JP2012102131A - Prophylaxis and treatment for amyloidogenic disease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a pharmaceutical composition for prophylaxis and treatment of a large number of amyloid diseases including an Alzheimer's disease, a prion disease, a familial amyloid neuropathy and the like, and a method therefor.SOLUTION: The pharmaceutical composition contains a reactant effective for inducing an immunological response to an amyloid component in a patient, and a pharmaceutical excipient therefor. The reactant is derived from a fibril precursor protein selected from the group composed of AA, AL, ATTR, A apo-A1, Alys, Agel, Acys, Aβ, AB2M, AScr, Acal, AIAPP and synuclein-NAC fragments.

Description

  The present invention relates to compositions and methods for treating conditions related to amyloid in humans and other mammalian vertebrates.

  Amyloidosis is a general term describing a number of diseases characterized by extracellular deposition of protein fibrils that form a large number of "amyloid deposits" that can occur at a localized site or systemically . The fibril composition of these deposits is a feature that identifies various forms of amyloid disease. For example, intracerebral and cerebrovascular deposits composed primarily of fibrils of β-amyloid peptide (β-AP) are characteristic of Alzheimer's disease (both familial and sporadic forms), and islet amyloid protein peptide (IAPP; amylin) is characteristic of fibrils in islet cell amyloid deposits associated with type II diabetes, and β2-microglobulin is the major component of amyloid deposits resulting from long-term hemodialysis treatment. is there. More recently, prion-related diseases such as Creutzfeldt-Jakob disease have also been recognized as amyloid diseases.

  The various forms of disease are divided into multiple classes based primarily on whether or not amyloidosis is associated with or underlying the underlying systemic disease. Thus, certain disorders are considered to be primary amyloidosis, where there is no evidence of pre-existing or coexisting diseases. In general, primary amyloidosis of the disease is characterized by the presence of “amyloid light chain type” (AL type) protein fibrils, and the N-terminal region of AL fibrils against various fragments of immunoglobulin light chain (κ chain or λ chain) So named because of homology.

Secondary or “reactive” amyloidosis is characterized by the deposition of type AA fibrils derived from serum amyloid A protein (apo SSA). These forms of amyloidosis are characterized by an underlying chronic inflammatory or infectious disease state (eg, rheumatoid arthritis, osteomyelitis, tuberculosis, leprosy).
Characterized by an underlying chronic inflammatory or infectious disease state (eg, rheumatoid arthritis, osteomyelitis, tuberculosis, leprosy).

Family hereditary amyloidosis can have associated neuropathic, renal or cardiovascular deposits of the ATTR transthyretin type. Other familial hereditary amyloidosis encompasses other syndromes and can have different amyloid components (eg, familial Mediterranean fever characterized by AA fibrils). Other forms of amyloidosis include local forms characterized by focal, often tumor-like deposits that occur in isolated organs. Other amyloidosis is associated with aging and is universally characterized by plaque formation in the heart or brain. Amyloid deposits associated with long-term hemodialysis are also universal. These and other forms of amyloid disease are summarized in Table 1. (Tan, S. Y. and Pepys, Histopathology 25: 403-414, 1994; Harrison's
Handbook of Internal Medicine, 13th edition, Isselbacher, K.M. J. et al. Et al., McGraw-Hill, San Francisco, 1995).

  Often, the fibrils that form the mass of amyloid deposits are derived from one or more primary precursor proteins or peptides and are usually accompanied by sulfated glycosaminoglycans. In addition, amyloid deposits can include various types of less proteins and peptides, along with other components such as proteoglycans, gangliosides and other sugars, as described in more detail in the sections that follow.

  Currently, there is no treatment directed at specific amyloid for any of the amyloid diseases. In the presence of an underlying or related disease state, treatment is directed to reducing the production of amyloidogenic protein by treating the underlying disease. This is exemplified by the treatment of tuberculosis with antibiotics, thereby reducing mycobacterial burden, resulting in suppression of inflammation and associated reduction of SSA protein. In the case of AL amyloid due to multiple myeloma, the patient is administered a chemotherapeutic drug, causing a decrease in plasma cells and a decrease in myeloma immunoglobulin levels. As these levels decrease, AL amyloid can disappear. Commonly owned (co-owned) US patent application USSN 09 / 201,430 filed November 30, 1998, and USSN 09 filed May 28, 1999. No./322,289 significantly reduces the load of amyloid plaques associated with Alzheimer's disease by administration of agents that generate or confer an immune response directed against β-amyloid peptide (Aβ) and fragments thereof Show that you can (and prevent). It is a discovery of the present invention that induction of an immune response against various amyloid plaque components is effective in the treatment of a wide range of amyloid diseases.

The present invention is directed to pharmaceutical compositions and methods for treating a number of amyloid diseases. According to one aspect, the present invention includes a pharmaceutical composition comprising as the active ingredient an agent that is effective in inducing an immune response against the amyloid component in a patient. Such compositions generally can also include excipients, and in preferred embodiments can include adjuvants. In further preferred embodiments, the adjuvant includes, for example, aluminum hydroxide, aluminum phosphate, MPL ™, QS-21 (Stimulon ™) or incomplete adjuvant of Freund. According to one related aspect, such pharmaceutical compositions can include a plurality of agents effective to induce an immune response against one or more amyloid components in a patient.

In one related aspect, the agent is effective to generate an immune response directed against the amyloid component of the fibril peptide or protein. Preferably, such fibril peptides or proteins are derived from fibril precursor proteins known to be associated with certain forms of amyloid disease, as described herein. Such precursor proteins include, but are not limited to, serum amyloid A protein (apo SSA), immunoglobulin light chain, immunoglobulin heavy chain, apo AI, transthyretin, lysozyme, fibrogen alpha chain, gelsolin , Cystatin C, amyloid β protein precursor (β-APP), β ₂ microglobulin, prion precursor protein (PrP), atrial natriuretic factor, keratin, islet amyloid polypeptide, peptide hormone and synuclein be able to. Such precursors also include mutant proteins, protein fragments and proteolytic peptides of such precursors. In a preferred embodiment, the agent is effective to induce an immune response directed against a new epitope formed by the fibril protein or peptide with respect to the fibril precursor protein. That is, as described in more detail herein, many fibril forming peptides or proteins are fragments of such precursor proteins, such as those listed above. When such a fragment is formed, such as by proteolytic cleavage, there is an epitope that is not present on the precursor and therefore not immunologically available to the immune system when the fragment is part of the precursor protein. Can appear. Agents directed to such epitopes can be preferred therapeutic agents. Because they are probably less likely to induce an autoimmune response in the patient.

According to one related aspect, the pharmaceutical composition of the present invention includes, but is not limited to, the following fibril peptide or protein: AA, AL, ATTR, A ApoA1, Alys, Agel, Acys, Aβ _{encompasses, AB 2 M, AScr, Acal} , agents directed to amyloid components, such as those selected from the group including the AIAPP and synuclein -NAC fragments. The complete names and compositions of these peptides are described herein. Such peptides can be made according to methods known in the art as described herein.

  According to a further related aspect, the agents included in such pharmaceutical compositions also include certain sulfated proteoglycans. In one related aspect, the proteoglycan is a heparin sulfate glycosaminoglycan, preferably perlecan, dermatan sulfate, chondroitin-4-sulfate, or pentosan polysulfate.

According to another related aspect, the invention includes a method for the prevention or treatment of disorders characterized by amyloid deposition in a mammalian subject. In accordance with this aspect of the invention, a subject is given a dosage of an agent effective to generate an immune response against the amyloid component characteristic of the amyloid disorder that the subject is suffering from. In essence, the method involves administering a pharmaceutical composition containing an immunogenic amyloid component specific for a disorder such as those described above. Such methods are further characterized by their effectiveness in inducing an immunogenic response in a subject. According to a preferred embodiment, the method is effective to generate an immunological response characterized by a serum titer of at least 1: 1000 for the amyloid component to which the immunogenic agent is directed. In yet a further preferred embodiment, the serum titer is at least 1: 5000 for the fibril component. According to one related aspect, the immune response is characterized by an immunoreactive serum volume corresponding to greater than about 4 times greater than the immunoreactive serum level measured in a control serum sample prior to treatment. . This latter characterization is particularly relevant when serum immunoreactivity is measured by ELISA techniques, but can be applied to any relative or absolute measure of serum immunoreactivity. According to one preferred embodiment, immunoreactivity is measured at about 100-fold serum dilution.

  According to yet a further related aspect, the invention encompasses a method for determining the prognosis of a patient undergoing treatment for an amyloid disorder. Here, serum levels of patients immunoreactive to the amyloid component characteristic of the selected disorder are measured, and serum levels of patients who are immunoreactive at least four times the baseline control level of serum immunoreactivity Is intended to indicate an improved prognosis for a particular amyloid disorder. According to a preferred embodiment, the amount of immunoreactivity for a selected amyloid component present in the patient's serum is characterized by a serum titer of at least about 1: 1000, or at least 1: 5000 for the amyloid component.

  According to a further related aspect, the invention also encompasses so-called “passive immunization” methods and pharmaceutical compositions for preventing or treating amyloid diseases. In accordance with this aspect of the present invention, the patient has an effective dosage that specifically binds to a selected amyloid component, preferably a fibril component present in an amyloid deposit characteristic of the disease to be treated. Given antibody. In general, such antibodies are selected for their ability to specifically bind the various proteins, peptides and components described with respect to the pharmaceutical compositions and methods described in the preceding paragraphs of this section. According to one related aspect, such methods and compositions can include a combination of antibodies that bind at least two amyloid fibril components. In general, the pharmaceutical composition provides an amount of serum that is immunoreactive for the target amyloid component that is at least about 4 times higher than the serum level of immunoreactivity for the component measured in the control serum sample. Administer. The antibody can also be administered with a carrier as described herein. In general, according to this aspect of the invention, such antibodies may be administered (or formulated for administration) intraperitoneally, orally, intranasally, subcutaneously, intramuscularly, topically or intravenously. Can, however, be administered by any pharmaceutically effective route (ie, effective to produce the indicated therapeutic level, as indicated above and herein) Or can be formulated for administration therewith.

  According to one related aspect, the therapeutic antibody can be administered by administering to the patient a polynucleotide encoding at least one antibody chain. According to this aspect of the invention, the polynucleotide is expressed in a patient and causes the antibody chain to be produced in a pharmaceutically effective amount in the patient. Such polynucleotides can encode the heavy and light chains of the antibody, thereby producing heavy and light chains in the patient.

  According to a preferred embodiment, the immunization regime described above is administered according to methods known in the art or according to the patient's needs as assessed by immunological response, followed by an initial immunization followed by a 6 week interval. Administration of agents including antibodies at multiple dosages, such as over a 6 month period, such as infusion of booster stimulation at such time intervals can be included. Alternatively, or in addition, such regimens can include the use of “sustained release” formulations, as are known in the art.

  These and other objects and features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings.

(Detailed description of the invention)
A. Definitions Unless otherwise indicated, all terms used herein have the same meaning as they would have for one of ordinary skill in the art of the invention. Practitioners, among others, for definitions, technical terms and standard methods known in the biochemical and molecular biology arts, see Sambrook et al. (1989) Molecular Cloning: A Laboratory.
Manual (2nd edition), Cold Spring Harbor Press, Plainview, NY, and Ausubel, F.M. M.M. (1998) Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY. It is understood that the present invention is not limited to the particular methodologies, protocols and reagents described. This is because they can be varied to produce the same result.

  The term “adjuvant” refers to a compound that enhances the immune response to an antigen when administered with an antigen, but does not produce an immune response to the antigen when administered alone. Adjuvants can enhance the immune response by several mechanisms, including lymphocyte recruitment, B and / or T cell stimulation, and macrophage stimulation.

  “Amyloid disease” or “amyloidosis” refers to any of a number of disorders that have accumulated or formed amyloid plaques as a symptom or part of its pathology.

  “Amyloid plaques” are extracellular deposits composed mainly of proteinaceous fibrils. In general, fibrils are composed of one dominant protein or peptide; however, the plaque may also include additional components that are peptide or non-peptide molecules as described herein. it can.

  An “amyloid component” is the entity of any molecule present in amyloid plaques that includes the antigenic portion of such molecule. Amyloid components can include, but are not limited to proteins, peptides, proteoglycans and carbohydrates. A “specific amyloid component” refers to a molecular entity found primarily or exclusively in a amyloid plaque of interest.

  An “agent” is a chemical molecule of synthetic or biological origin. In the context of the present invention, an agent is a molecule that can generally be used in a pharmaceutical composition.

  An “anti-amyloid agent” is an agent that is capable of producing an immune response against an amyloid plaque component in a vertebrate subject when administered by active or passive immunization techniques.

  The terms “polynucleotide” and “nucleic acid” as used interchangeably herein refer to a polymer molecule having a backbone that supports a base capable of hydrogen bonding to a typical polynucleotide, Here, the backbone of the polymer presents bases in a manner that allows such hydrogen bond formation in a sequence specific manner between the polymer molecule and a typical polynucleotide (eg, single stranded DNA). Such bases are typically inosine, adenosine, guanosine, cytosine, uracil and thymidine. Polymer molecules include double- and single-stranded RNA and DNA, as well as modifications to its backbone (eg, methyl phosphonate linkages).

As used herein, the term “polypeptide” refers to a compound composed of a single chain of amino acid residues linked by peptide bonds. The term “protein” can be synonymous with the term “polypeptide” or can refer to a complex of two or more polypeptides.

  The term “peptide” also refers to a compound composed of amino acid residues linked by peptide bonds. Generally, peptides are composed of 100 or fewer amino acids, while polypeptides or proteins have 100 or more amino acids. The term “protein fragment” as used herein can also be read to mean a peptide.

  “Fibrotic peptide” or “fibrillar protein” refers to a monomeric or aggregated form of a protein or peptide that forms fibrils present in amyloid plaques. Examples of such peptides and proteins are provided herein.

  “Pharmaceutical composition” refers to a chemical or biological composition suitable for administration to a mammalian individual. Such compositions may include, but are not limited to, one or more of a number of routes including oral, parenteral, intravenous, intraarterial, subcutaneous, intranasal, sublingual, intravertebral, intraventricular and the like. Can be specially formulated for administration via

  A “pharmaceutical excipient” or “pharmaceutically acceptable excipient” is a carrier (usually a liquid) in which an effective therapeutic agent is formulated. Excipients generally do not provide any pharmacological activity to the formulation, but it can provide chemical and / or biological stability, release characteristics, and the like. Exemplary formulations are described, for example, in Remington's Pharmaceutical Sciences, 19th Edition, Grennaro, A .; Ed., 1995.

  A “glycoprotein” is a protein to which at least one carbohydrate chain (oligopolysaccharide) is covalently linked.

  A "proteoglycan" is a long linear polymer of repeating disaccharides in which at least one carbohydrate chain is a glycosaminoglycan (one member of the pair is usually a sugar acid (uronic acid) and the other is an amino sugar) ) Is a glycoprotein.

The term “immunological” or “immune” or “immunogenic” response refers to humoral (antibody-mediated) and / or cellular (antigen-specific) directed against antigens in vertebrate individuals. It refers to the generation of responses (mediated by T cells or their secreted products). Such a response can be an active response induced by administration of the immunogen or a passive response induced by administration of antibody or primed T cells. The cellular immune response is elicited by the presentation of polypeptide epitopes in conjunction with class I or class II MHC molecules that activate antigen-specific CD4 ⁺ T helper cells and / or CD8 ⁺ cytotoxic T cells. . The response may also require activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia, eosinophils or other components of endogenous immunity. . The presence of a cell-mediated immunological response can be measured by standard proliferation assays (CD4 ⁺ T cells) or CTL (cytotoxic T lymphocyte) assays known in the art. The relative contribution of humoral and cellular responses to the protective or therapeutic effect of immunogens is the isolation of immunoglobulin (IgG) and T cell fractions separately from immunized common gene animals, and It can be identified by measuring the protective or therapeutic effect in the second subject.

An “immunogenic agent” or “immunogen” or “antigen” can induce an immunological response to itself upon administration to a patient, either with or without an adjuvant. It is a molecule that is possible. Such molecules include, for example, amyloid fibril peptides or fragments thereof conjugated to a carrier protein such as KLH, Cd3 or tetanus toxin.

  An “epitope” or “antigenic determinant” is the portion of an antigen that binds to the antigen-binding region of an antibody.

  The terms “Aβ”, “Aβ peptide”, and “amyloid β” peptide are synonymous and are approximately 38-43 amino acids from β-amyloid precursor protein (β-APP) as described herein. Refers to a species or more peptide composition. “Aβxx” refers to amyloid β peptide 1-xx, where xx is a number indicating the number of the amino acid in the peptide; for example, Aβ42 is identical to Aβ1-42, which is referred to herein as “AN1792”. Aβ40 is also identical to Aβ1-40, which is also referred to herein as “AN1578”.

  The component or monomeric Aβ means a soluble monomeric peptide unit of Aβ. One method for preparing monomeric Aβ is to dissolve the lyophilized peptide in neat DMSO using sonication. The resulting solution is centrifuged to remove any insoluble particulates. Aggregated Aβ is a mixture of oligomers in which monomeric units are held together by non-covalent bonds.

  The term “naked polynucleotide” refers to a polynucleotide that is not complexed with a colloidal material. Naked polynucleotides are sometimes cloned into plasmid vectors.

  The term “patient” includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.

  The terms “significantly different”, “statistically significant”, “significantly higher (or lower)” and similar phrases refer to comparisons between data or other measurements, where The difference between the two compared individuals or groups is clearly or reasonably different for the trained observer or is statistically significant (the phrase is referred to as “statistically” If the term is included, or if there is some indication of a statistical test such as p-value, or if analyzed, the data will be statistically different by standard statistical tests known in the art. To give rise to).

  Compositions or methods “comprising” one or more of the listed elements can include other elements not specifically recited. For example, a composition comprising a fibril component peptide encompasses both an isolated peptide and a peptide as a component of a larger polypeptide sequence. By way of further example, a composition comprising elements A and B also includes a composition comprising A, B and C.

B. Amyloid disease Overview and Etiology Amyloid disease or amyloidosis encompasses a number of disease states with a wide range of appearance symptoms. These disorders universally have the presence of abnormal extracellular deposits of protein fibrils known as “amyloid deposits” or “amyloid plaques”, which are usually about 10-100 μm in diameter, It is localized in a specific organ or tissue region. These plaques are mainly composed of naturally occurring soluble proteins or peptides.
These insoluble deposits are generally composed of fibrillar lateral aggregates approximately 10-15 nm in diameter. Amyloid fibrils produce a characteristic clear yellow-green birefringence in polarized light when stained with Congo red dye. The disorders are classified based on the major fibril components that form plaque deposits, as described below.

Peptides or proteins that form plaque deposits are often produced from larger precursor proteins. More specifically, the pathogenesis of amyloid fibril deposits generally requires proteolytic cleavage into fragments of “abnormal” precursor proteins. These fragments generally aggregate into antiparallel β-pleated sheets; however, in familial amyloid polyneuropathy (mutant transthyretin fibrils) and dialysis-related amyloidosis (β ₂ microglobulin fibrils) Have been reported that certain undegraded forms of precursor proteins aggregate and form fibrils (Tan et al., 1994, supra).

2. Clinical Syndrome This section provides a description of the major types of amyloidosis, including their characteristic plaque fiber composition. It is a general discovery of the present invention that amyloid diseases can be treated by administering an agent that acts to stimulate an immune response against one or more components of various disease-specific amyloid deposits. It is. As discussed in more detail in Section C below, these components are preferably fibril components that form plaques. The lower section serves to illustrate the major forms of amyloidosis and is not intended to limit the invention.

a. AA (Reactive) Amyloidosis In general, AA amyloidosis is a manifestation of a number of diseases that elicit a sustained acute phase response. Such diseases include chronic inflammatory disorders, chronic local or systemic microbial infections, and malignant neoplasms.

  AA fibrils are generally found in serum amyloid A protein (apo SSA) (a circulating apoliposome present in HDL particles and synthesized in hepatocytes in response to cytokines such as IL-1, IL-6 and TNF. It is composed of 8000 dalton fragments (AA peptide or protein) formed by proteolytic cleavage of the protein. Deposition is accompanied by a preference for the parenchyma and can be widely spread throughout the body. The spleen is usually the site of deposition, and the kidneys can also be affected. Deposition is also universal in the heart and gastrointestinal tract.

  AA amyloid diseases include, but are not limited to, inflammatory diseases such as rheumatoid arthritis, juvenile arthritis, ankylosing spondylitis, psoriasis, psoriatic arthropathy, Reiter's syndrome, adult Still's disease, Behcet's syndrome and Crohn's disease Can be mentioned. AA deposits also occur as a result of chronic microbial infections such as leprosy, tuberculosis, bronchiectasis, pressure ulcers, chronic pyelonephritis, osteomyelitis and Whipple's disease. Certain malignant neoplasms can also result in AA fibril amyloid deposits. These include conditions such as Hodgkin lymphoma, renal cancer, cancer of the intestine, lung and urogenital tract, basal cell carcinoma, and hairy cell leukemia.

b. AL Amyloidosis AL amyloid deposition is generally associated with almost any disease of the B lymphocyte lineage, ranging from malignant lesions of plasma cells (multiple myeloma) to benign monoclonal gammaglobulinemia. Sometimes the presence of amyloid deposits can be the main indicator of the underlying disease.

The fibrils of AL amyloid deposits are composed of a single clone immunoglobulin light chain or a fragment thereof. More specifically, the fragment is derived from the N-terminal region of the light chain (kappa or lambda chain) and contains all or part of its variable (V _L ) domain. Deposits are generally present in mesenchymal tissue and are peripheral and autonomous neuropathy, carpal tunnel syndrome, macroglossia, restrictive cardiomyopathy, arthropathy of large joints, immune disease, myeloma, and subclinical disease cause. However, it should be noted that almost any tissue, especially visceral organs such as the heart, can be involved.

c. Hereditary systemic amyloidosis There are many forms of hereditary systemic amyloidosis. Although they are relatively rare disease states, the onset of symptomatic adults and their genetic pattern (usually autosomal dominant) lead to the persistence of such disorders in the general population. In general, the syndrome can be attributed to point mutations in the precursor protein leading to the production of mutant amyloidogenic peptides or proteins. Table 2 summarizes the fibril composition of exemplary forms of these disorders.

  The data provided in Table 2 is exemplary and is not intended to limit the scope of the invention. For example, over 40 distinct point mutations in the transthyretin gene have been described, all of which give rise to clinically similar forms of familial amyloid polyneuropathy.

Transthyretin (TTR) is a 14 kilodalton protein sometimes referred to as prealbumin. It is produced by the liver and choroid plexus, which functions in the transport of thyroid hormone and vitamin A. Each characterized by a single amino acid change,
At least 50 variant forms of the protein are responsible for various forms of familial amyloid polyneuropathy. For example, substitution of proline instead of leucine at position 55 results in a progressive form of neuropathy; substitution of methionine instead of leucine at position 111 results in severe heart damage in Danish patients. An amyloid deposit isolated from the heart tissue of a patient with systemic amyloidosis is said to be a TTR, and fragments thereof collectively referred to as ATTR, whose full-length sequence is characterized. It was shown to be composed of a heterogeneous mixture. The fibrillar component of ATTR can be extracted from such plaques and their structure and sequence can be determined according to methods known in the art (eg, Gustavsson, A. et al., Laboratory Invest. 73: 703). -708, 1995; Kametani, F. et al., Biochem. Biophys. Res. Commun. 125: 622-628, 1984; Pras, M. et al., PNAS 80: 539-42, 1983).

  A person with a point mutation in the molecular apolipoprotein AI (eg Gly → Arg26; Trp → Arg50; Leu → Arg60) has some form characterized by deposits of the protein apolipoprotein AI or a fragment thereof (A apoAI) Represents amyloidosis (ester tag type). These patients have low levels of high density lipoprotein (HDL) and appear with peripheral neuropathy or renal failure.

  Mutations in the α chain of the enzyme lysozyme (eg, Ile → Thr56 or Asp → His57) are the basis for another form of non-neuropathic hereditary amyloid that has been reported in British families. It is. Here, mutant lysozyme protein fibrils (Alys) are deposited, and patients generally exhibit impaired renal function. This protein differs from most of the fibril-forming proteins described herein and usually exists in the entire (unfragmented) form (Benson, MD et al. CIBA Fdn. Symp. 199: 104 -131, 1996).

  β-amyloid peptide (Aβ) is a 39-43 amino acid peptide that can be generated by proteolysis from a large protein known as β-amyloid precursor protein (βAPP). Mutations in βAPP result in familial forms of Alzheimer's disease, Down's syndrome and / or senile dementia characterized by brain deposits of plaques composed of Aβ fibrils and other components (more details below) ). Known mutations in APP associated with Alzheimer's disease are present in the immediate vicinity of or within the cleavage site of β or γ secretase. For example, position 717 is close to the site of APP gamma-secretase cleavage in its processing to Aβ, and position 670/671 is close to the site of β-secretase cleavage. Mutations at any of these residues can lead to Alzheimer's disease, possibly by causing an increase in the amount of Aβ in the form of 42/43 amino acids generated from APP. The structure and sequence of Aβ peptides of various lengths are known in the art. Such peptides can be made according to methods known in the art (eg, Glenner and Wong, Biochem Biophys. Res. Comm. 129: 885-890, 1984; Glenner and Wong, Biochem Biophys. Res. Comm. 122). : 1131-1135, 1984). In addition, various forms of peptides are commercially available.

  Synuclein is a synapse-related protein that resembles alipoprotein and is abundant in the cytoplasm and presynaptic terminals of neurons. A peptide fragment derived from α-synuclein, named NAC, is also a component of Alzheimer's disease amyloid plaques. (Clayton et al., 1998). This component also acts as a target for the immunologically based therapy of the present invention, as detailed below.

  Gelsolin is a calcium binding protein that binds to and fragment actin filaments. A mutation at position 187 of the protein (eg Asp → Asn; Asp → Tyr) results in some form of hereditary systemic amyloidosis commonly found in Finnish patients as well as those of Dutch or Japanese origin. In afflicted individuals, the fibrils formed from gelsolin fragments (Agel) usually consist of amino acids 173-243 (68 kDa carboxy-terminal fragment) and are deposited in blood vessels and basement membranes, corneal degeneration, and It results in cranial neuropathy that progresses to peripheral neuropathy, dystrophic skin changes, and deposition in other organs. (Kangas, H. et al. Human Mol. Genet. 5 (9): 1237-1243, 1996).

  Other mutated proteins such as the alpha chain of the fibrinogen mutant (AfibA) and the mutant cystatin C (Acys) also form fibrils and give rise to characteristic genetic disorders. AfibA fibrils form deposits characteristic of non-neuropathic hereditary amyloid with renal disease; Acys deposits are characteristic of hereditary cerebral amyloid angiopathy reported in Iceland. (Isselbacher et al., Harrison's Principles of Internal Medicine, McGraw-Hill, San Francisco, 1995; Benson et al., Supra). In at least some cases, patients with cerebral amyloid angiopathy (CAA) have been shown to have amyloid fibrils containing the non-mutant form of cystatin C along with the β protein. (Nagai, A. et al., Molec. Chem. Neuropathol. 33: 63-78, 1998).

