term "AEF" refers to the amyloid enhancing factor characterized by Axelrad et al. (1982). Amyloid deposition is an irreversible process and the continual infiltration by this material leads to organ failure and eventually death. The development of AA amyloidosis in the AEF-enhanced transgenic animal model provides a system not only for investigating the disease process, but also as a tool to evaluate the efficacy of drugs and other agents designed to prevent and treat this disease.

2. Transgenic Animals

The procedure for producing a transgenic animal is known in the art (B. Hogan et al., (1986); and U.S. Pat. No. 4,873,191). As used herein, a "transgenic animal" is any animal, preferably a non-human mammal, a bird or an amphibian, in which one or more of the cells of the animal contain heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art. The nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. The term genetic manipulation refers to the introduction of a recombinant DNA molecule. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA. In the typical transgenic animals described herein, the transgene causes cells to express a recombinant form of IL-6, preferably human IL-6. The "non-human animals" of the invention include vertebrates, such as rodents, non-human primates, sheep, dog, cow, chickens, amphibians, reptiles, etc. Preferred non-human animals are selected from the rodent family including rat and mouse, most preferably mouse, though transgenic amphibians, such as members of the Xenopus genus, and transgenic chickens can also provide important tools for understanding, for example, embryogenesis and tissue patterning.

As used herein, the term "transgene" means a nucleic acid sequence (encoding, e.g., an IL-6 polypeptide), which is partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout). A transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of a selected nucleic acid.

The transgene encoding a desired polypeptide, for example an IL-6 polypeptide, is linked to one or more regulatory regions. Selection of the appropriate regulatory region or regions is a routine matter, within the level of ordinary skill in the art. The regulatory regions may comprise a promoter region for functional transcription, as well as a region situated 3' of the gene of interest, and which specifies a signal for termination of transcription and a polyadenylation site. It may also include an enhancer region. Examples of enhancer include E.mu. enhancer, the RSV enhancer, and SV40 enhancer. As shown in the examples, the E.mu. enhancer is sufficient for expression of IL-6 in transgenic animal.

Promoters that may be used in the present invention include both constitutive promoters and regulated (inducible) promoters. The promoter may be naturally responsible for the expression of the nucleic acid. It may also be from a heterologous source. In particular, it may be promoter sequences of eukaryotic or viral genes. For example, it may be promoter sequences derived from the genome of the cell which it is desired to infect. Likewise, it may be promoter sequences derived from the genome of a virus, including the adenovirus used. In this regard, there may be mentioned, for example, the promoters of the EIA, MLP, HCMV, and RSV genes and the like. In addition, the promoter may be modified by addition of activating or regulatory sequences or sequences allowing a tissue-specific or predominant expression.

Tissue specific promoters include, for example, the keratin 5 (K5) (Missero et al., 1993) and keratin 14 (K14) (Wang et al., 1997) promoters for the basal layer of skin; keratin 1 (K1) (Johnson et al., 1985) and keratin 10 (K10) (Feng et al., 1997) promoters for the suprabasal layer of skin; loricrin (Yoneda et al., 1993), involucrin (Carroll et al., 1997; Carroll et al., 1995) and transglutaminase I (Lee et al., 1996) promoters for the granular layer of skin; cornifin .beta. promoter (Austin et al., 1996) for squamous epithelia, and mCC10 (Ray et al., 1996) and elastin (Hsu-Wong et al., 1994) promoters for the respiratory epithelium.