Certain forms of prion disease are now considered hereditary, responsible for up to 15% of cases, and were previously considered primarily infectious in nature. (Baldwin et al., Research Advances in Alzheimer's Disease and Related Disorders, John Wiley and Sons, New York, 1995). In such prion disorders, patients develop plaques composed of an abnormal isoform (PrP ^c ) of normal prion protein. Isoform PrP ^Sc of also referred predominant mutant and AScr, the resistance to protease degradation, insolubility after detergent extraction, deposition in secondary lysosomes, post-translational synthesis, and in a high β-pleated sheet content Different from normal cellular proteins. Genetic associations have been established for at least five mutations that result in Creutzfeldt-Jakob disease (CJD), Gerstmann-Stroisler-Scheinker syndrome (GSS) and lethal familial insomnia (FFI). Extraction of fibril peptides from (Baldwin) scrapie fibrils, sequencing, and methods of making such peptides are known in the art. (E.g., Beekes, M. et al., J. Gen. Virol. 76: 2567-76, 1995).

  For example, one form of GSS is associated with a PrP mutation at codon 102, while the telencephalon GSS segregates with a mutation at codon 117. Mutations at codons 198 and 217 result in a form of GSS where the neuritic plaques characteristic of Alzheimer's disease contain PrP instead of Aβ peptide. Certain forms of familial CJD involve mutations at codons 200 and 210; mutations at codons 129 and 178 have been found in both familial CJD and FFI. (Baldwin, supra).

d. Senile systemic amyloidosis Systemic or focal amyloid deposition increases with age. For example, wild type transthyretin (TTR) fibrils are universally found in the heart tissue of elderly individuals. They can be asymptomatic and clinically silent or can lead to heart failure. Asymptomatic fibril focal deposits can also be present in the brain (Aβ), prostate amyloid bodies (Aβ ₂ microglobulin), joints and seminal vesicles.

e. Brain Amyloidosis Local deposition of amyloid is universal in the brain, especially in older individuals. The most frequent type of amyloid in the brain is composed primarily of Aβ peptide fibrils, resulting in dementia or sporadic (non-hereditary) Alzheimer's disease. In fact, the incidence of sporadic Alzheimer's disease far exceeds the forms that have been shown to be hereditary. The fibril peptides that form these plaques are very similar to those described above with respect to the inherited form of Alzheimer's disease (AD).

f. Dialysis-related amyloidosis β ₂ microglobulin (Aβ ₂ M) fibrils commonly occur in patients who receive long-term hemodialysis or peritoneal dialysis. β ₂ microglobulin is a 11.8 kilodalton polypeptide and is the light chain of class I MHC antigens present on all nucleated cells. Under normal circumstances, it is continuously shed from the cell membrane and is usually filtered by the kidneys. Failure to disappear, as in the case of impaired renal function, leads to deposition in the kidney and other sites (mainly collagen-rich tissues of the joints). Aβ ₂ M molecules, unlike other fibrillar proteins, generally exist in a form that is not fragmented in fibrils. (Benson, supra).

g. Hormone-derived amyloidosis Endocrine organs can have amyloid deposits, especially in aging individuals. Hormone-secreting tumors can also contain hormone-derived amyloid plaques, whose fibrils are calcitonin (thyroid medullary carcinoma), islet amyloid polypeptide (amylin; most patients with type II diabetes) And polypeptide hormones such as atrial natriuretic peptide (isolated atrial amyloidosis). The sequences and structures of these proteins are known in the art.

h. Miscellaneous Amyloidosis There are a variety of other forms of amyloid disease that are usually evident as localized deposits of amyloid. In general, these diseases are probably the result of the limited production of specific fibril precursors and / or the lack of catabolism or the predisposition of specific tissues (such as joints) to fibril deposition. Examples of such idiopathic deposition include nodular AL amyloid, cutaneous amyloid, endocrine amyloid and tumor-related amyloid.

C. Pharmaceutical Compositions The discovery of the present invention that compositions capable of eliciting or providing an immune response directed against a component of amyloid plaques are effective in treating or preventing the development of amyloid diseases It is. In particular, the invention provided herein prevents the progression of amyloid plaque burden in an afflicted individual when an immunostimulating dose of an anti-amyloid agonist or a corresponding anti-amyloid immunoreagent is administered to a patient. It is possible to alleviate the symptoms and / or reduce it. This section describes exemplary anti-amyloid agents that produce active as well as passive immunity against amyloid plaques, and provides exemplary data showing the effect treatment using such compositions on amyloid plaque burden.

In general, the anti-amyloid agents of the present invention form specific plaque components, preferably plaques, which are usually characteristic proteins, peptides or fragments thereof, as described in the previous section and exemplified below. It is comprised from the component to do. More generally, a therapeutic agent for use in the present invention produces or induces an immune response against plaques, or more specifically its fibril components. Thus, such agents are specifically, but not limited to, the component itself and variants thereof, analogs and mimetics of components that induce and / or cross-react with antibodies to the component, and amyloid components. Mention may be made of antibodies or T cells that are reactive. According to one important feature, the pharmaceutical composition is not selected from non-specific ingredients, ie ingredients that are generally circulating or ubiquitous in the body. As an example, serum amyloid protein (SAP) is a circulating plasma glycoprotein that is produced in the liver and binds to most known forms of amyloid deposits. The therapeutic composition is preferably directed to this component.

  Induction of an immune response involves administration of an antibody that binds to the amyloid component in the patient, either actively, as in the case where an immunogen is administered to induce antibodies or T cells reactive with the component in the patient. Can be passive as if Exemplary agents for inducing or generating an immune response against amyloid plaques are described in the section below.

  The pharmaceutical composition of the present invention can include an effective amount of adjuvants and / or excipients in addition to the immunogenic agent (s). Pharmaceutically effective and useful adjuvants and excipients are known in the art and are described in more detail in subsequent sections.

1. Immune stimulant (active immune response)
a. Antigen Fiber Compositions One general class of preferred anti-amyloid agents consists of agents that are derived from amyloid fibril proteins. As mentioned above, a prominent feature of amyloid disease is the deposition in one organ or organ of amyloid plaques composed primarily of fibrils, which in turn are composed of characteristic fibril proteins or peptides. According to the present invention, such fibril protein or peptide components are useful agents for inducing an anti-amyloid immune response.

Antibody titer in transgenic mice after injection of Aβ1-42. Amyloid load in the hippocampus. The percentage of the area of the hippocampal area occupied by amyloid plaques, defined by reactivity with Aβ-specific monoclonal antibody 3D6, was determined by computer-assisted quantitative image analysis of immunoreacted brain sections. Individual mouse values are shown grouped by treatment group. The horizontal line for each grouping shows the median of the distribution. Neuritic dystrophy in the hippocampus. The percentage of the area of the hippocampal area occupied by dystrophic axons, defined by their reactivity with human APP-specific monoclonal 8E5, was determined by quantitative computer-assisted image analysis of immunoreacted brain sections. . Individual mouse values are shown for AN1792-treated group and PBS-treated control group. The horizontal line for each grouping shows the median of the distribution. Astrocytes in the posterior plate cortex. The percentage of cortical area occupied by glial fibrillary acidic protein (GFAP) positive astrocytes was determined by quantitative computer-assisted image analysis of immunoreacted brain sections. Individual mouse values are shown grouped by treatment group, and median group values are indicated by a horizontal line. Antibody titer against Aβ42 after immunization in the range of 8 doses of Aβ42 (“AN1792”) containing 0.14, 0.4, 1.2, 3.7, 11, 33, 100 or 300 μg Geometric mean. Kinetics of antibody response to AN1792 immunization. The titer is expressed as the geometric mean of the values of 6 animals in each group. Quantitative image analysis of cortical amyloid burden in mice treated with PBS and AN1792. Quantitative image analysis of neuritic plaque burden in mice treated with PBS and AN1792. Quantitative image analysis of the percentage of posterior lamellar cortex occupied by astrocytes in mice treated with PBS and AN1792. Lymphocyte proliferation assay on splenocytes from AN1792-treated (top) or PBS-treated (bottom) mice. Total Aβ levels in the cerebral cortex. Scatter plot of individual Aβ profiles in mice immunized with Aβ or APP derivatives combined with Freund's adjuvant. Mice immunized with Aβ peptide complexes Aβ1-5, Aβ1-12 and Aβ13-28; full-length Aβ aggregates Aβ42 (“AN1792”) and Aβ1-40 (“AN1528”), and PBS treated Amyloid burden in the cortex as determined by quantitative image analysis of immunoreacted brain sections of the control group. Geometric mean of Aβ-specific antibody titers in groups of mice immunized with Aβ or APP derivatives combined with Freund's adjuvant. Geometric mean of Aβ-specific antibody titers of a group of guinea pigs immunized with AN1792 or its palmitoylated derivatives in combination with various adjuvants. Aβ levels in the cortex of 12 month old PDAPP mice treated with AN1792 or AN1528 with various adjuvants. Mean titer of mice treated with polyclonal antibodies against Aβ. Mean titer of mice treated with monoclonal antibody 10D5 against Aβ. Mean titer of mice treated with monoclonal antibody 2F12 against Aβ.

Embodiment of the present invention
Tables 1 and 2 summarize exemplary fibril forming proteins that are characteristic of various amyloid diseases. In accordance with this aspect of the invention, administration of an immunostimulatory composition comprising a suitable fibril protein or peptide, including homologues or fragments thereof, to a suffering or susceptible individual, with respect to amyloid disease, Provide therapeutic or prophylactic drugs.

  By way of example, also known as β-amyloid peptide or A4 peptide (see US Pat. No. 4,666,829; see Glenner and Wong, Biochem. Biophys. Res. Commun. 120, 1131 (1984)). Aβ is a 39-43 amino acid peptide that is the main component of plaques characteristic of Alzheimer's disease. Aβ is produced by processing of the larger protein APP by two enzymes termed β and γ-secreting enzymes (see Hardy, TINS 20, 154 (1997)).

  Example I describes the results of experiments performed in support of the present invention, where Aβ42 peptide was administered to heterozygous transgenic mice overexpressing human APP with a mutation at position 717. . These mice, known as “PDAPP mice”, exhibit Alzheimer-like pathology and are considered animal models of Alzheimer's disease (Games et al., Nature 373: 523-7, 1995). As detailed in the examples, these mice represent a detectable Aβ plaque neuropathology in their brain starting at about 6 months of age, with plaque deposition progressing over time. In the experiments described herein, aggregated Aβ42 (AN1792) was administered to mice. The majority of treated mice (7/9) contrasted with control mice (injected with saline or untreated), all of which showed significant brain amyloid burden at 13 months of age. In addition, they had no detectable amyloid in their brain at this age (FIG. 2). These differences were even more pronounced in the hippocampus (Figure 3). Treated mice also exhibited significant serum antibody titers against Aβ (all greater than 1: 1000, 8/9 greater than 1 / 10,000; FIG. 1, Table 3A). In general, mice treated with saline exhibited less than 4-5 fold background levels of antibodies to Aβ at 100-fold dilution at all time points tested and were therefore significant with respect to controls There appeared to be no response (Table 3B). These studies have demonstrated that infusion of peptide Aβ forming certain fibrils provides protection against deposition of Aβ amyloid plaques.

  Serum amyloid protein (SAP) is a circulating plasma glycoprotein that is produced in the liver and binds in a calcium-dependent manner to all forms of amyloid fibrils, including fibrils of cerebral amyloid plaques in Alzheimer's disease. is there. As part of the previous experiment, a group of mice were injected with SAP; these mice developed significant serum titers (1: 1000-1: 300000) for SAP, but detectable for Aβ peptide Serum titers were not generated and neuropathology of brain plaques was generated (Figure 2).

  Further experiments detailed in Example II demonstrate the dose dependence of the immunogenic effect of Aβ injection in mice treated between 5 weeks of age and about 8 months of age. In these mice, the average serum titer of anti-Aβ peptide antibody increased with the number of immunizations and increasing dosage; however, measured after 4 immunizations and 5 days after immunization. Serum titers leveled off at a level of about 1: 10000 over higher doses (1-300 μg) (FIG. 5).

Additional experiments in support of the present invention are described in Example III, where PDAPP model mice are treated with Aβ42 starting at a time point (approximately 11 months of age) after amyloid plaques are already present in their brain. did. In these studies, animals were immunized with Aβ42 or saline and sacrificed for amyloid burden testing at 15 or 18 months of age. As specifically illustrated in FIG. 7, 18 month old mice treated with Aβ42 were either 18 month old controls (spot burden, 4.7%) or 12 month untreated animals treated with PBS. (0.28%) represents significantly lower average amyloid plaque load (Plate load, 0.01%), where plaque load is detailed in Example XIII, Part 8 Measure by image analysis as described. These experiments demonstrate the efficacy of the treatment methods of the present invention in reducing existing plaque burden and preventing progression of plaque burden in sick individuals.

  According to this aspect of the invention, the therapeutic agent is derived from a fibril peptide or protein comprising plaques characteristic of the disease of interest. Alternatively, such agents are antigenically similar to such components sufficiently to induce an immune response that also cross-reacts with fibril components. Tables 1 and 2 provide examples of such fibril peptides and proteins, the composition and sequence of which are known in the art or can be readily determined according to methods known in the art. (See references below and references cited in section B2 for references that specifically teach methods and / or compositions for the extraction of various fibril peptide components; further exemplary fibril components below. To state.)

  Thus, in accordance with the present invention, when a diagnosis of amyloid disease is made based on clinical and / or biopsy decisions, a skilled practitioner can ascertain the fibril composition of the amyloid deposit and fibril peptide or It may be possible to provide an agent that induces an immune response directed against the protein.

  By way of example, as described above, therapeutic agents used in the treatment of Alzheimer's disease, or other amyloid diseases characterized by Aβ fibril deposition, include naturally occurring forms of Aβ peptides, and especially human forms ( That is, it can be any one of Aβ39, Aβ40, Aβ41, Aβ42, or Aβ43). The sequences of these peptides and their relationship to the APP precursor are known in the art and are known in the art (eg Hardy et al., TINS 20, 155-158 (1997)). For example, Aβ42 has the sequence:

Have

  Aβ41, Aβ40 and Aβ39 differ from Aβ42 by omitting Ala, Ala-Ile and Ala-Ile-Val from the C-terminus of the peptide, respectively. Aβ43 differs from Aβ42 by the presence of one C-terminal threonine residue. According to one preferred aspect of the invention, the therapeutic agent can induce an immune response against all or a portion of the fibril component of the disease of interest. For example, preferred Aβ immunogenic compositions are agents that induce antibodies specific for the free N-terminus of Aβ. Such a composition has the advantage that it does not recognize the precursor protein β-APP, thereby possibly making it less likely to cause autoimmunity.

As a further example, patients suffering from diseases characterized by the deposition of AA fibrils, such as certain chronic inflammatory disorders, chronic local or systemic microbial infections and malignant neoplasms as described above, It is worthwhile to be able to be treated with a known 8 kilodalton fragment of serum amyloid A protein (Apo SSA), an AA peptide. Exemplary AA amyloid disorders include but are not limited to inflammation such as rheumatoid arthritis, juvenile arthritis, ankylosing spondylitis, psoriasis, psoriatic arthropathy, Reiter's syndrome, adult Still's disease, Behcet's syndrome, Crohn's disease Sexually transmitted diseases, leprosy, tuberculosis, bronchiectasis, pressure ulcers, chronic pyelonephritis, chronic microbial infections such as osteomyelitis and Whipple's disease, and Hodgkin lymphoma, renal cancer, intestinal, lung and genitourinary tract cancers, Mention may be made of basal cell carcinoma as well as malignant neoplasms such as ciliary cell leukemia.

  The AA peptide is derived from the N-terminus of precursor protein serum amyloid A (apo SSA), starting at residues 1, 2 or 3 of the precursor protein and ending at any point between residues 58 and 84. Refers to one or more of a heterogeneous group of peptides; universally, AA fibrils are composed of residues 1-76 of apo SSA. The exact structure and composition can be determined, and appropriate peptides can be synthesized according to methods known in the art (Liepnieks, JJ et al. Biochem. Biophys Acta 1270: 81-86, 1995) .

As a further example, fragments derived from the N-terminal region containing all or part of the variable (V _L ) domain of an immunoglobulin light chain (κ chain or λ chain) generally comprise amyloid deposits in mesenchymal tissue. It causes peripheral and autonomic neuropathy, carpal tunnel syndrome, macroglossia, restrictive cardiomyopathy, arthropathy of large joints, immune disease, myeloma, and subclinical disease. The compositions of the invention preferably induce an immune response against a portion of the light chain, preferably a “new epitope” (an epitope formed as a result of fragmentation of the parent molecule) to reduce possible autoimmune effects. be able to.

  A variety of hereditary amyloid diseases also respond sensitively to the treatment methods of the present invention. Such diseases are described in B. above. Described in Section 2. For example, various forms of familial amyloid polyneuropathy are associated with at least 50 mutants of transthyretin (TTR), a 14 kilodalton protein produced by the liver, each characterized by a single amino acid change. It is the result of morphology. Many of these forms of this disease are distinguishable based on their specific pathology and / or demographic origin, while therapeutic compositions generally provide useful therapeutic compositions It is also worth mentioning that it can also consist of agents that induce an immune response against one or more forms of TTR, such as a mixture of two or more forms of ATTR, including wild type TTR. .

  Amyloid deposits containing apo AI are found in people with point mutations in the molecular apolipoprotein AI. Patients with this form of disease generally present with peripheral neuropathy or renal failure. In accordance with the present invention, the therapeutic composition comprises one or more of the various forms of A ApoAI described herein or known in the art.

  Certain familial forms of Alzheimer's disease as well as Down's syndrome are the result of mutations in the β-amyloid precursor protein, resulting in the deposition of plaques with fibrils composed primarily of β-amyloid peptide (Aβ) . The use of Aβ peptides in the therapeutic compositions of the present invention is described above and exemplified herein.

Other formulations for treating hereditary forms of amyloidosis as discussed above are gelsolin fragments for the treatment of hereditary systemic amyloidosis, mutant lysozyme for the treatment of hereditary neuropathy Protein (Alys), alpha chain of mutant fibrinogen (AfibA) for non-neuropathic forms of amyloidosis manifested as kidney disease, for the treatment of certain forms of hereditary cerebrovascular disorders reported in Iceland And a composition that generates an immune response against cystatin C (Acys). In addition, certain inherited forms of prion diseases such as Creutzfeldt-Jakob disease (CJD), Gerstmann-Stroisler-Scheinker syndrome (GSS) and lethal familial insomnia (FFI) It is characterized by a protein mutant isoform, PrP ^Sc . This protein can be used in accordance with the present invention in therapeutic compositions for the treatment and prevention of PrP plaque deposition.

As discussed above, either systemic or focal amyloid deposition is also associated with aging. It is a further aspect of the present invention that such deposition can be prevented or treated by administering a composition comprising one or more proteins associated with such aging to susceptible individuals. . Therefore, plaques composed of wild-type TTR-derived ATTR are frequently found in the heart tissue of elderly people. Similarly, some elderly individuals can develop asymptomatic fibrillar focal deposits of Aβ in their brain; Aβ peptide treatment as detailed herein can be applied to these individuals. Can be justified. β ₂ microglobulin is a frequent component of the amyloid body of the prostate and is therefore a further candidate agent according to the present invention.

As a further example, but not as a limitation, there are a number of additional non-inherited forms of amyloid disease that are candidates for the treatment methods of the present invention. β ₂ microglobulin fibrillar plaques universally develop in patients who receive long-term hemodialysis or peritoneal dialysis. Such patients can be treated according to the present invention by treatment with a therapeutic composition directed to β2 microglobulin, or more preferably an immunogenic epitope thereof.

  Tumors that secrete hormones can also contain hormone-derived amyloid plaques, the composition of which is typically characteristic of the particular endocrine organ affected. Thus, these fibrils are found in polypeptides such as calcitonin (thyroid medullary carcinoma), islet amyloid polypeptide (present in most patients with type II diabetes) and atrial natriuretic peptide (isolated atrial amyloidosis). It can be composed of peptide hormones. Compositions directed to amyloid deposits that occur in the aortic intima in atherosclerosis are also contemplated by the present invention. For example, Westermark et al. Describe a 69 amino acid N-terminal fragment of apolipoprotein A that forms these plaques (Westermark et al., Am. J. Path. 147: 1186-92; 1995); therapeutic compositions of the invention Includes immunological reagents directed to such fragments, as well as the fragments themselves.

The foregoing discussion has focused on amyloid fibril components that can be used as therapeutic agents in the treatment or prevention of various forms of amyloid disease. The therapeutic agent is also an active fragment or analog of a naturally occurring or mutant fibril peptide or protein containing an epitope that induces a similar protective or therapeutic immune response upon administration to humans. You can also. Immunogenic fragments typically have a sequence of at least 3, 5, 6, 10 or 20 contiguous amino acids from the natural peptide. Exemplary immunogenic fragments of Aβ peptides are Aβ1-5, 1-6, 1-7, 1-10, 3-7, 1-3, 1-4, 1-12, 13-28, 17-. 28, 1-28, 25-35, 35-40 and 35-42. Fragments lacking at least one, and sometimes at least 5 or 10 C-terminal amino acids present in naturally occurring fibril components are used in several ways. For example, a fragment lacking 5 amino acids from the C-terminus of Aβ43 includes the first 38 amino acids from the N-terminus of AB. Fragments from the N-terminal half of Aβ are preferred in several ways. Analogs include allelic variants, species variants and derived variants. Analogs typically differ from naturally occurring peptides at one or several positions, often due to conservative substitutions. Analogs typically represent a minimum of 80 or 90% sequence identity with the native peptide. Some analogs also include unnatural amino acids or N- or C-terminal amino acid modifications. Examples of non-natural amino acids are α, α-disubstituted amino acids, N-alkyl amino acids, lactic acid, 4-hydroxyproline, γ-carboxyglutamic acid, γ-N, N, N-trimethyllysine, γ-N-acetyllysine O-phosphoserine, N-acetylserine, N-formylmethionine, 3
-Methylhistidine, 5-hydroxylysine, omega-N-methylarginine.

  In general, one of skill in the art will recognize fragments and analogs designed according to this aspect of the invention in a transgenic animal model as described below, and / or prevention of cross-reactivity with naturally occurring fibril components. Or the real value of being able to be screened for therapeutic efficacy. Such fragments or analogs are present in the therapeutic compositions of the present invention when their immunoreactivity and the efficacy of the animal model is approximately equal to or greater than the corresponding parameter measured for the amyloid fibril component. Can be used.

  Such peptides, proteins or fragments, analogs and other amyloidogenic peptides can be synthesized by solid phase peptide synthesis or recombinant expression according to standard methods known in the art, or natural sources Can be obtained from Exemplary fibril compositions, methods of fibril extraction, sequences of fibril peptide or protein components are provided by many of the references cited along with descriptions of specific fibril components provided herein. . In addition, other compositions, methods of sequence extraction and determination are available to those who desire to make and use such compositions known in the art. Automated peptide synthesizers can be used to make such compositions and are commercially available from a number of manufacturers such as Applied Biosystems (Perkin Elmer; Foster City, Calif.). And procedures for producing synthetic peptides are known in the art. The recombinant expression can be in bacteria such as E. coli, yeast, insect cells or mammalian cells; or the protein is an in vitro translation system that does not include cells known in the art. Can be manufactured using. The procedure for recombinant expression is described by Sambrook et al., Molecular Cloning: A Laboratory Manual (CSH HP Press, New York State, 2nd Edition, 1989). Is done. Certain peptides and proteins are also commercially available; for example, some forms of Aβ peptides are available from American Peptides Company, Inc., Sunnyvale, Calif., And California Peptide Research. Ink (California Peptide Research, Inc.), available from sources such as Napa, California.

  The therapeutic agent can also be comprised of a longer polypeptide, including, for example, active peptide fibril fragments or analogs along with other amino acids. For example, the Aβ peptide can exist as an intact APP protein, or a segment thereof such as a C-100 fragment that begins at the N-terminus of Aβ and continues to the end of APP. Such polypeptides can be screened for prophylactic or therapeutic efficacy in animal models as described below. Aβ peptides, analogs, active fragments or other polypeptides can be administered in an associated form (ie, as an amyloid peptide) or in a dissociated form. Therapeutic agents can also include monomeric immunogenic agent multimers or complexes, or carrier proteins, and / or broader anti-amyloid plaques, as listed above. It can be added to other fibril components to provide activity.

In a further variation, immunogenic peptides such as fragments of Aβ can be presented by viruses or bacteria as part of an immunogenic composition. Nucleic acid encoding an immunogenic peptide is integrated into the viral or bacterial genome or episome. In some cases, the nucleic acid is expressed in a manner such that the immunogenic peptide is expressed as a secreted protein, or a fusion protein with a viral outer surface protein or bacterial transmembrane protein such that the peptide is displayed. Incorporated. Viruses or bacteria used in such methods should be non-pathogenic or attenuated. Suitable viruses include adenovirus, HSV, Venezuelan equine encephalitis virus and other alphaviruses, vesicular stomatitis virus, and other rhabdoviruses, vaccinia and fowlpox. Suitable bacteria include Salmonella and Shigella. Fusion of immunogenic peptides with HBV to HBsAg is particularly suitable. Therapeutic agents also include peptides and other compounds that do not necessarily have significant amino acid sequence similarity to Aβ, but nevertheless act as mimics of Aβ and induce similar immune responses. For example, any peptides and proteins that form β-pleated sheets can be screened for suitability. Anti-idiotypic antibodies against monoclonal antibodies against Aβ or other amyloidogenic peptides can also be used. Such anti-Id antibodies mimic antigens and generate immune responses thereto (see Essential Immunology (Roit, Blackwell Scientific Publications, Palo Alto, 6th edition), p. 181. Wanna) Agents other than Aβ peptides induce an immunogenic response to one or more of the preferred segments of Aβ listed above (eg, 1-10, 1-7, 1-3 and 3-7) Should do. Preferably, such agents induce an immunogenic response that is specifically directed to one of these segments without being directed to other segments of Aβ.

  Random libraries of peptides or other compounds can also be screened for suitability. For many types of compounds that can be synthesized in a step-wise manner, a combinatorial library can be generated. Such compounds include polypeptides, beta-turn mimetics, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, heterocyclic compounds, benzodiazepines, oligomeric N-substituted glycines and oligocarbamates. A large combinatorial library of compounds is available from Affymax, International Patent Application No. WO 95/12608, Affymax, International Patent Application No. WO 93/06121, Columbia University, International Patent application WO 94/08051, Pharmacopeia, international patent application WO 95/35503, and Scripps, international patent application WO 95/30642, each of which Can be constructed by the encoded synthetic libraries (ESL) method described in (incorporated by reference for all purposes). Peptide libraries can also be generated by phage display methods. See, for example, Devlin, International Patent Application No. WO 91/18980.

Combinatorial libraries and other compounds initially have their ability to bind to antibodies or lymphocytes (B or T) known to be specific for Aβ or other amyloidogenic peptides such as ATTR. Screen for suitability by measuring. For example, an initial screen can be performed using any polyclonal serum or monoclonal antibody against Aβ, or any other amyloidogenic peptide of interest. The compounds identified by such screening are then further analyzed for the ability to induce antibodies or reactive lymphocytes against Aβ or other amyloidogenic peptides. For example, multiple dilutions of serum can be tested on microtiter plates pre-coated with fibril peptide and a standard ELISA can be performed to test for reactive antibodies to Aβ. . The compounds can then be tested for prophylactic and therapeutic efficacy in transgenic animals predisposed to certain amyloidogenic diseases, as described in the Examples. Such animals include, for example, mice carrying the APP 717 mutation described above, as well as McConlogue et al., US Pat. No. 5,612,486 and Hsiao et al., Science 274, 99 (1996). Mice carrying the Swedish mutation of APP 670/671 as described by Staufenbiel et al., Proc. Natl. Acad. Sci. USA 94, 13287-13292 (1997); Churchler-Pierrat et al., Proc. Natl. Acad. Sci. USA 94, 13287-13292 (1997); Borchelt et al., Neuron 19, 939-945 (1997)). The same screening approach can be used with other potential agents such as the fragments of Aβ, analogs of Aβ and longer peptides including Aβ described above.

b. Other plaque components It is found worthwhile that immunological responses directed against other amyloid plaque components can also be effective in preventing, interfering with or reducing plaque deposition in amyloid diseases. These components can be low fibrillar components or can be accompanied by fibrils or fibril formation in plaques, but compared to ubiquitous or amyloid deposits in the body Components that are non-specific are generally less suitable for use as therapeutic targets.

  Accordingly, it is a further discovery of the present invention that agents that induce an immune response against specific plaque components are useful in treating amyloid disease or preventing its progression. This section provides background for several exemplary amyloid plaque related molecules. Immune response to any of these molecules, alone or in combination with an immunogenic therapeutic composition against any of the fibril components described above or other non-fibril forming components described below. Induction of this provides an additional anti-amyloid treatment regimen in accordance with the present invention. Passive immunization regimens based on such plaque components as described herein also form part of the present invention.

  By way of example, synuclein is a protein that is structurally similar to apolipoprotein but is found, inter alia, in the cytoplasm of neurons near the presynaptic terminal. There are at least three forms of the protein termed α, β and γ synuclein. Recently, α and β synuclein have been shown to be involved in nucleation of amyloid deposits in certain amyloid diseases, particularly Alzheimer's disease. (Clayton, DF et al., TINS 21 (6): 249-255, 1998). More specifically, a fragment of the NAC domain of α and β synucleins (residues 61-95) has been isolated from amyloid plaques of Alzheimer's disease patients; in fact, this fragment is sodium dodecyl sulfate (SDS). About 10% of plaques that remain insoluble after solubilization of (George, JM et al., Neurosci. News 1: 12-17, 1995). Furthermore, both full-length α-synuclein and its NAC fragment have been reported to accelerate the aggregation of β-amyloid peptide to insoluble amyloid in vitro. (Clayton, supra).

Additional components associated with amyloid plaques include non-peptide components. For example, perlecan and perorcan-derived glycosaminoglycans are large heparin sulfates present in amyloid plaques containing Alzheimer's disease and other CNS including amylin plaques associated with diabetes and systemic amyloidosis Aβ. It is a proteoglycan. These compounds have been shown to enhance Aβ fibril formation. Both perlecan core protein and glycosaminoglycan chains have been shown to participate in binding to Aβ. Additional glycosaminoglycans, notably dermatan sulfate, chondroitin-4-sulfate and pentosan polysulfate, are commonly found in various types of amyloid plaques and have also been shown to enhance fibril formation. ing. Dextran sulfate also has this property. This enhancement is significantly reduced when the molecule is desulfated. Immunogenic therapeutics directed against sulfated forms of glycosaminoglycans, including the specific glycosaminoglycans themselves, can be used as additional treatments of the invention as either primary or secondary treatments. Form an aspect. The manufacture of such molecules as well as suitable therapeutic compositions containing such molecules is within the skill of the artisan.

2. Agents that Induce a Passive Immune Response The therapeutic agents of the present invention also include immunoreagents such as antibodies that specifically bind to fibril peptides or other components of amyloid plaques. Such antibodies can be monoclonal or polyclonal and have a binding specificity consistent with the type of amyloid disease to be targeted. Therapeutic compositions and treatment regimens can include antibodies directed against a single binding domain or epitope on a particular fibrillar or non-fibrillar component of the plaque, or two on the same component Alternatively, antibodies directed against epitopes above or beyond, or antibodies directed against epitopes on multiple components of plaques can be included.

  For example, in experiments performed in support of the present invention, as detailed in Example XI herein, PDAPP mice aged 8 1/2 to 10 1/2 months were identified with polyclonal anti-Aβ42 or Aβ peptide. Monoclonal anti-Aβ antibodies prepared against these epitopes, or intraperitoneal (ip) injection of saline. In these experiments, boosting antibody concentrations are monitored as required, and boosting of antibody boosts is required to maintain circulating antibody concentrations greater than 1: 1000 for the specific antigen for which the antibody was made. Gave. Compared to controls, a decrease in total Aβ levels was observed in the cortex, hippocampus and cerebellum regions of mice treated with antibodies; the highest reduction was represented in mice treated with polyclonal antibodies in these studies .

  In further experiments conducted in support of the present invention, a predictive ex vivo assay (Example XIV) was used to test the disappearance of antibodies to a fragment of synuclein termed NAC. Synuclein has been shown to be an amyloid plaque associated protein. Antibodies against NAC were contacted with brain tissue samples containing amyloid plaques and microglia. Rabbit serum was used as a control. Subsequent monitoring showed a marked decrease in the number and size of plaques implying that antibody activity disappeared.

From these data, immunoreagents that are directed against Aβ peptide epitopes or NAC fragments of synuclein are effective in reducing the burden of amyloid plaques, which are associated with Alzheimer's disease and other amyloid diseases. It is clear that this can be greatly reduced by administration of. It is further understood that a wide variety of antibodies can be used in such compositions. Antibodies that specifically bind to the aggregated form of Aβ without binding to the dissociated form are as antibodies that specifically bind to the dissociated form without binding to the aggregated form Suitable for use in the present invention. Other suitable antibodies bind to both aggregated and dissociated forms. Some such antibodies bind to the naturally occurring short form of Aβ (ie Aβ39, 40 or 41) without binding to the naturally occurring long form of Aβ (ie Aβ42 and Aβ43). Some antibodies bind to the long form without binding to the short form. Some antibodies bind to Aβ without binding to full-length amyloid precursor protein. Some antibodies bind Aβ with a binding affinity greater than or equal to about 10 ⁶ , 10 ⁷ , 10 ⁸ , 109 or 10 ¹⁰ M ⁻¹ .

Polyclonal sera typically contain a mixed population of antibodies that bind several epitopes along the length of Aβ. Monoclonal antibodies bind to specific epitopes within Aβ that can be conformational or non-conformational epitopes. Some monoclonal antibodies bind to an epitope within residues 1-28 of Aβ (the first N-terminal residue of natural Aβ is referred to as 1). Other monoclonal antibodies bind to an epitope with residues 1-10 of Aβ. There are also monoclonal antibodies that bind to an epitope having residues 1-16 of Aβ. Other monoclonal antibodies bind to an epitope with residues 1-25 of Aβ. Some monoclonal antibodies bind to an epitope within amino acids 1-5, 5-10, 10-15, 15-20, 25-30, 10-20, 20, 30, or 10-25 of Aβ. The prophylactic and therapeutic efficacy of antibodies can be tested using the transgenic animal model procedures described in the Examples.

  More generally, from the teachings provided herein, practitioners may use the compositions described herein to make other, such as diseases described in Section 2 of this specification. Antibodies directed against fibrillar proteins or peptides characteristic of amyloid disease, as well as antibodies against other amyloid components can be designed, manufactured and tested.

a. General features of immunoglobulins The basic antibody structural unit is known to comprise a tetramer of subunits. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” chain (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region that is primarily responsible for effector function.

  Light chains are classified as either kappa or lambda chains. Heavy chains are classified as γ, μ, α, δ, or ε chains, and define the antibody's isotype as IgG, IgM, IgA, IgD, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are joined by a “J” region of about 12 or more amino acids, and the heavy chain also includes a “D” region of more than about 10 amino acids. (See, generally, Fundamental Immunology (Paul, W. Ed., 2nd edition, Raven Press, New York, 1989), Chapter 7 (incorporated by reference for all purposes)).

  The variable regions of each light / heavy chain pair form the antibody binding site. Thus, an intact antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are identical. The strands all represent the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from each pair of two strands are aligned by a framework region, allowing binding to a particular epitope. From the N-terminus to the C-terminus, both the light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain can be found in Kabat, Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, MD, 1987 and 1991), or Chothia and Lesk, J. et al. Mol. Biol. 196: 901-917 (1987); Chothia et al., Nature 342: 878-883 (1989).

b. Production of non-human antibodies Production of non-human monoclonal antibodies (eg, mouse, guinea pig, rabbit or rat) can be accomplished by immunizing animals with plaque components such as Aβ or other fibril components. it can. Longer polypeptides comprising Aβ or an immunogenic fragment of Aβ, or anti-idiotypic antibodies against antibodies to Aβ can also be used. See, for example, Harlow and Lane, Antibodies, A Laboratory Manual (CSHP NY, 1988), incorporated by reference for all purposes. Such immunogens can be obtained from natural sources, by peptide synthesis, or by recombinant expression. In some cases, the immunogen can be administered fused or otherwise complexed with a carrier protein as described below. In some cases, the immunogen can be administered with an adjuvant. Several types of adjuvants as described below can be used. Freund's complete adjuvant followed by incomplete adjuvant is preferred for immunization of experimental animals. Rabbits or guinea pigs are typically used for making polyclonal antibodies. Mice are typically used for the production of monoclonal antibodies. Antibodies are screened for specific binding to the immunogen. In some cases, antibodies are further screened for binding to specific regions of the immunogen. For example, in the case of Aβ peptide as the immunogen, the screen measures antibody binding to a collection of deletion mutants of Aβ peptide and determines which deletion mutant binds to the antibody. Can be achieved. Binding can be assessed, for example, by Western blot or ELISA. The smallest fragment showing specific binding to the antibody defines the epitope of the antibody. Alternatively, epitope specificity can be determined by a competition assay in which the test antibody and reference antibody compete for binding to the component. When the test and reference antibodies compete, they bind to the same epitope, or a sufficiently close epitope that the binding of one antibody prevents the binding of the other.

C. Chimeric and humanized antibodies Chimeric and humanized antibodies have the same or similar binding specificity and affinity to mice or other non-human antibodies that provide the starting material for the construction of chimeric or humanized antibodies. A chimeric antibody is an antibody whose light and heavy chain genes are typically constructed from immunoglobulin gene segments belonging to different species by genetic engineering. For example, a variable (V) segment of a gene from a mouse monoclonal antibody can be linked to human constant (C) segments such as IgG1 and IgG4. Thus, a typical chimeric antibody is a hybrid protein consisting of a V or antigen binding domain from a murine antibody and a C or effector domain from a human antibody.

  Humanized antibodies have substantially variable region framework residues from human antibodies (named acceptor antibodies) and complementarity-determining regions from mouse antibodies, termed donor immunoglobulins. . Queen et al., Proc. Natl. Acad. Sci. USA 86: 10029-10033 (1989), and International Patent Application No. WO 90/07861, U.S. Pat. Nos. 5,693,762, 5,693,761, and 5,585,089. No. 5,530,101 and Winter, US Pat. No. 5,225,539 (incorporated by reference in their entirety for all purposes). The constant region (s), if present, are also substantially or completely from human immunoglobulin. Human variable domains are usually selected from human antibodies whose framework sequences exhibit a high degree of sequence identity with the murine variable region domain from which the CDRs were derived. The framework residues of the heavy and light chain variable regions can be derived from the same or different human antibody sequences. The human antibody sequence can be the sequence of a naturally occurring human antibody or it can be the consensus sequence of several human antibodies. See Carter et al., International Patent Application No. WO 92/22653. Certain amino acids from human variable region framework residues are selected for substitution based on their possible impact on CDR conformation and / or binding to antigen. Examining these possible effects is by modeling, examining the characteristics of amino acids at specific positions, or empirically observing the effects of substitution or mutagenesis of specific amino acids.

For example, if an amino acid differs between a murine variable region framework residue and a selected human variable region framework residue, the human framework amino acid is usually:
(1) direct non-covalent binding of antigen,
(2) adjacent to the CDR region,
(3) interact with the CDR region in a different way (eg, within about 6A of the CDR region) or (4) if it is reasonably expected to participate in the VL-VH interface, Substitution with equivalent framework amino acids from the antibody should be made.

  Other candidates for substitution are acceptor human framework amino acids that are unusual for a human immunoglobulin at that position. These amino acids can be substituted with amino acids from the equivalent position of the mouse donor antibody or from the equivalent position of more typical human immunoglobulins. Other candidates for substitution are acceptor human framework amino acids that are unusual for a human immunoglobulin at that position. Humanized immunoglobulin variable region frameworks usually exhibit at least 85% sequence identity to the human variable region framework sequences or a consensus of such sequences.

d. Human antibodies Human antibodies against Aβ are provided by a variety of techniques described below. Some human antibodies are selected by competitive binding experiments or other methods to have the same epitope specificity as a particular mouse antibody, such as that of the mouse monoclonal described in Example XI. Human antibodies can also be screened for specific epitope specificity by using only fragments of Aβ as immunogens and / or screening antibodies against a collection of deletion mutants of Aβ. .

(1) Trioma Methodology The basic approach, and one exemplary cell fusion partner for use in this approach, SPAZ-4, is described by Oestberg et al., Hybridoma 2: 361-367 (1983); Oestberg, US Pat. 634,664; and Engleman et al., US Pat. No. 4,634,666, each of which is incorporated by reference in its entirety for all purposes. Antibody-producing cell lines obtained by this method are called triomas because they are derived from three types of cells: two humans and one mouse. First, a mouse myeloma line is fused with human B lymphocytes to obtain non-antibody producing heterogeneous hybrid cells such as the Ostz, cell line described by Ostberg, supra. The xenogeneic cells are then fused with immunized human B lymphocytes to obtain an antibody producing trioma cell line. Triomas have been found to produce antibodies more stably than normal hybridomas made from human cells.

Immunized B lymphocytes are obtained from human donor blood, spleen, lymph nodes or bone marrow. If antibodies against a particular antigen or epitope are desired, it is preferable to use that antigen or its epitope for immunization. Immunization can be either in vivo or in vitro. For in vivo immunization, B cells are typically isolated from humans immunized with anti-idiotypic antibodies to Aβ, fragments thereof, larger polypeptides containing Aβ or fragments, or antibodies to Aβ. Release. In some methods, B cells are isolated from the same patient to whom antibody therapy is ultimately to be administered. For in vitro immunization, B lymphocytes are typically cultured in a medium such as RPMI-1640 (Engleman, see above) supplemented with 10% human plasma for a period of 7-14 days. During the exposure to antigen.

  Immunized B lymphocytes are fused to heterologous hybrid cells such as SPAZ-4 by known methods. For example, cells are treated with 40-50% polyethylene glycol of MW 1000-4000 at about 37 ° C. for about 5-10 minutes. Cells are separated from the fusion mixture and propagated in media selective for the desired hybrid (eg, HAT or AH). Clones that secrete antibodies with the required binding specificity are identified by assaying trioma media for the ability to bind to Aβ or a fragment thereof. Triomas producing human antibodies with the desired specificity are subcloned by limiting dilution techniques and grown in vitro in media. The resulting trioma cell line is then tested for the ability to bind Aβ or a fragment thereof.

  Although triomas are genetically stable, they do not produce antibodies at very high levels. Expression levels can be determined by cloning the antibody gene from the trioma into one or more expression vectors and transducing the vector into standard mammalian, bacterial or yeast cell lines according to methods known in the art. It can be increased by conversion.

(2) Transgenic non-human mammals Human antibodies against Aβ can also be produced from non-human transgenic mammals having a transgene encoding at least one segment of the human immunoglobulin locus. Usually, the endogenous immunoglobulin loci of such transgenic mammals are functionally inactivated. Preferably, the segment of the human immunoglobulin locus includes non-rearranged sequences of heavy and light chain components. Both inactivation of endogenous immunoglobulin genes and introduction of exogenous immunoglobulin genes can be achieved by targeted homologous recombination or introduction of the YAC chromosome. The transgenic mammal resulting from this method is capable of functionally rearranging the sequences of immunoglobulin components and expressing antibodies of various isotypes encoded by human immunoglobulin genes without expressing endogenous immunoglobulin genes. It is possible to develop a repertoire. Production and properties of mammals having these properties are described, for example, in Lonberg et al., International Patent Application No. WO 93/12227 (1993); US Pat. Nos. 5,877,397, 5,874,299. No. 5,814,318 No. 5,789,650 No. 5,770,429 No. 5,661,016 No. 5,633,425 No. 5 No. 5 , 625,126, 5,569,825, 5,545,806, Nature 148, 1547-1553 (1994), Nature Biotechnology 14, 826 (1996), Kucherlapati, international patent application. WO 91/10741 (1991) (each of which is incorporated by reference in its entirety for all purposes) Will be described in greater detail is). Transgenic mice are particularly suitable in this regard. Anti-Aβ antibodies are obtained by immunizing a transgenic non-human mammal as described by Lonberg or Kucherlapati, supra, with Aβ or a fragment thereof. Monoclonal antibodies are produced, for example, by fusing B cells from such mammals to a suitable myeloma cell line using conventional Kohler-Milstein technology. Human polyclonal antibodies can also be provided in the form of serum from a human immunized with an immunogenic agent. In some cases, such polyclonal antibodies can be concentrated by affinity purification using Aβ or other immunogenic amyloid peptide as an affinity reagent.

(3) Phage display method One further approach to obtain human anti-Aβ antibodies is according to the general protocol outlined by Huse et al., Science 246: 1275-1281 (1989).
Screening a DNA library from human B cells. For example, as described for the trioma methodology, such B cells can be obtained from humans immunized with Aβ, fragments, longer polypeptides containing Aβ or fragments, or anti-idiotype antibodies. In some cases, these B cells are obtained from a patient who should ultimately receive antibody therapy. An antibody that binds to an epitope of the amyloid component of interest such as Aβ or a fragment thereof is selected. The sequence encoding such an antibody (or binding fragment) is then cloned and amplified. The protocol described by Huse is made more efficient in combination with phage display technology. For example, Dower et al., International Patent Application No. WO 91/17271 and McCafferty et al., International Patent Application No. WO 92/01047, US Pat. Nos. 5,877,218, 5,871,907, 5,858,657, 5,837,242, 5,733,743 and 5,565,332 (each of which is incorporated by reference in its entirety for all purposes) See). In these methods, a library of phage is produced in which members display different antibodies on their outer surface. Antibodies are usually displayed as Fv or Fab fragments. Phages displaying antibodies with the desired specificity are selected by affinity enrichment for Aβ peptides or fragments thereof.

In a variation of the phage display method, human antibodies having the binding specificity of selected mouse antibodies can be produced. See Winter, International Patent Application No. WO 92/20791. In this method, the variable region of either the heavy or light chain of the selected mouse antibody is used as a starting material. For example, if a light chain variable region is selected as the starting material, a phage library is constructed in which members display the same light chain variable region (ie, the mouse starting material) and different heavy chain variable regions. The heavy chain variable region is obtained from a library of rearranged human heavy chain variable regions. Phages that exhibit strong specific binding to the component of interest (eg, a minimum of 10 ⁸ and preferably a minimum of 10 ⁹ M ⁻¹ ) are selected. The human heavy chain variable region from this phage then serves as a starting material for constructing additional phage libraries. In this library, each phage displays the same heavy chain variable region (ie, the region identified from the first display library) and a different light chain variable region. The light chain variable region is obtained from a library of rearranged human variable light chain regions. Again, phages that exhibit strong specific binding to the amyloid peptide component are selected. These phage display the variable regions of fully human anti-amyloid peptide antibodies. These antibodies usually have the same or similar epitope specificity as the mouse starting material.

e. Selection of Constant Regions The heavy and light chain variable regions of chimeric, humanized or human antibodies can be linked to at least a portion of the human constant region. The choice of constant region depends in part on whether antibody-dependent complement and / or cell-mediated toxicity is desired. For example, isotypes IgG1 and IgG3 have complement activity and isotypes IgG2 and IgG4 do not. Isotype selection can also affect the passage of antibodies into the brain. The light chain constant region can be a λ or κ chain. Antibodies can be produced as tetramers containing two light chains and two heavy chains, as separate heavy chains, as light chains, as Fab, Fab′F (ab ′) ₂ and Fv, or as heavy and light chains. It can be expressed as a single chain antibody in which the variable domains of the chains are linked by a spacer.

f. Recombinant Antibody Expression Chimeric, humanized and human antibodies are typically produced by recombinant expression. A recombinant polynucleotide construct typically includes expression control sequences operably linked to the coding sequence of the antibody chain, including naturally associated or heterologous promoter regions. Preferably, the expression control sequence is a eukaryotic promoter system in a vector capable of transforming or transfecting a eukaryotic host cell. Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high level expression of the nucleotide sequence and collection and purification of cross-reacting antibodies.

  These expression vectors are typically replicable in the host organism, either as episomes or as an integral part of the host chromosomal DNA. Universally, expression vectors contain a selectable marker (eg ampicillin resistance or hygromycin resistance) that allows detection of cells transformed with the desired DNA sequence.

  E. coli is one prokaryotic host that is particularly useful for cloning the DNA sequences of the present invention. Microorganisms such as yeast are also useful for expression. Saccharomyces is a preferred yeast host and suitable vectors have expression control sequences, origins of replication, termination sequences, etc. as desired. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for the utilization of maltose and galactose.

  Mammalian cells are a preferred host for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes to Clones, (VCH Publishers, New York, 1987). A number of suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art and include CHO cell lines, various COS cell lines, HeLa cells, L cells and myeloma cell lines. Include. Expression vectors for these cells include origins of replication, promoters, expression control sequences such as enhancers (Queen et al., Immunol. Rev. 89:49 (1986)), as well as ribosome binding sites, RNA splice sites, polyadenylation. Necessary processing information sites such as sites and transcription terminator sequences can be included. Preferred expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papilloma virus and the like. Co et al. Immunol. 148: 1149 (1992).

  Alternatively, the antibody coding sequence may be used for introduction into the genome of the transgenic animal and subsequent expression in the milk of the transgenic animal (see, eg, US Pat. Nos. 5,741,957, 5,304). , 489, 5,849,992 (according to the method described in their entirety, incorporated herein by reference in their entirety). Suitable transgenes include light chain and / or heavy chain coding sequences in operable linkage with promoters and enhancers from mammary gland specific genes such as casein or β-lactoglobulin.

  A vector containing the target DNA segment can be transferred to a host cell by a known method depending on the type of the cell host. For example, calcium chloride transfection is commonly used for prokaryotic cells, whereas calcium phosphate treatment, electroporation, lipofection, biolistic or virus-based transfection can be used for other cellular hosts. it can. Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation and microinjection methods (see generally Sambrook et al., Supra). . For the production of transgenic animals, the transgene can be microinjected into a fertilized oocyte, or can be integrated into the genome of an embryonic stem cell, and the nucleus of such a cell becomes an enucleated oocyte. Can be imported.

  Once expressed, the antibody can be purified according to standard procedures in the art, including HPLC purification, column chromatography, gel electrophoresis and the like (generally, Scopes, Protein Purification (Springer Fairerk). -Verlag), New York, 1982)).

4). Other Therapeutic Agents Therapeutic agents for use in the present methods also include T cells that bind to plaque components such as Aβ peptides. For example, T cells can be activated against Aβ peptides by expressing human MHC class I genes and human β-2 microglobulin genes from insect cell lines so that empty complexes are on the surface of the cells. And can bind to Aβ peptide. T cells contacted with the cell line appear to be specifically activated for the peptide. See Peterson et al., US Pat. No. 5,314,813. Insect cell lines expressing MHC class II antigens can be used as well to activate CD4 T cells.

5. Carrier Proteins Some agents for inducing an immune response contain the appropriate epitope to induce an immune response against amyloid deposits, but are too small to be immunogenic. In this situation, the peptide immunogen can be linked to a suitable carrier to help elicit an immune response. Suitable carriers are serum albumin, KLH, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, or diphtheria, E. coli, cholera or H. coli. Includes toxoids from other pathogenic bacteria such as H. pylori, or attenuated toxin derivatives. Other carriers include T cell epitopes that bind to multiple MHC alleles (eg, at least 75% of all human MHC alleles). Such carriers are sometimes known in the art as “universal T cell epitopes”. Examples of universal T cell epitopes are:
Influenza hemagglutinin: HA _307-319 PKYVKQNTLKLAT (SEQ ID NO: 1)
PADRE (universal residues are bolded) AKXVAAWTLKAAA (SEQ ID NO: 2) Malaria CS: T3 epitope EKKIAKMEKASSVFFNV (SEQ ID NO: 3)
Hepatitis B surface antigen: HBsAg _19-28 FFLLTRILTI (SEQ ID NO: 4)
Heat shock protein 65: hsp65 _153-171 DQSIGDLIAAMDKVGGNEG (SEQ ID NO: 5)
Calmette-Guerin QVHFQPLPPAVVKL (SEQ ID NO: 6)
Tetanus toxoid: TT _830-844 QYIKANSKFIGITEL (SEQ ID NO: 7)
Tetanus toxoid: TT _947-967 FNNFTVSFWLRVPKVSASHLE (SEQ ID NO: 8)
HIV gp120 T1: KQIINMWQEVVGKAYA. (SEQ ID NO: 9)
Is included.

  Other carriers for stimulating or enhancing the immune response include IL-1, IL-1α and β peptides, cytokines such as IL-2, γINF, IL-10, GM-CSF, and MIP1α and β and RANTES Such chemokines. The immunogenic agent may also be linked to a peptide that enhances transport across the tissue as described in O'Mahony, International Patent Applications WO 97/17613 and WO 97/17614. it can.

The immunogenic agent can be linked to the carrier by chemical crosslinking. Techniques for linking an immunogen to a carrier include disulfides using N-succinimidyl-3- (2-pyridylthio) propionate (SPDP) and succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC). Includes bond formation (if the peptide lacks a sulfhydryl group, this can be provided by the addition of a cysteine residue). These reagents have disulfide bonds between themselves and peptide cysteine residues on one protein and amide bonds by ε-amino on lysine or other free amino groups in other amino acids. Create. A variety of such disulfide / amide forming agents are described in Immun. Rev. 62, 185 (1982). Other bifunctional coupling agents form thioethers rather than disulfide bonds. Many of these thioether formers are commercially available and the reactivity of 6-maleimidocaproic acid, 2-bromoacetic acid and 2-iodoacetic acid, 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid Includes esters. The carboxyl group can be activated by combining them with succinimide or 1-hydroxy-2-nitro-4-sulfonic acid sodium salt.

  An immunogenic peptide can also be expressed as a fusion protein with a carrier (ie, a heterologous peptide). The immunogenic peptide can be linked to a carrier at its amino terminus, its carboxy terminus, or both. In some cases, multiple repetitive sequences of immunogenic peptides can be present in the fusion protein. In some cases, an immunogenic peptide can be linked to multiple copies of a heterologous peptide, eg, at both the N and C termini of the peptide. Some carrier peptides act to induce a helper T cell response against the carrier peptide. Induced helper T cells in turn induce a B cell response to the immunogenic peptide linked to the carrier peptide.

  Some agents of the invention comprise a fusion protein in which an N-terminal fragment of Aβ is linked at its C-terminus to a carrier peptide. In such agents, the N-terminal residue of the Aβ fragment constitutes the N-terminal residue of the fusion protein. Accordingly, such fusion proteins are effective in inducing antibodies that bind to epitopes that require the N-terminal residue of Aβ to be in a free form. Some agents of the invention comprise multiple repetitive sequences of the N-terminal segment of Aβ linked at the C-terminus to one or more copies of a carrier peptide. N-terminal fragments of Aβ incorporated into such fusion proteins sometimes begin with Aβ1-3 and end with Aβ7-11. Aβ1-7, Aβ1-3, 1-4, 1-5 and 3-7 are preferred N-terminal fragments of Aβ. Some fusion proteins comprise different N-terminal segments of tandem Aβ. For example, the fusion protein can include Aβ1-7, then Aβ1-3, and then a heterologous peptide.