Additional promoters useful for practice of this invention are the ubiquitous promoters HPRT (Rincon-Limas et al., 1994), vimentin (Vicart et al., 1994), actin (Bronson et al., 1996), and tubulin (Gloster et al., 1994); the intermediate filament promoters desmin (Lee et al., 1995), neurofilaments, keratin, and GFAP (Galou et al., 1994); the therapeutic gene promoters MDR (Yang et al., 1996), CFTR (Matthews et al., 1996), and factor VIII (McGlynn et al., 1996); promoters which are preferentially activated in dividing cells; promoters which respond to a stimulus such as steroid hormone receptor (Cicatiello et al., 1995) and retinoic acid receptor (Mendelsohn et al., 1994) promoters; tetracycline-regulated transcriptional modulators (Furth et al., 1994); cytomegalovirus immediate-early; retroviral LTR (Choate et al., 1996), metallothionein-1 (Fattori et al., 1994); SV-40; E1a and MLP promoters. Tetracycline-regulated transcriptional modulators and CMV promoters are described in WO 96/01313, U.S. Pat. Nos. 5,168,062 and 5,385,839, the contents of which are incorporated herein by reference.

Generally, a method for producing a transgenic animal, which can be stably bred to produce offspring containing the gene, comprises the following steps: (a) isolating a fertilized oocyte from a first female animal; (b) transferring the transgene into the fertilized oocyte; (c) transferring the fertilized oocyte containing the transgene to the uterus of the same species as the first animal; (d) maintaining the second female animal such that (i) the second female animal becomes pregnant with the embryo derived from the fertilized oocyte containing the transgene, (ii) the embryo develops into the transgenic animal, and (iii) the transgenic animal is viably born from the second female animal; wherein the transgenic animal has the genetic sequence for the desired protein and is capable of being bred to produce offsprings having cells stably containing the desired genetic sequence.

3. Method of Using the TRIAD Mouse for Identifying Potential Agents for Prevention or Treatment of Amyloidosis

As set forth above, the present invention includes a method for identifying potential therapeutic agents for preventing and treating inflammation-associated, AA amyloidosis. The present invention also includes a method for identifying agents that inhibit the aggregation of AA amyloid fibrils.

In one assay format, a test agent is administered to transgenic animals, preferably six weeks old, carrying an IL-6 gene, preferably the human IL-6, under the control of a promoter, preferably the metallothionein-1 promoter, or an enhancer, the E.mu. enhancer. For control, transgenic animals carrying an IL-6 gene under the control of a promoter or enhancer and receiving no test agent or receiving a control agent similar in nature to the test agent are raised under the same environmental conditions as the transgenic animals administered with the test agent. The life span of the transgenic animals is ascertained and compared. A longer life span for transgenic animals administered with a test agent as compared to the control transgenic animals indicates that the test agent is an effective therapeutic agent in preventing AA amyloidosis.

In another assay format, a test agent and AEF is administered to transgenic animals, preferably six weeks old, carrying an IL-6 gene, preferably the human IL-6 gene, under the control of a promoter, preferably the metallothionein-1 promoter, and/or an enhancer, preferably the E.mu. enhancer. Similar to the assay above, control transgenic animals are administered with only AEF or AEF and a control agent similar in nature to the test agent and are raised under identical environmental conditions as transgenic animals administered with both the test agent and AEF. The life span of the transgenic animals is ascertained and compared. A longer life span for transgenic animals administered with a test agent as compared to control transgenic animals indicates that the test agent is an effective therapeutic agent in preventing AA amyloidosis.

In a third assay format, a test agent is administered to transgenic animals carrying an IL-6 gene, preferably the human IL-6 gene, under the control of a promoter, preferably the metallothionein-1 promoter, and/or an enhancer, preferably the E.mu. enhancer, and having amyloid deposits in its body. For control, transgenic animals carrying an IL-6 gene under the control of a promoter or enhancer and receiving no test agent or receiving a control agent similar in nature to the test agent are raised under the same environmental conditions as the transgenic animals administered with the test agent. The life span of the transgenic animals is ascertained and compared. A longer life span for transgenic animals administered with a test agent as compared to the control transgenic animals indicates that the test agent is an effective therapeutic agent in treating AA amyloidosis.