  In some fusion proteins, the N-terminal segment of Aβ is fused at its N-terminus to a heterologous carrier peptide. The same diversity of N-terminal segments of Aβ can be used as in the C-terminal fusion. Some fusion proteins comprise a heterologous peptide linked to the N-terminus of the N-terminal segment of Aβ, which in turn is linked in tandem to one or more additional N-terminal segments of Aβ.

Some examples of fusion proteins suitable for use in the present invention are shown below. Some of these fusion proteins include Aβ linked to an epitope of tetanus toxoid as described in US Pat. Nos. 5,196,512, EP 378,881 and 427,347. Comprising segments. Some fusion proteins comprise a segment of Aβ linked to a carrier peptide described in US Pat. No. 5,736,142. Some heterologous peptides are universal T cell epitopes. In some methods, the agent for administration is simply a single fusion protein with an Aβ segment linked to a heterologous segment in a linear arrangement. In some methods, the agent is multimer of fusion proteins represented by the formula 2 ^x, where x is an integer from 1-5. Preferably x is 1, 2 or 3, with 2 being most preferred. When x is 2, such a multimer has four fusion proteins linked in a preferred configuration called MAP4 (see US Pat. No. 5,229,490). The epitope for Aβ is underlined.

  The arrangement of MAP4 is shown below, where a branched structure is generated by initiating peptide synthesis at both the N-terminus and the side chain amine of lysine. Depending on the number of times lysine is incorporated into the sequence and allowed to branch, the resulting structure can present multiple N-termini. In this example, four identical N-termini are generated on a branched lysine-containing core. Such multiplicity greatly enhances the responsiveness of cognate B cells.

  Another example of a fusion protein (Aβ immunogenic epitope is bolded)

Is included.

  The same or similar carrier protein and linking method can be used to generate an immunogen to be used in generating antibodies against Aβ for use in passive immunization. For example, Aβ or a fragment linked to a carrier can be administered to a laboratory animal in the production of monoclonal antibodies against Aβ.

6). Nucleic acids encoding therapeutic agents An immune response against amyloid deposits can also be induced by administration of selected peptide immunogens, or nucleic acids encoding antibodies and their component chains used for passive immunization. . Such nucleic acids can be DNA or RNA. The nucleic acid segment encoding the immunogen is typically linked to regulatory elements such as promoters and enhancers that allow expression of the DNA segment in the intended target cell of the patient. For expression in blood cells, as desired for the induction of an immune response, promoter and enhancer elements from light or heavy chain immunoglobulin genes, or CMV's major mid-early promoter and enhancer direct expression. Suitable for doing. Linked regulatory elements and coding sequences are often cloned into vectors. For administration of double chain antibodies, the two chains can be cloned into the same or separate vectors.

Retroviral systems (see, eg, Lawrie and Tumin, Cur. Opin. Genet. Develop. 3, 102-109, 1993); adenoviral vectors (see, eg, Bett et al., J. Virol. 67, 5911, 1993). Adeno-associated viral vectors (see, for example, Zhou et al., J. Exp. Med. 179, 1867, 1994), viral vectors from the Pox family, including vaccinia viruses and tripox viruses, Sindbis Due to viruses and Semliki Forest virus (see eg Dubensky et al., J. Virol. 70, 508-519, 1996), Venezuelan equine encephalitis virus (see US Pat. No. 5,643,576) Viral vectors from the genus Alphavirus, such as those of vesicular stomatitis virus (see International Patent Application No. WO 96/34625) and papillomavirus (Ohe et al., Human Gene Therapy 6, 325-333, 1995); Woo et al., International Patent Application No. WO 94/12629 and Xiao and Brandsma, Nucleic Acids. Res. A number of viral vector systems are available including rhabdoviruses such as 24, 2630-2622, 1996).

  DNA encoding the immunogen, or a vector containing it, can be packaged in liposomes. Suitable lipids and related analogs are described by US Pat. Nos. 5,208,036, 5,264,618, 5,279,833, and 5,283,185. The The vector and DNA encoding the immunogen can also be adsorbed to or associated with a microparticle carrier, examples of which include polymethyl methacrylate polymers and polylactides and poly (lactide coglycolides). For example, McGee et al. Micro Encap. (1996).

  Gene therapy vectors or naked DNA are typically administered systemically (eg, intravenous, intraperitoneal, intranasal, gastric, intradermal, intramuscular, subcutaneous or intracranial injection) or topical application (eg, US Pat. , 399, 346), and can be delivered in vivo. Such vectors further include facilitating agents such as bupivacaine (US Pat. No. 5,593,970). DNA can also be administered using a gene gun. See Xiao and Brandsma, supra. DNA encoding the immunogen is precipitated on the surface of the microscopic metal beads. Microprojectiles are accelerated with shock waves or expanding helium gas and penetrate tissue to a depth of several cell layers. For example, the Accel ™ gene delivery device manufactured by Agracetus, Inc. (Middleton, Wis.) Is suitable. Alternatively, naked DNA can be passed through the skin into the bloodstream by simply spotting the DNA on the skin using chemical or mechanical stimulation (see International Patent Application No. WO 95/05853). See).

  In a further variation, the vector encoding the immunogen is ex vivo for cells such as cells explanted from individual patients (eg, lymphocytes, bone marrow aspirates, tissue biopsies) or universal donor hematopoietic stem cells. Following delivery and then selection for cells that normally incorporate the vector, the cells can be reimplanted into the patient.

7). Screening Antibodies for Vanishing Activity Example XIV describes a method for screening antibodies for activity in disappearing amyloid deposits. To screen for activity against amyloid deposits, tissue samples from patients with amyloidosis, such as brain tissue of Alzheimer's disease, or animal models with characteristic amyloid pathology, such as microglia Contact in medium with phagocytes carrying Fc receptors and the antibody under test. Phagocytic cells can be primary cultures or cell lines such as BV-2, C8-B4 or THP-1. These components can be combined on a microscope slide to facilitate microscopic monitoring, or multiple reactions can be performed in parallel in the wells of a microtiter dish. In such formats, separate reduced microscope slides can be placed in separate wells, or non-microscopic detection formats such as ELISA detection of Aβ can be used. Preferably, the series of measurements consists of the amount of amyloid deposits in the in vitro reaction mixture starting from a baseline value before the reaction proceeds, and one or more test values during the reaction. The antigen can be detected, for example, by staining with a fluorescently labeled antibody against Aβ or other components of amyloid plaques. The antibody used for staining may or may not be the same as the antibody being tested for disappearance activity. A decrease in the basis during the reaction of amyloid deposits indicates that the antibody under test has a clearing activity. Such antibodies are likely to be useful in the prevention or treatment of Alzheimer's disease and other amyloidogenic diseases. As described above, experiments conducted in support of the present invention show that using such an assay, antibodies against the NAC fragment of synuclein are effective in clearing amyloid plaques characteristic of Alzheimer's disease. It was.

D. Patients Responsive to Anti-Amyloid Treatment Regimens Patients who respond sensitively to treatment include individuals who are at risk of disease but do not show symptoms, as well as patients who are currently showing symptoms of amyloidosis. In the case of Alzheimer's disease, virtually anyone is at risk of suffering from Alzheimer's disease if he or she lives long enough. Thus, the method can be administered prophylactically to the general population without the need for any assessment of the subject patient's risk. The method is particularly useful for individuals with a known genetic risk of either Alzheimer's disease or other hereditary amyloid diseases. Such individuals include those with relatives who have experienced the disease and those whose risk is determined by analysis of genetic or biochemical markers. Genetic markers of risk for Alzheimer's disease include mutations in the APP gene, especially mutations at positions 717 and 670 and 671, referred to as Hardy and Swedish mutations, respectively (Hardy, TINS, see above). Other markers of risk are presenilin genes PS1 and PS2, and mutations in apoE4, family history of AD, hypercholesterolemia or atherosclerosis. Individuals currently suffering from Alzheimer's disease can be recognized from characteristic dementia as well as the presence of the risk factors described above. In addition, a number of diagnostic tests are available to identify individuals with AD. These include measurement of CSF tau and Aβ42 levels. Elevated tau and reduced Aβ42 levels indicate the presence of AD. Individuals suffering from Alzheimer's disease can also be diagnosed by MMSE or ADRDA criteria, as discussed in the Examples section.

  In asymptomatic patients, treatment can begin at any age (eg, 10, 20, 30 years). However, it is usually not necessary to begin treatment until the patient reaches 40, 50, 60 or 70 years of age. Treatment typically requires multiple doses over a period of time. Treatment is monitored by assaying an activated T cell or B cell response to an antibody or therapeutic agent (eg, Aβ peptide) over time along the lines described in Examples I and II herein. can do. If the response falls, a booster dosage is indicated. For patients with potential Down's syndrome, treatment can be initiated during pregnancy by administering a therapeutic agent to the mother or immediately after birth.

Other forms of amyloidosis often progress undiagnosed unless a specific prediction of the disease is suspected. The primary symptom is the presence of heart or kidney disease in middle-aged and elderly patients who also have signs of other organ involvement. Electrocardiogram low potential or extreme axial excursions, and thickened ventricular tissue can imply cardiac involvement. Proteinuria is a symptom of renal involvement. Liver involvement can also be suspected if hepatomegaly is detected by physical examination of the patient. Peripheral neuropathy is also a universal event in some forms of amyloidosis; autonomic neuropathy characterized by postural hypotension can also be found. Anyone with progressive neuropathy of indeterminate origin should be suspected of amyloidosis. Tissue biopsy can be used to make a definitive diagnosis of the disease, where the affected organ (s) are available. For systemic amyloidosis, an aspirated fat pad or rectal biopsy sample can be used. The biopsy material is stained with Congo Red and positive samples exhibit clear yellow-green birefringence under a polarized light microscope.

E. Therapeutic regimen In prophylactic applications, a pharmaceutical composition or medicament is excluded or reduced to a patient susceptible to or otherwise at risk of a particular disease, or the disease In an amount sufficient to delay the onset of In therapeutic applications, the composition or medicament is used to cure or at least partially stop the symptoms of the disease and its complications in a patient suspected of such disease or who is already suffering from such disease. Administer in sufficient quantity. An amount adequate to accomplish this is defined as a therapeutically or pharmaceutically effective dose. In both prophylactic and therapeutic regimes, the agent is usually administered in several dosages until a sufficient immune response is achieved. Typically, the immune response is monitored and repeated dosages are given when the immune response begins to weaken.

  Effective doses of the compositions of the present invention for the treatment of the above-mentioned medical conditions are the means of administration, the target site, the physiological state of the patient, whether the patient is a human or animal, other pharmaceuticals to be administered, And vary depending on many different factors, including whether the treatment is prophylactic or therapeutic. Usually, the patient is a human, but in some diseases such as mad cow disease associated with prion protein, the patient can be a non-human mammal such as a cow. The therapeutic dosage needs to be determined to optimize safety and efficacy. The amount of immunogen depends on whether an adjuvant is also administered, and higher dosages are generally required in the absence of adjuvant. Depending on the immunogenicity of the particular formulation, the amount of immunogen for administration can vary from 1 μg to 500 μg per patient, and more usually from 5 to 500 μg per injection for human administration. Sometimes higher doses of 0.5-5 mg per infusion are used. Typically, a minimum of about 10, 20, 50 or 100 μg is used for each human injection. The timing of infusion can vary widely from once a day to once a year, once every 10 years, and a continuous “boost” of the immunogen is somewhat preferred. In general, in accordance with the teaching provided herein, an effective dosage is obtained from a patient to obtain a liquid sample (generally a serum sample), as well as a specific antigen to be known and measured in the art. Can be monitored by measuring the titer of antibodies raised against the immunogen using methods that can be readily adapted to the immunogen. Ideally, samples are taken before the first dose; subsequent samples are taken and titrated after each immunization. In general, a dose or dosing schedule that provides a detectable titer at least 4 times greater than a control or “background” level at a 100-fold serum dilution is desirable, where the background is the control serum or ELISA assay. Define with respect to the background of the plate. According to the invention, a titer of at least 1: 1000 or 1: 5000 is preferred.

On any given day that gives a dose of immunogen, the dose is usually greater than about 1 μg per patient and preferably greater than 10 μg per patient when adjuvant is also administered, and the non-adjuvant of the adjuvant. In the presence, it is greater than at least 10 μg per patient and usually greater than 100 μg per patient. The dose of an individual immunogen selected according to the present invention is determined according to standard administration and titration methods employed in conjunction with the teachings provided herein. A typical regimen consists of immunization followed by booster injections at time intervals such as 6 week intervals. Another regimen consists of immunization followed by booster injections after 1, 2 and 12 months. Another regimen requires an infusion every two months for life. Alternatively, booster injections can be irregular as indicated by monitoring the immune response.

  For passive immunization with antibodies, dosages range from about 0.0001 to 100 mg / kg of host body weight and more usually in the range of 0.01 to 5 mg / kg. For example, the dosage can be 1 mg / kg body weight or 10 mg / kg body weight. An exemplary treatment regime entails administration once every two weeks, or once a month, or once every 3 to 6 months. In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered is within the range indicated. Antibodies are usually administered on multiple occasions. The interval between single dosages can be weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of antibodies to Aβ in the patient. Alternatively, the antibody can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency will vary depending on the half-life of the antibody in the patient. In general, human antibodies show the longest half life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes used until disease progression is suppressed or stopped, and preferably until the patient exhibits partial or complete improvement of disease symptoms. Is needed to. Thereafter, the patient can be administered a prophylactic regime.

  The dose of nucleic acid encoding the immunogen ranges from about 10 ng to 1 g, 100 ng to 100 mg, 1 μg to 10 mg, or 30 to 300 μg of DNA per patient. The dose of infectious viral vector varies from 10 to 100 or more virions per administration.

  Agents for inducing an immune response can be administered by parenteral, topical, intravenous, oral, subcutaneous, intraperitoneal, intranasal or intramuscular means for prophylactic and / or therapeutic treatment. Typical routes of administration of immunogenic agents are intramuscular (im), intravenous (iv) or subcutaneous (sc), although other routes are possible. Can be equally effective. Intramuscular injection is most typically performed in the arm or leg muscles. In some methods, the agent is injected directly into a particular tissue where deposits have accumulated (eg, intracranial injection). Intramuscular or intravenous injection is preferred for antibody administration. In some methods, particular therapeutic antibodies are injected directly into the cranium. In some methods, the antibody is administered as a sustained release composition or device, such as a Medipad ™ device.

The agents of the present invention can optionally be administered with other agents that are at least partially effective in the treatment of amyloidogenic diseases. In the case of Alzheimer's disease and Down's syndrome, where amyloid deposits are present in the brain, administering the agent of the present invention together with other agents that increase the passage of the agent of the present invention across the blood-brain barrier Can also. In addition, therapeutic cocktails comprising immunogens designed to elicit an immune response against one or more amyloid components are also combinations of antibodies directed against one plaque component and immunogens directed against different plaque components As such, it is contemplated by the present invention.

  An immunogenic agent of the invention, such as a peptide, is sometimes administered with an adjuvant. A variety of adjuvants can be used with peptides such as Aβ to elicit an immune response. Preferred adjuvants enhance the intrinsic response to the immunogen without causing a conformational change in the immunogen that affects the qualitative form of the response. Preferred adjuvants are aluminum hydroxide and aluminum phosphate, 3 de-O-acylated monophosphoryl lipid A (MPL ™) (UK Patent 2220 211 (RIBI ImmunoChem Research Inc.), See Hamilton, Montana, now part of Corixa). Stimulon (TM) QS-21 is a triterpene glycoside or saponin isolated from the bark of Quillaja saponaria Molina found in South America (Kensil et al., Vaccine Design: The Subunit and Adjuvant Approach (Edited by Powell and Newman, Plenum Press, New York, 1995); U.S. Pat. See). Other adjuvants are oil-in-water emulsions (such as Stoure et al., N. Engl. J. Med. 336), optionally in combination with an immunostimulant such as monophosphoryl lipid A (such as squalene or peanut oil). 86-91 (1997)). Another adjuvant is CpG (International Patent Application No. WO 98/40100). Alternatively, Aβ can be conjugated to an adjuvant. However, such binding should not substantially alter the Aβ conformation to affect the nature of the immune response thereto. Adjuvants can be administered as a component of a therapeutic composition containing the active ingredient, or can be administered separately, prior to, in conjunction with, or after administration of the therapeutic agent. it can.

One preferred class of adjuvants is aluminum salts (alum) such as aluminum hydroxide, aluminum phosphate, aluminum sulfate. Such adjuvants may be used with or without other specific immunostimulants such as polymeric or monomeric amino acids such as MPL or 3-DMP, QS-21, polyglutamic acid or polylysine. it can. Another class of adjuvants are oil-in-water emulsion formulations. Such adjuvants include muramyl peptides (eg, N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetylnormuryl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) ethylamine (MTP-PE), N-acetylglu Such as xaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP) terramid ™, or other bacterial cell wall components) Can be used with or without other specific immunostimulants The oil-in-water emulsion was formulated into particles consisting of submicron particles using a microfluidizer such as (a) a Model 110Y microfluidizer (Microfluidics, Newton, Mass.). MF59 (International Patent Application No. WO 90/14837) containing% squalene, 0.5% Tween 80 and 0.5% Span 85 (possibly containing various amounts of MTP-PE) (B) 10% squalene, 0.4% Tween, either microfluidized into an emulsion of submicron grains or vortexed to produce a larger grain size emulsion. 80) 5% pluronic blocked polymer L121 and thr-M SAF containing DP, and (c) 2% squalene, 0.2% Tween 80, and one from the group consisting of monophosphoryl lipid A, trehalose dimycolate (TDM) and cell wall skeleton (CWS) Or Ribi (TM) adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.) Containing bacterial cell wall components, preferably MPL + CWS (Detox (TM)) Include. Another class of preferred adjuvants is saponin adjuvants such as Stimulon ™ (QS-21; Aquila, Flamingham, Mass.), Or Iscom (immunostimulatory complex) and iscomatrix. Particles derived therefrom such as (ISCOMATRIX). Other adjuvants include cytokines such as Freund's incomplete adjuvant (IFA), interleukins (IL-1, IL-2 and IL-12), macrophage colony stimulating factor (M-CSF) and tumor necrosis factor (TNF). Is included. Such adjuvants are generally available from commercial sources.

  The adjuvant can be administered with the immunogen as a single composition, or can be administered before, in conjunction with, or after administration of the immunogen. The immunogen and adjuvant can be packaged and supplied in the same vial, or can be packaged in separate vials and mixed prior to use. Immunogens and adjuvants are typically packaged with a label indicating the intended therapeutic application. If the immunogen and adjuvant are packaged separately, the packaging typically includes instructions for mixing prior to use. The choice of adjuvant and / or carrier depends on factors such as the stability of the formulation containing the adjuvant, the route of administration, the administration schedule, and the efficacy of the adjuvant against the species being vaccinated. In humans, preferred pharmaceutically acceptable adjuvants are those approved for human administration by appropriate modulators. Examples of such preferred adjuvants for humans include alum, MPL and QS-21. In some cases, two or more different adjuvants can be used simultaneously. Preferred combinations include alum comprising MPL, alum comprising QS-21, MPL comprising QS-21, and alum together, QS-21 and MPL. Also, in some cases, Freund's incomplete adjuvant can be used in combination with Alum, any of QS-21 and MPL, and all of them (Chang et al., Advanced Drug Delivery Reviews 32, 173-186 (1998). )).

  The agents of the present invention are often administered as pharmaceutical compositions comprising an effective therapeutic agent and a variety of other pharmaceutically acceptable ingredients. See Remington's Pharmaceutical Sciences (19th edition, 1995). The preferred form depends on the intended mode of administration and therapeutic application. The composition is a pharmaceutically acceptable non-toxic carrier or diluent, depending on the desired formulation (these are universally used to formulate pharmaceutical compositions for animal or human administration). (Defined as the vehicle used) can also be included. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate buffered saline, Ringer's solution, dextrose solution and Hank's solution. In addition, the pharmaceutical composition or formulation can also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.

The pharmaceutical composition comprises proteins, polysaccharides such as chitosan, polylactic acid, polyglycolic acid, and copolymers (such as latex functionalized sepharose, agarose, cellulose etc.), polymeric amino acids, amino acid copolymers, As well as large, slowly metabolized macromolecules such as lipid aggregates (such as oil droplets or liposomes). In addition, these carriers are immunostimulants (ie, adjuvants)
Can function as.

  For parenteral administration, an agent of the invention is a physiologically acceptable dilution comprising a pharmaceutical carrier which can be a sterile liquid such as water, oil, saline, glycerol or ethanol. It can be administered as an injectable dosage of a solution or suspension of the substance in the agent. In addition, auxiliary substances such as wetting or emulsifying agents, surfactants, pH buffering substances and the like can be present in the composition. Other ingredients of the pharmaceutical composition are of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil and mineral oil. In general, glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, especially for injectable solutions. The antibody can be administered in the form of a depot infusion or implant that can be formulated in a manner to allow sustained release of the active ingredient. One exemplary composition comprises 5 mg / mL monoclonal antibody formulated in an aqueous buffer consisting of 50 mM L-histidine, 150 mM NaCl, adjusted to pH 6.0 with HCl.

  Typically, the compositions are manufactured as injectables, either as liquid solutions or suspensions; solid forms suitable for solutions or suspensions in liquid vehicles prior to injection are also present. Can be manufactured. The formulation can also be emulsified or encapsulated in liposomes or microparticles such as polylactides, polyglycolides or copolymers for enhanced adjuvant effects, as discussed above (Langer , Science 249, 1527 (1990) and Hanes, Advanced Drug Delivery Reviews 28, 97-119 (1997)). The agents of the present invention can be administered in the form of a depot infusion or implant that can be formulated in a manner that allows for a sustained or pulsatile release of the active ingredient.

  Additional formulations suitable for other modes of administration include oral, nasal and pulmonary formulations, suppositories, and transdermal applications.

  For suppositories, binders and carriers include, for example, polyalkylene glycols or triglycerides; such suppositories contain active ingredient in the range of 0.5% to 10%, preferably 1% -2%. It can be formed from the mixture it contains. Oral formulations include excipients such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose and magnesium carbonate. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10% to 95% of active ingredient, preferably 25% to 70% To do.

  Topical application can result in transdermal or intradermal delivery. Topical administration can be facilitated by co-administration of the agent with cholera toxin or a detoxified derivative or subunit thereof, or other similar bacterial toxin (see Glenn et al., Nature 391, 851 (1998)). I want to be) Co-administration can be achieved by using the components as a mixture or linked molecule obtained by chemical cross-linking or expression as a fusion protein.

  Alternatively, transdermal delivery is performed using a skin patch or by transferosomes (Paul et al., Eur. J. Immunol. 25, 3521-24 (1995); Cevc et al., Biochem. Biophys. Acta 1368. 201-15 (1998)).

F. Methods of Diagnosis The present invention provides a method of detecting an immune response against Aβ peptide in a patient suffering from or suspected of having Alzheimer's disease. The method is particularly useful for monitoring a course of therapy being administered to a patient. The method can be used to monitor both therapeutic treatment in symptomatic patients and prophylactic treatment in asymptomatic patients. The method is useful for monitoring both active immunization (eg, antibodies produced in response to administration of an immunogen) and passive immunization (eg, measuring the level of administered antibody). It is.

1. Active immunization Some methods require determining a baseline value of the patient's immune response before administering a dose of the agent and comparing it to the value of the immune response after treatment. A significant increase in the value of the immune response (ie, greater than typical acceptable experimental error in repeated measurements of the same sample, expressed as one standard deviation from the average of such measurements) is positive (Ie, administration of the agent achieved or increased an immune response). If the value of the immune response does not change significantly or decreases, a negative treatment result is indicated. In general, patients undergoing an initial course of treatment with an immunogenic agent are expected to show an increased immune response with continuous dosing, which eventually reaches a stable level. Administration of the agent is generally continued while the immune response is increasing. Reaching a stable level indicates that treatment can be interrupted or the dosage or frequency can be reduced.

  In another method, control values (ie, mean and standard deviation) of the immune response are determined for the control group. Typically, the control group of individuals received no pretreatment. The value of the patient's immune response measured after administration of the therapeutic agent is then compared to a control value. A significant increase (eg, greater than 1 standard deviation from the mean) relative to the control value indicates a positive treatment result. A signal with no significant increase or decrease indicates a negative treatment outcome. Administration of the agent is generally continued while the immune response is increasing relative to the control value. As noted above, once a stable level is reached compared to the control value, it is an indication that treatment administration can be interrupted or the dosage or frequency can be reduced.

  In another method, control values (eg, mean and standard deviation) of the immune response are determined from a control group of individuals who have received treatment with a therapeutic agent and whose immune response has reached a stable level in response to treatment. . A measure of the patient's immune response is compared to a control value. If the level measured in the patient is not significantly different from the control value (eg, greater than 1 standard deviation), the treatment can be discontinued. If the patient's level is significantly below the control value, continued administration of the agent is observed. If the patient's level remains below the control value, it may indicate a change in treatment plan, such as the use of a different adjuvant.

  In another method, patients who are not currently receiving treatment but who have already undergone a course of treatment are monitored for an immune response to determine if resumption of treatment is necessary. The measured value of the patient's immune response can be compared to the value of the immune response that the patient has previously achieved after the previous course of treatment. A significant decrease compared to previous measurements (ie, greater than the typical tolerance range with repeated measurements of the same sample) indicates that treatment can be resumed. Alternatively, patient measurements can be compared to control values (mean plus standard deviation) determined in a group of patients after undergoing a course of treatment. Alternatively, patient measurements can be compared to control values for a group of prophylactically treated patients that remain disease free or a group of therapeutically treated patients that exhibit improved disease characteristics. In all cases, a significant decrease (ie, greater than the standard deviation) relative to the control level indicates that patient treatment should be resumed.

The tissue sample for analysis is typically blood, plasma, serum, mucosa or cerebrospinal fluid from the patient. Samples are analyzed for indicators of immune response to the desired amyloid component, such as any form of Aβ peptide. The immune response can be determined from the presence of, for example, antibodies or T-cells that specifically bind to components of interest such as Aβ peptides. ELISA methods for detecting antibodies specific for Aβ are described in the Examples section and can be applied to other peptide antigens. Methods for detecting reactive T-cells are well known in the art.

2. Passive immunization In general, the procedure for monitoring passive immunization is similar to the procedure for monitoring active immunization described above. However, the antibody profile after passive immunization typically shows an immediate peak of antibody concentration followed by an exponential decrease. Without further dosage, this decrease reaches pretreatment levels within a period of days to months, depending on the half-life of the administered antibody. For example, some human antibodies have a half-life of 20 days.

  In some methods, a baseline measurement of antibody to the patient's Aβ is performed prior to administration, a second measurement is performed immediately thereafter to determine peak antibody levels, and one or more additional measurements are performed to reduce antibody levels. At intervals for monitoring. Additional antibody doses are administered when the level of antibody reaches a baseline or a pre-determined percentage peak reduction baseline (eg, 50%, 25% or 10%). In some methods, a level below the peak or subsequently measured background is compared to a previously established reference level to construct a prophylactic or therapeutic treatment plan that is beneficial to other patients. If the measured antibody level is significantly lower than the reference level (eg, lower than the mean minus one standard deviation of the reference value of the group of patients enjoying the treatment benefit), administration of additional doses of antibody Show.

3. Diagnostic Kit The present invention further provides a diagnostic kit for performing the above-described diagnostic method. Typically, such a kit comprises an agent that specifically binds to an antibody against an amyloid plaque component, such as Aβ, or reacts with a T-cell specific for that component. The kit can also include a label. In order to detect antibodies against Aβ, the label is typically in the state of a labeled anti-idiotype antibody. For antibody detection, the agent can be supplied pre-bound to a solid phase, such as a well of a microtiter dish. For detection of reactive T-cells, a label that measures the proliferative response can be supplied as 3H-thymidine. The kit also typically includes a label that directs the use of the kit. This label may comprise a chart or other corresponding regime that correlates the level of antibody measured against Aβ or T-cells reactive with Aβ with the level of label measured. The term label refers to the written or recorded data attached to or associated with the kit at any time during manufacture, transportation, sale or use. For example, the term labels include advertising leaflets and booklets, packaging materials, instructions for use, audio or video cassettes, computer discs, and descriptions printed directly on the kit.

Example
I. Prophylactic Efficacy of Aβ against Alzheimer's Disease (AD) These examples are transgenic mice overexpressing APP with a mutation at position 717 (APP _717V → _F ) _predisposed to developing Alzheimer-like neuropathology The administration of Aβ42 peptide to is described. The generation and characteristics of these mice (PDAPP mice) are described in Games et al., Nature , ibid. These animals, in their heterozygous state, begin to deposit Aβ first at 6 months of age. By 15 months of age, they represent a level of Aβ deposition equal to that found in Alzheimer's disease. PDAPP mice were injected with aggregated Aβ ₄₂ (aggregated Aβ ₄₂ ) or phosphate buffered saline. Aggregated A [beta] ₄₂ were selected from the ability to induce antibodies to many epitope of A [beta].

A. Method 1. Mouse origin
Thirty PDAPP foreign female mice were randomly divided into the following groups: the 10 mice injected with aggregated A [beta] ₄₂ (1 animal died during the movement), the five is PBS / adjuvant or PBS 10 were injected and 10 were uninjected controls. Five mice were injected with peptides derived from the sequence of serum amyloid protein (SAP).

2. Preparation of immunogen Preparation of aggregated Aβ42: 2 milligrams of Aβ42 (US Peptide: lot K-42-12) was dissolved in 0.9 ml of water and 0.1 ml of 10 × PBS was added to make 1 ml. This was mixed by vortexing and incubated overnight at 37 ° C., where the peptide aggregated. Unused Aβ was stored at −20 ° C. as a dry lyophilized powder until next injection.

  When using such commercially available peptides for injection, the dry weight may include salt weight; the weights reported in the examples herein include the weight of the salt unless otherwise indicated. The exact mass of the peptide can be determined using standard assays for the preparation, such as nitrogen measurement in combination with a known composition.

3. Preparation for injection For each injection, 100 μg aggregated Aβ42 in PBS per mouse, 1: 1 for the first immunization, emulsified in complete Freund's adjuvant (CFA) and a final volume of 400 μl, followed by the same volume The immunogens were boosted at 2 weeks in complete Freund's adjuvant (IFA). Two more doses during IFA were given at 1 month intervals. Subsequent immunizations were performed in 500 μl PBS at 1 month intervals. Injections were delivered intraperitoneally (ip).

  PBS injections followed the same schedule, and mice were injected with a 1: 1 mixture of PBS / adjuvant at 400 μl per mouse or 500 μl PBS per mouse. SAP injections similarly followed the same schedule using a dose of 100 μg per injection.

4). Mouse blood titration, tissue preparation and immunohistochemistry The above methods are described in the following general materials and methods.

B. result
PDAPP mice were injected with either aggregated Aβ42 (aggregated Aβ42), SAP peptide or phosphate buffered saline. The group of PDAPP mice also remained as a non-injected positive control. The force value of mice against aggregated Aβ42 was monitored every other month from the 4th boost until the mice were 1 year old. Mice were sacrificed at 13 months. At all time points investigated, 8 out of 9 aggregated Aβ42 mice produced high antibody titers that remained high throughout the series of injections (titers higher than 1/10000). The ninth mouse was low but had a measurable titer of about 1/1000 (Figure 1, Table 1). SAPP-injected mice had titers from 1: 1,000 to 1: 30,000 for this immunogen, with only one mouse exceeding 1: 10,0000.

  PBS-treated mouse sera were titrated against aggregated Aβ42 at 6, 10 and 20 months. At 1/100 dilution, PBS mice were titrated against aggregated Aβ42, only exceeding 4 times background at one data point, and 4 times less than background at all other times (Table 3). The SAP-specific response could be ignored for all such time points where all titers were less than 300.

  Seven out of nine mice in the aggregated Aβ1-42 treatment group had no detectable amyloid in their brain. In contrast, brain tissue from mice in the SAP and PBS groups contained numerous amyloid deposits in the hippocampus and frontal and cingulate cortex. The pattern of deposition was similar to that of untreated controls, with characteristic involvement of vulnerable subregions such as the outer molecular layer of the dentate gyrus. One mouse from the Aβ1-42 injection group had a large decrease in amyloid burden limited to the hippocampus. Isolated plaques were confirmed in another Aβ1-42 treated mouse.

  Quantitative image analysis of amyloid burden in the hippocampus demonstrated that a dramatic reduction was achieved in Aβ42 (AN1792) -treated animals (FIG. 2). The median amyloid burden in the PBS group (2.22%) and the untreated control group (2.65%) was significantly greater than the group immunized with AN1792 (0.00%, p = 0.0005). In contrast, the median of the group immunized with SAP peptide (SAPP) was 5.74%. Brain tissue from untreated, control mice contained numerous Aβ amyloid deposits visualized with Aβ-specific monoclonal antibody (mAb) 3D6 in the hippocampus as well as in the retrocostal cortex. A similar amyloid deposition pattern was seen in mice immunized with SAPP or PBS (FIG. 2). In addition, these three latter groups have characteristic involvement of fragile subregions of the brain that are classically seen in AD, such as the outer molecular layer of the dentate gyrus in all three such groups. there were.

Brains that did not contain Aβ deposits typically also had no neuritic aspect visualized with human APP antibody 8E5 in PDAPP mice. All of the remaining groups of brains (SAP-injected, PBS and non-injected mice) had numerous neuritis aspects typical of untreated PDAPP mice. A small number of neuritis aspects were present in one mouse treated with AN1792, and one cluster of dystrophic neurites was seen in a second mouse treated with AN1792. As seen in hippocampal image analysis and FIG. 3, compared to the PBS receptor (median 0.28%, p = 0.0005), the substantial elimination of dystrophic neurites in AN1792-treated mice is substantially Indicated (median 0.00%).

  There was also no sign of inflammation-related astrocytosis in the brain of the Aβ1-42 injection group. Brains from other groups of mice contained GFAP-positive astrocytes typical of gliosis involving abundant and clustered Aβ plaques. A subset of GFAP-reacted slides was counterstained with thioflavin S to locate Aβ deposits. GFAP-positive astrocytes were associated with Aβ plaques in SAP, PBS and untreated controls. Such an association was not seen in plaque-negative Aβ1-42 treated mice, while gliosis involving minimal plaque was confirmed in one mouse treated with AN1792.

  Image analysis for the posterior calcaneus cortex shown in FIG. 4 shows that the reduction in astrocytosis is significant at a median of 1.56% for the group treated with AN1792, but the group immunized with SAP peptide, PBS, or not. The treatment confirmed a median value greater than 6% (p = 0.0017).

  Evidence from a subset of Aβ1-42 and PBS-injected mice showed that MHCII immunoreactivity involving plaques was absent in Aβ1-42-injected mice, consistent with a lack of Aβ-related inflammatory responses.

  Mouse brain slices also reacted with mAb specific for MAC-1, a monoclonal antibody specific for cell surface proteins. MAC-1 (CD11b) is a member of the integrin family and exists as a heterodimer with CD18. The CD11b / CD18 complex is present on monocytes, macrophages, neutrophils and natural killer cells (Mak and Simard). MAC-1-reactive cell types present in the brain will be microglia based on similar phenotypic morphology in sections immunoreactive with MAC-1. MAC-1 labeling involving plaques was lower in the brains of mice treated with AN1792 compared to the PBS control group, which was found to be consistent with the lack of inflammatory response induced by Aβ.

C. Conclusion Absence of Aβ plaques and changes in reactive neurons and glues in the brains of Aβ1-42-injected mice have no or very little amyloid deposited in their brains, such as gliosis and neuritic conditions It indicated that there was no pathological outcome. PDAPP mice treated with Aβ1-42 show essentially the same absence of pathology as control non-transgenic mice. Thus, injection of Aβ1-42 is highly effective in preventing or eliminating human Aβ deposition from brain tissue and subsequently eliminating neuronal and inflammatory degenerative changes. That is, administration of Aβ peptide can have both prophylactic and therapeutic benefits in preventing AD.

II. Dose-response experiment 300, 100, 33, 11, 3.7, 1.2, 0.4 in 5-week-old female Swiss Webster mice (N = 6 per group) formulated in CFA / IFA administered intraperitoneally Alternatively, immunization was performed with 0.13 μg of Aβ. Three doses were given at biweekly intervals, followed by a fourth one month later. The first dose was emulsified with CFA and the remaining dose was emulsified with IFA. The animals were bled 4-7 days after each immunization starting from the second dose to measure antibody titer. Three subsets of animals immunized with 11, 33 or 300 μg of antigen were further monitored for 4 months after the fourth immunization to further monitor the reduction in antibody response over the immunogen dose range. Blood was collected at approximately one month intervals. These animals received a fifth final immunization 7 months after the start of the experiment. Animals were sacrificed one week later to measure antibody responses to AN1792 and to perform toxicological analysis.

  A decreasing dose response was observed from 300 to 3.7 μg, with no response at the two lower doses. The average antibody titer is about 1: 1000 after 3 doses of 11-300 μg of antigen and about 1: 10,000 after 4 doses (see FIG. 5).

  Antibody titers increased dramatically for all, but the lowest dose group had a GMT increase in the range of 5- to 25-fold after the third immunization. A low antibody response could be detected with 0.4 μg receptor. The 1.2 and 3.7 μg groups had titers comparable to about 1000 GMT, and the highest 4 doses were grouped together at about 25,000 GMT, while the 33 μg dose group was 3000 low GMT. It was. After the fourth immunization, the increase in titer was more moderate in most groups. There was a clear dose response in the range of 0.14 μg to 11 μg lower antigen dose group, from 0.14 μg receptor with no detectable antibody to 11 μg receptor at 36,000 GMT. Again, the titers of the four highest dose groups of 11-300 μg were combined. Thus, after two immunizations, antibody titers depending on the antigen dose ranged from 0.4 to 300 μg. By the third immunization, the titers of the four highest doses were all equivalent and they remained at a stable level after further immunization.

  One month after the fourth immunization, the titer was 2- to 3-fold higher in the 300 μg group than measured from blood 5 days after immunization (FIG. 6). This observation suggests that the peak native antibody response occurs later than 5 days after immunization. At this point, a more modest (50%) increase was seen in the 33 μg group. The 300 μg dose group sharply decreased GMT to about 70% 2 months after the last dose. After a further month, the slope weakened to 45% (100 μg) and about 14% at 33 and 11 μg. That is, the rate of circulating antibody titer ramps after immunization ends appears sharply in the first month after the peak response, and then appears in two aspects, followed by a slower rate of decline.

  Antibody titers and the kinetics of the response of these Swiss Webster mice are similar to those of young heterozygous PDAPP transgenic mice immunized in a parallel fashion. Effective dosages for inducing an immune response in humans are typically similar to effective dosages in mice.

III. Screening for therapeutic efficacy against stable AD This assay is designed to test immunogenic agents for activity to stop or improve the neuropathological features of AD in aging mice. Immunization with 42 amino acids long Aβ (AN1792) was started when amyloid plaques were already present in the brain of PDAPP mice.

During the period of this experiment, untreated PDAPP mice develop numerous neurodegenerative changes similar to those seen in AD ( Games et al. , Ibid . And Johnson-Wood et al., Proc. Natl. Acad. Sci. USA 94, 1550-1555 (1997)). Aβ deposition in amyloid plaques is accompanied by a degenerative neuronal response consisting of vagal axons and dendritic elements called dystrophic neurites. An amyloid deposit surrounded by and containing dystrophic neurites is referred to as a neuroinflammatory aspect. In both AD and PDAPP mice, dystrophic neurites have a prominent globular structure, are immunoreactive with a panel of antibodies that recognize APP and cytoskeletal components, and complex subcellular at the ultrastructural level Represents a degenerative change in These features allow the measurement of the formation of selective and reproducible neuroinflammatory aspects associated with disease in the PDAPP brain. The dystrophic neuronal component of the PDAPP neuroinflammatory aspect is easily visualized using an antibody specific for human APP (monoclonal antibody 8E5) and can be readily measured by computerized image analysis. Therefore, in addition to measuring the effect of AN1792 on amyloid plaque formation, we monitored the effect of this treatment on the development of neuroinflammatory dystrophy.

  Astrocytes and microglia are non-neuronal cells that respond to and influence the degree of neuronal injury. GFAP-positive astrocytes and MHC II-positive microglia are usually observed in AD, and their activation increases the severity of the disease. We therefore also monitored the occurrence of reactive astrocytosis and microgliosis in AN1792-treated mice.

A. Materials and Methods 48, 11-11.5 month old heterozygous female PDAPP mice from Charles River were randomly divided into two groups: 24 mice were immunized with 100 μg AN1792. And 24 mice were immunized with PBS (each combined with Freund's adjuvant). The AN1792 and PBS groups were again divided when they reached -15 months of age. At 15 months of age, approximately half of each AN1792- and PBS-treated animal was euthanized (n = 10 and 9 respectively) and the rest continued to be immunized until ending at ˜18 months (n = 9 and respectively 12). A total of 8 animals (5 AN1792 and 3 PBS) died during the experiment. In addition to immunized animals, untreated PDAPP mice of 1 age (n = 10), 15 months of age (n = 10) and 18 months of age (n = 10) were measured for Aβ and APP levels in the brain by ELISA. Included in the comparison; 1-year-old animals were also included in the immunohistochemical analysis.

  The method is as in Example 1 unless otherwise indicated. AN1792 US Peptide Lot 12 and California Peptide Lot ME0339 were used to prepare 6 immunizing antigens administered prior to 15 months. California peptide lots ME0339 and ME0439 were used for 3 further immunization doses between 15 and 18 months.

  For immunization, 100 μg AN1792 or PBS alone in 200 μl PBS is emulsified 1: 1 (volume / volume) in complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA) or PBS at a final dose of 400 μl. did. The first immunization was delivered using CFA as an adjuvant, the next 4 doses were given using IFA, and the last 4 doses were given using PBS without adjuvant only. . A total of 9 immunizations were given, the first 3 doses on a 2 week schedule over 7 months, followed by the remaining injections at 4 week intervals. The 4-month treatment group euthanized at 15 months of age received only the first 6 immunizations.

B. Result 1. Effect of Aβ (AN1792) treatment on amyloid burden The results of AN1792 treatment on cortical amyloid burden determined by quantitative image analysis are shown in FIG. The median cortical amyloid burden was 0.28% in the untreated 12-month-old PDAPP mouse group and was a value representing the plaque burden in the mice at the start of the experiment. At 18 months, amyloid burden increased more than 17-fold in PBS-treated mice to 4.87%, while AN1792-treated mice greatly decreased to only 0.01% amyloid burden, 12 months untreated and It was significantly lower than both 15- and 18-month PBS-treated groups. This amyloid burden was significantly reduced at both the 15- (96% decrease; p = 0.003) and 18-month (> 99% decrease; p = 0.0002) AN1792 receptors.

  Typically, cortical amyloid deposits in PDAPP mice begin with the frontal and retrotarsal cortex (RSC) and progress in the ventral-lateral direction to include the temporal-entorhinal cortex (EC). Little or no amyloid was found in ECs of 12-month-old mice, approximately when AN1792 was first administered. Four months after AN1792 treatment, amyloid deposition was greatly reduced in RSC, and progressive EC involvement was completely eliminated by AN1792. The latter observation indicated that AN1792 completely stopped the progression of amyloid, usually affecting the temporal and temporal cortex, and also stopped or improved the deposition in RSC.

  The enormous effect of AN1792 treatment on the development of cortical amyloid burden in PDAPP mice was further demonstrated in groups treated for 7 months up to 18 months. Almost complete absence of cortical amyloid was seen in AN1792-treated mice, with a general lack of diffuse plaque and reduced dense deposits.

2. Cellular and morphological changes associated with Aβ (AN1792) -treatment Aβ-positive cell populations were typically found in areas of the brain containing amyloid deposits. Of note, there were few or no cortical amyloid plaques in some brains of the AN1792 receptor. The majority of Aβ immunoreactivity appeared to be contained within cells containing large leaflets or aggregated cell bodies. Phenotypically, these cells resemble activated microglia or monocytes. They were immunoreactive with antibodies that recognize ligands expressed by activated monocytes and microglia (MHCII and CD11b) and were optionally associated with the walls and lumens of blood vessels. Comparison of near-adjacent sections labeled with Aβ and MHCII-specific antibodies revealed that similar patterns of these cells were recognized by both classes of antibodies. Detailed investigation of AN1792-treated brains revealed that MHCII-positive cells are confined to the limited amyloid vicinity that remains in such animals. Under the fixation conditions used, cells were not immunoreactive with antibodies recognizing T cell (CD3, CD3e) or B cell (CD45RA, CD45RB) ligands or leukocyte common antigen (CD45), but cross-reacted with monocytes. It was reactive with an antibody that recognizes leucosialin (CD43). Such cells were not found in PBS-treated mice.

  PDAPP mice consistently develop heavy amyloid deposits in the outer molecular layer of the hippocampal dentate gyrus. This deposition forms a clear streak under the perforant pathway, an area classically containing amyloid plaques in AD. The characteristic appearance of these deposits in PBS-treated mice was similar to that already characterized in untreated PDAPP mice. Amyloid deposits consisted of both diffuse and densified plaques within a continuous band. In contrast, this pattern changed dramatically in many brains from AN1792-treated mice. Hippocampal amyloid deposits did not contain diffuse amyloid, and the banded pattern was completely destroyed. Instead, there were numerous abnormal punctate structures reactive with anti-Aβ antibodies, some of which appeared to be cells containing amyloid.

  MHCII-positive cells were often observed near the extracellular amyloid in AN1792-treated animals. The association pattern of Aβ-positive cells and amyloid was very similar in several brains derived from AN1792-treated mice. Such monocytic cell distribution was confined proximal to amyloid deposits and was not present at all in other brain regions without Aβ plaques.

  Quantitative image analysis of MHCII- and MACI-labeled sections showed that immunoreactivity was found in R179 and hippocampus of AN1792-treated mice compared to the PBS group reached significantly using measurements of MACI reactivity in the hippocampus. The upward trend was clarified.

  These results indicate active cellular removal of amyloid in areas of the brain with plaques.

3. AN1792 effect on Aβ levels: ELISA measurements (a) Cortical levels In untreated PDAPP mice, the median level of total Aβ in the cortex at 12 months was 1,600 ng / g, which was 8,700 ng / g by 15 months (Table 4). At 18 months, the value was 22,000 ng / g, increasing more than 10 times during the experiment. PBS-treated animals had 8,600 ng / g total Aβ at 15 months, which rose to 19,000 ng / g at 18 months. In contrast, AN1792-treated animals had 81% less total Aβ than the PBS-immunized group at 15 months (1,600 ng / g). Significantly less (p = 0.0001)
) Total Aβ (5,200 ng / g) was found when comparing the AN1792 and PBS groups at 18 months (Table 4) and represents a 72% reduction in Aβ when present. Similar results were obtained when comparing cortical levels of Aβ42, ie the AN1792-treated group contained less Aβ42, in which case the difference between the AN1792 and PBS groups was 15 months (p = 0.04) and 18 Significant at both months (p = 0.0001, Table 4).

(B) Hippocampal level In untreated PDAPP mice, the median total hippocampal level of Aβ was 15,000 ng / g at 12 months of age, which was 51,000 ng / g at 15 months and 81,000 ng / g at 18 months. (Table 5). Similarly, PBS immunized mice showed values of 40,000 ng / g and 65,000 ng / g at 15 and 18 months, respectively. AN1792 immunized animals exhibited low total Aβ, especially 25,000 ng / g and 51,000 ng / g, respectively, at 15 and 18 months. The 18-month AN1792-treated group values were significantly lower than the PBS-treated group (p = 0.0105; Table 5). Measurement of Aβ42 gave the same pattern of results, ie the level of the AN1792-treated group was significantly lower than the PBS group at the 18-month assessment (39,000 ng / g vs. 57,000 ng / g, respectively; p = 0.002) ( Table 3).

(C) Cerebellar level
In 12 month naïve PDAPP mice, the median cerebellar level of total Aβ was 15 ng / g (Table 6). At 15 months, this median increased to 28 ng / g and by 18 months to 35 ng / g. PBS-treated animals showed a median total Aβ value of 21 ng / g at 15 months and 43 ng / g at 18 months. AN1792-treated mice were found to have 22 ng / g total Aβ at 15 months and significantly lower (p = 0.002) total Aβ (25 ng / g) than the corresponding PBS group at 18 months. (Table 6).

4). Effect of AN1792 treatment on APP level
APP-α and full-length APP molecules both contain all or part of the Aβ sequence, ie may be affected by the generation of an immune response to which AN1792- is directed. Studies to date have recorded a slight increase in APP levels as neuropathology increases in PDAPP mice. In the cortex, levels of either APP-α / FL (full length) or APP-α are essentially not except that APP-α was reduced to 19% with AN1792-treated vs. PBS-treated at 18 months. Did not change. The 18-month AN1792-treated APP value was not significantly different from the 12-month and 15-month untreated and 15-month PBS groups. In all cases, APP values remained within the normal range seen in PDAPP mice.

5. Effect of AN1792 treatment on neurodegenerative and gliotic pathology The loading of the neuroinflammatory phase was 15 months of age (84%; p = 0.03) in the frontal cortex of AN1792-treated mice compared to the PBS group And significantly decreased at both 18 months of age (55%; p = 0.01) (FIG. 8). The median load of the neuroinflammatory phase increased from 0.32% to 0.49% in the PBS group between 15 and 18 months of age. This was in stark contrast to the large reduction in the occurrence of neuroinflammatory aspects in the AN1792 group, which had a median neuroinflammatory aspect burden of 0.05% and 0.22% in the 15 and 18 months old groups, respectively.

  Immunization with AN1792 is well tolerated, and reactive astrocytosis was also observed in the PBS group at both 15 months of age (56%; p = 0.011) and 18 months of age (39%; p = 0.028). When compared to the AN1792-treated mice, there appears to be a significant decrease in RSC (FIG. 9). The median percent of astrocytosis in the PBS group increased from 4.26% to 5.21% between 15 and 18 months. AN1792-treatment suppressed the occurrence of astrocytosis at both time points to 1.89% and 3.2%, respectively. This suggests that the neural network was not damaged by the removal process.

6). Antibody Response As described above, 11 month old heterozygous PDAPP mice (N = 24) were challenged with 100 μg AN1792 emulsified with Freund's adjuvant in a series of 5 immunizations at 0, 2, 4, 8, and 12 weeks. Sensitization was administered intraperitoneally and the sixth time was immunized with PBS only (no Freund's adjuvant) at 16 weeks. As a negative control, a set of 24 parallel age-matched transgenic mice were immunized with PBS emulsified with the same adjuvant and delivered on the same schedule. The animals were bled within 3-7 days after each immunization starting from the second dose. The antibody response to AN1792 was measured by ELISA. The geometric mean titer (GMT) for animals immunized with AN1792 was approximately 1,900, 7,600 and 45,000 after the second, third and last (sixth) doses, respectively. After the sixth immunization, Aβ-specific antibodies in the control animals were not measured.

About half of the animals were further immunized at about 20, 24 and 27 weeks for 3 months. Each of these doses was delivered with PBS vehicle alone without Freund's adjuvant. Average antibody titers remained unchanged during this period. In practice, antibody titers appeared to be stable over the 4th to 8th blood draws corresponding to the period covering the 5th to 9th injections.

  Determining whether Aβ-specific antibodies detected in the serum of AN1792-treated mice were induced by immunization is also related to deposited brain amyloid, a subset of sections of AN1792- and PBS-treated mice Was reacted with an antibody specific for mouse IgG. In contrast to the PBS group, Aβ plaques in the AN1792-treated brain were covered with endogenous IgG. Differences between the two groups were seen in both 15- and 18-month groups. Despite the presence of heavy amyloid burden in these mice, it was particularly notable that the PBS group was unlabeled. These results indicate that immunization with synthetic Aβ protein generates antibodies that recognize and bind Aβ in amyloid plaques in vivo.

7). Cellular immune response Spleens were removed from 9 AN1792-immunized mice and 12 PBS-immunized 18 month old PDAPP mice 7 days after the 9th immunization. Splenocytes were isolated and cultured for 72 hours in the presence of Aβ40, Aβ42 or Aβ40-1 (reverse protein order). Mitogen CoA served as a positive control. The optimal response was obtained with> 1.7 μM protein. Cells from all nine AN1792-treated animals proliferated in response to either Aβ1-40 or Aβ1-42 protein and were taken up at equal levels for both proteins (FIG. 10, upper panel). . It did not react with Aβ40-1 reverse protein. Cells from control animals did not respond to any Aβ protein (Figure 10, lower panel).

C. Conclusions The results of this experiment show that AN1792 immunization of PDAPP mice with existing amyloid deposits delays and prevents progressive amyloid deposition, and consequently neuropathology in the brain of aged PDAPP mice To delay the change. Immunization with AN1792 essentially stops structural amyloid development, usually overwhelmed by amyloidosis. That is, administration of Aβ peptide has a therapeutic benefit in the treatment of AD.

IV. Screening for Aβ Fragments 100 PDAPP mice, 9-11 months old, were immunized with 9 different regions of APP and Aβ to determine which epitopes produced a potent response. Nine different immunogens and one control were injected ip as described above. The immunogen included four human Aβ peptide conjugates 1-12, 13-28, 32-42, 1-5, all coupled to sheep anti-mouse IgG via a cysteine linkage; APP polypeptide amino acid 592 -695, aggregated human Aβ1-40 and aggregated human Aβ25-35 and aggregated rodent Aβ42. Aggregated Aβ42 and PBS were used as positive and negative controls, respectively. Ten mice were used per treatment group. Titers were monitored as described above and mice were euthanized at the end of 4 months after injection. Histochemistry, Aβ levels and toxicological analysis were measured postmortem.

A. Materials and Methods Immunogen Preparation Bound Aβ Peptide Preparation: Four human Aβ peptide conjugates (amino acid residues 1-5, 1-12, 13-28 and 33-42 each bound to sheep anti-mouse IgG) Prepared by coupling through an artificial cysteine added to the Aβ peptide using the crosslinking reagent sulfo-EMCS. The Aβ peptide derivative was synthesized using the following final amino acid sequence. In each case, the position of the inserted cysteine residue is underlined. The Aβ13-28 peptide derivative also had two glycine residues before the indicated carboxyl terminal cysteine.