In a fourth assay format, AEF is administered to transgenic animals carrying an IL-6 gene, preferably the human IL-6 gene, under the control of a promoter, preferably the metallothionein-1 promoter, and/or an enhancer, preferably the E.mu. enhancer. After AEF injection, for instance at three weeks, a test agent is administered to the transgenic animal injected with AEF. Similar to the assay above, control transgenic animals, administered with only AEF or AEF and a control agent similar in nature to the test agent, are raised under identical environmental conditions as transgenic animals administered with both AEF and the test agent. The life span of the transgenic animals is ascertained and compared. A longer life span for transgenic animals administered with AEF and a test agent as compared to control transgenic animals indicates that the test agent is an effective therapeutic agent in treating AA amyloidosis.

To determine whether a test agent inhibits amyloid formation, the four assays described above may be modified. Instead of ascertaining the life span of the transgenic animals, the animals are sacrificed, for instance at eight weeks after AEF administration or eight months if AEF is not administered. Histochemical and/or immunohistochemical analyses of organs from the animals, such as the spleen, liver, kidney, heart, and pancreas, are performed to confirm the presence, absence, or extent of amyloid deposits. Alternatively, radiographic imaging via MRI, CT, or SPECT scan on small transgenic animals may be performed to detect amyloid deposits without sacrificing the animal, and the life span of the transgenic animal can still be determined.

Agents that are capable of preventing, delaying the onset, or retarding the development of AA amyloidosis are potentially effective in preventing the aggregation of AA into amyloid fibrils. Agents that are-capable of removing established AA amyloid deposits and/or decreasing the extent of amyloid deposition within the organs are potentially effective as mediators in the removal of amyloid fibrils.

As used herein, the term "a test agent" refers to any agent that is to be tested. A test agent can be, but is not limited to, a chemical compound, a peptide, a polypeptide, a carbohydrate, or an antibody. A skilled artisan can readily recognize that there is no limit as to the structural nature of the test agents of the present invention.

A test agent that is assayed by the above method can be randomly selected or rationally selected or designed. As used herein, an agent is said to be randomly selected when the agent is chosen randomly without considering the specific structures or mechanisms involved in the development, treatment, or prevention of AA amyloidosis or inhibition of amyloid fibrillogenesis (i.e. the aggregation of amyloidogenic proteins into fibrils). An agent is said to be rationally selected or designed when the agent is chosen on a nonrandom basis which takes into account the structures, i.e. compares the structures to those of known agents that are effective in treating and preventing AA amyloidosis or inhibiting amyloid aggregation. For example, a rationally selected peptide could be a molecule that binds AA amyloid fibrils and prevents the growth of those fibrils by blocking the addition of protein to the amyloid fibril.

4. Administration of Test Agent to Transgenic Animal

The test agents of the present invention can be administered to the transgenic animal via parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdernal, or buccal routes. Alternatively, or concurrently, administration may be by the oral route. The dosage administered will be dependent upon the age and weight of the transgenic animal, frequency of treatment, the nature of the effect desired, as well as the toxicity of the agent and the pharmacokinetics of the agent in the animal. For example, as a means of inhibiting amyloid fibril formation by preventing the aggregation of protein, removing established amyloid deposits, or preventing amyloidosis by other means, the test agent is administered systemically or locally to the transgenic animal being treated. As described below, there are many methods that can readily be adapted to administer such agents.

While individual transgenic animal may vary, a determination of optimal ranges of effective amounts of each component in the composition is within the skill of the art. Typical dosages comprise 0.1 to 100 mg/kg body wt. The preferred dosages comprise 0.1 to 10 mg/kg body wt. The most preferred dosages comprise 0.1 to 1 mg/kg body wt.

In addition to the pharmacologically active agent, the compositions of the present invention may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically for delivery to the site of action. Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts. In addition, suspensions of the active compounds as appropriate oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include dimethyl sulfoxide, or fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension may also contain stabilizers. Liposomes can also be used to encapsulate the agent for delivery into the cell.

The pharmaceutical formulation for systemic administration according to the invention may be formulated for enteral, parenteral, or topical administration. Indeed, all three types of formulations may be used simultaneously to achieve systemic administration of the active ingredient.

Suitable formulations for oral administration include pulverized preparations mixed with food, hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups, or inhalations and controlled release forms thereof.