  To prepare the coupling reaction, 10 mg sheep anti-mouse IgG (Jackson ImmunoResearch Laboratories) was dialyzed overnight against 10 mM sodium borate buffer, pH 8.5. The dialyzed antibody was then concentrated to a volume of 2 mL using an Amicon Centriprep tube. 10 mg sulfo-EMS.

  [N (γ-maleimidocaproyloxy) succinimide] (Molecular Science Co.) was dissolved in 1 mL of deionized water. A 40-fold molar excess of sulfo-EMCS was added dropwise to the sheep anti-mouse IgG with stirring and the solution was stirred for an additional 10 minutes. Activated sheep anti-mouse IgG was purified and the buffer was equilibrated with 0.1 M NaPO4, 5 mM EDTA, pH 6.5, 10 mL gel filtration column (Pierce Presto Column from Pierce Chemicals). Antibody-containing fractions identified by absorption at 280 nm were pooled and diluted to a concentration of about 1 mg / mL using 1.4 mg per OD as the extinction coefficient, 40-fold molar excess of Aβ peptide Was dissolved in 20 mL of 10 mM NaPO4, pH 8.0, but for the Aβ33-42 peptide, 10 mg was first dissolved in 0.5 mL DMSO and then diluted to 20 mL with 10 mM NaPO4 buffer. In addition to sheep anti-mouse IgG and shaken for 4 hours at room temperature, the resulting conjugate was concentrated to a final volume of less than 10 mL using Amicon Centriprep tubes and then dialyzed against PBS. The buff The conjugate was exchanged and free peptide was removed The conjugate was passed through a 0.22 μm-pore size filter for sterilization and divided into 1 mg fractions and stored frozen at −20 ° C. Concentration of conjugate Was determined using equine IgG as a standard curve using the BCA protein assay (Pierce Chemicals) Binding was evidenced by an increase in the molecular weight of the bound peptide relative to activated sheep anti-mouse IgG. The 5 sheep anti-mouse conjugate was a pool of two bindings, the rest from one preparation.

2. Preparation of Aggregated Aβ Peptides Human 1-40 (AN1528: California Peptide, Lot ME0541), Human 1-42 (AN1792: California Peptide, Lots ME0339 and ME0439), Human 25-35 and Rodent 1-42 (California Peptide, Lot ME0218) Peptides were freshly solubilized in the preparation of each set of injections from lyophilized powder that had been dried and stored at -20 ° C. For this purpose, 2 mg of peptide was added to 0.9 ml of deionized water and the mixture was vortex mixed to make a relatively homogeneous solution or suspension. Of these, AN1528 was the only peptide soluble at this stage. An aliquot of 100 μl 10 × PBS (1 × PBS: 0.15 M NaCl, 0.01 M sodium phosphate, pH 7.5) was then added, at which point AN1524 began to precipitate. The suspension was vortexed again and incubated overnight at 37 ° C. for use the next day.

Preparation of pBx6 protein: Expression plasmid encoding pBx6 (100 amino acid bacteriophage MS-2 polymerase N-terminal leader sequence followed by fusion protein consisting of amino acids 592-695 of APP (βAPP)) It was constructed as described by et al., J. Biol. Chem. 265, 4492-4497 (1990). The plasmid was transfected into E. coli and the protein was expressed by induction of a promoter. Bacteria were lysed in 8M urea and pBx6 was partially purified by preparative SDS PAGE. Fractions containing pBx6 were confirmed by Western blot using rabbit anti-pBx6 polyclonal antibody, pooled, concentrated using Amicon Centriprep tubes, and dialyzed against PBS. The purity of the preparation estimated by Coomassie Blue stained SDS PAGE was about 5-10%.

B. Results and discussion Experimental Design 100 male and female heterozygous PDAPP transgenic mice 9- to 11 months of age were obtained from Charles River Laboratories and Taconic Laboratory. Mice were divided into 10 groups immunized with different regions of Aβ or APP combined with Freund's adjuvant. Animals were divided as much as possible to match gender, age, pedigree and source within the group. The immunogen contained four Aβ peptides derived from sequences 1-5, 1-12, 13-28 and 33-42 of human origin, each bound to sheep anti-mouse IgG; four aggregates Aβ peptide, human 1-40 (AN1528), human 1-42 (AN1792), human 25-35, and rodent 1-42; and a fusion polypeptide, pB × 6, APP amino acid residue 592 Includes -695. The tenth group was immunized with PBS combined with adjuvant as a control.

  For each immunization, 100 μg of each Aβ peptide (in 200 μl of PBS), or 200 μg of APP derivative pB × 6 (in the same volume of PBS) or PBS alone, 1: 1 (volume) : Volume) of complete Freund's adjuvant (CFA) to a final volume of 400 μl, followed by booster immunization with the same amount of immunogen in incomplete Freund's adjuvant (IFA) for the next 4 doses, and PBS was used for the last administration. Immunizations were delivered intraperitoneally on a biweekly schedule for the first three doses and then monthly thereafter. To measure antibody titers, animals were bled 4-7 days after each immunization, starting after the second dose. The animals were euthanized approximately 1 week after the last dose.

2. Aβ and APP levels in the brain Approximately 4 months after immunization with various Aβ peptides or APP derivatives, the brain was removed from the salt-perfusate. One hemisphere was prepared for immunohistochemical analysis and the second was used for quantification of Aβ and APP levels. To determine the concentration of various forms of β-amyloid peptide and amyloid precursor protein, hemispheres were dissected and homogenates of hippocampal, cortical and cerebellar regions were prepared with 5M guanidine. These were diluted and the levels of amyloid and APP were quantified by comparison with a series of standard dilutions of known concentrations of Aβ peptide or APP in an ELISA format.

  The median concentration of total Aβ for the control group immunized with PBS was 5.8 times higher in the hippocampus than in the cortex (4,221 ng / g of cortex, 24,318 ng / g median hippocampus). The median level (23.4 ng / g tissue) in the cerebellum of the control group was about 1,000 times lower than the hippocampus. These levels were similar to those previously reported for this age heterozygous PDAPP transgenic mouse (Johnson-Woods et al., 1997, ibid).

  With respect to the cortex, a subset of treatment groups had median total Aβ and Aβ1-42 levels that were significantly different from the control group (p <0.05), and these animals were AN1792, rodent Aβ1 as shown in FIG. -42 or Aβ1-5 peptide conjugate was received. Median levels of total Aβ for these treatment groups were reduced to 75%, 79%, and 61%, respectively, compared to controls. There was no discernable correlation between Aβ-specific antibody titers and Aβ levels in the cortical regions in the brain in any group.

  In the hippocampus, the median decrease in total Aβ associated with AN1792 treatment (46%, p = 0.0543) was greater than that observed in the cortex (75%, p = 0.0002). However, the magnitude of the decrease was greater in the hippocampus than in the cortex, with a net decrease of 3,171 ng / g tissue in the cortex, compared with a net decrease of 11,186 ng / g tissue in the hippocampus. For groups of animals that received rodent Aβ1-42 or Aβ1-5, the median total Aβ levels were reduced to 36% and 26%, respectively. However, considering the small size of the group and the high variability in amyloid peptide levels between animals in both groups, such a decrease was not significant. None of the treatment-induced reductions became significant when Aβ1-42 levels were measured in the hippocampus. That is, because of the smaller Aβ loading in the cortex, changes in this region are a more sensitive indicator of treatment effect. The changes in Aβ levels measured by ELISA in the cortex are similar, but are not identical to the results from immunohistochemical analysis (see below).

  Total Aβ was also measured in the cerebellum, which is typically least affected by AD disease. The median Aβ concentration of any group immunized with various Aβ peptides or APP derivatives was not different from the control group in this area of the brain. This result suggests that treatment does not affect non-pathological levels of Aβ.

  APP concentrations were also determined by ELISA in the cortex and cerebellum from treated and control mice. Two different APP assays were used. The first is called APP-α / FL, which recognizes APP-alpha (α, secreted from APP cleaved within the Aβ sequence), and the full-length form of APP (FL), while the second is Recognize only APP-α. In contrast to the treatment-related decrease in Aα in the treatment group subset, APP levels did not change with all treatments compared to control animals. These results indicate that immunization with Aβ peptide does not reduce APP; rather, the treatment effect is specific to Aβ.

  In summary, total Aβ and Aβ1-42 levels were significantly reduced in the cortex by treatment with AN1792, rodent Aβ1-42 or Aβ1-5 conjugate. In the hippocampus, total Aβ was significantly reduced only with AN1792 treatment. Changes in Aβ or APP levels involving other treatments in the hippocampus, cortex or cerebellum area were not significant.

2. Histochemical analysis Three groups immunized with Aβ peptide conjugates Aβ1-5, Aβ1-12 and Aβ13-28, prepared brains from a subset of six groups for histochemical analysis; full length Aβ aggregation 2 groups immunized with objects AN1792 and AN1528, and a control group treated with PBS. The results of image analysis of amyloid burden in brain sections from these groups are shown in FIG. There was a significant decrease in amyloid burden in the cortical areas of the three treatment groups versus the control animals. The greatest reduction in amyloid burden was observed in the group receiving AN1792, where the mean value decreased to 97% (p = 0.001). Significant reduction was also observed in animals treated with AN1528 (95%, p = 0.005) and Aβ1-5 peptide conjugate (67%, p = 0.02).

The results obtained by quantification of total Aβ or Aβ1-42 by ELISA and amyloid burden by image analysis were somewhat different. Treatment with AN1528 significantly affected the level of cortical amyloid burden as measured by quantitative image analysis, but not the total Aβ concentration in the same area as measured by ELISA. The difference between these two results may be due to the specificity of the assay. Image analysis measures only insoluble Aβ aggregates in plaques. In contrast, ELISA measures all Aβ forms of both soluble and insoluble monomers and aggregates. Since disease pathology is thought to be a form of Aβ associated with insoluble plaques, image analysis is more sensitive to reveal the effects of treatment. However, ELISA is a faster and easier assay and is very useful for screening purposes. Further ELISA
Can reveal the reduction associated with treatment of Aβ greater in relation to plaque than in total Aβ.

  In order to determine if Aβ-specific antibodies induced by immunization of treated animals reacted with amyloid deposited in the brain, a subset of sections from treated animals and control mice were specific for mouse IgG. And reacted with specific antibodies. In contrast to the PBS group, Aβ-containing plaques are endogenous for animals immunized with the Aβ peptide conjugates Aβ1-5, Aβ1-12 and Aβ13-28; and full-length Aβ aggregates AN1792 and AN1582. Covered with IgG. Brains from animals immunized with other Aβ peptides or APP peptide pB × 6 were not analyzed in this assay.

3. Measurement of antibody titer Mice were bled 5 times in total starting from the second immunization, 4-7 days after each immunization. Antibody titers were measured as Aβ1-42 binding antibodies using a sandwich ELISA using a plastic multi-well plate coated with Aβ1-42. As shown in FIG. 13, peak antibody titers were induced after the fourth dose for the four immunizing agents, and the maximum titer of AN1792-specific antibody was induced: AN1792 (peak GMT: 94,647), AN1528 (peak GMT: 88,231), Aβ1-12 conjugate (peak GMT: 47,216) and rodent Aβ1-42 (peak GMT: 10,766). The titers in these groups decreased somewhat after the 5th and 6th doses. For the remaining 5 immunogens, peak titers were reached after the 5th or 6th dose and these were much lower than the 4 highest titer groups: Aβ1-5 conjugate ( Peak GMT: 2,356), pB × 6 (peak GMT: 1,986), Aβ13-28 conjugate (peak GMT: 1,183), Aβ33-42 conjugate (peak GMT: 658), Aβ25-35 conjugate (peak GMT: 125 ). Antibody titers against homologous peptides were also measured for a subset of immunogens using the same ELISA sandwich format and such groups were identified as Aβ1-5, Aβ13-28, Aβ25-35, Aβ33-42 and rodent Aβ1- Immunized with 42. These titers were approximately the same as the titers measured against Aβ1-42, except for the titers associated with rodent Aβ1-42 immunogens that were about twice as high as antibody titers against homologous immunogens. The magnitude of individual animal AN1792-specific antibody titers, or the mean of the treatment group, did not correlate with potency measured as a decrease in Aβ in the cortex.

4). Lymphocyte proliferative response Aβ-dependent lymphocyte proliferation was measured using spleen cells collected approximately 1 week after the last 6 immunizations. Freshly collected cells (105 / well) were cultured for 5 days in the presence of 5 μM stimulatory concentration of Aβ1-40. A subset of cells from 7 out of 10 groups was also cultured in the presence of reverse peptide Aβ40-1. As a positive control, additional cells were cultured with T cell mitogen (PHA) and as a negative control, cells were cultured without the addition of peptide.

  Lymphocytes from most animals proliferated in response to PHA. There was no significant response to the Aβ40-1 reverse peptide. Cells from animals immunized with the larger aggregated Aβ peptide, AN1792, rodent Aβ1-42 and AN1528, proliferate well when stimulated with Aβ1-40, with the highest cpm being the receptor for AN1792. there were. One animal from each group immunized with Aβ1-12 conjugate, Aβ13-28 conjugate and Aβ25-35 grew in response to Aβ1-40. The remaining groups that received Aβ1-5 conjugate, Aβ33-42 conjugate pB × 6 or PBS had no animals with Aβ-stimulated responses. These results are summarized in Table 7 below.

  These results indicate that AN1792 and AN1528 stimulate a strong T cell response, most often the CD4 + phenotype. The absence of an Aβ-specific T cell response in animals immunized with Aβ1-5 is that the peptide epitope recognized by CD4 + T cells is usually about 15 amino acids long, but shorter peptides are less efficient. It's not surprising because it can function. That is, the majority of helper T cell epitopes for the four binder peptides appear to be in the IgG binder partner rather than in the Aβ region. This hypothesis is supported by the very low incidence of proliferative responses for animals in each treatment group. Since Aβ1-5 conjugates are effective in significantly lowering Aβ levels in the brain, the key effector induced by immunization with this peptide in the obvious absence of Aβ-specific T cells. The immune response will be an antibody.

  The lack of T-cells and a low antibody response from the fusion peptide pBx6 encompassing APP amino acids 592-695 including all Aβ residues may be due to the poor immunogenicity of this particular preparation. The poor immunogenicity of Aβ25-35 aggregates may be because the peptides are too small to contain good T cell epitopes that help to induce an antibody response. If this peptide is bound to a carrier protein, it is likely to be more immunogenic.

V. Preparation of polyclonal antibodies for passive protection
125 non-transgenic mice were immunized with adjuvant added to Aβ and euthanized at 4-5 months. Blood was collected from immunized mice. IgG was separated from other blood components. Antibodies specific for the immunogen were partially purified by affinity chromatography. Approximately 0.5 to 1 mg of immunogen specific antibody was obtained per mouse, giving a total of 60 to 120 mg.

VI. Passive immunization with antibodies against Aβ Each group of 7-9 month old PDAPP mice was injected with 0.5 mg of polyclonal anti-Aβ or specific anti-Aβ monoclonal in PBS as indicated below. All antibody preparations were purified to have low endotoxin levels. Monoclonal injects mice with Aβ fragments or longer forms of Aβ, prepares hybridomas, and hybridomas of antibodies that specifically bind to the desired fragments of Aβ without binding to other non-overlapping fragments of Aβ. By screening, fragments can be prepared.

  Mice are injected ip as needed for a period of 4 months to maintain circulating antibody concentrations as measured by ELISA titers greater than 1/1000 as determined by ELISA against Aβ42 or other immunogens. It was. Titers were monitored as described above and mice were euthanized at the end of 6 months of injection. Histochemistry, Aβ levels and toxicology were performed postmortem. Ten mice were used per group. Further experiments on passive immunization are described in Examples XI and XII below.

VII. Comparison of various adjuvants This example compares CFA, alum and oil-in-water emulsions and MPLs for their ability to stimulate an immune response.

A. Materials and Methods Experimental Design 100 6-week-old female Hartley guinea pigs from Elm Hill Breeding Laboratories (Chelmsford, Mass.) Were combined with AN1792 or their palmitoylated derivatives in combination with various adjuvants. Divided into 10 groups for immunization. Seven groups are AN1792 (unless otherwise specified) in combination with a) PBS, b) Freund's adjuvant, c) MPL, d) squalene, e) MPL / squalene, f) low dose alum or g) high dose alum (300 μg AN1792) 33 μg). Two groups received an injection of a palmitoylated derivative of AN1792 (33 μg) in combination with a) PBS or b) squalene. Finally, the tenth group received only PBS without antigen and further adjuvant. The group receiving Freund's adjuvant emulsified the first dose with CFA and the remaining 4 doses with IFA. Antigen was administered at a dose of 33 μg in all groups except the high dose alum group, and the high dose alum group received 300 μg AN1792. Injections were intramuscularly administered intraperitoneally for CFA / IFA, and alternately for the right and left hind quadriceps for all other groups. The first three doses were given on a biweekly schedule, followed by monthly intervals. In order to determine the antibody titer, blood was collected 6 to 7 days after each immunization starting after the second administration.

2. Immunogen preparation 2 mg Aβ42 (California Peptide Lot ME0339) was added to 0.9 ml deionized water and the mixture was vortex mixed to make a comparative suspension. A 100 μl aliquot of 10 × PBS (1 × PBS, 0.15 M NaCl, 0.01 M sodium phosphate, pH 7.5) was added. This suspension was vortexed again and incubated overnight at 37 ° C. for use the next day. Unused Aβ1-42 was stored with the desiccant as a lyophilized powder at -20 ° C.

  The palmitoylated derivative of AN1792 was obtained by coupling palmitic anhydride dissolved in dimethylformamide to the amino terminal residue of AN1792, and then removing the attached peptide from the resin by treatment with hydrofluoric acid. .

  To prepare an immunogenic combination dose with complete Freund's adjuvant (CFA) (group 2), 33 μg AN1792 (in 200 μl PBS) for the first immunization, 1: 1 (volume: volume) Emulsified with CFA to a final volume of 400 μl. For subsequent immunization, the antigen was similarly emulsified using incomplete Freund's adjuvant (IFA).

  For Groups 5 and 8, lyophilized powder (Liby Immunochemistry Research, Hamilton, Mass.) Is added to 0.2% aqueous triethylamine to a final concentration of 1 mg / ml to prepare a compound dose using MPL. And vortex mixed. The mixture was heated to 65-70 ° C. for 30 seconds to create a slightly opaque and uniform micelle suspension. Solutions were prepared fresh for each set of injections. For each injection in Group 5, 33 μg AN1792 (in 16.5 μl PBS), 50 μg MPL (50 μl) and 162 μl PBS were mixed in a borosilicate tube immediately prior to use.

  To prepare the formulation using a low oil-in-water emulsion, AN1792 in PBS is added to 5% squalene, 0.5% Tween80, 0.5% Span85 in PBS, and the final AN1792 at a concentration of 33 μg in 250 μl. A single dose was reached (Group 6). The mixture is transferred to a two-chambered hand-held device until the emulsion droplets are approximately the same diameter as a 1.0 μm diameter standard latex bead when viewed under a microscope. It emulsified by passing 20 times. The resulting suspension was an opaque milky white. Emulsions were freshly prepared for each series of injections. For Group 8, MPL in 0.2% triethylamine was added to the surfactant mixture for emulsification as described above at a concentration of 50 μg per dose. For palmitoylated derivatives (Group 7), 33 μg of palmitoyl-NH-Aβ-1-42 was added to squalene and vortex mixed. Tween 80 and Span 85 were then added with vortex mixing. The mixture was added to PBS to reach a final concentration of 5% squalene, 0.5% Tween 80, 0.5% Span 85, and the mixture was emulsified as described above.

  To prepare a compound dose using alum (groups 9 and 10), AN1792 in PBS was added to Alhydrogel (Aluminum Hydroxide Gel, Accurate, Westbury, NY) and 250 μl final dose A concentration of 33 μg (low dose, group 9) or 300 μg (high dose, group 10) per 5 mg alum in the dose was reached. The suspension was gently mixed for 4 hours at RT.

3. Measurement of antibody titer Guinea pigs collected blood a total of 4 times starting from the second immunization and 6 to 7 days after the immunization. Antibody titers against Aβ42 were measured by ELISA as described in General Materials and Methods.

4). Tissue preparation After approximately 14 weeks, all guinea pigs were euthanized by CO ₂ administration. Cerebrospinal fluid was collected and the brain removed and three brain regions (hippocampus, cortex and cerebellum) dissected and used to measure total Aβ protein concentration using ELISA.

B. Result 1. Antibody response Various adjuvants had a wide range of potencies when measured as antibody responses to AN1792 after immunization. As shown in FIG. 14, when AN1792 was administered in PBS, no antibody was detected after 2 or 3 immunizations and a negligible response was only about 45 geometric mean power after the 4th and 5th doses. Detected by value (GMT). The o / w emulsion induced a slight titer after 3 doses (GMT 255), which was maintained after the 4th dose (GMT 301) and dropped at the last dose (GMT 54). There was a clear antigen dose response for AN1792 bound to alum, with 300 μg more immunogenic than 33 μg at all time points. At the peak of the antibody response, after the fourth immunization, the difference between the two doses was approximately 1940 (33 μg) and 3400 (300 μg) in GMT. The antibody response to 33 μg AN1792 plus MPL was very similar to the response generated with an almost 10-fold higher dose of antigen (300 μg) bound to alum. When MPL was added to the o / w emulsion, the efficacy of the formulation was reduced by up to 75% compared to MPL as a simple adjuvant. The palmitoylated derivative of AN1792 is completely non-immunogenic when administered in PBS and has a slight 340 and 105 GMT for the third and fourth bleeds when present in the o / w emulsion. Gave the titer. The highest antibody titer was generated with Freund's adjuvant, with a peak GMT of about 87,000, and almost 30 times greater than the GMT of the next most potent formulation MPL and high dose AN1792 / alum.

  The most promising adjuvants confirmed in this experiment are MPL and alum. Of these two, MPL is preferred because the antigen dose required to produce the same antibody titer as obtained with alum is 10 times lower. The response can be increased by increasing the dose of antigen and / or adjuvant and by optimizing the immunization schedule. The o / w emulsion is a very weak adjuvant for AN1792, and the addition of the o / w emulsion to the MPL adjuvant reduced the inherent adjuvant activity of MPL alone.

2. Aβ level in the brain About 14 weeks, guinea pigs are deeply anesthetized, cerebrospinal fluid (CFS) is drawn, and alum containing Freund's adjuvant (group 2), MPL (group 5), high dose, 300 μg AN1792 Brains were removed from a subset of animals in (Group 10) and a control group (Group 3) immunized with PBS. To determine the level of Aβ peptide, one hippocampus was dissected and a hippocampal, cortical and cerebellar region homogenate was prepared with 5M guanidine. These were diluted and quantified by comparison with a series of known concentrations of dilutions of Aβ standard protein in an ELISA format. The levels of Aβ protein in the hippocampus, cortex and cerebellum were very similar in all four groups, despite a wide range of antibody responses to Aβ induced by these formulations. Average Aβ levels of about 25 ng / g tissue were measured in the hippocampus, 21 ng / g in the cortex and 12 ng / g in the cerebellum. That is, the presence of high circulating antibody titers against Aβ did not alter total Aβ levels in the brain in some of these animals for approximately 3 months. Aβ levels in CFS were also similar between groups. The lack of a significant effect of AN1792 immunization on endogenous Aβ indicates that the immune response is concentrated in the formation of the pathological state of Aβ.

VIII. Immune Response to Various Adjuvants in Mice Six week old female Swiss Webster mice were used in this experiment with 10-13 mice per group. Immunization was given subcutaneously on days 0, 14, 28, 60, 90 and 20 at a dose of 200 μl. PBS was used as a buffer for all formulations. The animals were bled 7 days after each immunization starting after the second dose for analysis of antibody titers by ELISA. The treatment plan for each group is summarized in Table 9.

For group 8, no adjuvant and antigen were included.

  The ELISA titers of antibodies against Aβ42 in each group are shown in Table 10 below.

  The table shows that the highest titers were obtained in groups 4, 5 and 18, where the adjuvant was QS-21 plus 125 μg MPL, 50 μg MPL and MPL.

IX. Therapeutic Efficacy of Various Adjuvants Therapeutic efficacy experiments are suitable adjuvants in human use to determine the ability of adjuvants to enhance immune responses against Aβ and induce immune removal of amyloid deposits in the brain. This was done with PDAPP transgenic mice.

  180 male and female 7.5-8.5 month old heterozygous PDAPP transgenic mice were obtained from Charles River Laboratories. Mice were divided into 9 groups with 15-23 animals per group immunized with AN1792 or AN1528 in combination with various adjuvants. Animals were distributed within the group as closely as possible to match gender, age and animal breed. Adjuvants included alum, MPL and QS-21, each combined with both antigens, and Freund's adjuvant (FA) combined with AN1792. An additional group was immunized with AN1792 formulated in PBS buffer with the addition of the preservative thimerosal and no adjuvant. Group 9 was immunized with PBS alone as a negative control.

Aggregated Aβ peptide preparation: human Aβ1-40 (AN1528; California Peptide, Napa, CA; lot ME0541) and human Aβ1-42 (AN1792; California Peptide; lot ME0439) peptides were dried at −20 ° C. The freshly stored lyophilized powder was freshly solubilized in the preparation of each set of injections. For this purpose, 2 mg of peptide was added to 0.9 ml of deionized water and the mixture was vortex mixed to make a relatively homogeneous solution or suspension. In contrast to AN1792, AN1528 was soluble at this stage. An aliquot of 100 μl 10 × PBS (1 × PBS: 0.15 M NaCl, 0.01 M sodium phosphate, pH 7.5) was then added, at which point AN1528 began to precipitate. The suspension was vortexed again and incubated overnight at 37 ° C. for use the next day.

  Aβ peptide in PBS to Alhydrogel (2% aqueous aluminum hydroxide gel, Sargent Inc., Clifton, NJ) to prepare compounded doses using alum (groups 1 and 5) In addition, an Aβ peptide concentration of 100 μg per mg of alum was reached. 10 × PBS was added to give a final dose of 200 μl in 1 × PBS. The suspension was then gently mixed for about 4 hours at RT before injection.

  To prepare compound doses using MPL (groups 2 and 6), lyophilized powder (Liby Immunochemistry Research, Hamilton, Mass .: Lot 67039-E0896B) was added to 0.2% aqueous triethylamine at 1 mg / ml. And vortex mixed. The mixture was heated to 65-70 ° C. for 30 seconds to create a slightly opaque and uniform micelle suspension. The solution was stored at 4 ° C. For each set of injections, 100 μg peptide per dose (in 50 μl PBS), 50 μg MPL per dose (50 μl) and 100 μl PBS per dose were mixed in a borosilicate tube immediately prior to use.

  To prepare compound doses with QS-21 (groups 3 and 7), lyophilized powder (Aquila: Aquila, Framingham, Mass .: Lot A7018R) was added to PBS, pH 6.6-6.7 to give 1 mg / Final concentration of ml and vortex mixed. The solution was stored at -20 ° C. For each set of injections, 100 μg peptide per dose (in 50 μl PBS), 25 μg QS-21 per dose (in 25 μl PBS) and 125 μl PBS per dose were mixed in a borosilicate tube just prior to use. did.