5. Synthetic Amyloid Fibrils for Removing In Vivo Amyloid Fibrils

The present invention is based in part on the finding that the use of anti-amyloid antibodies together with the body's cellular immune defense system can effect the removal of the pathologic amyloid deposits. Immunizing mice with synthetic AL amyloid fibrils formed in vitro from a recombinantly derived immunoglobulin light chain variable-region protein generates in vivo anti-fibril antibodies reactive with AL, as well as other types of amyloid (e.g., AA, A, etc). The mice may also be immunized with the complete light chain protein, i.e. the variable and the constant region together. The immunogen may be composed of any protein, peptide, poly-amino acid, or other synthetic polymer which is capable of forming amyloid-like fibrils defined by having the correct ultrastructural and biochemical properties. When such mice are injected subsequently with human AL extracts, this material is removed within 5 days as compared to more than 30 days in non-immunized animals. The use of this particular synthetic AL amyloid fibril and related AL amyloid fibrils as a vaccine is of potential clinical benefit in the treatment and prevention of primary (AL) amyloidosis as well as other types of amyloid-associated disorders, e.g., adult onset (type 2) diabetes and Alzheimer's Disease.

As used herein, the term "homologous amyloid fibrils" refers to synthetic fibrils used as the immunogen in the vaccine that are of the same type as those found in the amyloid in the patient, e.g., light chain fibrils used to treat a patient with AL amyloidosis or synthetic AA-fibrils used to treat AA-amyloid. In contrast, as used herein, the term "heterologous amyloid fibrils" refers to synthetic fibrils used in the vaccine that are different from the amyloid in the patient, i.e. light chain fibrils used to treat AA-amyloidosis.

The present invention provides a method of removing in vivo amyloid fibrils from a patient comprising administering to the patient an effective amount of synthetic amyloid fibril or related amyloid fibril, structurally comparable (or similar) to the fibril found in the amyloid deposit of the patient. There is evidence that both homologous and heterologous amyloid deposits in the mice can be cleared by immunization with synthetic light chain fibrils.

As used herein, the term "an effective amount of amyloid fibril" refers to an amount of amyloid fibrils that for example, may be effective to perform an activity, such as to increase removal of endogenous or in vivo amyloid fibrils by about 10%, 20%, 30%, 40%, or preferably 50% or more compared to the untreated condition. In the most preferred embodiment, the effective amount increases the efficacy of removal so that essentially all of the amyloid fibrils are removed.

Other isolated proteins identified in amyloid deposits including immunoglobulin light chains, serum amyloid A protein, .beta.2-microglobulin, transthyretin, cystatin C variant, gelsolin, procalcitonin, PrP protein, ApoAI, .beta.-amyloid protein (.beta./A4), and lysozyme can also be used to immunize patients with amyloidosis and to remove in vivo amyloid fibrils from the patients body and to prevent subsequent accumulation of amyloid deposits.

As used herein, the term "amyloid fibril" refers to protein aggregates which possess the ultrastructural, biochemical, physical, or tinctorial properties of amyloid fibrils formed in vivo. As used herein, the term "amyloid protein" refers to proteins or peptides that are capable of forming amyloid fibrils, to the protein present in the amyloid deposit of a patient, to allelic variants thereof, variants thereof, and to peptides thereof. Allelic variants, though possessing a slightly different amino acid sequence than a naturally occurring amyloid polypeptides, will still have the requisite ability to aggregate and form amyloid fibrils. The term also includes variants of amyloid proteins having amino acid alterations that do not adversely affect the ability of the amyloid fibril to aggregate. A substitution, insertion, or deletion is said to adversely affect the amyloid protein when the altered sequence prevents it from aggregating into fibrils. For example, the overall charge, structure or hydrophobic/hydrophilic properties of amyloid protein can be altered without adversely affecting the activity of the amyloid protein. Accordingly, the amino acid sequence of amyloid protein can be altered, for example, to render the protein more hydrophobic or hydrophilic, without adversely affecting the activity of the protein. The term, "amyloid fibril", encompasses, but is not limited to, fibrils composed of immunoglobulin light chains, serum amyloid A protein, .beta.2-microglobulin, transthyretin, cystatin C variant, gelsolin, procalcitonin, PrP protein, amyloid .beta.-protein, peptides thereof, variants thereof, and allelic variants thereof.