  To prepare the compound dose using Freund's adjuvant (Group 4), 100 μg AN1792 (in 200 μl PBS) for the first immunization, 1: 1 (volume: volume) and complete Freund's adjuvant (CFA) Emulsified to a final volume of 400 μl. For subsequent immunization, the antigen was similarly emulsified using incomplete Freund's adjuvant (IFA). For formulations containing adjuvant alum, MPL or QS-21, combine 100 μg AN1792 or AN1528 per dose with alum (1 mg per dose) or MPL (50 μg per dose) at a final dose of 200 μl PBS, and scapula Delivered by subcutaneous inoculation. For the group receiving FA, 100 μg AN1792 is emulsified with 1: 1 (volume: volume) complete Freund's adjuvant (CFA) to a final volume of 400 μl and delivered intraperitoneally for the first immunization. Subsequent 5 doses were boosted with the same amount of immunogen in incomplete Freund's adjuvant (IFA). For the group receiving AN1792 without adjuvant, 10 μg AN1792 was combined with 5 μg thimerosal in a final volume of 50 μl PBS and delivered subcutaneously. The ninth control group received only 200 μl of PBS delivered subcutaneously. Immunizations were given on a biweekly schedule for the first three doses, then on a monthly schedule on days 0, 16, 28, 56, 85 and 112 thereafter. The animals were bled 6-7 days after each immunization for the first time after the second dose in order to determine antibody titers. The animals were euthanized approximately 1 week after the last dose. Results were measured by ELISA assay for Aβ and APP levels in the brain and by immunohistochemical assessment of the presence of amyloid plaques in brain sections. In addition, Aβ-specific antibody titers, and Aβ-dependent proliferation and cytokine responses were determined.

Table 10 shows that the best antibodies against Aβ1-42 are induced by FA and AN1792, the titer peaks after the 4th immunization (peak GMT: 75,386) and the last 6 immunizations By sensitization, it decreased to 59%. The peak mean titer induced by MPL containing AN1792 is 62% lower than that generated with FA (peak GMT: 28,867) and also reaches the beginning of the immunization schedule after the third dose, followed by 28 peak after the 6th immunization
%. The peak mean titer (GMT: 1,511) produced by QS-21 combined with AN1792 was about 5 times lower than that obtained by MPL. Furthermore, the response kinetics were slow as further immunization was required to reach the peak response. The titer generated by alum-bound AN1792 was slightly greater than that obtained with QS-21 and the response kinetics were more rapid. For AN1792 delivered in PBS containing thimerosal, the frequency and size of titers were barely greater than PBS alone. The peak titers generated by MPL and AN1528 (peak GMT 3099) were about 9 times lower than those using AN1792. alum-conjugated AN1792 was a very bad immunogen and low titers only occurred in a few animals. There was no antibody response observed in control animals immunized with PBS alone.

The results of AN1792 or AN1528 treatment with various adjuvants or thimerosal on cortical amyloid burden in 12 month old mice determined by ELISA are shown in FIG. In PBS control PDAPP mice, the median level of total Aβ in the cortex at 12 months was 1,817 ng / g. Significant levels of Aβ reduction were observed in mice treated with AN1792 with CFA / IFA, AN1792 with alum, AN1792 with MPL and QS-21 with AN1792. The reduction reached statistical significance only at the p <0.05 level for AN1792 plus CFA / IFA. However, as shown in Examples I and III, the effect of immunization on the reduction of Aβ levels was substantially greater in 15 and 18 month mice. Thus, further formulations, particularly AN1792 with alum, AN1792 with MPL and AN1792 with QS-21, would provide a positive effect on the treatment of older mice. In contrast, AN1792, with the preservative thimerosal,
Approximately the same median level of Aβ as PBS-treated mice was shown. Similar results were obtained when comparing cortical levels of Aβ42. The median level of Aβ in the PBS control was 1624 ng / g. Significant decreases in median levels of 403, 1149, 620 and 714 were observed and decreased in AN1792 with CFA / IFA, AN1792 with alum, AN1792 with MPL and AN1792 with QS-21, respectively. Was statistically significant (p = 0.05) for the AN1792 treated group plus CFA / IFA. The median level in AN1792 thimerosal treated mice was 1619 ng / g Aβ42.

X. Toxicity analysis At the end of the experiments described in Examples II, III and VII, tissues were collected for histopathological investigations. Further hematology and clinical chemistry were performed on the final blood samples from Examples III and VI. Most of the major organs including the brain, lungs, lymph, gastrointestinal tract, liver, kidneys, thyroid and gonads were evaluated. Although sporadic damage was seen in experimental animals, there was no apparent difference in the affected tissue or severity of damage between AN1792 treated and untreated animals. There were no unique histopathologic damage recorded in AN-1528-immunized animals compared to PBS-treated or untreated animals. In Example VI, there was no difference in the clinical chemistry profile between the adjuvant group and the PBS treatment group. Although there was a significant increase in some hematological parameters between animals treated with AN1792 and Freund's adjuvant in Example VI compared to PBS-treated animals, these types of effects are expected from treatment with Freund's adjuvant And is associated with peritonitis and does not show a negative effect from AN1792 treatment. Although not part of the toxicological assessment, PDAPP mouse brain pathology was thoroughly assessed as part of the efficacy endpoint. There were no signs of adverse effects related to treatment on brain morphology in any of the experiments. These results indicate that AN1792 treatment is well tolerated and at least substantially free of side effects.

XI. Therapeutic treatment with anti-Aβ antibodies The experiments described in this chapter test the ability of various monoclonal and polyclonal antibodies to Aβ to suppress Aβ deposition in the brains of heterozygous transgenic mice. Went for.

A. Experiment 1
1. Experimental design
Six male and female heterozygous PDAPP transgenic mice, 8.5-10.5 months old, were obtained from the Charles River Laboratory. Mice were divided into 6 groups treated with various antibodies against Aβ. Animals were distributed as closely as possible to match the sex, age, pedigree and source of the animals in the group. As shown in FIG. 10, the antibodies include four mouse Aβ-specific monoclonal antibodies, 2H3 (directed to Aβ residues 1-12), 10D5 (directed to Aβ residues 1-16), 266 21F12 (directed to Aβ residues 33-42) was included (directed to Aβ residues 13-28 and binds to monomers but not aggregated AN1792). The fifth group was treated with an Aβ-specific polyclonal antibody fraction (generated by immunization with aggregated AN1792). The negative control group received only diluted PBS without antibody.

  Monoclonal antibody was injected in a volume of approximately 10 mg / kg (mouse was assumed to be 50 g). Injections were administered intraperitoneally every 7 days to maintain an average anti-Aβ titer higher than 1000. Low titers were measured because mAb266 did not bind well to the aggregated AN1792 used as capture antigen in this assay, but the same dosing schedule was maintained in this group. The group that received monoclonal antibody 2H3 was discontinued within the first 3 weeks because the antibody was cleared too rapidly in vivo. Animals were bled before each dose to determine each antibody titer. Treatment continued for a total of 196 days for 6 months. Animals were euthanized one week after the last dose.

2. Materials and methods
a. Antibody Preparation Anti-Aβ polyclonal antibodies were prepared from blood collected from two groups of animals. Group 1 consisted of 100 female Swiss Webster mice 6-8 weeks old. They were immunized with 100 μg AN1792 combined with CFA / IFA on days 0, 15 and 29. The fourth injection gave a 1/2 dose of AN1792 on day 36. Animals were bled at 42 days of sacrifice, serum was prepared, and serum was pooled to a total of 64 ml. The second group was syngeneic with PDAPP mice but consisted of 24 6-9 week old females that were non-transgenic for the human APP gene. They were immunized with 100 μg AN1792 combined with CFA / IFA on days 0, 14, 28 and 56. These animals were also bled at 63 days of sacrifice, serum was prepared, and a total of 14 ml of serum was pooled. Two lots of serum were pooled. The antibody fraction was purified using two 50% saturated ammonium sulfate precipitates in succession. The final precipitate was dialyzed into PBS and tested for endotoxin. Endotoxin levels were less than 1 EU / mg.

Anti-Aβ monoclonal antibody was prepared from ascites. The fluid was first defatted by adding concentrated dextran sulfate sodium to ice-cold ascites with stirring on ice to a final concentration of 0.238%. Concentrated CaCl ₂ was then added with stirring to a final concentration of 64 mM. The solution was centrifuged at 10, x000 g and the pellet was discarded. The supernatant was stirred on ice while adding an equal volume of saturated ammonium sulfate dropwise. The solution was again centrifuged at 10, x000 g and the supernatant was discarded. The pellet was resuspended and dialyzed against 20 mM Tris-HCl, 0.4 M NaCl, pH 7.5. This fraction was applied to a Pharmacia FPLC Sepharose Q column and eluted from a reverse gradient from 0.4 M to 0.275 M NaCl in 20 mM Tris-HCl, pH 7.5.

  Antibody peaks were identified by absorbance at 280 nm and appropriate fractions were pooled. The purified antibody preparation was measured for protein concentration using the BCA method and for purity using SDS-PAGE. This pool was also tested for endotoxin. Endotoxin levels were less than 1 EU / mg. The titer was arbitrarily assigned as a titer of 25 with a titer of less than 100.

3. Aβ and APP levels in the brain Approximately 6 months after treatment with various anti-Aβ antibody preparations, brains were removed from the animals after saline perfusion. One hemisphere was prepared for immunohistochemical analysis and the second was used for quantification of Aβ and APP levels. To measure the concentration of various forms of β-amyloid peptide and amyloid precursor protein (APP), hemispheres were dissected and homogenates of hippocampal, cortical and cerebellar regions were prepared with 5M guanidine. These were serially diluted and the levels of amyloid peptide or APP were quantified by comparison with a series of known concentrations of Aβ peptide or APP dilutions in an ELISA format.

  The levels of total Aβ and Aβ1-42 in cortical and hippocampal homogenates measured by ELISA and the levels of total Aβ in the cerebellum are shown in Tables 11, 12, and 13, respectively. The median concentration of total Aβ in the control group inoculated with PBS was 3.6 times higher in the hippocampus than in the cortex (hippocampal tissue was a median of 63,389 ng / g compared to 17,818 ng / g in the cortex). The median level (30.6 ng / g tissue) in the cerebellum of the control group was 2,000 times higher than in the hippocampus. These levels are similar to the values we have previously reported for this age heterozygous PDAPP transgenic mouse (Johnson-Wood et al., 1997).

  For the cortex, one treatment group has a median Aβ level measured as Aβ 1-42 that is significantly different from the control group (p <0.05), and these animals receive the polyclonal anti-Aβ antibody shown in Table 13 Accepted. The median level of Aβ1-42 was reduced to 65% compared to the control for this treatment group. The median level of Aβ 1-42 was reduced to 55% compared to controls in one additional treatment group, and these animals received mAb 10D5 (p = 0.0433).

In the hippocampus, the median percent reduction in total Aβ associated with treatment with polyclonal anti-Aβ antibody (50%, p = 0.0005) was not greater than that observed in the cortex (65%) (Table 14). ). However, the absolute magnitude of the decrease in the hippocampus was almost three times greater than that of the cortex, with a net decrease of 31,683 ng / g tissue in the hippocampus versus 11,658 ng / g tissue in the cortex. The reduction achieved using the polyclonal antibody was significant when measured as the level of Aβ 1-42 in the more amyloidogenic state of Aβ but not total Aβ (p = 0.0025). Median levels in the groups treated with mAbs 10D5 and 266 were reduced to 33% and 21%, respectively.

Total Aβ was also measured in the cerebellum (Table 15). Those groups that received polyclonal anti-Aβ and 266 antibodies showed a significant reduction in total Aβ levels (43% and 46%, respectively, p = 0.
0033 and p = 0.0184), and the group treated with 10D5 showed almost significant reduction (29%, p = 0.0675).

  APP concentrations were also determined by ELISA in the cortex and cerebellum from antibody-treated and control, PBS-treated mice. Two different APP assays were used. First, the assay termed APP-α / FL recognizes APP-alpha (α, a secreted form from APP cleaved within the Aβ sequence), and the full-length form (FL) of APP, while 2 The second assay recognizes APP-α only. In contrast to the treatment-related decrease in Aβ, a subset of treatment groups showed little change in APP levels in all treatments compared to control animals. These results indicate that immunization with Aβ antibody reduces Aβ without reducing APP.

  In summary, Aβ levels were significantly reduced in the cortex, hippocampus and cerebellum of animals treated with a polyclonal antibody against AN1792. To a lesser extent, monoclonal antibodies against the amino terminal region of Aβ1-42, particularly amino acids 1-16 and 13-28, also showed significant treatment effects.

4). Histochemical analysis:
The morphology of Aβ-immunoreactive plaques in a subset of brains from mice in PBS, polyclonal Aβ42, 21F12, 266 and 10D5 treated groups was compared with previous experiments following standard immunization procedures with Aβ42. Quantitative comparison was made.

The greatest change in both the extent and appearance of amyloid plaques occurred in animals immunized with polyclonal Aβ42 antibody. The reduction in amyloid burden, declining plaque morphology and cellular Aβ immunoreactivity were very similar to the effects produced by standard immunization procedures. These observations support the ELISA results in which a significant decrease in both total Aβ and Aβ42 was achieved by administration of polyclonal Aβ42 antibody.

  In a similar quantitative evaluation, the amyloid plaques in the 10D5 group also decreased in number and appearance, indicating some evidence of cellular Aβ immunoreactivity. There were no major differences when comparing the 21F12 and 266 groups with the PBS control.

5. Antibody titer measurement:
Blood was drawn from a total of 30 animals immediately before each intraperitoneal inoculation from three randomly selected subsets of mice from each group. Antibody titers were measured as Aβ1-42 binding antibodies by sandwich ELISA using plastic multi-well plates coated with Aβ1-42 as described in detail in General Materials and Methods. The average titer of each blood collection is shown in Figures 16-18 for the polyclonal and monoclonal antibodies 10D5 and 21F12, respectively. For polyclonal antibody preparations, the titer over this period averaged over about 1: 1000, and this level was slightly higher than 10D5- and 21F12-treated animals.

6). Lymphocyte proliferative response:
Aβ-dependent lymphocyte proliferation was measured using splenocytes collected 8 days after the last antibody injection. Freshly collected cells (105 / well) were cultured for 5 days in the presence of 5 μM stimulatory concentration of Aβ1-40. As a positive control, additional cells were cultured with T cell mitogen (PHA) and as a negative control, cells were cultured without the addition of peptide.

  Splenocytes from aged PDAPP mice passively immunized with various anti-Aβ antibodies were stimulated with AN1792 in vitro and proliferation and cytokine responses were measured. The purpose of these assays was to determine whether passive immunization allows antigen presentation, ie, priming a T cell response specific for AN1792. AN1792-specific proliferation or cytokine response was observed in mice passively immunized with anti-Aβ antibody.

B. Experiment 2
In the second experiment, treatment with antibody 10D5 was repeated and two additional anti-Aβ antibodies, monoclonal antibody 3D6 (Aβ _1-5 ) and 16C11 (Aβ _33-42 ) were tested. Control groups received either PBS or an irrelevant isotype matched antibody (TM2a). Mice were older than previous experiments (11.5-12 months old heterozygosity), otherwise the experimental design was the same. Again, after 6 months of treatment, 10D5 reduced plaque burden by more than 80% relative to either PBS or an isotype matched antibody control (p = 0.003). One of the other antibodies against Aβ, 3D6, was equally effective, resulting in a 86% reduction (p = 0.003). In contrast, the third antibody against the peptide, 16C11, had no effect on plaque burden. Similar findings were obtained using Aβ ₄₂ ELISA measurements. These results indicate that the antibody response to Aβ peptide is sufficient to reduce amyloid deposition in PDAPP mice in the absence of T cell immunity, but not all anti-Aβ antibodies are effective. Show. Antibodies against epitopes comprising amino acids 1-5 or 3-7 of Aβ are particularly potent.

These experiments showed that passively administered antibodies to Aβ reduce the extent of plaque deposition in a mouse model of Alzheimer's disease. When kept at moderate serum concentrations (25-70 μg / ml), the antibody gained access to the CNS at a level sufficient to decorate β-amyloid plaques. Evans blue (Evans blue) in PDAPP mice
Since the vascular permeability measured by Blue) did not increase, it was not due to abnormal leakage of the blood vessel-brain barrier. Furthermore, the antibody concentration in the brain parenchyma of aged PDAPP mice was the same as that in non-transgenic mice, representing an antibody concentration of 0.1% in serum (regardless of isotype).

C. Experiment 3: Monitoring of antibody binding To determine whether antibodies against Aβ could actually act in the CNS, brains removed from mice perfused with salt at the end of Example XII were used for the administration of peripherally administered antibodies. The existence was investigated. Non-fixed cryopreserved brain sections were exposed to a fluorescent reagent for mouse immunoglobulin (goat anti-mouse IgG-Cy3). The plaques in the 10D5 and 3D6 groups were strongly modified by the antibody, while the 16C11 group had no staining. To reveal the complete degree of plaque deposition, serial sections of each brain were first immunoreacted with anti-Aβ antibody and then reacted with a secondary reagent. After peripheral administration, 10D5 and 3D6 gained access to most plaques in the CNS. The added amount of plaque was greatly reduced in such treatment group compared to 16C11 group. These data indicate that peripherally administered antibodies can enter the CNS where they can directly trigger amyloid removal. 16C11 can also access the plaques, but it seems that they could not bind to the plaques.

XII. Prevention and Treatment of Human Individuals To determine human safety, a single dose Phase I study is performed. Therapeutic agents are administered at different dosages to different patients by starting at a level of about 0.01 where efficacy is expected and increasing by a factor of 3 until reaching a level about 10 times the effective mouse dosage. To do.

  Phase II trials are conducted to determine therapeutic efficacy. Select patients with early to intermediate Alzheimer's disease as determined using Alzheimer's disease and associated disability criteria (ADRDA) for Alzheimer's potential. Suitable patients range from 12 to 26 points in the Mini-Mental-Status Test (MMSE). Another selection criterion is that patients do not have complications such as the use of concomitant drugs that may survive and interfere with the study. Baseline assessment of patient function is performed using classic psychometrics such as ADAS, a comprehensive measure for assessing patients with MMSE and Alzheimer's disease status and function. Such a mental scale provides a measure of the progression of Alzheimer's symptoms. Appropriate qualitative life scales can also be used to monitor treatment. Disease progression can also be monitored by MRI. Patient blood profiles can also be monitored, including immunogen-specific antibodies and T-cell response assays.

  After the baseline measurement, the patient begins to receive treatment. They are randomized and treated in a blinded manner with either therapeutic agents or placebo. Patients are monitored at least every 6 months. Efficacy is determined by a significant decrease in the progression of the treatment group relative to the placebo group.

  The second phase II trial is based on early memory loss of non-Alzheimer's disease (sometimes referred to as age-related memory impairment (AAMI)) or mild cognitive impairment (MCI) to perhaps Alzheimer's disease as defined by the ADRDA criteria. To assess the conversion of Patients at high risk of conversion to Alzheimer's disease can be identified from the non-clinical group by screening the reference group for early signs of memory loss or other difficulties associated with the overall symptoms of pre-Alzheimer's disease. Select from the family of the disease, genetic risk factors, age, gender, and other characteristics known to be at high risk for Alzheimer's disease. Baseline scoring of appropriate metrics, including MMSE and ADAS, is collected along with other metrics designed to evaluate more normal groups. These patient groups are divided into appropriate groups, and placebos are compared against medication options that include drugs. These patient groups are followed at approximately 6-month intervals, and the end point of each patient is whether he or she is improving the likelihood of Alzheimer's disease as defined by the ADRDA criteria at the end of observation.

XIII. General materials and methods Antibody titer determination Mice were bled by making a small wound in the tail vein and approximately 200 μl of blood collected in a microcentrifuge tube. Guinea pigs were bled by first shaving the posterior knee region, using an 18 gage needle to injure the metatarsal vein, and collecting the blood into a microcentrifuge tube. The blood was allowed to clot at room temperature (RT) for 1 hour, vortexed and then centrifuged at 14,000 xg for 10 minutes to separate the clot from serum. The serum was then transferred to a clean microcentrifuge tube and stored at 4 ° C. until titration.

Antibody titer was measured by ELISA. A 96-well microtiter plate (Costar EIA plate) is a 100 μl solution containing 10 μg / ml of either Aβ42 or SAPP or other antigen (well coating buffer: 0.1 M sodium phosphate, pH 8.5, 0.1% sodium azide) as described in individual reports and kept overnight at RT. Wells were aspirated and serum was added to wells starting at 1/100 dilution with specimen diluent (0.014 M sodium phosphate, pH 7.4, 0.15 M NaCl, 0.6% bovine serum albumin, 0.05% thimerosal). Seven serial dilutions of three times the sample were made directly on the plate, resulting in a final dilution of 1 / 218,700. This dilution was incubated for 1 hour at RT in coated plate wells. The plates were then washed 4 times with PBS containing 0.05% Tween 20. A secondary antibody, goat anti-mouse Ig conjugated to horseradish peroxidase (obtained from Boehringer Mannheim) is added to the wells as 100 μl of a 1/3000 dilution (in specimen dilution) and 1 hour at RT. Incubated. The plate was washed again 4 times with PBS, Tween 20. To develop the chromogen, 100 μl of Slow TMB (3,3 ′, 5,5′-tetramethylbenzidine obtained from Pierce Chemicals) was added to each well and incubated for 15 minutes at RT. The reaction was stopped by adding 25 μl of 2M H ₂ SO ₄ . The color intensity was then read at the Vmax of Molecular Devices (450 nm-650 nm).

  Titers were defined as mutual dilutions of sera, giving half the maximum OD. The maximum OD was generally taken from the first 1/100 dilution except for very high titers (in which case a higher initial dilution was required to establish the maximum OD). When the 50% point fell between the two dilutions, the final titer was calculated by performing a linear extrapolation. To calculate geometric mean antibody titers, titers less than 100 were arbitrarily assigned to 25 titers.

2. Lymphocyte proliferation assay Mice were anesthetized with isoflurane. The spleen is removed and washed twice with 5 ml PBS (PBS-FBS) containing 10% heat-inactivated fetal bovine serum and the Centricon unit at 50 ° C (Daco A / S, Denmark) Homogenized in 1.5 ml PBS-FBS for 10 seconds at 100 rpm with a Medimachine (Dako), followed by filtration through a 100 micron pore size nylon mesh. Splenocytes were washed once with 15 ml PBS-FBS and then pelleted by centrifugation at 200 × g for 5 minutes. Red blood cells were lysed by resuspending the pellet in 5 mL of buffer (containing 0.15 M NH4Cl, 1 M KHCO3, 0.1 M NaEDTA, pH 7.4) for 5 min at RT. The leukocytes were then washed as described above. Freshly isolated splenocytes (10 ⁵ cells per well) were transferred to 2.05 mM L-glutamine, 1% penicillin in 96-well U-bottom tissue culture-treated microtiter plates (Corning, Cambridge, Mass.). / RPMI1640 medium supplemented with streptomycin and 10% heat-inactivated FBS (JHR Biosciences, Lenexa, KS) was prepared in triplicate and cultured at 37 ° C. for 96 hours. Various Aβ peptides, Aβ1-16, Aβ1-40, Aβ1-42 or Aβ40-1 reverse sequence protein were also added in doses ranging from 5 to 0.18 micromolar in 4 steps. Cells in control wells were cultured with Concanavalin A (ConA) (Sigma, catalog # C-5275, 1 microgram / ml) without protein addition. Cells were 3H-thymidine (Amersham Corp., Arlington, Ill.).
1 μCi / well obtained from) was applied for the last 24 hours. Cells were then harvested onto UniFilter plates and counted on a Top Count Microplate scintillation counter (Packard Instruments, Downers Grove, Ill.). Results are shown as a count of radioactivity incorporated into the insoluble polymer per minute (cpm).

4). Brain tissue preparation The brain is removed after anesthesia and one hemisphere is prepared for immunohistochemical analysis, while three brain regions (hippocampus, cortex and cerebellum) are dissected from another hemisphere and various Aβ proteins And APP form concentrations were used to measure using a specific ELISA (Johnson-Wood et al., Ibid).

  The tissue for ELISA was homogenized in 10 volumes of ice-cold guanidine buffer (5.0 M guanidine-HCl, 50 mM Tris-HCl, pH 8.0). The homogenate was mixed using an Adams Nutator (Fisher) at RT for 3-4 hours with gentle agitation and then stored at −20 ° C. prior to quantification of Aβ and APP. In previous experiments, subjects were stable under these storage conditions, and synthetic Aβ protein (Bachem) was quantitatively injected into control brain tissue homogenates derived from mouse littermates. It was recovered (Johnson-Wood et al., Ibid).

5. Measurement of Aβ level The brain homogenate was diluted 1:10 with ice-cold casein dilution (0.25% casein, PBS, 0.05% sodium azide, 20 μg / ml aprotinin, 5 mM EDTA pH 8.0, 10 μg / ml leupeptin), Centrifuged at 16,000 × g for 20 minutes at 4 ° C. Synthetic Aβ protein standards (1-42 amino acids) and APP standards were prepared to include 0.5 M guanidine and 0.1% bovine serum albumin (BSA) in the final composition. The “all” Aβ sandwich ELISA consists of a monoclonal antibody monoclonal antibody 266 specific for amino acids 13-28 of Aβ as a capture antibody and a biotinylated monoclonal antibody 3D6 specific for amino acids 1-5 of Aβ as a reporter antibody (Johnson-Wood et al. al.). The 3D6 monoclonal antibody does not recognize secreted APP or full-length APP and detects only Aβ species containing amino-terminal aspartic acid. This assay has a lower sensitivity limit of ˜50 ng / ml (11 nM) and does not show cross-reactivity with endogenous mouse Aβ protein up to a concentration of 1 ng / ml (Johnson-Wood et al., Ibid).

The Aβ1-42 specific sandwich ELISA utilizes mAβ21F12 specific for amino acids 33-42 of Aβ as a capture antibody. This assay also uses biotinylated mAβ3D6 as the reporter antibody with a lower sensitivity limit of about 125 μg / ml (28 μM, Johnson-Wood et al.). For Aβ ELISA, either 100 μl of mAβ266 (at 10 μg / ml) or mAβ21F12 (at 5 μg / ml) was coated on 96-well immunoassay plates (Costar) by incubating overnight at RT. The solution was aspirated and the wells blocked by adding 200 μl of 0.25% human serum albumin to PBS buffer for at least 1 hour at RT. The blocking solution was removed and the plates were stored dry at 4 ° C. until use. Plates were rehydrated before use with wash buffer [Tris-buffered saline (0.15M NaCl, 0.01M Tris-HCl, pH 7.5) plus 0.05% Tween20]. Samples and standards were added in triplicate aliquots of 100 μl per well and then incubated overnight at 4 ° C. Plates were washed at least three times with wash buffer between each step of the assay. Biotinylated mAβ 3D6 diluted to 0.5 μg / ml in casein assay buffer (0.25% casein, PBS, 0.05% Tween 20, pH 7.4) was added and incubated in the wells for 1 hour at RT. Avidin-horseradish peroxidase conjugate (Avidin-HRP obtained from Vector, Burlingham, Calif.) Diluted 1: 4000 in casein assay buffer was added to the wells for 1 hour at RT. Add a colorimetric substrate, Slow TMB-ELISA (Pierce),
The reaction was then allowed to proceed for 15 minutes at RT, after which the enzyme reaction was stopped by adding 25 μl of 2N H2SO4. The reaction product was quantified by measuring the Vmax of the molecular device with different absorptions at 450 nm and 650 nm.