As used herein, the term "synthetic amyloid fibril" or "synthetic amyloid protein" refers to an amyloid fibril or constituent protein obtained by synthetic means. The term encompasses amyloid fibril-forming-proteins and polypeptides having the same amino acid sequence as the proteins or polypeptides present in amyloid deposits, allelic variants thereof, variants thereof, and peptides thereof obtained by synthetic means.

As used herein, the term "recombinant amyloid protein" refers to a protein or peptide capable of forming amyloid fibrils that is obtained by recombinant means. The term encompasses but is not limited to, proteins and polypeptides having the same amino acid sequence as the polypeptides present in the amyloid deposit, allelic variants thereof, variants thereof, and peptides thereof obtained by synthetic means.

The present invention can be practiced with amyloid fibrils composed of naturally occurring amyloid protein obtained by synthetic or recombinant means, or isolated from its native source. The present invention can also be practiced with amyloid fibrils composed of a peptide of an amyloid protein, an allelic variant of an amyloid protein, or a variant of an amyloid protein, provided that the peptide or variant when in fibrillar form generates an immune response that promotes the removal of amyloid fibrils from the patient.

6. Pharmaceutical Compositions that Generate an Immune Response for Removal of Amyloids Fibrils

The pharmaceutical compositions for therapeutic treatment according to the present invention are intended for any form of administration including, parenteral, oral, or local administration. Preferably, the pharmaceutical compositions are administered orally or parenterally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. Delivery of amyloid fibrils across the blood-brain barrier (BBB) may be achieved through liposomal or micellar delivery. Alternatively, the amyloid fibrils of this invention can be delivered directly into the cerebrospinal fluid (Walker et al., 1994). For other delivery mechanisms, refer to P. M. Friden, 1996 U.S. Pat. No. 5,527,527 and W. M. Pardridge, 1991 U.S. Pat. No. 5,004,697. All of the above documents are incorporated herein by reference.

Thus, the invention provides compositions and vaccines for parenteral administration which comprise a solution of amyloid fibril dissolved or suspended in a pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be used, e.g., water, buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized,'the lyophilized preparation being combined with a sterile solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

The concentration of amyloid fibrils of the invention in the pharmaceutical formulations can vary widely, i.e., from less than about 1%, usually at or at least about 10-15% to as much as 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.

Without undue experimentation, one of ordinary skill in the art could determine the quantity of amyloid fibril that would be effective to adequately generate an immune response for removal of the in vivo amyloid fibrils. Amounts effective for this use will depend on, e.g., the nature of the amyloid protein composition, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, the toxicity of the preparation, the pharmacokinetics of catabolism and clearance, and the judgment of the prescribing physician. A typical single dose of 5 mg/kg per injection could generally be used. It must be kept in mind that the amyloid protein and peptide compositions derived therefrom may be employed in serious disease states, that is, life-threatening or potentially life-threatening situations. In such cases it is possible and may be felt desirable by the treating physician to administer substantial excesses of these compositions.

As noted earlier, any amyloid fibril that generates an immune response sufficient to remove amyloid fibrils from the patient may be administered to the patient. Amyloid fibrils may also be conjugated to a carrier, such as bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH) or thyroglobulin, to increase their immunogenicity. Various adjuvants may also be used to increase immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, and dinitrophenol. Among adjuvants used in humans, BCG (bacilli Calmette-Guerin), Corynebacterium parvum, and aluminum hydroxide (ALUM) are especially preferable.

The particular manner in which an amyloid fibril of this invention can be bonded to a carrier-protein will depend on the functionalities which are available on the peptide and the carrier, the number of peptide groups to be conjugated, etc. Groups which find use include amino groups; or carboxyl groups which can be activated by employing the mixed carbonic acid anhydride or carbodiimide; imidates; diazo groups; alpha-haloketones; and the like. Peptides or peptide-protein conjugates can be injected in the fluid state; adsorbed to insoluble particles, such as alumina; or incorporated in matrix materials such as agar, calcium alginate, or Freund's adjuvants ("complete" or "incomplete"). Other adjuvants include polyacrylamide gel, bentonite, and proteins such as methylated bovine serum albumin. Complete Freund's adjuvant, a suspension of mycobacteria in oil, is given with an aqueous preparation of the immunogen in the form of an emulsion stabilized with lanolin, lanolin derivatives, e.g., Aquaphor, mannide mono-oleate or Arlacel A. The complete adjuvant is distinguished from the incomplete adjuvant, by having mycobacteria, e.g., M. butyricum or M. tuberculosis.

The fibril conjugates can be injected interperitoneally, intramuscularly, subcutaneously, etc. When employing Freund's adjuvants, usually in combination with saline, the amount of immunogen employed will vary depending on the particular immunogenic material and the number and period of prior injections. Usually, about 0.1 to 5 mg of immunogenic material will be employed per ml of solution. The total amount of immunogenic material and solution will depend on the size, nature, and weight of the subject. The initial injection will normally be at a number of sites, aliquots of the composition being employed.

Treatment of humans with amyloidosis according to the present invention could also be applied to animals susceptible to amyloidosis, such as cows, sheep, goats, dogs, cats, or chickens. Thus, references to human patients and transgenic mice herein apply also to non-human patients.

7. Chemical Identification of Amyloid using Ultra-Thin Sections of Formalin-Fixed, Paraffin-Embedded Tissue Sections

In many cases, only microscopic slides or paraffin embedded tissue blocks are available for further examination of amyloid fibrils found in a patient. Normally, tissues samples are formalinized and dehydrated before they are embedded in paraffin. The present invention discloses a method that uses ultra-thin sections of formalin fixed, paraffin-embedded tissue biopsy specimens for precise identification of the nature of the tissue deposits. The extraction, purification, and digestion procedures has been miniaturized in order to obtain usable information about the amino acid sequence of amyloid fibril forming proteins from microscopic slides or paraffin embedded tissue. The present invention provides a method to rapidly determine the type of protein deposited as amyloid.

8. In vitro Microplate Assay to monitor Immunoglobulin Light Chain Fibrillogenesis in Real-Time

The present invention is also based in part on a microplate based method to quantitate the rate and extent of fibril formation. Samples are placed in a 48- or 96 well microplate containing the amyloidophilic fluorescent dye thioflavin T (ThT). The fluorescence intensity of ThT at a specific wavelength is proportional to the fibril content of the solution. The fluorescence intensity is measured.

This assay is rapid and reproducible and is useful for investigating the basic principles governing light chain fibrillogenesis. This assay can also be used to study other types of amyloid-associated proteins, such as .beta./A4, TTR, etc., or to investigate whether a compound is fibrillogenic.

This assay also provides a method of screening for therapeutic inhibitors of light chain fibril formation in a rapid, reproducible fashion. A test agent is incubated with a polypeptide, preferably immunoglobulin light chain, known to form fibrils and ThT. The fluorescence intensity is measured as a function of time. As a control, the fluorescence intensity of a sample containing only the polypeptide and ThT is measured as a function of time. The test agent is an inhibitor, if the fluorescence intensity does not increase with time. A variety of test agents can be screened at the same time using microplates containing a fibrillogenic polypeptide and ThT.
 

Claim 1 of 19 Claims

1. A method of removing amyloid deposits from a subject comprising administering to the subject amyloid fibrils comprising an immunoglobulin light chain polypeptide or a whole immunoglobulin light chain polypeptide, heterologous to the amyloid fibrils in the subject, in an effective amount to generate an immune response, wherein the immune response promotes the removal of amyloid deposits from the subject.

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