6). Measurement of APP levels Two types of APP assays were utilized. The first is called APP-α / FL and recognizes both APP-alpha (α) and the full-length (FL) form of APP. The second assay is specific for APP-α. The APP-α / FL assay recognizes secreted APP containing the first 12 amino acids of Aβ. Since reporter antibody (2H3) is not specific for the α-clip-site, it occurs between amino acids 612-613 of APP695 (Esch et al., Science 248, 1122-1124 (1990); this assay is based on full-length APP ( In a preliminary experiment using APP antibody immobilized on the cytoplasmic tail of APP-FL to consume brain APP-FL homogenate, about 30-40 of APP-α / FL % Is FL (data not shown) The capture antibody for both APP-α / FL and APP-α assays is mAb 8E5, at amino acids 444-592 in APP695 form (Games et al., Ibid.) The reporter mAb for the APP-α / FL assay is mAb 2H3 specific for amino acids 597-608 of APP695 (Johnson-Wood et al., Ibid.) And the reporter antibody for APP-α assay is a biotinylated derivative of mAb 16H9 raised against amino acids 605 to 611 of APP under the sensitivity of APP-α / FL assay. Is approximately 11 ng / ml (150 pM) (Johnson-Wood et al.) And the lower limit of APP-α specific assay is 22 ng / ml (0.3 nM) For both APP assays, mAb 8E5 is 96-well EIA plate wells were coated as described for mAb 266. Purified recombinant secreted APP-α was used as a reference standard for APP-α and APP-α / FL assays (Esch et al., Supra). Dilute brain homogenate sample in 5M guanidine 1:10 in ELISA specimen diluent (0.014M phosphate buffer, pH7.4, 0.6% bovine serum albumin, 0.05% thimerosal, 0.5M NaCl, 0.1% NP40) They were then diluted 1: 4 with specimen diluent containing 0.5 M guanidine The diluted homogenates were then centrifuged at 16,000 × g for 15 seconds at RT The APP standards and samples were duplicated on plates. Added in aliquots and incubated at RT for 1.5 hours. Otinylated reporter antibody 2H3 or 16H9 was incubated with the sample for 1 hour at RT and streptavidin-arikariphosphatase (Boehringer Mannheim) diluted 1: 1000 in specimen diluent for 1 hour at RT in the well. Incubated. The fluorescent substrate, 4-methyl-umbelliferyl-phosphate, was added to the incubation for 30 minutes at RT, and the plate was read on a Cytofluor tm 2350 fluorimeter (Millipore) with 365 nm excitation and 450 nm emission.

7). Immunohistochemistry Brains were fixed with 4% paraformaldehyde (in PBS) for 3 days at 40 C and then stored for 1-7 days at 4 ° C. until dissection in 1% paraformaldehyde, PBS. A 40 micron thick coronal section is cut at RT on a vibratome and -20 under cryoprotection (30% glycerol, 30% ethylene glycol in phosphate buffer) before immunohistochemical treatment Stored at ° C. For each brain, 6 sections, each separated by a continuous 240 μm interval at the dorsal hippocampal level, were incubated overnight with one antibody: (1) in PBS and 1% horse serum Biotinylated anti-Aβ diluted to a concentration of 2 μg / ml (specific for mAb, 3D6, human Aβ); or (2) Specific for human APP diluted to a concentration of 3 μg / ml in PBS and 1.0% horse serum Original biotinylated mAb 8E5; or (3) glial source diluted 1: 500 in 0.25% TritonX-100 and 1% horse serum (in Tris-buffered saline, pH 7.4 (TBS)) MAb specific for fibrillary acidic protein (GFAP; Sigma Chemical); or (4) CD11b, MAC-1 antigen diluted 1: 100 with 0.25% TritonX-100 and 1% rabbit serum (in TBS) Specific mAbs; or (5) mAbs specific for the MHC II antigen diluted 1: 100 in 0.25% TritonX-100 and 1% rabbit serum (in TBS) (Family Gen); or (6) mAb (Phamingen) specific for CD43 diluted 1: 100 with 1% rabbit serum in PBS or (7) diluted 1: 100 with 1% rabbit serum in PBS Rat mAb specific for CD45RA (Famingen); or (8) Rat monoclonal Aβ specific for CD45RB (Famingen) diluted 1: 100 with 1% rabbit serum in PBS; or (9) PBS Specific to CD3e, diluted 1: 100 with rat monoclonal Aβ (Famingen) specific for CD45, diluted 1: 100 with 1% rabbit serum in; or (10) diluted 1: 100 with 1% rabbit serum in PBS Specific biotinylated polyclonal hamster Aβ (Phamingen) or (11) rat mAb specific for CD3 (Serotec) diluted 1: 200 with 1% rabbit serum in PBS; or (12) 1% normal PBS solution lacking primary antibody containing horse serum.

  The sections reacted with the antibody solutions listed in the above 1, 2, and 6-12 were pretreated with 1.0% TritonX-100, 0.4% hydrogen peroxide (in PBS) for 20 minutes at RT, and endogenous peroxidase was removed. Blocked. They were then incubated overnight with primary antibody at 4 ° C. Sections reacted with 3D6 or 8E5 or CD3e mAb were then subjected to a horseradish peroxidase-avidin-biotin-complex containing kit components “A” and “B” diluted 1:75 in PBS for 1 hour at RT. Reacted with Vector Elite Standard kit, Vector Labs, Bellingham, CA. Sections reacted with PBS solutions without antibodies specific to CD45RA, CD45RB, CD45, CD3 and primary antibody were diluted with biotinylated anti-rat IgG (vector) diluted 1:75 in PBS for 1 hour at RT. Or biotinylated anti-mouse IgG (vector) diluted 1:75 in PBS, respectively. Sections were then reacted for 1 hour at RT with horseradish peroxidase-avidin-biotin-complex containing kit components “A” and “B” diluted 1:75 in PBS (Vector Elite Standard kit, Vector Lab Bellingham, California).

  Sections were developed in 0.01% hydrogen peroxide, 0.05% 3,3′-diaminobenzidine (DAB) at RT. Sections for incubation with GFAP-, MAC-1- and MHCII-specific antibodies were pretreated with 0.6% hydrogen peroxide at RT to block endogenous peroxidase and 4 with primary antibody. Incubate overnight at ° C. The sections reacted with the GFAP antibody were reacted with biotinylated anti-mouse IgG (Vector Laboratories; Vectastain Elite ABC kit) prepared with horse diluted 1: 200 with TBS for 1 hour at RT. The sections were then reacted for 1 hour with avidin-biotin-peroxidase complex (Vector Laboratories; Vectastain Elite ABC kit) diluted 1: 1000 with TBS. Sections incubated with MAC-1 or MHCII-specific monoclonal antibodies as primary antibodies are subsequently reacted with biotinylated anti-rat IgG made in rabbits diluted 1: 200 in TBS for 1 hour at RT. This was followed by 1 hour incubation with avidin-biotin-peroxidase complex diluted 1: 1000 in TBS. Sections incubated with GFAP-, MAC-1- and MHCII-specific antibodies were then treated with 0.05% DAB, 0.01% hydrogen peroxide, 0.04% nickel chloride, TBS at RT for 4 and 11 minutes, respectively. To visualize.

  Immunolabeled sections were placed on glass slides (VWR, Superfrost slides), air dried overnight, soaked in Propar (Anatech), and covered with a coverslip using Permount as the mounting medium.

  To counterstain Aβ plaques, a subset of GFAP-positive sections were placed on Superfrost slides and incubated for 7 minutes in aqueous 1% Thioflavin S (Sigma) after immunohistochemical treatment. The sections were then dehydrated and cleaned in Propar and covered with a coverslip using Permount.

8). Image Analysis The Nikon Microphot-FX microscope connected to a CCD video camera and Sony Trinitron monitor, the Videometric 150 Image System (Oncor, Inc., Gettysburg, Md.) Was used for immunoreactive slides. Used for quantification. Section images were stored in a video buffer and the total pixel area occupied by the immunolabeled structure was selected and a color- and saturation-based threshold was determined for calculation. For each section, the hippocampus was contoured by hand and the total pixel area occupied by the hippocampus was calculated. Percent amyloid burden was measured as follows: (fraction of hippocampal region containing mAb 3D6 and immunoreactive Aβ deposits) × 100. Similarly, percent neuritis burden was measured as follows: (fraction of hippocampal region containing monoclonal antibody 8E5 and reactive dystrophic axons) × 100. Simple
32 Software A C-Imaging System (Compix, Inc., Cranberry Township, PA) operating an application program is connected to a Nikon Microphot-FX microscope via an optical camera and GFAP-positive astrocytes And used to quantitate the proportion of posterior cortical bone occupied by MAC-1- and MHCII-positive microglia. Images of the immunoreacted sections were stored in a video buffer and the total pixel area occupied by the immunolabeled cells was selected and a monochrome based threshold was determined for calculation. For each section, the posterior radial cortex (RSC) was manually outlined and the total pixel area occupied by the RSC was calculated. Percent astrocytosis was defined as follows: (fraction of RSC occupied by GFAP-reactive astrocytes) × 100. Similarly, percent microgliosis was defined as follows: (the fraction of RSC occupied by MAC-1- or MHCII-reactive microglia) x 100. For all image analyses, 6 sections (each separated by successive 240 μm intervals) at the dorsal hippocampal level were quantified for each animal. In all cases, the animal's treatment status is not known to the observer.

XIV. Ex vivo screening assay for antibody activity against amyloid deposition To investigate the effect of antibody on plaque removal, an ex vivo assay was established in which primary microglia cells were cultured with non-fixed cryopreserved sections of PDAPP mice or human AD brain. Microglial cells were obtained from cerebral cortex of newborn DBA / 2N mice (1-3 days). The cortex was mechanically dissected in HBSS ⁻ (Hanks Balanced Salt Solution, Sigma) containing 50 μg / ml DNaseI (Sigma). Dissociated cells were filtered through a 100 μm cell stainer (Falcon) and centrifuged at 1000 rpm for 5 minutes. The pellet was resuspended in growth medium (high glucose DMEM, 10% FBS, 25 ng / ml GM-CSF) and cells were seeded at a brain density of 2 per T-75 plastic culture flask. After 7-9 days, the flask was rotated on an orbital shaker at 200 rpm for 2 hours at 37 ° C. The cell suspension was centrifuged at 1000 rpm and resuspended in assay medium.

10-μm cryopreserved sections of PDAPP mice or human AD brains (post-mortem interval <3 hours) were thawed on glass coverslips coated with poly-lysine and placed in 24-well tissue culture plates. Coverslips were washed twice with assay medium consisting of H-SFM (hybridoma-serum-free medium, Gibco-BRL), 1% FBS, glutamine, penicillin / streptomycin and 5 ng / ml rmGM-CSF (R & D). . Control or anti-Aβ antibody was added at 2 × concentration (final 5 μg / ml) for 1 hour. Microglial cells were then seeded at a density of 0.8 × 10 ⁶ cells / ml assay medium. The culture was maintained in a humidified incubator (37 ° C., 5% CO ₂ ) for at least 24 hours. At the end of the incubation, the culture was fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton-X100. Sections were stained with biotinylated 3D6 followed by streptavidin / Cy3 conjugate (Jackson ImmunoResearch). Exogenous microglia cells were visualized by nuclear staining (DAPI). Cultures were observed with a reverse fluorescence microscope (Nicon, TE300) and photomicrographs were taken with a SPOT digital camera using SPOT software (Diagnostic instruments). Cultures were extracted with 8M urea, diluted 1: 1 in reduced Tricine sample buffer and loaded onto 16% Tricine gel (Novex) After transfer onto immobilon, blots were 5 μg / ml. Of pabAβ42 followed by HRP-conjugated anti-mouse antibody and developed with ECL (Amersham).

When the assay was performed on PDAPP brain sections in the presence of antibody 16C11 (one of the antibodies against Aβ, which is not effective in vivo), β-amyloid plaques remained intact and no phagocytosis was observed. In contrast, when adjacent sections were cultured in the presence of 10D5, the amyloid deposits were largely absent and the microglial cells showed numerous phagocytic vesicles containing Aβ. Identical results were obtained with AD brain sections; 10D5 induced phagocytosis of AD plaques, but 16C11 was not effective. In addition, the assay provided comparable results when performed with either mouse or human microglia on either cells and mouse, rabbit or primate antibodies against Aβ.

  Table 16 shows the results obtained with several antibodies against Aβ, comparing the ability of antibodies to induce phagocytosis in an ex vivo assay and reduce in vivo plaque burden in passive transport experiments. 16C11 and 21F12 bind with high affinity to aggregated synthetic Aβ peptides, these antibodies cannot react with β-amyloid plaques in non-fixed brain sections and cannot elicit phagocytosis in an ex vivo assay, And was not effective in vivo. 10D5, 3D6 and polyclonal antibodies against Aβ were active in all these measurements. The 22C8 antibody binds more strongly to the natural homologous form of Aβ where aspartic acid at positions 1 and 7 is replaced by iso-aspartic acid. These results indicate that in vitro efficacy is due to direct antibody-mediated removal of plaques in the CNS, and that ex vivo assays predict in vivo efficacy.

  The same assay was used to test antibody removal against a fragment of synuclein called NAC. Synuclein has been shown to be a protein associated with amyloid plaques. Antibodies to NAC are contacted as before with brain tissue samples containing amyloid plaques, microglia cells. Rabbit serum was used as a control. Subsequent monitoring showed a marked decrease in the number and size of plaques that show antibody ablation activity.

A confocal microscope was used to confirm that Aβ was internalized during the ex vivo assay. In the presence of the control antibody, exogenous microglial cells remained in the confocal plate on the tissue, there were no phagocytic vesicles containing Aβ, and the plaque remained completely in the section. In the presence of 10D5, almost all plaque material was contained in vesicles within exogenous microglial cells. To determine the fate of the internalized peptide, 10D5 treated cultures were extracted with 8M urea at various time points and examined by Western blot analysis. At 1 hour, when phagocytosis had not yet occurred, a strong 4 kD band (corresponding to Aβ peptide) was revealed by reaction with polyclonal antibody against Aβ. Aβ immunoreactivity decreased in 1 day and disappeared by 3 days. That is, the phagocytosis of antibody Aβ leads to its degradation.

  To determine if phagocytosis in the ex vivo assay is Fc-mediated, an F (ab ′) 2 fragment of anti-Aβ antibody 3D36 was prepared. Although F (ab ′) 2 fragments retain their full ability to react with plaques, they were unable to trigger phagocytosis by microglial cells. Furthermore, the phagocytosis of all antibodies was blocked by a reagent for mouse Fc receptor (anti-CD16 / 32). These data indicate that in vivo removal of Aβ occurs through Fc-receptor-mediated phagocytosis.

XV. This experiment described herein provides the ability of the antibody to pass through the brain after being injected intravenously and into the brain after being injected intravenously into peripheral tissues of normal or PDAPP mice. This was done to provide a means of measuring the concentration of antibody delivered.

  PDAPP or control normal mice were perfused with 0.9% NaCl. The brain area (hippocampus or cortex) was dissected and frozen rapidly. The brain was homogenized in 0.1% Triton + protease inhibitor. Immunoglobulin was detected in the extract by ELISA. Fab'2 goat anti-mouse IgG as a capture reagent was coated on RIA plates. Serum or brain extract was incubated for 1 hour. Isotypes were detected with anti-mouse IgG1-HRP or IgG2a-HRP or IgG2b-HRP (Caltag). Regardless of isotype, the antibody was present in the CNS at a concentration of 1: 1000 found in blood. For example, when the concentration of IgG1 is 3 times that of IgG2a in the blood, it is also present in the brain at 3 times that of IgG2a, and both are present at 0.1% of their respective blood levels. Since this result was observed in both transgenic and non-transgenic mice, PDAPP did not leak the vascular brain barrier on its own.

  Although the foregoing invention has been described in detail for purposes of clarity of understanding, it will be apparent that certain modifications may be practiced within the scope of the appended claims. All publications and patent specifications cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each was individually listed. Moreover, although not limited, the main aspects or features of the present invention are listed below.

  Embodiment 1: A pharmaceutical composition comprising an agent effective in inducing an immune response against an amyloid component in a patient and a pharmaceutical excipient.

  Aspect 2: The pharmaceutical composition according to aspect 1, wherein the amyloid component is a fibril peptide or protein.

Aspect 3: Amyloid component is serum amyloid A protein (Apo SSA), immunoglobulin light chain, immunoglobulin heavy chain, apo AI, transthyretin, lysozyme, fibrogen α chain, gelsolin, cystatin C, amyloid β protein precursor (Β-APP), β ₂ microglobulin, prion precursor protein (PrP), atrial natriuretic factor, keratin, islet amyloid polypeptide, peptide hormones and synuclein (these mutant proteins, protein fragments and proteolytic peptides The pharmaceutical composition of embodiment 2, wherein the pharmaceutical composition is derived from a fibril precursor protein selected from the group consisting of a protein or peptide consisting of:

  Aspect 4: The pharmaceutical composition according to aspect 3, wherein said agent induces an immune response directed against a new epitope formed by said fibril protein or peptide with respect to fibril precursor protein.

Aspect 5: Aspect 3 wherein said amyloid component is selected from the group consisting of AA, AL, ATTR, A apoA1, Alys, Agel, Acys, Aβ, AB ₂ M, AScr, Acal, AIAPP and synuclein-NAC fragment The pharmaceutical composition as described.

Aspect 6: The aspect according to aspect 5, wherein the agent is selected from the group consisting of AA, AL, ATTR, A ApoA1, Agel, Acys, Aβ, AB ₂ M, Ascr, Acal, AIAPP and a synuclein-NAC fragment. Pharmaceutical composition.

  Embodiment 7: The pharmaceutical composition according to embodiment 1, wherein said composition comprises an agent effective to induce an immunogenic response to at least two different amyloid components.

  Aspect 8: The pharmaceutical composition according to aspect 1, wherein the agent is a peptide linked to a carrier protein.

  Aspect 9: The pharmaceutical composition according to any of aspects 1 to 8, wherein the composition comprises an adjuvant.

  Embodiment 10: The pharmaceutical composition according to embodiment 9, wherein said adjuvant is selected from the group consisting of QS21, monophosphoryl lipid, alum and Freund's adjuvant.

  Embodiment 11: comprising administering to said subject a dosage effective to produce an immune response against an amyloid component characteristic of a disorder characterized by amyloid deposition in a mammalian subject, How to prevent or treat a disorder.

  Aspect 12: The method according to aspect 11, wherein the amyloid component is a fibrillar protein or peptide.

Aspect 13: The immune response is serum amyloid A protein (apo SSA), immunoglobulin light chain, immunoglobulin heavy chain, apo AI, transthyretin, lysozyme, fibrogen α chain, gelsolin, cystatin C, amyloid β protein precursor Body (β-APP), β ₂ microglobulin, prion precursor protein (PrP), atrial natriuretic factor, keratin, islet amyloid polypeptide, peptide hormone and synuclein (these mutant proteins, protein fragments or peptides 13. The method of embodiment 12, directed to a fibril component derived from a precursor protein selected from the group consisting of:

  Embodiment 14: The method of embodiment 13, wherein the agent induces an immune response directed against a new epitope formed by the amyloid component with respect to the precursor protein.

Aspect 15: The amyloid component is selected from the group consisting of AA, AL, ATTR, A apoA1, Alys, Agel, Acys, Aβ, AB ₂ M, AScr, Acal, AIAPP and a synuclein-NAC fragment. The method described.

Aspect 16: The aspect according to aspect 15, wherein the agent is selected from the group consisting of AA, AL, ATTR, A apoA1, Agel, Acys, Aβ, AB ₂ M, Ascr, Acal, AIAPP and a synuclein-NAC fragment. Method.

  Embodiment 17: A method according to embodiment 11, wherein said agent is effective to induce an immunogenic response to at least two different amyloid components.

  Embodiment 18: A method according to embodiment 17, wherein said administering comprises administering at least two amyloid fibril components.

  Embodiment 19: The method according to embodiment 11, wherein the agent is a peptide linked to a carrier protein.

  Embodiment 20: A method according to any of embodiments 11 to 19, wherein said administering further comprises an adjuvant.

  Embodiment 21: A method according to embodiment 20, wherein said adjuvant is selected from the group consisting of QS21, monophosphoryl lipid, alum and Freund's adjuvant.

  Embodiment 22: A method according to embodiment 11, wherein said immunological response is characterized by a serum titer of at least 1: 1000 with respect to said amyloid component.

  Embodiment 23: A method according to embodiment 22, wherein said serum titer is at least 1: 5000 for said fibril component.

  Aspect 24: The immunological response is characterized by an immunoreactive serum amount corresponding to a greater than about 4 times greater than the immunoreactive serum level measured in a control serum sample prior to treatment. The method according to embodiment 11.

  Embodiment 25: A method according to embodiment 24, wherein said immunoreactive serum amount is measured at about 100-fold serum dilution.

  Aspect 26: Sera of a patient immunoreactive comprising measuring the amount of serum of a patient immunoreactive to a selected amyloid component and having an immunoreactivity of at least 4 times the immunoreactivity of a baseline control level serum A method for determining the prognosis of a patient undergoing treatment for an amyloid disorder, wherein the amount is indicative of an improved prognosis of the condition with respect to the amyloid disorder.

  Embodiment 27: A method according to embodiment 26, wherein the serum volume of said immunoreactive patient for said selected amyloid component is characterized by a serum titer of at least about 1: 1000.

  Embodiment 28: The method according to embodiment 27, wherein the serum volume of said immunoreactive patient to said selected amyloid component is characterized by a serum titer of at least 1: 5000.

  Embodiment 29: of a disease characterized by amyloid deposits in a patient comprising administering to the patient an effective dosage of an antibody or antibody fragment that specifically binds to an amyloid component present in the patient's amyloid deposit Prevention or treatment method.

  Aspect 30: The method according to aspect 29, wherein the amyloid component is a fibril component.

  Embodiment 31: A method according to embodiment 30, wherein said antibody or antibody fragment binds to an epitope of said fibril component.

  Embodiment 32: A method according to embodiment 31, wherein the antibody or antibody fragment specifically binds to said fibril component without binding to a precursor of said fibril component.

  Embodiment 33: The method according to embodiment 30, wherein the antibody is a human antibody against said fibril component prepared from a human-derived B cell immunized with an epitope of the fibril component.

Aspect 34: The amyloid fibril component is serum amyloid A protein (apo SSA), immunoglobulin light chain, immunoglobulin heavy chain, apo AI, transthyretin, lysozyme, fibrogen α chain, gelsolin, cystatin C, amyloid β protein precursor (β-APP), β ₂ microglobulin, prion precursor protein (PrP), atrial natriuretic factor, keratin, islet amyloid polypeptide, peptide hormones and synuclein (these mutants protein, protein fragment or The method of embodiment 30, wherein the method is derived from a precursor protein selected from the group consisting of:

Aspect 35: The amyloid fibril component is selected from the group consisting of AA, AL, ATTR, A ApoA1, Alys, Agel, Acys, Aβ, AB ₂ M, AScr, Acal, AIAPP, and a synuclein-NAC fragment. 35. A method according to embodiment 34.

  Embodiment 36: A method according to embodiment 29, wherein said administering comprises administering an antibody that binds at least two amyloid fibril components.

  Aspect 37: Level in a patient with an immunoreactive serum amount to said component that is at least about 4 times higher than said serum level of immunoreactivity to said amyloid component measured in said control serum sample prior to treatment 30. A method according to aspect 29, characterized in that

  Embodiment 38: A method according to embodiment 29, wherein the antibody or antibody fragment is administered as a pharmaceutical composition with a carrier.

  Embodiment 39: A method according to embodiment 29, wherein the antibody or antibody fragment is administered intraperitoneally, orally, subcutaneously, intramuscularly, intranasally, topically or intravenously.

  Embodiment 40: A method according to embodiment 29, wherein the antibody is administered by administering to the patient a polynucleotide encoding at least one antibody chain, and the polynucleotide is expressed in the patient to produce an antibody chain.

  Embodiment 41: A method according to embodiment 40, wherein the polynucleotide encodes the heavy and light chains of the antibody, and the polynucleotide is expressed in the patient to produce the heavy and light chains.

  Embodiment 42: A method according to embodiment 29, wherein the antibody or antibody fragment is administered in multiple doses over a period of at least 6 months.

  Embodiment 43: A method according to embodiment 29, wherein the antibody is administered as a sustained release composition.

  Aspect 44: A pharmaceutical for the prevention or treatment of a disease characterized by a deposit in a patient comprising an effective dosage of an antibody or antibody fragment that specifically binds to an amyloid component present in the amyloid deposit Composition.

  Aspect 45: The pharmaceutical composition according to aspect 44, wherein the amyloid component is a fibril component.

  Embodiment 46: A pharmaceutical composition according to embodiment 45, wherein said antibody binds to an epitope of said fibril component.

  Embodiment 47: A pharmaceutical composition according to embodiment 46, wherein the antibody specifically binds to said fibril component without binding to a precursor of said fibril component.

  Embodiment 48: A pharmaceutical composition according to embodiment 46, wherein the antibody is a human antibody against said fibril component prepared from B cells from a human immunized with an epitope of the fibril component.

Aspect 49: The amyloid fibril component is serum amyloid A protein (apo SSA), immunoglobulin light chain, immunoglobulin heavy chain, apo AI, transthyretin, lysozyme, fibrogen α chain, gelsolin, cystatin C, amyloid β protein precursor (β-APP), β ₂ microglobulin, prion precursor protein (PrP), atrial natriuretic factor, keratin, islet amyloid polypeptide, peptide hormones and synuclein (these mutants protein, protein fragment or 46. The pharmaceutical composition of embodiment 45, wherein the pharmaceutical composition is derived from a precursor protein selected from the group consisting of: (including peptides).

Embodiment 50: The amyloid fibril component is selected from the group consisting of AA, AL, ATTR, A apoA1, Alys, Agel, Acys, Aβ, AB ₂ M, AScr, Acal, AIAPP and a synuclein-NAC fragment. 50. A pharmaceutical composition according to embodiment 49.

  Embodiment 51: The pharmaceutical composition according to embodiment 44, wherein said composition comprises an antibody or antibody fragment that binds at least two amyloid fibril components.

  Embodiment 52: In said patient sera immunoreactive to said component, said effective dosage is at least about 4 times higher than the serum level of immunoreactivity to said amyloid component measured in a control serum sample prior to treatment 45. A pharmaceutical composition according to embodiment 44, characterized by the amount of antibody or antibody fragment effective to produce a level.

  Embodiment 53 The pharmaceutical composition according to embodiment 44, wherein the pharmaceutical composition comprises a carrier.

  Embodiment 54: The pharmaceutical composition according to embodiment 44, wherein the pharmaceutical composition is formulated for administration intraperitoneally, orally, subcutaneously, intramuscularly, intranasally, topically, or intravenously. object.

  Embodiment 55: The pharmaceutical composition according to embodiment 44, wherein the pharmaceutical composition comprises a polynucleotide encoding at least one antibody chain effective to express the antibody chain in a patient.

  Embodiment 56: A pharmaceutical composition according to embodiment 55, wherein the polynucleotide encodes the heavy and light chains of the antibody, and the polynucleotide is capable of expression to produce heavy and light chains in the patient.

  Embodiment 57 The pharmaceutical composition according to embodiment 44, wherein said pharmaceutical composition is formulated as a sustained release composition.

  Since the present invention is effective for the prevention or treatment of diseases characterized by deposits of amyloid components in patients, it can be used, for example, in the pharmaceutical manufacturing industry.

Claims (1)

  A pharmaceutical composition comprising an agent effective to induce an immune response against an amyloid component in a patient and a pharmaceutical excipient.

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